29 high performance liquid chromatography-clarkes - cópia
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29 High Performance Liquid Chromatography
T Kupiec, M Slawson, F Pragst and M Herzler
Sub-sections
Introduction
Practical aspects of HPLC theory
Hardware
Columns
Maintenance
Eluent preparation
Separation techniques
Quantitative analysis
Validation
New emerging trends
Systems for drug analysis
References
Introduction
The ability to separate and analyse complex samples is integral to the biological and
medical sciences. Classic column chromatography has evolved over the years, with
chromatographic innovations introduced at roughly decade intervals. These techniques
offered major improvements in speed, resolving power, detection, quantification,
convenience and applicability to new sample types. The most notable of these
modifications was high performance liquid chromatography (HPLC). Modern HPLC
techniques became available in 1969; however, they were not widely accepted in the
pharmaceutical industry until several years later. Once HPLC systems capable of
quantitative analysis became commercially available, their usefulness in pharmaceutical
analysis was fully appreciated.
By the 1990s, HPLC had begun an explosive growth that made it the most popular
analytical method judged according to sales of instruments and also scientific
importance. Its present popularity results from its convenient separation of a wide range
of sample types, exceptional resolving power, speed and nanomolar detection levels. It
is presently used in pharmaceutical research and development:
To purify synthetic or natural products. To characterise metabolites. To assay active ingredients, impurities, degradation products and in dissolution
assays. In pharmacodynamic and pharmacokinetic studies.
Improvements made in HPLC in recent years include:
Changes in packing material, such as smaller particle size, new packing and column materials.
High–speed separation. Micro-HPLC, automation and computer–assisted optimisation. Improvements in detection methods, including the so–called hyphenated
detection systems.
These innovations will be discussed in the appropriate sections of this chapter.
Practical aspects of HPLC theory
Sub-sections
Chromatographic principles
Chromatographic mechanisms
The practical application of HPLC is aided by an awareness of the concepts of
chromatographic theory, in particular the measurement of chromatographic retention
and the factors that influence resolution.
Chromatographic principles
The retention of a drug with a given packing material and eluent can be expressed as a
retention time or retention volume, but both of these are dependent on flow rate, column
length and column diameter. The retention is best described as a column capacity ratio
(k), which is independent of these factors. The column capacity ratio of a compound (A)
is defined by Equation (29.1):
where VA is the elution volume of Aand V0 is the elution volume of a non–retained
compound (i.e. void volume). At constant flow rate, retention times (tA and t0) can be
used instead of retention volumes. The injection of a solvent or salt solution can be used
to measure V0, but the solute used should always be recorded along with reported k data.
The importance of selecting suitable solutes for the measurement of V0 has been
discussed (Wells and Clark 1981).
It is sometimes convenient to express retention data relative to a known internal
standard (B). The ratio of retention times (tA/tB) can be used, but the ratio of adjusted
retention times, (tA – t0)/(tB – t0), is better when data need to be transferred between
different chromatographs (Ettre 1980).
Resolution is the parameter that describes the separation power of the complete
chromatographic system relative to the particular components of the mixture. By
convention, resolution (R) is expressed as the ratio of the distance between two peak
maxima to the mean value of the peak width at the base line, Equation (29.2):
If we approximate peaks by symmetric triangles, then if R is equal to or more than 1, the
components are completely separated. If R is less than 1, the components overlap.
Sensitivity in chromatographic analysis is a measure of the smallest detectable level of a
component in a chromatographic separation and is dependent on the signal–to–noise
ratio in a given detector. Sensitivity can be increased by derivatisation of the compound
of interest, optimisation of chromatographic system or miniaturisation of the system.
The limit of detection is normally taken as three times the signal–to–noise ratio and the
limit of quantification as ten times this ratio.
Chromatographic mechanisms
The systems used in chromatography are often described as belonging to one of four
mechanistic types: adsorption, partition, ion exchange and size exclusion. Adsorption
chromatography arises from interactions between solutes and the surface of the solid
stationary phase. Generally, the eluents used for adsorption chromatography are less
polar than the stationary phases and such systems are described as ‘normal phase’.
Partition chromatography involves a liquid stationary phase that is immiscible with the
eluent and coated on an inert support. Partition systems can be normal phase (stationary
phase more polar than eluent) or reversed–phase chromatography, referred to as RPC
(stationary phase less polar than eluent).Ion–exchange chromatography involves a solid
stationary phase with anionic or cationic groups on the surface to which solute
molecules of opposite charge are attracted. Size–exclusion chromatography involves a
solid stationary phase with controlled pore size. Solutes are separated according to their
molecular size, with the large molecules unable to enter the pores elute first. However,
this concept of four separation modes is an over–simplification. In reality, there are no
distinct boundaries and several different mechanisms often operate simultaneously.
Other types of chromatographic separation have been described. Ion–pair
chromatography is an alternative to ion–exchange chromatography. It involves the
addition of an organic ionic substance to the mobile phase, which forms an ion pair with
the sample component of opposite charge. This allows a reversed–phase system to be
used to separate ionic compounds. Chiral chromatography is a method used to separate
enantiomers, which can be achieved by various means. In one case, the mobile phase is
chiral and the stationary phase is non–chiral. In another, the liquid stationary phase is
chiral with the mobile phase non–chiral or, finally, the solid stationary phase may be
chiral with a non–chiral mobile phase.
Hardware
Sub-sections
Mobile phase reservoir
Pumps
Injectors
Thermostats
Sub-sections
Column switches
Detectors
Data systems
HPLC instrumentation includes a pump, injector, column, detector and recorder or data
system (Fig. 29.1). The heart of the system is the column in which separation occurs.
Since the stationary phase is composed of micrometer–size porous particles, a high–
pressure pump is required to move the mobile phase through the column. The
chromatographic process begins by injecting the solute onto the top of the column.
Separation of components occurs as the analytes and mobile phase are pumped through
the column. Eventually, each component elutes from the column and is registered as a
peak on the recorder. Detection of the eluting components is important; this can be
either selective or universal, depending upon the detector used. The response of the
detector to each component is displayed on a chart recorder or computer screen and is
known as a chromatogram. To collect, store and analyse the chromatographic data,
computers, integrators and other data–processing equipment are used frequently.
Figure 29.1.
Figure 29.1. Typical HPLC system.
Mobile phase reservoir
The most common type of solvent reservoir is a glass bottle. Most of the manufacturers
supply these bottles with special caps, Teflon tubing and filters to connect to the pump
inlet and to the sparge gas (helium) used to remove dissolved air. When the mobile
phase contains excessive gas that remains dissolved at the pressure produced by the
column, the gas may come out of the solution at the column exit or in the detector,
which results in sharp spikes. Spikes are created by microscopic bubbles that change the
nature of the flowing stream to make it heterogeneous, while drift may occur as these
microscopic bubbles gradually collect and combine in the detector cell. The main culprit
is oxygen (from the air) that dissolves in polar solvents, particularly water. Degassing
may be accomplished by one or a combination of the following methods: apply a
vacuum to the liquid, boil the liquid, place the liquid in an ultrasonic bath, bubble a fine
stream of helium through the liquid (sparging) or by commercial on–line degassing
units
Pumps
High–pressure pumps are needed to force solvents through packed stationary phase
beds. Smaller bed particles (e.g. 3 μm) require higher pressures. There are many
advantages to using smaller particles, but they may not be essential for all separations.
The most important advantages are higher resolution, faster analyses and increased
sample load capacity. However, only the most demanding separations require these
advances in significant amounts. Many separation problems can be resolved with larger
particle packings (e.g. 5 μm) that require less pressure.
Flow–rate stability is another important pump feature that distinguishes pumps.
Constant–flow systems are generally of two basic types: reciprocating piston and
positive displacement (syringe) pumps. The basic advantage of both systems are their
ability to repeat elution volume and peak area, regardless of viscosity changes or
column blockage, up to the pressure limit of the pump. Although syringe–type pumps
have a pressure capability of up to 540 000 kPa (78 000 psi), they have a limited ability
to form gradients. Reciprocating piston pumps can maintain a liquid flow for an
indefinite length of time, while a syringe pump needs to be refilled after the syringe
volume has been displaced. Dual–headed reciprocating piston pumps provide more
reproducible and pulse–free delivery of solvent, which reduces detector noise and
enables more reliable integration of peak area. Reciprocating pumps now dominate the
HPLC market and are even useful for micro-HPLC applications, as they can maintain a
constant flow at flow rates in μL/min ranges.
An additional pump feature found on the more elaborate pumps is external electronic
control. Although it adds to the expense of the pump, external electronic control is a
very desirable feature when automation or electronically controlled gradients are to be
run. Alternatively, this becomes unnecessary when using isocratic methods. The degree
of flow control also varies with pump expense. More expensive pumps include such
state–of–the–art technology as electronic feedback and multiheaded configurations.
Modern pumps have the following parameters:
Flow–rate range, 0.01 to 10 mL/min. Flow–rate stability, not more than 1% (short term). For size exclusion chromatography (SEC), flow–rate stability should be <0.2%. Maximum pressure, up to 34 500 kPa (5000 psi).
Injectors
An injector for an HPLC system should provide injection of the liquid sample within
the range of 0.1 to 100 mL of volume with high reproducibility and under high pressure
(up to 27 600 kPa). The injector should also minimise disturbances to the flow of the
mobile phase and produce minimum band broadening. Sample introduction can be
accomplished in various ways. The injection valve has, in most cases, replaced syringe
injection. Valve injection offers rapid, reproducible and essentially operator–
independent delivery of a wide range of sample volumes. The most common valve is a
six–port Rheodyne valve in which the sample fills an external stainless steel loop. A
clockwise turn of the valve rotor places the sample–filled loop into the mobile–phase
stream, which deposits the sample onto the top of the column. These valves can be
operated manually or actuated via computer–automated systems. One minor
disadvantage of valve injection is that the sample loop must be changed to obtain
various sample volumes. However, this is a simple procedure that requires a few
minutes only. In more sophisticated HPLC systems, automatic sampling devices are
incorporated. These autosamplers have a piston–metering syringe–type pump to suck
the preset sample volume into a line and transfer it to a sample loop of adequate size in
a standard six–port valve. Most autosamplers are computer controlled and can serve as
the master controller for the whole system.
In HPLC, liquid samples may be injected directly and solid samples need only be
dissolved in an appropriate solvent. The solvent need not be the mobile phase, but
frequently it is wise to choose the mobile phase to avoid detector interference, column–
component interference, loss in efficiency or all of these. It is always best to remove
particles from the sample by filtration or centrifugation, since continuous injections of
particulate material eventually cause blockage of injection devices or columns.
Sample sizes may vary widely. The availability of highly sensitive detectors frequently
allows the use of small samples that yield the highest column performance.
Thermostats
It often is advantageous to run ion exchange, size–exclusion and reversed–phase
columns above room temperature and to control precisely the temperature of liquid–
liquid columns. Therefore, column thermostats are a desirable feature in modern HPLC
instruments. Temperature variation within the HPLC column should generally be held
within ±0.2°. To maintain a constant temperature is especially important in quantitative
analysis, since changes in temperature can seriously affect peak–size measurement. It is
often important to be able to work at higher temperatures for size–exclusion
chromatography of some synthetic polymers because of solubility problems. High–
velocity circulating air baths, which usually consist of high–velocity air blowers plus
electronically controlled thermostats, are the most convenient for HPLC. Alternatively,
HPLC columns can be jacketted and the temperature controlled by contact heaters or by
circulating fluid from a constant–temperature bath. This latter approach is practical for
routine analyses, but is less convenient when columns must be changed frequently
Column switches
These valve devices are used to divert the flow from one column to another within a
single HPLC system. Column–switching techniques can be used during method
development when several columns are to be evaluated for their efficiency, retention,
etc. More recently, the use of column switching has been employed in the on–line
analysis of biological matrices. Raw plasma or other sample matrix is injected directly
onto the first column. Chromatographic conditions are optimised such that interfering
substances are eluted from the column while the analytes of interest are retained. The
column switch then diverts the eluent that contains the analytes of interest from the
‘clean–up column’ onto the analytical column, which then separates the analytes of
interest for quantification or characterisation. Another use of column switches is in
gradient chromatography for which high throughput is essential. The first column is
switched off–line to re–equilibrate to initial conditions, while the second column is
brought on–line for the next injection. This conserves valuable analysis time that would
otherwise be wasted waiting for the column to re–equilibrate. The most up–to–date
information on the use of column switching can be found by searching the current
literature.
Detectors
Today, optical detectors are used most frequently in HPLC systems. These detectors
pass a beam of light through the flowing column effluent as it passes through a flow–
cell. Flow–cells are available in preparative, analytical and micro–analytical sizes The
variations in light intensity, caused by ultraviolet (UV) absorption, fluorescence
emission or change in refractive index (depending on the type of detector used) from the
sample components that pass through the cell, are monitored as changes in the output
voltage. These voltage changes are recorded on a strip–chart recorder and frequently are
fed into an integrator or computer to provide retention time and peak–area data.
Most applications in drug analysis use detectors that respond to the absorption of UV
radiation (or visible light) by the solute as it passes through the flow–cell. Absorption
changes are proportional to concentration, following the Beer–Lambert Law. Flow–cells
generally have path–lengths of 5 to 10 mm with volumes between 5 and 10 μL. These
detectors give good sensitivities with many compounds, are not affected by slight
fluctuations in flow rate and temperature, and are non–destructive, which allows solutes
to be collected and further analysed if desired.
The simplest detectors are of the fixed–wavelength type and usually contain low–
pressure mercury lamps that have an intense emission line at 254 nm. Some instruments
offer conversion kits that allow the energy at 254 nm to excite a suitable phosphor to
give a new detection wavelength (e.g. 280 nm). Variable–wavelength detectors have a
deuterium lamp with a continuous emission from 180 to 400 nm and use a manually
operated diffraction grating to select the required wavelength. Tungsten lamps (400 to
700 nm) are used for the visible region.
Many organic compounds absorb at 254 nm and hence a fixed–wavelength detector has
many uses. However, a variable–wavelength detector can be invaluable to increase the
sensitivity of detection by using the wavelength of maximum absorption. This is
particularly useful when analysing proteins that absorb at 280 nm, or peptides that are
detected commonly at 215 nm. Using a variable–wavelength detector can also increase
the selectivity of detection by enhancing the peak of interest relative to interfering
peaks.
Eluents must have sufficient transparency at the selected detection wavelength. Buffer
salts can also limit transparency. The spectra of some drugs change with pH and the
sensitivity and selectivity of an assay can sometimes be controlled by changing the
eluent pH. The influence of such changes on the chromatography must also be
considered.
Other detectors commonly used include diode array, refractive index (RI), fluorescence
(FL), electrochemical (EC) and mass spectrometry (MS). Infra–red (IR) and nuclear
magnetic resonance (NMR) spectrometers may also be used as detectors.
Photodiode array detectors
The photodiode array detector (DAD) is an advanced type of UV detector. Depending
on the wavelength, a tungsten lamp and a deuterium lamp are used as light sources. The
polychromatic light beam is focused on a flow–cell (volume 8 to 13 μL) and
subsequently dispersed by a holographic grating or quartz prism. The spectral light then
reaches a chip that contains 100 to 1000 light–sensitive diodes arranged side by side.
Each diode only registers a well–defined fraction of the information and in this way all
wavelengths are measured at the same time. Note that although having more diodes in
an array increases the resolution of UV spectra, it lowers the absolute sensitivity since
less radiation is absorbed by each individual diode. The wavelength resolution of up–
to–date detectors is of the order of 1 nm per diode, with a wavelength accuracy of better
than ±1 nm and a sensitivity below 10−4 absorbency units. All operations of the detector
are controlled by a computer: correction of fluctuations of the lamp energy, collection of
signals (Iλ) from all the diodes, storage of the data of the mobile phase (I0λ, measured at
the start of the chromatogram) and calculation of the absorbance according to the Beer–
Lambert Law from Iλ to I0λ. The number of spectra recorded per second can be chosen
from between 0.1 and 10; usually one spectrum/sec is optimum with respect to
chromatographic resolution and noise. At the end of the run, a three–dimensional
spectrochromatogram (absorbance as a function of wavelength and time) is stored on
the computer and can be evaluated qualitatively and quantitatively. A detailed
description of the DAD operation is given in Huber and George (1993).
Diode array detection offers several advantages. Knowledge of the spectra of
compounds of interest enables interfering peaks to be eliminated such that an accurate
quantification of peaks of interest can be achieved despite less than optimal resolution.
Simultaneous detection at two wavelengths allows calculation of an absorbance ratio. If
this ratio is not constant across a peak, the peak is not pure, regardless of its appearance.
An additional advantage of diode array detection is the subtraction of a reference
wavelength. This reduces baseline drift during gradient elution. HPLC–DAD systems
linked to libraries of UV spectra are particularly useful in clinical and forensic
toxicology in screening for drugs in biological samples and its use in this context is
described in detail in later (Pragst and Herzler, personal communication).
Refractive index detector
The RI detector is a universal detector, in that changes in RI (either positive or negative)
that arise from the presence of a compound in the eluent are recorded. However, it is
also the least–sensitive detector (as much as 100 times less sensitive than UV detection).
RI detectors may be used for excipients such as sugars in pharmaceuticals. Many factors
influence RI and must be controlled during separation, such as temperature, eluent
composition and pressure. The chromatography is best facilitated using a
thermostatically controlled cabinet and high–quality pump to minimise pressure
fluctuations.
Fluorescence detector
In FL detectors, the solute is excited with UV radiation and emits radiation at a longer
wavelength. Most detectors allow the selection of both excitation and emission
wavelengths. There are only a few drugs and natural compounds that have strong
natural fluorescence (e.g. ergot alkaloids), however, many drug derivatives are
fluorescent compounds. FL detection can offer great selectivity, since excitation and
emission wavelengths as well as retention time can be used to identify drugs. It is
necessary to choose eluents carefully when using FL detection. The eluent must neither
fluoresce nor absorb at the chosen wavelengths. It is also necessary to consider the pH
of the system, in that some drugs only show fluorescence in certain ionic forms.
Electrochemical detectors
EC detectors measure the current that results from the electrolytic oxidation or reduction
of analytes at the surface of an electrode. These detectors are quite sensitive (down to
10–15 mole) and also quite selective. Two types of detector are available. The
coulometric detector has a large electrode surface at which the electrochemical reaction
is taken to completion. The amperometric detector has a small electrode with a low
degree of conversion. Despite the difference in conversion rate, in practice these two
types have approximately the same sensitivity. Eluents for EC detection must be
electrically conductive. This is accomplished by the addition of inert electrolytes. EC
detection is most easily used in the oxidative mode, as use in the reductive mode
requires the removal of dissolved oxygen from the eluent.
Hyphenated techniques
The recent development of the so–called hyphenated techniques has improved the
ability to separate and identify multiple entities within a mixture. These techniques
include HPLC–MS, HPLC–MS–MS, HPLC–IR and HPLC–NMR. These techniques
usually involve chromatographic separation followed by peak identification with a
traditional detector such as UV, combined with further identification of the compound
with the MS, IR or NMR spectrometer.
MS as a detector for an HPLC system has gained wide popularity over the past several
years. Advances in data systems and the simplification of the user interface have
facilitated the ease of use of a mass spectrometer as an HPLC detector. The most
common types of mass spectrometers used in HPLC are quadrupoles and ion traps.
Tandem mass spectrometers (also called triple quadrupoles) are also commonly
available and are widely used in the pharmaceutical industry for the quantitative
analysis of trace concentrations of drug molecules.
The process of mass analysis is essentially the same as in any other mass spectrometric
analyses that utilise quadrupole or ion–trap technology. The unique challenge to
interfacing an HPLC to a mass spectrometer is the need to convert a liquid–phase eluent
into a gas phase suitable for mass spectral analysis. Modern mass spectrometers
commonly utilise a technique known as atmospheric pressure ionisation (API) to
accomplish this. API can be subdivided into electrospray (ionspray) ionisation (ESI)
and atmospheric pressure chemical ionisation (APCI). Each technique has its own
advantages. ESI is particularly useful for the analysis of a wide variety of compounds,
especially proteins and peptides. APCI is also very well suited for the analysis of a large
variety of compounds, particularly the less polar organic molecules. Both techniques are
very rugged and well suited to pharmaceutical analysis.
An important consideration when using API is the need for volatile mobile–phase
modifiers in the chromatographic separation. Acetic acid, formic acid, etc., are
commonly used as acidic modifiers. Ammonium formate and ammonium acetate salts
can also be used when more pH control is required for the separation. Organic modifiers
are most often methanol or acetonitrile. One very important issue that must be
considered when developing a method using API (electrospray, in particular) is the
phenomenon of ion suppression. Co–eluting contaminants compete with the analyte of
interest for ionisation, which results in a loss of signal for the analyte of interest. This
can be very problematic if extremely small quantities of analyte are to be measured (as
is often the case when MS is being used). Additional sample cleanup or adjustment of
the chromatography to prevent coelution of the contaminant is often necessary to correct
this problem.
HPLC–MS–MS is commonly used in the pharmaceutical industry and in forensic
science to analyse trace concentrations of drug and/or metabolite. MS–MS offers the
advantage of increased signal–to–noise ratio, which in turn lowers the limits of
detection and quantification easily into the sub ng/mL range. MS–MS is also a very
useful technique in the qualitative identification of previously unidentified metabolites
of drugs, which thus makes MS–MS a very powerful technique in research laboratories.
Several recently published studies have utilised MS–MS as a high–throughput
analytical technique in the pharmaceutical industry.
HPLC–IR has proved to be an effective method to detect degradation products in
pharmaceuticals. IR provides spectral information that can be used for compound
identification or structural analysis. The IR spectra obtained after HPLC separation and
IR analysis can be compared to the thousands of spectra available in spectral libraries to
identify compounds, metabolites and degradation products. An advantage of IR
spectroscopy is its ability to identify different isomeric forms of a compound based on
the different spectra that result from alternative locations of a functional group on the
compound. Unlike MS, IR is a non–destructive technique in which the original
compound is deposited on a plate as pure, dry crystals and can be collected afterwards if
desired.
HPLC–NMR is also growing in popularity for the identification of various components
in natural products and other disciplines. Although a relatively new hyphenated system,
HPLC–NMR has several applications on the horizon. The miniaturisation of the system
and the possibility of measuring picomole amounts of material are both areas currently
attracting a large amount of attention. Also, in the future HPLC–NMR systems will be
interfaced with other detectors, such as Fourier transform IR and mass spectrometers.
This will provide a wide range of possibilities for further applications, which could
include the analysis of mixtures of polymer additives and the ability to identify
unknowns without first having to isolate them in a pure form.
Data systems
Since the detector signal is electronic, use of modern data–acquisition techniques can
aid in the signal analysis. In addition, some systems can store data in a retrievable form
for highly sophisticated computer analysis at a later time.
The main goal in using electronic data systems is to increase analysis accuracy and
precision, while reducing operator attention. There are several types of data systems,
each of which differ in terms of available features. In routine analysis, where no
automation (in terms of data management or process control) is needed, a pre–
programmed computing integrator may be sufficient. If higher control levels are
desired, a more intelligent device is necessary, such as a data station or minicomputer.
The advantages of intelligent processors in chromatographs are found in several areas.
Firstly, additional automation options become easier to implement. Secondly, complex
data analysis becomes more feasible. These analysis options include such features as
run–parameter optimisation and deconvolution (i.e. resolution) of overlapping peaks.
Finally, software safeguards can be designed to reduce accidental misuse of the system.
For example, the controller can be set to limit the rate of solvent switching. This acts to
extend column life by reducing thermal and chemical shocks. In general, these stand–
alone, user–programmable systems are becoming less expensive and increasingly
practical.
Other more advanced features can also be applied to a chromatographic system. These
include computer–controlled automatic injectors, multi–pump gradient controllers and
sample fraction collectors. These added features are not found on many systems, but
they do exist, and can save much time and effort for the chromatographer
Columns
Sub-sections
Column dimensions
Packing materials
Sub-sections
Zirconia packing materials
Polymer–based packing materials
Monolithic columns
Typical HPLC columns are 10, 15 and 25 cm in length and are fitted with extremely
small diameter (3, 5 or 10 μm) particles. The columns may be made of stainless steel,
glass–lined stainless steel or polyetheretherketone (PEEK). The internal diameter of the
columns is usually 4.0 or 4.6 mm for traditional detection systems (UV, FL, etc.); this is
considered the best compromise between sample capacity, mobile phase consumption,
speed and resolution. However, if pure substances are to be collected (preparative
scale), larger diameter columns may be needed. Smaller diameter columns (2.1 mm or
less) are often used when HPLC is coupled with MS. The smaller diameter columns
also have the advantage of consuming less solvent because of their lower optimal flow
rates. HPLC systems sold today can often be plumbed with narrower tubing diameters
to take advantage of the benefits of these smaller column diameters.
Packed capillary microcolumns are also gaining wider use when interfacing the HPLC
to a mass spectrometer and extremely low flow rates (nL/min) are needed to maximise
sensitivity for the analysis of proteins and peptides.
Packing of the column tubing with small diameter particles requires high skill and
specialised equipment. For this reason, it is generally recommended that all but the most
experienced chromatographers purchase pre–packed columns, since it is difficult to
match the high performance of professionally packed HPLC columns without a large
investment in time and equipment.
In general, HPLC columns are fairly durable and one can expect a long service life
unless they are used in some manner that is intrinsically destructive, such as with highly
acidic or basic eluents, or with continual injections of ‘dirty’ biological or crude
samples. It is wise to inject some test mixture (under fixed conditions) into a column
when new and to retain the chromatogram. If questionable results are obtained later the
test mixture can be injected again under specified conditions. The two chromatograms
are compared to establish whether or not the column is still useful.
Column dimensions
The description of column dimensions and assignment of a category to that size varies
greatly depending on the reference cited. The following categories were suggested by
Rozing et al. (2001), and may be more stratified than other categories.
Preparative
Preparative columns generally are larger bore than analytical columns. Some have inner
diameters as large as 100 mm and may have lengths up to 600 mm. These columns are
usually packed with packing materials of larger particle size that may range from 10 to
50 μm particle size. The flow rate used with these columns normally exceeds 5 mL/min.
Normal bore
The normal bore for an analytical column can range from 3.9 mm to 5.0 mm inner
diameter, but the most common is 4.6 mm. This diameter is the best compromise
between sample capacity, mobile phase consumption, speed and resolution. The normal
flow rate for this type of column is 1.5 to 5 mL/min.
Minibore
A mini or narrow bore column has an inner diameter of 2.1 mm to 3.9 mm. The flow
rate for this column size ranges from 500 to 1500 μL/min.
Microbore
Microbore columns have a 1.0 mm to 2.1 mm inner diameter and have flow rates of 100
to 500 μL/min. These small columns save solvent, are popular when HPLC is interfaced
with MS and provide increased sensitivity in situations of limited sample mass.
Capillary
Capillary columns have inner diameters of 50 μm to 1.0 mm and have a typical flow
rate of 0.2 to 100 μL/min. So–called ‘nanobore’ columns usually fall into the lower end
of this size range. The inner surface of these very narrow columns must be extremely
smooth. Since this is difficult to obtain with stainless steel columns, many of these
columns are glass–lined stainless steel. Fused silica columns also fall into this category.
Packing materials
Silica–based packing materials
Silica (SiO2,xH2O) is the most widely used substance for the manufacture of packing
materials. It consists of a network of siloxane linkages (Si–O–Si) in a rigid three–
dimensional structure that contains interconnecting pores. The size of the pores and the
concentration of silanol groups (Si–OH), which line the pores, can be controlled in the
manufacturing process. Thus, a wide range of commercial products is available with
surface areas that range from 100 to 800 m2/g and average pore sizes from 4 to 33 nm.
Spherical packing materials are now the only types being introduced for analytical
HPLC. Irregular shaped materials are still being used to pack preparative columns. The
silanol groups on the surface of silica give it a polar character, which is exploited in
adsorption chromatography using organic eluents. Silanol groups are also slightly acidic
and hence basic compounds are adsorbed particularly strongly. Unmodified silicas can
thus be used with aqueous eluents for the chromatography of basic drugs.
Silica can be altered drastically by reaction with organochlorosilanes or
organoalkoxysilanes to give Si–O–Si–R linkages with the surface. The attachment of
hydrocarbon chains to silica produces a non–polar surface suitable for RPC in which
mixtures of water and organic solvents are used as eluents. The most popular material is
octadecylsilica (ODS), which contains C18 chains, but materials with C1, C2, C4, C6, C8
and C22 chains are also available. The latest silica–based bonded phase to be introduced
is a long C30 phase, which has 24% carbon coverage to make it one of the most retentive
phases available.
During manufacture, such materials may be reacted with a small monofunctional silane
(e.g. trimethylchlorosilane) to reduce further the number of silanol groups that remain
on the surface (endcapping). Recent advances in column technology include multiple
reactant endcapping, use of Type B (high purity, low trace metal, low acidity) silica and
encapsulating the surface with a polymeric phase. These silicas are often referred to as
‘base–deactivated’ and are especially useful in RPC in the pH range of 4 to 8 when
many basic compounds are partially ionised. Variations in elution order on different
commercial packing materials of the same type (e.g. ODS) are often attributed to
differences in surface coverage and the presence of residual silanol groups. For this
reason it must not be assumed that a method developed with one manufacturer’s ODS
column can be transferred easily to another manufacturer’s ODS column.
Speciality silicasA vast range of materials have intermediate surface polarities that arise from the
bonding to silica of organic compounds that contain groups such as phenyl, cyano,
nitro, amino, fluoro, sulfono and diols. There are also miscellaneous chemical moieties
bound to silica, as well as polymeric packings, designed to purify specific compounds.
PhenylPropylphenylsilane ligands attached to the silica gel show weak dipole–induced dipole
interactions with polar analytes. Usually this type of bonded phase is used for group
separations of complex mixtures. Newer phases have phenyl backbones that allow π–π
(stacking) interactions. These are recommended for peptide mapping applications.
Amino–compounds show some specific interactions with phenyl–modified adsorbents.
CyanoA cyano–modified surface is very slightly polar. Columns with this phase are useful for
fast separations of mixtures that consist of very different components. These mixtures
may show a very broad range of retention times on the usual columns.
Cyano–columns can be used on both normal- and reversed–phase modes of HPLC.
AminoAmino–phases are weak anion–exchangers. This type of column is mainly used in
normal–phase mode, especially for protein separation and also the selective retention of
aromatic compounds.
FluoroA newer type of silica packing has fluorinated surfaces. This phase is generally more
hydrophilic than phases with hydrocarbons of similar chain length. It has increased
retention and unique selectivity for halogenated organic compounds and lipophilic
compounds.
SulfonoSulfonic functional groups separate compounds on the basis of hydrophobic
interactions. These packing materials allow the isocratic separation of mixtures that
normally require gradient elution.
DiolsDiols are slightly polar adsorbents for normal–phase separations. These are useful to
separate complex mixtures of compounds with different polarities that usually have a
strong retention on unmodified silica.
MiscellaneousCyclodextrins, amylose, avidin, ristocetin, nitrophenylethyl, carbamate, ester,
diphenylethyldiamine and Pirkle–type functional groups are all bound to silica packing
material to enable enantiomeric separations. These columns are often referred to as
chiral columns. Strong ion–exchangers are also available, in which sulfonic acid groups
or quaternary ammonium groups are bonded to silica. These packing materials are
useful to separate proteins. There are also proprietary functional groups added to silica
packing materials for a variety of uses. These include petrochemical analysis,
environmental analysis, detection of deoxyribose nucleic acid (DNA) adducts,
purification of double stranded DNA, separation of cationic polymers and separation of
nitro–aromatic explosives.
For size–exclusion chromatography, a special type of silica is available that has a
narrow range of pore diameters. Size–exclusion chromatography can be complicated by
adsorption, but this can be reduced by treating the surface with trimethylchlorosilane.
pH rangeThe useful pH range for silica-based columns is 2 to 8, since siloxane linkages are
cleaved below pH 2 while at pH values above 8 silica may dissolve. However, the pH
range may be extended above 8 if a precolumn packed with microparticulate silica is
included between the pump and injector to saturate the eluent before it enters the
analytical column.
Zirconia packing materials
Zirconia is a metal oxide that is more chemically and thermally stable than silica. It can
be used for separations conducted at temperatures as high as 200° and is unaffected by
changes in ionic strength or organic content of the mobile phase. Zirconia packings have
a wider pH range and are especially useful for basic separations at pH 10 or higher,
where silica gel starts to dissolve. Zirconia can be used for RPC and is extremely stable
and efficient through surface modification with polymer or carbon coatings. Other
chemical modifications of zirconia produce packing materials suitable for normal–phase
or ion–exchange chromatography
Polymer–based packing materials
Several packing materials based on organic polymers are available. For example,
unmodified styrene–divinylbenzene co–polymers have a hydrophobic character and can
be used for RPC. Although they traditionally give lower column efficiencies than ODS-
silica, this has improved greatly in the past few years. Polymeric materials are best
when separation conditions require a mobile phase that can go beyond the upper pH
limits of silica gel (usually pH 6.5 to 7), as they have the advantage of being stable over
a wide pH range. Polymeric materials also provide different selectivity and retention
characteristics to silica–based reversed phase packings. They also avoid problems
associated with residual silanol groups (e.g. peak tailing). Ion–exchange materials of the
styrene–divinylbenzene type are also available in which sulfonic acids, carboxylic acids
or quaternary ammonium groups are incorporated in the polymeric matrix.
Monolithic columns
Monoliths are chromatographic columns that are cast as continuous homogenous phases
rather than packed as individual particles, creating porous rods of polymerised silica
that are mechanically stable. Monolithic phases have flow–through pores with
macroporosity (approx. 2 μm) and mesopores, which are diffusive pores with an
average pore diameter that can be controlled. To create the column, a silica gel polymer
is formed, which, after ageing, is dried into the form of a straight rod of highly porous
silica with the bimodal pore structure. The rod is then encased (or clad) in a PEEK
cover, ensuring that there is absolutely no void space between the silica and PEEK
material. The pore structure yields a very large internal surface area and ensures high–
quality separations. In addition, the high porosity of the column means very high flow
rates can be used with lower pressures. This enables separations in a fraction of the time
needed when using a column with conventional packing materials.
Recently, a polymeric monolithic column was introduced. It contains a
poly(glycidylmethacrylate–ethyleneglycol-dimethacrylate) co–polymer that has
functional groups added to make various types of stationary phases.
Maintenance
Sub-sections
Columns
Pumps
Injection valves
An effective maintenance programme is essential to keep an HPLC system in proper
working order. The maintenance programme should include preventative, periodical and
necessary repairs of the HPLC system. This programme is essential to ensure that all of
the components of the system are in proper working condition. In this section, the
general maintenance of columns, pumps, injection valves and detectors is discussed. For
information on the functions and uses of these components, refer to the earlier sections
of this chapter.
It is always recommended that the maintenance guidelines provided with the system
should be consulted to ensure compliance with the manufacturers' suggestions. This
guide should be utilised whenever maintenance is required.
Columns
The column is an essential key to good chromatography and its maintenance ensures
proper functionality of the HPLC system. High back pressures, poor resolution, non–
uniform peak symmetry and decreasing retention times are several signs that may
indicate the column is in need of repair or is failing.
Column degradation is inevitable, but column life can be prolonged if it is maintained
properly. Flushing a column with a mobile phase of high elution strength after sample
runs is essential. When a column is not in use, it should be capped to prevent it from
drying out. Particulate samples should be filtered and, when possible, a guard column
should be utilised. Column regeneration can instil some life into a column, but
preventative maintenance is the vital key to prevent premature degradation.
Pumps
The pump forces the mobile phase through the HPLC system. A steady pump pressure
is needed to ensure reproducibility and accuracy. Inability to build pressure, high
pressures or leakage may indicate that the pump is not functioning correctly.
Pumps are typically known to be robust, but adequate maintenance must be performed
to maintain that characteristic. Good maintenance practice includes replacing
components, such as inlet check valves, outlet check valves, frits, pump seals and piston
rods, on a routine schedule, based on the amount of usage. Proper maintenance of the
pump system minimises down time.
Injection valves
Injection valves play the role of directing injected volumes into the mobile phase, where
they then travel onto the column. Proper valve function is a necessity to ensure
reproducibility between injections. The symptoms of injection valve failure are low
pump pressure, leakage or inadequate inert gas pressure to the switch valve.
The seals of the injection valve may eventually falter, after numerous injections.
Replacement of these seals is necessary to maintain system reproducibility with respect
to injections made.
Detectors
Detector maintenance is generally performed as needed. Baseline drift, erratic baseline
and decreasing response may be indicators of a failing detector.
A malfunctioning or contaminated flow cell can also cause baseline drift. The cell
should be flushed regularly with water to remove salts when using mobile phases of
high salt concentration. An organic mobile phase of high elution strength should be
used to remove any organic residue that may remain in the cell. An erratic baseline can
occur because of an air bubble in the flow cell. Increasing the flow rate may push the
bubble out of the cell. Decreasing responses can also result from a decrease in lamp
intensity.
Eluent preparation
The quality of solvents and inorganic salts is an important consideration. Soluble
impurities can give noisy baselines and spurious peaks or can build up on the surface of
the packing material, eventually changing chromatographic retention. Furthermore, the
eluate may need to be collected for further experimentation and all contamination must
be avoided. In addition, particulate matter should be removed, otherwise pump filters,
frits and tubing can become blocked.
Now commercially available is a wide range of HPLC-grade solvents that are free from
particulate matter, have low residues on evaporation and have guaranteed upper limits
of UV-absorbing and fluorescent impurities. However, if a detector is not to be operated
at its maximum sensitivity, analytical grade solvents may be used. A general rule of
thumb is to use the highest purity of solvent that is available and practical depending on
the particular application.
Air dissolved in the mobile phase can lead to problems. The formation of a bubble in a
pump head usually reduces or stops eluent flow, while bubbles formed in the detector
can give spurious peaks. One commonly used remedy is to degas the eluent using an in–
line vacuum chamber. HPLC solvents are pumped from the reservoirs into a vacuum
chamber in–line with the HPLC eluent flow. This method ensures continuous and
efficient degassing of the mobile phase. Vacuum degassing can also be performed off–
line by applying a weak vacuum to the mobile phase reservoir while sonicating. Off–
line techniques do not offer the advantage of continuous degassing throughout the
analysis. Eluents can also be degassed by purging with helium, which has a very low
solubility and drives the air out. This technique can be performed on–line and be
controlled by the HPLC system, or off–line. Care must always be taken when degassing
eluents that contain volatile components to avoid changing the composition.
It is convenient to prepare eluents as volume plus volume mixtures of solvents (i.e. the
volume of each solvent is measured separately and then mixed). Volume changes can
occur when solvents are mixed (e.g. methanol and water show a contraction in volume),
which must be remembered if the volume of only one solvent is measured and the
second solvent added to make up to volume (v/v).
True pH values can only be measured in aqueous solutions and any measurements made
with a pH meter in aqueous–organic solvents should be described as ‘apparent pH’. In
general, the apparent pH of a buffer solution rises as the proportion of organic solvent in
the aqueous mixture increases. When an eluent is prepared it is usually best to dissolve
the required buffer salts in water at the appropriate concentrations, adjust the pH and
then mix this solution (v/v) with the organic solvents.
Separation techniques
Sub-sections
Isocratic
Gradient elution
Derivatisation
Chiral separation
High–speed/high–temperature HPLC
Isocratic
When the mobile–phase composition does not change throughout the course of the run,
it is said to be isocratic. A mixed mobile phase can be delivered at a constant ratio by
the pumps themselves or the solvent mixture can be prepared prior to analysis and
pumped through a single reservoir. This is the simplest technique and should be the
method of first choice when developing a separation.
Gradient elution
HPLC can be performed with changes in composition over time (gradient elution). The
elution strength of the eluent is increased during the gradient run by changing polarity,
pH or ionic strength. Gradient elution can be a powerful tool to separate mixtures of
compounds with widely different retention. A direct comparison can be drawn with
temperature programming in gas chromatography (GC; see Chapter 28).
Eluent gradients are usually generated by combining the pressurised flows from two
pumps and changing their individual flow rates with an electronic controller or data
system, while maintaining the overall flow rate constant. Alternatively, a single pump
with a low sweep volume can be used in combination with a proportioning valve, which
controls the ratio of two liquids that enter the pump from two liquid reservoirs.
Equipment and data systems that allow the gradient to take almost any conceivable form
(e.g. step gradients, concave and convex gradient curves) are commonly available. The
gradient can be programmed to return the system to the original eluent composition for
the next analysis.
While most, if not all, commercially available pumps are capable of performing reliable
gradient elutions, there are some potential difficulties. The technique can be very time
consuming, as the column must be reconditioned with the initial eluent between runs.
This drawback can be overcome by utilising a column–switching apparatus (see
elsewhere in this chapter). In addition, drifting of the detector response and the
appearance of spurious peaks that arise from solvent impurities may occur. While
isocratic elution is usually favoured over gradients for simplicity, gradient elution can
be a very important and useful technique in the separation of complex mixtures.
Recently, the use of ‘fast gradient’ separation has enabled the implementation of high
throughput analysis in laboratories with a high sample load.
Derivatisation
Derivatisation involves a chemical reaction that alters the molecular structure of the
analyte of interest to improve detection and/or chromatography. In HPLC, derivatisation
of a drug is usually unnecessary to achieve satisfactory chromatography. This applies to
compounds of all polarities and molecular weights and is an important advantage of
HPLC over GC. Derivatisation is used to enhance the sensitivity and selectivity of
detection when available detectors are not satisfactory for the underivatised compounds.
Both UV-absorbing and fluorescent derivatives have been used widely. UV
derivatisation reagents include N-succinimidyl-p-nitrophenylacetate (SNPA),
phenylhydrazine and 3,5–dinitrobenzoyl chloride (DNBC), while fluorescent
derivatives can be formed with reagents such as dansyl chloride (DNS-Cl), 4–
bromomethyl–7–methoxycoumarin (BMC) and fluorescamine. The characteristics of a
good derivative in HPLC are similar to those in GC (i.e. stability, low background,
convenience, etc.).
Derivative formation can be carried out before the sample is injected on to the column
(pre–column) or by on–line chemical reactions between the column outlet and the
detector. Such post–column reactions generally involve the addition of reagents to the
eluent. With pre–column derivatisation there are no restrictions on reaction conditions
(e.g. solvent, temperature) and a large excess of reagent can be used, as this can be
separated from the derivatives during the chromatography. The major drawback of pre–
column reactions is the need to obtain reproducible yields for accurate quantification,
which is best achieved when the reactions proceed to completion. Furthermore, it is
important that the products of pre–column derivatisation reactions be characterised
fully. With post–column derivatisation, the reaction is well controlled by the flow rates
of eluate and reagents, temperature, etc. Hence, it is less necessary for the reaction to
proceed to completion or even for the chemistry to be understood as the system is
calibrated by the injection of known quantities of the reference standards. A much more
detailed discussion can be found in Snyder et al. (1997).
Chiral separation
Separation of compounds by chiral chromatography began in the early 1980s. At that
time, the separation of enantiomeric compounds was one of the most challenging
problems in chromatography. However, in recent years more than 100 chiral columns
have been made available. These columns are based on several different approaches to
solve the many enantiomeric separation problems. Chiral columns are used in a variety
of different applications that range from pharmacokinetic and pharmacodynamic studies
to measuring enantiomeric impurity of amino acids.
Chiral stationary phases (CSPs) are designed to separate optical isomers. The use of
these columns provides an efficient and economical way to separate optical isomers by
HPLC. CSPs are used for both resolving optical isomers to determine enantiomeric
purity and for isolating enantiomerically pure compounds. Fig. 29.2 shows the
separation of enantiomers of flurbiprofen.
The columns can be classified according to two categories, class or origin. The class
category is based on the structural properties of the chiral selector. The category is made
up of five different column types (macrocyclic, polymeric, π–π associations, ligand
exchange, miscellaneous) and hybrids. The macrocyclic chiral columns have had the
largest impact on analytical enantiomeric separations. The origin category separates
columns according to their source and classifies them into three types (naturally
occurring, semisynthetic and synthetic chiral selectors).
Figure 29.2.
Figure 29.2. Chiral separation of the (+) and (–) enantiomers of flurbiprofen. Enantiomers were separated on a CHIRALPAKbAD-RHTM column using methanol–0.1%
trifluoroacetic acid (TFA) as the mobile phase. This separation was performed at 15° to improve selectivity.
High–speed/high–temperature HPLC
The speed of a chromatographic method directly affects the economy and operating cost
of the separation. High–speed HPLC is accomplished by using short microbore columns
packed with small particles (3 μm). In addition, the use of higher temperatures increases
the speed of HPLC separations through the 5- to 10–fold decrease in eluent viscosity
upon an increase of the eluent’s temperature from 25 to 200°. High–temperature/high–
speed HPLC is not universally useful because of several limitations. Silica–based
stationary phases are unstable in aqueous media at temperatures above 50 to 60°. Some
detectors are also not able to tolerate hot temperatures.
Quantitative analysis
Sub-sections
External standard
Internal standard
Standard addition method
The quantification methods incorporated in HPLC derive mostly from GC methods. The
basic theory for quantification involves the measurement of peak height or peak area.
To determine the concentration of a compound, the peak area or height is plotted versus
the concentration of the substance (Fig. 29.3). For peaks that are well resolved, both
peak height and area are proportional to the concentration. Three different calibration
methods, each with its own benefits and limitations, can be utilised in quantitative
analysis, external standard, internal standard and the standard addition method.
Figure 29.3.
Figure 29.3. Example of a calibration curve for pseudohypericin.
External standard
The external standard method is the simplest of the three methods. The accuracy of this
method is dependent on the reproducibility of the injection of the sample volume. To
perform this method, a standard solution of known concentration of the compound of
interest is prepared. A fixed amount, which should be similar in concentration to the
unknown, is injected. Peak height or area is plotted versus the concentration for each
compound. The plot should be linear and go through the origin. The concentration of
the unknown is then determined according to Equation (29.3),
The calibrator concentrations should cover the range of the likely concentration in the
unknown sample. Only concentrations read within the highest and lowest calibration
levels are acceptable. Concentrations read from an extrapolated regression line may not
be accurate. This applies to all of the quantification methods.
Internal standard
Although each method is effective, the internal standard method tends to yield the most
accurate and precise results. In this method, an equal amount of an internal standard, a
component that is not present in the sample, is added to both the sample and standard
solutions. The internal standard selected should be chemically similar to the analyte,
have a retention time close to that of the analyte and derivatise in a similar way to the
analyte. For biological samples, the internal standard should extract similarly to the
analyte without significant bias toward the internal standard or the analyte.
Additionally, it is important to ensure that the internal standard is stable and that it does
not interfere with any of the sample components. The internal standard should be added
before any preparation of the sample so that extraction efficiency can be evaluated.
Quantification is achieved by using ratios of peak height or area of the component to the
internal standard, Equation (29.4):
Standard addition method
The third method for quantification is the standard addition approach. This is especially
useful when there is a problem with interference from the sample matrix, since it
cancels out these effects. To perform this quantification, the sample is divided into two
portions, so that a known amount of the analyte (a spike) can be added to one portion.
These two samples, the original and the original–plus–spike, are then analysed. The
sample with the spike shows a larger analytical response than the original sample
because of the additional amount of analyte added to it. The difference in analytical
response between the spiked and unspiked samples results from the amount of analyte
in the spike. This provides a calibration point to determine the analyte concentration in
the original sample. The method has a drawback if only a small volume of sample is
available. Equation (29.5) is used for this method:
Validation
It is important to use a validated HPLC method when carrying out analyses. Typical
analytical characteristics evaluated in an HPLC validation may include precision,
accuracy, specificity, limit of detection, limit of quantification, linearity and range.
Some appropriate suggestions for LC validation for postmortem and body fluids
samples are published in the SOFT/AAFS Forensic Toxicology Laboratory Guidelines
(http://www.soft-tox.org). It is important to consider the US Food and Drug
Administration (FDA; http://www.fda.gov/cder/guidance) and US Pharmacopoeia
(USP; http://www.usp.org) guidelines when validating HPLC methods used for
pharmaceutical samples. USP 24 section <1225> provides guidance on the validation of
compendial methods including definitions and determination. International Conference
on Harmonisation (ICH) guidelines (http://www.ich.org) provide suggestions
concerning the validation of pharmaceuticals. Valuable sources of information
providing regulatory guidance may be found in the FDA website at
http://www.fda.gov/cder/guidance.
System suitability tests evaluate the function of the overall HPLC system. This includes
all parts that make up a system, such as the instrument, reagents, packing material,
details of the procedure and even the analyst. These tests imply that the all the
components of a system constitute a single system in which the overall function can be
tested. These tests are very valuable and have been accepted in general application
because reliable and reproducible chromatographic results are based on a wide range of
specific parameters.
Most laboratories have a standard operating procedure that outlines the specifications of
running a systems suitability test. For example, in pharmaceutical analysis at least five
replicate injections should be made of a single solution that contains 100% of the
expected active and excipient ingredients level. The peak response is measured and the
standard deviation of that response should not exceed the limit set by the testing
monograph or 2%, whichever of the two is the lowest. Using the USP method, the
tailing factors of the analytes should be determined. The values should not exceed 2.0.
Peak–to–peak resolutions are also determined by using the USP calculations and the
value should not be lower than 1.5. The system test should be used to ensure the quality
of the data and of the analysis.
New emerging trends
Sub-sections
On–line sample preparation
Rapid screening
Several new trends, including hyphenated systems and micro-HPLC, are discussed in
other sections of this chapter. Two other trends that deserve mention are described
below.
On–line sample preparation
The preparation of samples typically demands a large amount of time, work and cost in
an analytical laboratory. The innovation of on–line sample preparation makes the
process more efficient and reduces the cost. On–line sample preparation techniques
usually involve direct elution of the extract from a solid–phase extraction (SPE)
cartridge into the system by the mobile phase. The on–line method gives superior
analytical results and can be automated fully. Another benefit is that the sample
preparation is reliable, reproducible and robust. This sample preparation method is also
discussed in the column–switching section of this chapter.
Rapid screening
The need for high throughput in a laboratory environment is ever increasing. The use of
short (2 mm), highly efficient analytical columns, rapid gradients and column–
switching apparatus in HPLC systems is helping to facilitate this. Sample turnaround
time can often be reduced to a few minutes or less in highly automated and optimised
systems. Other information on this topic is given earlier in this chapter in the gradients
and column switching section.
Systems for drug analysis
Sub-sections
Eluent systems
Selection of chromatographic systems
Analysis of drugs in pharmaceutical preparations
Analysis of drugs in biological fluids and tissues
Identification of drugs by HPLC with photodiode array detection and UV spectra library search
Sub-sections
Recommended HPLC systems
General screens
Amfetamines, other stimulants and anorectics
Amfetamines, other stimulants and anorectics
Analgesics, non-steroidal anti-inflammatory drugs
Analgesics, NSAIDs
Anti-fungals
Antibacterials
Anticholinergics
Anticholinergics
Anticonvulsants and Barbiturates
Anticonvulsants, barbiturates and antiepileptics
Antidepressants
Antidepressants and antipsychotics
Antihistamines
Antimalarials
Antineoplastics
Antitussives
Antivirals
Benzodiazepines
Sub-sections
Benzodiazepines
Cannabinoids
Cannabinoids
Cardiac glycosides
Cardiac glycosides
Cardioactive drugs
Diuretics
Diuretics
Drugs of abuse
Drugs of abuse
Ergot alkaloids
Ergot alkaloids
Local anaesthetics
Local anaesthetics
Narcotic analgesics
Narcotic analgesics and narcotic antagonists
Oral hypoglycemics and antidiabetics
Pesticides
Phenothiazines and other tranquilisers
Steroids
Sub-sections
Sulfonamides
Sulfonamides
Xanthine stimulants
Additional systems
Eluent systems
A large number of eluent and/or packing material combinations have been used for drug
analysis. However, currently most are performed on silica or one of the hydrocarbon–
bonded silicas (usually ODS-silica). Other types of packing are employed when these
conventional materials fail. The majority of drug analyses can be carried out with the
four types of system described next.
Silica with non–polar eluents
With silica normal–phase systems the principal mechanism is adsorption
chromatography. Separation is controlled by the competition between solute molecules
and molecules of the mobile phase for the adsorption sites on the silica surface. Polar
groups are attracted most strongly to these sites and hence polar compounds are retained
more strongly than non–polar ones. Retention can be decreased by increasing the
polarity of the eluent.
Adsorption energies of numerous solvents on alumina (ε° values given in Table 29.1)
have been measured and this scale can be used as a good guide to the elution strengths
of eluents on silica as well as alumina (Snyder 1968).
Mixtures of solvents can be employed to give elution strengths between those of the
pure solvents. Furthermore, different solvent mixtures that have the same ε° value often
give different separations of a group of compounds.
Water is strongly bound to silica and thus the water content of the eluent must be
controlled strictly to maintain constant activity of the silica surface and hence
reproducible retention times. This is most critical when the eluent is of very low
polarity. However, because anhydrous systems are difficult to maintain, a low
concentration of water can be used in the eluent, sufficient to deactivate the most active
sites without deactivating the whole surface. Typical water concentrations range from
0.01 to 0.2% (v/v). The most satisfactory method used to prepare a solvent of known
water content is to mix anhydrous and water–saturated solvents in known proportions.
Anhydrous hydrocarbon or halohydrocarbon solvents can be prepared by passing them
through a bed of activated silica or alumina (200 μm) in a glass column. The problems
associated with the control of water concentration mean that commonly alcohols, such
as methanol (0.01 to 0.5% v/v), are employed to moderate the silica surface (Engelhardt
1977).
Silica with polar eluents
Several systems have been described that involve the use of silica with eluents of
moderate–to–high polarity that contain alcohols and/or water as major components.
With such eluents, adsorption chromatography is most probably not the principal
mechanism. The mechanisms are poorly understood, which makes the prediction of
retention behaviour difficult; nevertheless, many of these systems are very useful for
drug analysis.
An eluent that consists of methanol:ammonium nitrate buffer (90:10) is suitable for a
wide range of basic drugs (e.g. amfetamines and opiates). Retention can be controlled
by changes to the pH, ionic strength or methanol:water ratio, or by the addition of other
organic solvents such as methylene chloride. With these alkaline eluents the silica
surface must bear a negative charge and the principal mechanism is probably cation
exchange.
Benzodiazepines can be chromatographed with methanolic eluents that contain
perchloric acid (typically 0.001 M). Retention can be modified by the addition of other
organic solvents (e.g. ether) or by changes to the acid concentration.
Both acidic and basic drugs can be chromatographed on silica using aqueous methanolic
eluents that contain cetyltrimethylammonium bromide (Hansen 1981). Hydrophobic
quaternary ammonium ions are strongly adsorbed on silica to give a dynamically coated
stationary phase. Retention may be controlled by varying the concentration or nature of
the quaternary ammonium ion, changing the ionic strength or pH of the buffer or
changing the concentration or nature of the organic component.
ODS with polar eluents
Eluents for RPC on ODS are usually mixtures of methanol or acetonitrile with an
aqueous buffer solution. Retention is controlled mainly by the hydrophobic interactions
between the drugs and the alkyl chains on the packing material. Retention increases as
the analytes decrease in polarity (i.e. polar species are eluted first). Hence, the elution
time is increased by increasing the polarity of the eluent (i.e. increasing the water
content). The pH of the eluent and the pKa of the drug are also important, since non–
ionised species show greater retention. Thus, acids show an increase in retention as the
pH is reduced while bases show a decrease. It is important to use a buffer of sufficient
capacity to cope with any injected sample size, otherwise tailing peaks can arise from
changes in ionic form during chromatography. Phosphate buffers (0.05 to 0.2 M) are
widely used as they have a good pH range and low UV absorbance.
Drugs that contain basic nitrogen atoms sometimes show poor efficiencies and give
tailing peaks caused by interactions with residual silanol groups on the packing
material. This can often be improved by the addition of an amine or quaternary
ammonium compound to the eluent, which competes with the analytes for adsorption
sites on the silica. Amines of small molecular weight (e.g. diethylamine) can be used as
part of the buffer system. Alternatively, low concentrations (0.001M) of long–chain
hydrophobic modifiers (e.g. N,N-dimethyloctylamine) can be added to eluents together
with conventional buffers.
Other hydrocarbon–bonded packing materials can be used in RPC. A decrease in
retention is associated with a decrease in the alkyl chain length.
ODS with polar eluents that contain hydrophobic cations or anions
Drugs that bear positive or negative charges are retained poorly in reversed–phase
systems. If the pH of the eluent cannot be changed to convert the drug into its non–
ionised form, a hydrophobic ion of opposite charge can be added to form a neutral ion
pair and increase retention. Hence, for a basic drug an acidic eluent is chosen and a
hydrophobic anion added. This technique is referred to as reversed–phase ion–pair
chromatography.
The sodium salts of alkylsulfonic acids (RSO–3 Na+, where R = pentyl, hexyl, heptyl or
octyl) are used widely as ion–pair reagents for basic drugs, while quaternary ammonium
compounds (e.g. tetrabutylammonium salts) are used for acidic drugs. Ion–pair reagents
are generally added to eluents in the concentration range 0.001 to 0.005 M, and within
this range an increase in concentration leads to an increase in retention. When
detergents such as sodium lauryl sulfate or cetyltrimethylammonium bromide are used
as the ion–pair reagents, the method is sometimes referred to as ‘soap chromatography’.
With these salts, ions build up on the surface of the packing material and produce a
stationary phase, which behaves like an ion–exchanger. This type of mechanism has
been described as ‘dynamic ion–exchange’ and probably also occurs with less
hydrophobic ion–pair reagents. It is virtually impossible to remove an ion–pair reagent
completely from a hydrocarbon–bonded phase, and such columns should not, therefore,
be reused with other reversed–phase eluents.
Table 29.1.
Solvent ε°
Pentane 0.00
Hexane 0.01
Iso–octane 0.01
Cyclohexane 0.04
Toluene 0.29
1-Chlorobutane 0.30
Ether 0.38
Chloroform 0.40
Methylene chloride 0.42
Tetrahydrofuran 0.45
Solvent ε°
Acetone 0.56
Ethyl acetate 0.58
Diethylamine 0.63
Acetonitrile 0.65
Isopropyl alcohol 0.82
Ethanol 0.88
Methanol 0.95
Acetic acid large
Water large
Table 29.1. ε° values for numerous solvents on alumina (Snyder 1968)
Selection of chromatographic systems
Many different combinations of packing material and eluent may be suitable for the
analysis of a particular compound or group of compounds and the final choice can be
influenced by many factors. The time required to develop a new system can be
shortened if it is possible to predict the way in which changes in eluent composition
influence chromatographic retention. Systems that use hydrocarbon–bonded phases are
particularly attractive from this viewpoint as a large range of parameters can be adjusted
(pH, organic solvent, ionic strength, ion–pair reagents) with largely foreseeable
consequences. Predictions for silica are generally less reliable. Silica is good for
separating drugs that belong to different chemical classes, while hydrocarbon–bonded
silicas are preferred for separations of drugs with closely related structures (e.g.
barbiturates).
Most of the endogenous materials in biological extracts that can interfere with the
analysis of a drug are fairly polar. In reversed–phase systems this material generally
elutes before the drug and can obscure the drug peak. In these circumstances, reversed–
phase ion–pair chromatography can be valuable to increase selectively the retention of
the drug relative to the interfering peaks. Normal–phase systems that use silica do not
generally suffer from this problem, as most of the endogenous material usually elutes
after the drug. However, these slow–eluting compounds can lead to a noisy baseline or
may remain adsorbed to the packing material and thus eventually lead to a loss in
column performance.
The vast majority of compounds are separated using a silica–based column with C18, and
fine–tuning of the separation can be made by selecting a column with a shorter bonded
phase, such as C8 (see later).
Specially endcapped columns designed to minimise the tailing common with nitrogen–
containing weak bases are available. These are often marketed as a ‘basic’ column (e.g.
Metachem’s MetaSil Basic). There are also specially endcapped columns designed to
withstand extremely high concentrations of aqueous mobile phase (95 to 100%). These
columns are endcapped with a hydrophilic moiety that ensures proper ‘wetting’ of the
silica to prevent bonded–phase collapse. The columns are typically marketed as ‘AQ’
for aqueous (e.g. YMC’s ODS-AQ).
Analysis of drugs in pharmaceutical preparations
HPLC has found widespread use for the quantitative analysis of drugs in preparations of
pharmaceutical and illicit manufacture. Drug concentrations are generally high enough
to allow dissolution of the sample (tablet, powder, ointment, etc.) in a suitable solvent
followed by injection. UV, visible, FL, RI or mass spectrometric detection methods are
used often. These techniques are well–suited to provide specific data as to the chemical
composition of the sample in question (e.g. a UV spectrum, mass spectrum, etc.).
Within the pharmaceutical industry, HPLC is used at various stages of drug
development, such as the optimisation of synthetic reactions and stability testing.
Furthermore, it is used extensively for quality control during production to monitor the
purity of drugs and excipients. HPLC systems can be automated easily (including
injection and data handling), which allows large numbers of samples to be analysed
rapidly and economically. HPLC is particularly valuable for the analysis of drugs that
are polar (e.g. aspirin), thermally unstable (e.g. benzodiazepines) or present in oil–based
formulations for which analysis by GC can be very difficult. Similarly, HPLC can be
used for the forensic analysis of illicit preparations to aid the identification of an
unknown drug by the measurement of retention times and UV spectra and comparison
to spectral libraries. Furthermore, as the technique can be non–destructive, depending
on the detection system used, the eluted compounds can be collected for further
analysis.
Example of a drug analysis system
Opiates have been separated by many methods in the past, and the system described
here was developed for this purpose. The three opiates separated were morphine sulfate,
hydrocodone bitartrate and oxycodone hydrochloride. The column used was a
Phenomenex Luna C18 (2), 150 mm × 4.60 mm × 5 μm. The mobile phase was 39mM
dipotassium hydrogen phosphate (K2HPO4) and methanol in a 40:60 ratio. The final pH
was 10 and the mobile phase flow rate was 1.0 mL/min. The retention times obtained
(Fig. 29.4) for morphine sulfate, hydrocodone bitartrate and oxycodone hydrochloride
were 2.799 min, 4.696 min and 6.143 min, respectively.
Figure 29.4.
Figure 29.4. Separation of opiates by HPLC. Conditions for separation are described in the text.
Analysis of drugs in biological fluids and tissues
Several factors determine the ability of HPLC to detect a drug among the endogenous
compounds present in biological material. Clearly, selective detection of the drug
relative to the endogenous material is advantageous. In addition, the stationary phase
and/or mobile phase can be altered to separate the drug peak from interfering peaks (e.g.
using ion–pair reagents). Finally, the sample may be extracted before HPLC to
concentrate the drug relative to the endogenous material.
The chromatographic system and detector should always be chosen to minimise the time
needed for sample preparation. The complexity of the sample preparation procedure is
controlled by several factors, which include the nature of the sample (urine, blood, liver,
etc.), the condition of the sample and the concentration of the drug. Interference from
endogenous compounds is most acute when drug concentrations are low (e.g.
therapeutic drug monitoring), so more extensive sample preparation and more sensitive
and specific detectors are often required. Such assays can be very susceptible to changes
in the condition of the sample (e.g. a method developed for fresh blood may not be
satisfactory for urine or hair samples), which can present severe difficulties in forensic
toxicology. Thus, methods should be tested and validated with the most difficult
samples that may be encountered. In contrast, the analysis of biological samples that
contain high drug concentrations (e.g. fatal drug overdose) by HPLC may require much
less sample preparation and is less susceptible to changes in sample condition.
Sample preparation for HPLC is essentially the same as for other methods of drug
analysis. A drug that is physically trapped within solid tissue (e.g. liver), or chemically
bound to the surface of proteins, must be released; then the protein is precipitated to
leave the drug in aqueous solution. The protein may be degraded by strong acids or
enzymes, precipitated by various chemicals (e.g. tungstic acid, ammonium sulfate) or
removed by ultra–filtration. Some drugs are destroyed by protein degradation methods,
while ultra–filtration and precipitation can lead to drug losses through protein binding.
No single procedure works well for all drugs and the method should be selected to give
the maximum recovery of the drug being analysed.
When drug concentrations are high (typically μg/mL) and systems with polar mobile
phases are used, the direct injection of deproteinised solutions may be acceptable.
Proteins must be removed to protect the column from irreversible contamination. A
rapid procedure is to mix the biological fluid with at least two volumes of methanol or
acetonitrile, centrifuge to remove the precipitated protein, evaporate the organic
supernatant and reconstitute the sample in a volume of mobile phase. Urine can be
treated similarly to guard against the precipitation of salts on the column. Great care and
consideration should be taken when injecting minimally prepared biological samples
onto a HPLC system. Particulates are more likely to become trapped in the system
plumbing and a more rapid degradation of column performance may be observed from
contaminant build up on the head of the column. To help maximise column performance
and lifetime, it is good policy to use a guard column between the injector and analytical
column. This is packed with the same material as the analytical column and replaced at
frequent intervals. The configuration of guard columns ranges from easily replaceable
and relatively inexpensive frit–like filters and/or cartridges to shorter versions of the
analytical column itself. All are designed to protect the analytical column by acting as a
trap for components that would otherwise irreversibly bind to the analytical column, and
thus decrease the useable life of the column.
Extraction of drugs and other analytes away from endogenous materials prior to analysis
is a common procedure for all types of biological samples. This may also entail a
concentration step, which increases the sensitivity of the method. Solvent extraction
remains the most popular approach, as many factors can be modified to optimise the
extraction. These modifications include changing the polarity of the organic solvent, the
pH and ionic strength of the aqueous phase and the use of ion–pairing agents. It is
generally recommended that the collected organic phase be evaporated to dryness and
the residue dissolved in a suitable solvent, typically something greater than or equal to
the polarity and composition of the initial mobile phase before injection. Care must be
taken that volatile drugs are not lost by evaporation and that lipid material in the residue
does not prevent the drug from dissolving in the new solvent.
Example protocol for the extraction of a wide variety of weak bases
To 1 mL of plasma, urine or other homogenised matrix add 100 μL concentrated ammonium hydroxide.
Extract the sample with 4 mL of a mixture of n–butyl chloride:acetonitrile (4:1) for 20 min.
Centrifuge at high speed for 20 to 30 min to partition the phases. Carefully collect the organic phase into a clean tube. Evaporate the organic phase under a stream of air or nitrogen at 25 to 40°,
depending on the volatility of the analytes (a small volume of acidified methanol can be added to prevent the loss of amfetamine–type analytes).
Reconstitute the residue in an HPLC mobile phase that is more polar than the LC mobile phase to be used for analysis (e.g. if the HPLC elution ratio is 60%
aqueous, reconstitute the sample in >60% aqueous). This ensures that, when injected, the sample is focused on the front end of the column and minimises band (peak) broadening.
An example of a chromatogram that utilises this extraction technique is shown in Fig.
29.5. The urine was fortified with analytes and deuterated internal standards for
amfetamine and methamfetamine (dashed chromatograms) and extracted as described
above. The sample was eluted using a MetaSil Basic 3 × 100 mm × 3 μm column. The
mobile phase was 85% (0.1% formic acid in water), 15% (methanol), pumped
isocratically at 0.2 mL/min. The instrument used was an Agilent 1100 LC/MSD with
ESI.
SPE columns are also widely used to extract drugs from biological samples. The
column is washed with suitable solvents to remove endogenous material before the drug
is removed by passing through a solvent of higher elution strength. Such columns are
usually attached to extraction manifolds utilising either positive or negative pressure to
draw the liquids through the sorbent beds. Extraction selectivity can be controlled by
adjustments to the biological fluid before extraction (e.g. pH, ionic strength) and the
choice of washing solvents. Most, if not all, manufacturers of SPE columns offer
methods and columns optimised for a particular drug class and/or matrix. As less
traditional biological matrices are used for drug analysis (e.g., sweat, hair, oral fluids),
some modifications of the sample preparation scheme are needed. Hair requires
solubilisation prior to extraction; oral fluids and sweat may need to be isolated from
their respective collection devices. Consideration of the pH and solubility may be
needed prior to sample preparation, but in general the principles in place for the
extraction of blood, urine, etc., apply to these alternative matrices. Some important
issues unique to these matrices are:
Sample volume is typically much less than blood or urine. The amount of drug extracted from a particular matrix may be much less than
from traditional matrices, so that much more sensitive detectors (e.g. MS or MS–MS) are required.
Figure 29.5.
Figure 29.5. Separation of amfetamines by HPLC–MS. Conditions for separation are described in the text.
Identification of drugs by HPLC with photodiode array detection and UV spectra library search
HPLC with DAD in combination with a UV spectra library has proved to be a very
successful ‘systematic toxicological analysis’ (STA) technique for use in clinical and
forensic toxicology (see Chapter 1). Any drugs or other poisons in the sample are
identified by coincidence of the UV spectrum and of the retention time or another
chromatographic retention parameter with the library data; one system and its use is
described below (Pragst and Herzler, personal communication).
Chromatographic conditions
Since the method is used in combination with a database of UV spectra and retention
parameters, the chromatographic conditions must be reproducible and the same as used
to generate the database. The mobile phase must be suitable for the separation of a large
variety of organic substances and must be transparent in the wavelength range used.
These prerequisites are best met by reversed–phase columns (RP8 or RP18) and acidic
acetonitrile–buffer mixtures as mobile phases. Systems described in the literature
generally either use a gradient elution or two isocratic runs with different
buffer:acetonitrile ratios.
Gradient elution has the advantages that strongly polar and non–polar substances can be
analysed in one run, that peaks are not broadened with increasing retention time and that
the retention times of the toxicologically relevant compounds are distributed more
evenly over the run time, but it has some disadvantages (see above). A system of HPLC
retention indices was introduced by Bogusz et al. (1993) analogous to the Kovats
indices used in GC and based on the retention times of the nitroalkanes.
Isocratic HPLC has the advantage of higher reproducibility of the retention times,
greater ruggedness and a more economic use of the mobile phase by recycling.
Disadvantages are an unfavourable distribution of the retention times of toxicologically
relevant compounds with an increased number at the beginning of the chromatogram,
and the need for a second mobile phase for non–polar compounds. Nevertheless,
isocratic HPLC–DAD procedures are used successfully in many toxicological
laboratories for screening purposes. Suitable experimental conditions, also used in the
recording of an extensive UV spectra library, were as follows (Pragst et al. 2001):
HPLC column: RP8, endcapped, 5 μm, 250 × 4.0 mm. Mobile Phase A: 0.1 M phosphate buffer pH 2.3:acetonitrile (67:33 v/v). Mobile Phase B: 0.1 M phosphate buffer pH 2.3:acetonitrile (33:67 v/v). Flow rate: 1 mL/min.
Standard compounds are histamine hydrochloride to measure the time of an unretained
peak t0 (dead time), 5-(4–methylphenyl)-5–phenylhydantoin (MPPH) to calculate
relative retention times (RRTs) in mobile phase A and 4–phenylbenzophenone to
calculate the RRTs in mobile phase B.
The UV spectra of a large number of compounds listed in this book were measured
under these conditions. An overview of HPLC–DAD conditions used for STA is given
in Pragst et al. (2001).
Retention parameters
Absolute retention times are not suitable for peak identification purposes, since they
depend strongly on the configuration and experimental conditions of the HPLC device.
Moreover, the capacity ratio kA (see above) is sensitive to small fluctuations of the
experimental conditions and is not suitable for an identification system used in different
laboratories. Therefore, for gradient elution, retention indices are preferred (Bogusz et
al. 1993). Under isocratic conditions RRTs related to a standard compound are more
reproducible, Equation (29.6):
where RRTx is the RRT of compound x, tx is the absolute retention time of compound x,
t0 is the retention time of an unretained peak and ts is the retention time of the standard
compound.
The relatively small peak resolution of HPLC and the differences between charges of
the reversed–phase material mean the value of retention indices or of RRTs in the
identification of a compound from a large number of candidates is rather limited.
However, it is very useful for distinguishing between compounds with very similar UV
spectra. In this way an RRT window can be chosen as a pre–selection parameter for the
spectra library search.
UV spectra library search and specificity of UV spectraBefore peak identification a ‘peak purity check’ should be carried out. A pure peak
means that it originates only from one compound and that the UV spectrum does not
change over the whole peak width.
A UV spectra library search is based on the comparison of the spectrum of the unknown
peak with all spectra of the library. This comparison is not confined to UV maxima and
minima, but can comprise all absorbance–wavelength points measured by DAD.
Mathematical models to assess spectral similarity use the description of the spectrum as
a vector in n–dimensional space, where n is the number of absorbance–wavelength pairs
measured. For the complete identity of two spectra both vectors point in exactly the
same direction, that is the angle between them is θ = 0°. Different concentrations have
an effect on vector length, but not on its direction in space. The similarity index (SI) is
defined as cosθ and is calculated by Equation (29.7):
where s̄i is the vectorised spectrum of compound i.
UV spectra can be measured with extremely high reproducibility. Therefore, small
differences between spectra measured under identical conditions indicate that they
originate from different compounds. SI is 1.000 for completely identical spectra.
However, in practice two spectra with SI >0.9990 can be regarded as identical. At small
concentrations, and in the case of partly overlapping peaks, SI >0.990 may be a
sufficient criterion for identity.
It was shown in a systematic study on the selectivity of an HPLC–DAD method
(Herzler et al. 2003) that from 2888 toxicologically relevant compounds, 2682 (93%)
exhibited UV absorption above 195 nm. Out of these, 1619 (60.4%) had a unique UV
spectrum and could be identified unambiguously. By inclusion of the retention time this
portion was increased to 84.2%. Large UV spectra libraries can be divided into sub–
libraries, according to the retention parameter or the effect or use of the substance, to
facilitate a faster and more specific library search. The result can also be supported by
the presence of metabolites, while in doubtful cases complementary methods may be
used for confirmation (e.g. MS).
As an example, in Fig. 29.6 the results of the library search for a peak with
RRT = 0.811 in an intoxication case are shown. In this case a sub–library of all
compounds with RRT = 0.601 to 0.900 was used. Hit 1 was promethazine with
SI = 0.9992; hit 2 (promazine, SI = 0.9964) and hit 3 (dixyrazine, SI = 0.9961) also
originated from compounds of the phenothiazine type. The small difference between the
spectra of hits 1 and 2 may be because in these two compounds the amino group of the
side chain is separated from the phenothiazine ring by two and three saturated carbon
atoms, respectively. Dixyrazine could clearly be excluded by the much smaller retention
time. However, promethazine and promazine could not be distinguished by the RRT
values stored in the database. Therefore, to confirm the library search result, promazine
and promethazine standards were measured immediately after the sample, which
resulted in an exact agreement with promethazine.
As a prerequisite for the optimal use of a commercially available UV spectra library, the
same mobile phase must be used and the technical parameters of the DAD (wavelength
accuracy and resolution) need to be (and stay) sufficient. This can be controlled by daily
measurement of a compound with a vibration fine structure of the UV spectrum, such as
benzene.
UV spectra and retention times of metabolitesThe use of HPLC–DAD has the advantage that in many cases, metabolites can be
attributed easily to the parent drug by the UV spectrum. Depending on the site of
metabolism, the UV spectrum may be altered significantly (change of the UV-absorbing
unsaturated part of the molecule, the chromophore) or it may be the same as (or very
similar to) that of the parent drug (reaction at the aliphatic part of the molecule). As an
example, in Fig. 29.7 the spectrum of flunitrazepam is compared with that of its
metabolites, 7–aminoflunitrazepam (strong change of the chromophore by
transformation of the aromatically bound nitro group into the amino group) and 3–
hydroxyflunitrazepam (no essential change of the chromaphore by hydroxylation at the
aliphatic carbon atom 3).
The retention times of drugs on reversed–phase columns are shifted in a typical way by
metabolism. Metabolism to more hydrophilic products (e.g. hydroxylation, reduction of
the nitro to amino group; Fig. 29.7) leads to a decrease in retention time, whereas de–
amination strongly increases retention time, particularly in an acidic mobile phase
(removal of the strongly hydrophilic, protonated amino group). For many drugs, the
chromatograms obtained from blood or urine extracts have a typical metabolite pattern
that supports identification in the context of STA.
Sample pre–treatmentTablets, powders or residues in syringes can simply be dissolved in the mobile phase
and analysed by HPLC–DAD without further treatment. The investigation of biological
samples, such as whole blood (serum, plasma), stomach contents, urine or tissue
samples, is more complicated. In these cases the drug must be separated from the
biological matrix.
Although SPE has been much improved in the past decade, liquid–liquid extraction
(LLE) is still preferred if HPLC–DAD is used for toxicological screening, since it is less
susceptible to interferences, more reproducible and easier to handle for single samples.
An important advantage of UV detection is that cholesterol and fatty acids, co–extracted
to a high extent from human samples by lipophilic solvents, show no UV absorption and
therefore, in contrast to GC–MS, do not interfere with the analysis. Moreover,
derivatisation is not necessary. A sample pre–treatment method by extraction with n–
butyl chloride:acetonitrile (4:1), which can be used for a wide variety of basic
compounds, is given above. For systematic toxicological screening of blood (serum,
plasma) samples by HPLC–DAD, the measurement of two extracts obtained at pH 2
and pH 9 with dichloromethane and of the supernatant of a protein precipitation by
acetonitrile has proved to be very useful (Pragst et al. 2002).
Preparation of a basic and an acidic methylene chloride extract Dispense 500 μL of whole blood, serum or plasma into two 1.5 mL vials. To vial 1 add 100 μL of a 0.2 M solution of tri-(hydroxymethyl)-amine (basic
extract). To vial 2 add 100 μL of 0.1 M hydrochloric acid (acidic extract). To both vials add 400 μL of dichloromethane. Vortex mix the vials for 1 min and centrifuge.
Withdraw 200 μL of the dichloromethane extract and evaporate the solvent at room temperature under a stream of nitrogen.
Dissolve the residue in 100 μL of mobile phase. Analyse 50 μL of each extract (basic extract in mobile phase A and acidic
extract in mobile phase B).
Protein precipitation by acetonitrile To 500 μL of whole blood, serum or plasma add 500 μL of acetonitrile. Vortex the mixture for 2 min and centrifuge. Separate off the supernatant. Analyse 50 μL in mobile phase A.
Protein precipitation is particularly useful for hydrophilic drugs, which are extracted
poorly by the procedure mentioned above. These include paracetamol, salicylicacid and
lamotrigine. The limits of detection are between 0.01 and 0.1 μg/mL for
dichloromethane extraction (depending on the extinction coefficient and on the
extraction yield) and between 0.1 and 1 μg/mL for protein precipitation.
Application exampleIn STA, the library search must be applied to all peaks of the HPLC–DAD
chromatogram. As an example, the chromatogram at 225 nm of the basic extract from
the blood sample of a lethal drug poisoning case and the UV spectra of the highest
peaks are shown in Fig. 29.8. To determine RRT, the standard compound (MPPH, peak
No. 10, RRT = 1.000) was added. From the remaining eleven peaks of the
chromatogram, seven could be identified by both UV spectrum and RRT. As the result,
a high overdose of trimipramine and promethazine was found to be the cause of death.
The extensive metabolism indicated that there had been a long survival time after drug
ingestion. The similarities between the UV spectra of the parent drugs (peaks 9 and 12)
and some of their metabolites (peaks 8, and peaks 6 and 11, respectively) are also
demonstrated in this case. On the other hand, the sulfoxides of promethazine (peak 3)
and desmethylpromethazine (peak 2) show completely changed spectra because of the
transformation that takes place directly at the UV absorbing phenothiazine ring.
Caffeine (peak 1) is found in almost all samples. The poor separation of peaks 4, 5 and
7 meant the UV spectra were not suitable for a library search.
Figure 29.6.
Figure 29.6. Result of the HPLC–DAD library search for a peak in the chromatogram of an alkaline blood extract of a lethal trimipramine–promethazine intoxication. Hit 1
(promethazine) was confirmed by exact agreement of the retention time with the reference compound measured immediately after the sample. sa, sample; li, library.
Figure 29.7.
Figure 29.7. Change of the UV spectrum and the RRT of flunitrazepam by metabolism.
Figure 29.8.
Figure 29.8. HPLC–DAD investigation of a combined trimipramine–promethazine poisoning. Chromatogram of a basic extract of a venous blood sample, UV spectra of the
highest peaks, results of the library search and semiquantitatively determined concentrations
Recommended HPLC systems
There are general screening methods based on gradient elution and retention indices that
have proved value by many laboratories, and data from these are listed below (systems
HA, HX, HZ, HY, HAA). Another (system HBK) is based on a combination of isocratic
systems. The tabulated data are derived from systems in which groups of compounds
have been chromatographed either as part of a general screening procedure or from
systems that have been used specifically for that group of compounds. Other systems
for the chromatography of individual compounds, especially those used for
quantification, are given in the monographs.
Chromatographic retention data are presented as k values as well as retention times
(RT), retention indices (RI) and relative retention times (RRT).
NoteIn the tables, a dash indicates that no value is available for the compound, not that it
does not elute.
General screens
System HA
I. Jane et al. ,J. Chromatogr. 1985, 323, 191–225.
Column: Silica Spherisorb S5W (125 × 4.9 mm i. d., 5 μm). Mobile phase: Solution containing 1.175 g (0.01 M) of ammonium perchlorate
in 1 L methanol; adjust to pH 6.7 by the addition of 1 mL 0.1 M sodium hydroxide in methanol.
k values: Values for drugs in this system will be found in drug monographs and in the Indexes to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.
System HX
J. Hartstra, J. P. Franke, R. A de Zeeuw, personal communication.
Column: Lichrospher 60 RP-Select B (125 × 4.0 mm i.d., 5 μm) with pre-column Lichrospher 60 RP-Select B (4 × 4.0 mm i.d., 5 μm).
Mobile phase: (A:B) triethylammonium phosphate buffer (25 mM, pH 3.0):acetonitrile.
Elution programme: (A:B) (100:0) to (30:70) in 30 min, hold 10 min, back to initial conditions in 3 min with equilibration for 10 min before next injection.
Flow rate: 1 mL/min.
Detection: UV diode-array. Standards: Nitro-n-alkanes (C1 to C11) 10 μL in 10 mL acetonitrile. RI values: Values for drugs in this system will be found in the monographs and
in the Indexes to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.
System HY
R. K. Waters, R. A. Watt and A. C. Moffat, unpublished information.
Column: C18 symmetry (250 × 4.6 mm i.d., 5 μm). Column temperature: 40°. Mobile phase: (A:B) sulfuric acid (0.5 mL of 2.5 M) in water (500 mL):sulfuric
acid (0.5 mL of 2.5 M) in acetonitrile (500 mL). Elution programme: (98:2) for 3 min to (2:98) over 23 min, hold for 10 min
back to initial conditions over 2 min with equilibration of 8 min before next injection.
Detection: UV diode-array. Standards: Nitro-n-alkanes (C1 to C16) 10 μL in 10 mL acetonitrile. RI values: Values for drugs in this system will be found in the monographs and
in the Indexes to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.
System HZ
J. M. H. Conemans et al., http://home-2.worldonline.nl/~sint1166/stiptox.htm
Column: C18 endcapped LiChrospher 100 RP-18e, (125 × 4.0 mm i.d., 5 μm) with pre-column LiChrocart 124-4.
Mobile phase: Add 146 μL triethylamine and about 750 μL phosphoric acid to 530 mL water. Adjust pH to 3.3 using a 10% potassium hydroxide solution and finally add 470 mL acetonitrile.
Flow rate: 0.6 mL/min. Detection: UV diode-array. Retention times: Values for drugs in this system will be found in the monographs
and in the Indexes to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.
System HAA
Y. Gaillard and G.Pepin,J. Chromatogr. A. 1997, 763, 149–163.
Column: C8 Symmetry (250 × 4.6 mm i.d., 5 μm) with Symmetry C18 pre-column (20 mm).
Column temperature: 30°. Mobile phase: (A:B) phosphate buffer (pH 3.8):acetonitrile. Elution programme: (85:15) for 6.5 min to (65:35) until 25 min to (20:80) for
3 min and back to initial conditions for equilibration for 7 min.
Flow rate: 1 mL/min for 6.5 min, then linear increase to 1.5 mL/min for 6.5 to 25 min and hold for 3 min (re-equilibration is made at 1.5 mL/min).
Detection: UV diode-array. Retention times: Values for drugs in this system will be found in the monographs
and in the Indexes to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow
System HBK
F. Pragst, M. Herzler, S. Herre, B-T. Erxleben, M. Rothe, UV Spectra of Toxic
Compounds, Verlag Dr Dieter Helm, Heppenheim, 2001.
Column: Lichrospher RP-8ec (250 × 4.0 i.d., 5 μm). Mobile phase: Three different composition are used: A: acetonitrile:phosphate
buffer pH 2.3 (33:67). Internal standard: 5-(4-methylphenyl)-5-phenylhydantoin (for compounds eluting within 30 min); B: acetonitrile:phosphate buffer pH 2.3 (67:33). Internal standard: 4-phenylbenzophenone (for compounds eluting after 30 min); C: acetonitrile:phosphate buffer pH 2.3 (20:80). Internal standard: salicylamide (for compounds with RRTs below 0.2).
Flow rate: 1 mL/min. Detection: UV diode-array. Note: The phosphate buffer is prepared by dissolving 4.8 g phosphoric acid
(85%) and 6.66 g potassium dihydrogen phosphate in 1 L of water, adjust pH to 2.3. Values for drugs in this system will only be found in the Indexes to Analytical Data in Volume 2.
Amfetamines, other stimulants and anorectics
Systems HA, HX or HY previously described, may be used or Systems HB or HC,
below.
System HB
R. Gill et al. ,J. Chromatogr. 1981, 218, 639–646.
Column: ODS Hypersil (250 × 5 mm i.d., 5 μm). Mobile phase: Solution containing 19.60 g (0.2 M) phosphoric acid and 7.314 g
(0.1 M) diethylamine in 1 L of a 10% v/v solution of methanol; adjust the pH to 3.15 by the addition of sodium hydroxide solution.
System HC
B. Law et al. ,J. Chromatogr. 1984, 301, 165–172.
Column: Silica Spherisorb (250 × 5 mm i.d., 5 μm). Mobile phase: Methanol:ammonium nitrate buffer solution (90:10). To prepare
the buffer solution add 94 mL strong ammonia solution and 21.5 mL nitric acid
to 884 mL water and adjust to pH 10 by the addition of strong ammonia solution.
Amfetamines, other stimulants and anorectics
HA HB HC HX HY
k k k RI RI
Adrenaline – – 0.63 – –
Amfetamine 0.9 8.48 0.98 244 –
Benzfetamine 1.2 – 0.15 – –
Brucine 11.1 – – 312 267
Caffeine 0.2 – 0.26 – –
Cathine 1 4.39 0.83 – –
Chlorphentermine 0.9 – 0.82 – –
Diethylpropion 1.7 – 0.16 – 230
Dimethylamfetamine – 11.08 1.89 – –
DOM – – 1.13 – –
Ephedrine 1.0 5.68 1.79 – –
HA HB HC HX HY
k k k RI RI
Fencamfamin 1.3 – 0.72 354 309
Fenethylline – – 0.27 – –
Fenfluramine 1.3 – 0.88 371 315
norfenfluramine 1 – – – –
Fenproporex – – – – 226
Hordenine – 2.00 – – –
Hydroxyamfetamine – 2.24 1.11 – –
Hydroxyephedrine – 0.73 – – –
Mazindol 1.8 – 0.2 357 286
Mephentermine 1.5 – 2.48 – –
Mescaline 1.3 16.82 2.17 – –
Metamfetamine 2 10.52 2.07 262 216
Methoxyamfetamine – 14.95 – – –
HA HB HC HX HY
k k k RI RI
Methoxyphenamine 1.7 32.17 – – –
Methylamfetamine 2.0 10.52 2.07 – –
Methylenedioxymethamfetamine – – – 278 252
Methylephedrine 2.3 – 1.83 – –
Methylphenidate 1.7 – 0.36 – 277
Noradrenaline – 0.10 – – –
Normetanephrine – – 1.08 – –
Oxedrine – 0.27 – – –
Pemoline 0.2 – 0.1 307 271
Phendimetrazine 0.9 – 0.3 263 218
Phenelzine 1.0 5.91 0.37 – –
Phenethylamine 1.2 3.64 1.31 – –
Phenmetrazine 1.7 – – 258 241
HA HB HC HX HY
k k k RI RI
Phentermine 0.6 19.46 0.86 – 245
Phenylephrine 1.3 – 1.64 – –
Phenylpropanolamine 0.9 3.87 0.70 – –
Pipradrol 1.2 – 0.69 355 –
Prolintane 2 – 1.3 370 –
Pseudoephedrine 1.2 5.90 1.77 – –
Tranylcypromine 1.0 – 0.26 – –
Trimethoxyamfetamine – – 1.48 – –
Tyramine 1.2 0.81 1.47 – –
Analgesics, non-steroidal anti-inflammatory drugs
System HD
H. M. Stevens and R. Gill, unpublished data.
Column: ODS Hypersil (160 × 5 mm i.d., 5 μm). Mobile phase: Isopropyl alcohol:formic acid:0.1 M potassium dihydrogen
phosphate (13.61 g/L) (540:1:1000).
System HV
Column: ODS Spherisorb (200 × 4.6 mm i.d., 5 μm). Mobile phase: acetronitrile:acetic acid (45:55) for 2 min, to (75:25) at 3%/min,
hold 6 minutes. Flow rate: 1.7 mL/min.
System HW
H. M. Stevens and R. Gill, unpublished data.
Column: As for System HD, above. Mobile phase: Isopropyl alcohol:formic acid:0.1 M potassium dihydrogen
phosphate (13.61 g/L) (176:1:1000).
Analgesics, NSAIDs
HD HV HW HX HY HZ HAA
k RRT k RI RI RT RT
Acetanilide 0.5 – 2.3 – 281 – –
paracetamol 0.1 – 0.32 – – – –
Alclofenac 2.6 0.61 – – – – –
Aminophenazone 0.2 – 0.32 262 204 2.1 –
Aspirin 0.5 – 2.7 350 318 2.7 –
salicylic acid 0.7 – 4.6 – – – –
Benorilate 0.7 – 22.4 – – – –
aspirin 0.5 – 2.7 – – – –
HD HV HW HX HY HZ HAA
k RRT k RI RI RT RT
paracetamol 0.1 – 0.32 – – – –
Benoxaprofen 11.3 0.98 – – – – –
Clonixin – 0.87 – – 345 – –
Diclofenac 11.5 0.85 – 616 592 14.8
22.1 Diflunisal 4.1 0.77 – 508 583 5.4 –
Dipyrone 0.1 – 0.45 316 194 1.4 –
Etenzamide 0.55 – 4.6 – 303 – –
Fenbufen 4 0.81 – 520 461 – 19.3
Fenoprofen 7.9 – – 574 524 10.9 21.2
Floctafenine – – – – – 4.4 17.2
Flufenamic Acid 19.7 1 – 671 667 – –
Flunixin – 0.99 – – 414 – –
Flurbiprofen – 0.89 – 585 – 11.8 21.3
HD HV HW HX HY HZ HAA
k RRT k RI RI RT RT
Glafenine – – – 372 276 2.3 –
Ibuprofen 15.1 – – 616 598 16.5 23.8
Indometacin 6.95 0.87 – 607 590 14.4 21.7
Indoprofen 1.2 0.52 – – 406 – –
Ketoprofen 2.4 0.66 – 495 – 6.4 19.6
Ketorolac – – – – – 4.1 –
Meclofenamic Acid – – – 653 690 – –
Mefenamic Acid 21.1 0.95 – 661 686 – –
Methyl Salicylate 3.9 – – 480 449 – –
salicylic acid 0.7 – – – – – –
Morazone 0.4 – 2.05 – 294 – –
Naproxen 3.3 – – 501 468 6.8 –
Nefopam – – – – 313 – 12.7
HD HV HW HX HY HZ HAA
k RRT k RI RI RT RT
Nifenazone 0.1 – 0.45 310 – – –
Niflumic Acid – 0.93 – 595 530 – 22
Oxyphenbutazone 1.95 0.69 – 501 459 6.7 –
Paracetamol 0.1 – 0.32 264 241 1.9 5.6
Phenacetin 0.6 – 4.4 377 335 3.0 –
paracetamol 0.1 – 0.3 264 241 1.9 –
Phenazone 0.1 – 0.95 333 299 2.1 –
Phenylbutazone 6.5 0.95 – 672 643 19.5 24.1
oxyphenbutazone 1.95 0.7 – 501 459 6.7 –
Piroxicam 0.6 – 7.7 431 382 4.9 16.6
M (5-hydroxy) – – – – 446 – –
Propyphenazone 1.3 – 11 441 370 4.7 –
Salicylamide 0.4 – 2.5 327 289 – –
HD HV HW HX HY HZ HAA
k RRT k RI RI RT RT
Salsalate 3.6 0.69 – – – – –
Sulindac 1.25 0.78 – 488 462 3.9 16.6
sulindac sulfoxide – – – – – 7.2 –
Tenoxicam – – – 366 – – 12.7
Tiaprofenic Acid – – – 484 452 5.8 17.6
Tolfenamic Acid – – – 690 – 37.9 –
Tolmetin 2.05 0.60 and 0.99 – 470 434 5.4 –
Zomepirac 3.7 – – – 495 – –
Anti-fungals
The general screening systems, previously described, may be used.
HX HY HZ HAA
RI RI RT RT
Econazole 526 385 – 20.1
HX HY HZ HAA
RI RI RT RT
Fluconazole 340 289 – 11.4
Flucytosine 72 – 1.5 3.1
Griseofulvin – 488 – 18.4
Ketoconazole 439 464 5.2 15.7
Antibacterials
The general screening systems, previously described, may be used.
HX HY HAA
RI RI RT
Amoxicillin – 226 3.1
Ampicillin – 250 3.8
Azithromycin – – –
Ceftriaxone 239 – 5.3
Chloramphenicol 390 336 14.1
HX HY HAA
RI RI RT
Ciprofloxacin 318 260 9.1
Clarithromycin – – –
Clindamycin 354 291 12
Furazolidone 336 – 12.2
Isoniazid – 246 –
Metronidazole 257 226 6.8
Minocycline – 240 22.6
Nalidixic Acid – 380 16
Nitrofurantoin 319 288 –
Ofloxacin 314 260 8.6
Oxytetracycline Dihydrate 299 260 –
Rifampicin – 417 16.2
Roxithromycin – – 15.8
HX HY HAA
RI RI RT
Tetracycline 314 265 9.9
Trimethoprim 299 254 8.3
Anticholinergics
The general screening systems, previously described, may be used.
System HAX
E. M. Koves ,J. Chromatogr. A, 1995, 692, 103–119.
Column: Column: Supelcosil LC-DP (250 × 4.6 mm i.d., 5 μm). Eluent: (A:B:C) Acetonitrile:phosphoric acid (0.025% v/v):triethylamine buffer. Isocratic elution: (25:10:5). Flow rate: 0.6 mL/min. Detection: UV diode-array (λ=229 nm). Note: The triethylamine (TEA) buffer is prepared by adding 9 mL concentrated
phosphoric acid and 10 mL TEA to 900 mL water, adjusted to pH 3.4 with diluted phosphoric acid and made up to 1 L with water.
System HAY
E. M. Koves ,J. Chromatogr. A, 1995, 692, 103–119.
Column: LiChrospher 100 RP-8 (250 × 4.0 mm i.d., 5 μm). Eluent: (A:B:C) as per System HAX. Isocratic elution: (60:25:15). Flow rate: 0.6 mL/min. Detection: UV diode-array (λ=229 nm).
Anticholinergics
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Adiphenine 1.8 422 – – – – –
Atropine 3.9 306 251 2.2 10.4 7 3.8
Biperiden – – – 6.4 14.8 – –
Chlorphenoxamine 2.9 – 346 – – – –
Clidinium Bromide – 379 – – – – –
Clidinium – – – – 13.3 – –
Cyclopentolate 1.6 353 287 3.2 – – –
Dicycloverine 1.1 – 575 – – – –
Diethazine 3.4 – – – – 15.1 7.4
Emepronium Bromide 5.2 420 – – – – –
Homatropine 4.2 272 223 – – 6.8 3.6
Hyoscine 1.1 270 253 – 7.4 7 3.7
Hyoscyamine 3.7 – – – 9.7 – –
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Isopropamide Iodide 2.4 379 – – – – –
Metixene 3.6 451 – – – – –
Orphenadrine 3 418 323 6 – – –
N-monodesmethylorphenadrine 1.7 – – – – – –
N-oxide 1.1 – – – – – –
Oxyphencyclimine 2.8 424 – – – – –
Oxyphenonium Bromide 2.6 424 – – – – –
Piperidolate 1.7 429 – – – – –
Procyclidine 2 406 – 6.2 – >20 4.7
Profenamine 2.4 444 338 – – 16.6 8.3
Propantheline Bromide 4.4 454 – – – – –
xanthanoic acid – 499 – – – – –
Trihexyphenidyl 1.8 429 381 7.6 15.3 – –
Anticonvulsants and Barbiturates
System HE
J. A. Christofides and D. E.Fry,Clin. Chem. 1980, 26, 499–501.
Column: Alkyl-silica SAS-Hypersil (125 × 4.5 mm i.d., 5 μm). Mobile phase: Acetonitrile:tetrabutylammonium phosphate, 0.005 M, pH 7.5
(20:80).
System HG
R. Gill et al. ,J. Chromatogr. 1981, 204, 275–284.
Column: ODS Hypersil (150 × 4.6 mm i.d., 5 μm). Mobile phase: Methanol:0.1 M sodium dihydrogen phosphate (11.998 g/L)
(40:60); adjust to pH 3.5 by the addition of phosphoric acid.
System HH
R. Gill et al. , J. Chromatogr. 1981, 226; Biomed. Appl., 15, 117–123.
Column: As for System HG, above. Mobile phase: As for System HG except that the mixture is adjusted to pH 8.5
by the addition of sodium hydroxide solution.
Anticonvulsants, barbiturates and antiepileptics
HG HH HX HY HZ
k k RI RI RT
Allobarbital 2.46 1.33 346 – 2.7
Amobarbital 10.91 7.05 424 374 4
Aprobarbital 3.42 2.22 357 319 2.8
Barbital 1.11 0.63 308 258 2.2
HG HH HX HY HZ
k k RI RI RT
Benactyzine – – 382 – –
Brallobarbital 3.09 1.72 371 336 3
Butalbital 6.17 3.48 394 342 3.4
Butetamate – – 390 – –
Butobarbital 5.43 3.42 384 355 3.2
Carbamazepine – – 418 368 –
Clonazepam – – 465 403 4.6
Cyclobarbital 5.25 2.61 384 352 3.2
Cyclopentobarbital 6 3.84 391 352 –
Enallylpropymal 8.65 6.96 – 394 –
Ethosuximide – – 301 276 2.3
Flavoxate – – – – –
Heptabarb 9.9 4.93 416 377 3.9
HG HH HX HY HZ
k k RI RI RT
Hexethal 34.28 20.39 – 451 –
Hexobarbital 7.37 5.67 419 242 4.3
Ibomal 4.01 2.58 379 352 –
Idobutal 8.12 4.77 – 357 –
Mebeverine – – 448 – 7.1
Mephenytoin – – – 366 3.7
Mesuximide – – – 387 4.8
Metharbital 2.69 1.99 435 324 –
barbital 1.11 0.63 – – –
Methylphenobarbital 7.27 3.84 435 395 4.6
Nealbarbital 10.22 6.19 417 382 –
Papaverine – – 363 295 –
Pentobarbital 10.96 8.07 424 383 4.1
HG HH HX HY HZ
k k RI RI RT
Phenacemide – – 339 266 –
Phenobarbital 3.09 1.23 379 335 3
Phenytoin – – 431 381 3.7
Primidone – – 322 288 2.1
Secbutabarbital 4.9 3.3 377 331 –
Secobarbital 16.28 11.47 437 407 4.7
Sultiame – – 344 275 –
Talbutal 7.2 4.7 403 370 –
Thiamylal – – 516 476 –
Thiopental – – 485 433 6.9
Vinbarbital 4.83 2.32 379 363 –
Vinylbital – – 424 – 4.1
Antidepressants
The general screening systems, previously described, may be used or Systems HF and
HAZ below.
System HF
R. Gill, unpublished data, after P. M. Kabra et al., Clinica Chim. Acta, 1981, 111, 123–
132.
Column: ODS Hypersil (160 × 5 mm i.d., 5 μm). Mobile phase: Acetonitrile:phosphate buffer (pH 3.0) (30:70). To prepare the
phosphate buffer, add 0.6 mL nonylamine to 1 L 0.01 M sodium dihydrogen phosphate (1.1998 g/L) and adjust the pH to 3.0 by the addition of phosphoric acid.
System HAZ
K. Chiba et al. ,J. Chromatogr. B, 1995, 668, 77–84.
Column: C18 (250 × 4.0 mm i.d., 5 μm). Mobile phase: (A:B:C) Water:methanol:triethylamine adjusted to pH 5.5 with
phosphoric acid. Isocratic elution: (70:30:0.1). Flow rate: 0.7 mL/min. Detection: UV (λ=240 nm).
Antidepressants and antipsychotics
HA HF HX HY HZ HAA HAX HAZ
k k RI RI RT RT RT k
Amitriptyline 3.3 5.42 440 375 7.5 15.9 15.8 1.76
10-hydroxyamitriptyline 2.9 – – – – – – –
10-hydroxynortriptyline 1.8 – – – – – – –
nortriptyline 2 4.58 – – – – – 1.71
HA HF HX HY HZ HAA HAX HAZ
k k RI RI RT RT RT k
Amoxapine – – 398 – – 14.2 – –
Benperidol 1.1 – 393 324 3.6 – – –
Butriptyline 2.7 7.33 – 369 – – – –
norbutriptyline 1.7 – – – – – – –
Citalopram – – 403 – 4.5 – – –
desmethylcitalopram – – – – 3.7 – – –
Clomipramine 3.4 9.92 462 405 10.2 16.4 – –
monodesmethylclomipramine 2 – – – – – – –
Desipramine 2.1 3.6 424 361 5.9 14.9 13 1.52
didesmethylimipramine 1.3 – – – – – – –
2-hydroxydesipramine 1.2 – – – – – – –
M (2-OH-) – – – – – – – 0.39
Dibenzepin 2.8 0.5 361 300 – – – –
HA HF HX HY HZ HAA HAX HAZ
k k RI RI RT RT RT k
Dosulepin 3.2 3.6 428 367 5.7 – – –
M (sulfoxide) 4.6 – – – – – – –
M (nor-) 2.2 – – – – – – –
Doxepin 3.7 2.27 404 316 5 14.1 12.9 –
M (nor-) 2.2 – – – 4.6 – – –
Fluoxetine – – – 400 7.6 16.2 12.2 –
desmethylfluoxetine – – – – 6.7 – – –
Fluvoxamine – – 430 363 5.6 15.3 10 –
Imipramine 4.2 4.17 437 335 6.7 15.1 14.7 1.62
desipramine 2.1 3.6 – – – – – –
2-hydroxydesipramine 1.2 – – – – – – –
2-hydroxyimipramine 3.1 – – – – – – –
M (10-OH-) – – – – – – – 0.39
HA HF HX HY HZ HAA HAX HAZ
k k RI RI RT RT RT k
M (2-OH-) – – – – – – – 0.39
M (N-oxide) – – – – – – – 1.85
Iprindole 4.1 10.83 – – – – – –
Isocarboxazid – – 392 353 – – – –
Maprotiline 2.2 4.92 438 389 6.6 15.5 – 1.44
desmethylmaprotiline 1.1 – – – – – – –
Mianserin 1.8 – 391 342 4.6 13.8 – 1.18
M(nor-) 2.4 – – – – – – –
M (nor-) – – – – – – – 0.88
M (N-oxide) – – – – – – – 0.53
M (8-OH-) – – – – – – – 0.19
Moclobemide – – 295 – 2.4 10.2 6.9 –
Nialamide 1.2 – 334 – – – – –
HA HF HX HY HZ HAA HAX HAZ
k k RI RI RT RT RT k
Nomifensine 0.9 0.42 349 296 – – – –
Nortriptyline 2 4.58 – 338 6.6 15.6 13.7 1.71
10-hydroxynortriptyline 1.8 – – – – – – –
Noxiptiline – 1.63 – 330 – – – –
Opipramol 2.2 1.63 377 340 3.9 14.2 – –
Paroxetine – – 426 337 5.6 15.3 11.1 –
Phenelzine 1 – 184 – – – – –
Protriptyline 2.1 3.6 418 362 – – – –
Remoxipride – – 334 – 3 – 8.8 –
M(FLA-838) – – 316 – – – – –
M(NCM-001) – – 364 – – – – –
M(NCM-009) – – 341 – – – – –
Sertraline – – 460 – 8.2 – 14.5 –
HA HF HX HY HZ HAA HAX HAZ
k k RI RI RT RT RT k
(desmethylsertraline) – – – – 7.0 – – –
Tofenacin 1.7 – – – 5.3 – – –
Trazodone 0.6 – 378 305 3.3 12.7 – –
Trimipramine 2.7 6.17 454 345 8.3 15.9 15.5 –
M (nor-) 1.8 – – – – – – –
Viloxazine – 2.7 325 273 – 11 – –
Zimeldine 3.2 0.67 – 270 – – – –
M (nor-) 2.9 – – – – – – –
Antihistamines
The general screening systems, previously described, may be used.
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Alimemazine 3.1 420 – – – 14.9 7.1
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Antazoline 1.8 383 294 – – – –
Astemizole – – 286 3.9 13.2 – –
(astemizole) – 383 – – – – –
(M-nor) – 361 – – – – –
Bromazine 2.7 444 – – – – –
Brompheniramine 4.1 – 267 – 13.9 – –
Buclizine 0.7 – 454 – – – –
Carbinoxamine 4.7 359 – – 12.8 – –
Cetirizine – – – 3.6 15.7 8.89 5.29
Chlorcyclizine 2.3 – 340 – – – –
Chlorphenamine 3.9 356 264 3.5 12.9 10.8 5.3
Cinnarizine 0.8 560 – 22 19.3 – –
Clemastine 3.7 501 – 14 – – –
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Clemizole 4.8 420 – – – – –
Cyclizine 2.9 405 – 4.8 – 12.4 5.8
norcyclizine 2.2 – – – – – –
Cyproheptadine 3.2 – 354 6.5 15 – –
Deptropine 5 471 – 10.3 – – –
Dimetindene 5.1 338 288 – – – –
Diphenhydramine 3.3 393 336 – – 12.2 6
Diphenylpyraline 3.3 401 – – – – –
Doxylamine 4.4 – 259 – 11.1 – –
Hydroxyzine 1.4 437 326 5.7 15.3 11.4 6.3
Isothipendyl 3.8 390 – – 13.5 – –
Loratadine – 523 362 14.6 22.9 10.9 13.3
Mebhydrolin 3 411 – 5.3 – – –
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Meclozine 0.7 587 398 – 20 – –
Mepyramine 3.9 448 257 – – – –
Methapyrilene 4.1 342 197 – – – –
Methdilazine 6 – – – – 15.2 6.7
Phenindamine 2.5 397 – – – – –
Pheniramine 4.1 283 206 – – 9.5 4.5
Phenyltoloxamine 3.1 415 – – – – –
Pizotifen 3.4 435 – 6.6 15.2 – –
Promethazine 5 409 324 5.7 14.5 13.2 6.4
Propiomazine 2.1 440 359 – – 14.1 7.1
Pyrrobutamine 2.8 477 – – – – –
Thenyldiamine 4 317 – – – – –
Thiazinamium Metilsulfate – – – 6.4 – – –
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Trimethobenzamide 4.7 347 – – – – –
Tripelennamine 3.6 336 265 – – – –
Triprolidine 3.2 388 270 – 13.1 – –
Antimalarials
The general screening systems, previously described, may be used.
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Chloroquine 15.2 282 246 2.1 5.4 12.7 3.6
Cinchonidine 3.1 306 214 – – – –
Cinchonine – 304 209 – 10.2 – –
Halofantrine – 800 – – 23 – –
Hydroxychloroquine – 280 – 1.9 – 9.6 3.2
Primaquine 1.4 – 276 – – – –
HA HX HY HZ HAA HAX HAY
k RI RI RT RT RT RT
Proguanil – 379 – 3.8 13.6 – –
Pyrimethamine 1 – 289 – 12.5 – –
Quinine 2.4 327 246 2.6 11.3 8.3 4.5
Antineoplastics
The general screening systems, previously described, may be used.
HX HAA
RI RT
Diethylstilbestrol 592 20.9
Doxorubicin 370 12.1
Fluorouracil 70 3.4
Methotrexate 292 –
Vinblastine – 8.4
Antitussives
The general screening systems, previously described, may be used.
HA HX HY HAA
k RI RI RI
Bromhexine 0.4 417 334 –
Dextromethorphan 5.6 377 298 13.3
dextrorphan 4.7 – – –
Dextrorphan – 325 – –
Dropropizine – 240 – 7.2
Guaifenesin – 328 262 11.4
Noscapine 0.3 368 289 12.8
Pholcodine 6 65 92 2.7
Pipazetate 5.4 385 – –
Antivirals
The general screening systems, previously described, may be used.
System HAB
R. W. Sparidans et al. ,J. Chromatogr. B Biomed. Sci. Appl. 2000, 742, 185–192.
Column: C18 Symmetry (100 × 4.6 mm i.d., 3.5 μm) with Symmetry C18 pre-column (20 × 3.8 mm, 5 μm).
Mobile phase: Acetonitrile:sodium phosphate buffer (25 mM, pH 6.8) (40:60). Flow rate: 1.5 mL/min.
Detection: Fluorescence (λex=270 nm, λem=340 nm). Note: 8 min after each injection, flush column for 5 min at 1.5 mL/min with
aetonitrile:water (30:70). Equilibrate for about 8 min with the original eluent before injecting the next sample.
System HAC
G. Aymard et al. ,J. Chromatogr. B. Biomed. Sci. Appl. 2000, 744, 227–240.
Column: C18 Symmetry (250 × 4.6 mm i.d., 5 μm) with C18 pre-column (Guard-Pak, μBondapak).
Column temperature: 37°. Mobile phase: (A:B) Disodium hydrogen phosphate (0.04 M) with 4% (v/v)
octane sulfonic acid (0.25 M):acetonitrile. Isocratic elution: (50:50). Flow rate: 1.3 mL/min. Detection: UV diode-array. λ=261 nm between time 0 and 9 min; λ=241 nm
between time 9 and 20 min; λ=254 nm between time 20 and end of the run (32 min).
HAB HAC
RT k
Abacavir 1 –
Amprenavir 4 2.5
Efavirenz – 8.5
Indinavir 4.2 2
Benzodiazepines
System HI
R. Gill, unpublished data.
Column: ODS Hypersil (200 × 5 mm i.d., 5 μm). Mobile phase: Methanol:water:phosphate buffer (55:25:20). To prepare the
phosphate buffer dissolve 11.038 g (0.092 M) sodium dihydrogen phosphate and 1.136 g (0.008 M) disodium hydrogen phosphate in sufficient water to produce 1 L.
System HJ
R. Gill, unpublished data.
Column: As for System HI, above. Mobile phase: Methanol:water:phosphate buffer (as in System HI), (70:10:20).
System HK
R. Gill, unpublished data, after R. J. Flanagan et al., J. Chromatogr., 1980, 187, 391–
398.
Column: Silica Spherisorb (250 × 5 mm i.d., 5 μm). Mobile phase: Methanol to which has been added 100 μL perchloric acid per
litre.
Benzodiazepines
HI HJ HK HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
Acecarbromal – – – 429 374 – – – –
Alprazolam – – 2.79 – – – – – –
Bromazepam – – 2.99 – – – – – –
Bromisoval – – – 365 307 2.9 – – –
Brotizolam – – – 484 – 4.6 – 7.4 7.9
Carbromal – – – 410 377 3.9 – – –
Chlordiazepoxide – – 2.87 – – – – – –
Clobazam – – 0.03 – – – – – –
HI HJ HK HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
Clomethiazole – – – 395 292 – 16 – –
Clonazepam – – 0.35 – – – – – –
Clorazepic acid – – 2.00 – – – – – –
Demoxepam – – 0.03 – – – – – –
Diazepam – – 2.49 – – – – – –
Flumazenil – – – 387 327 2.6 – – –
Flunitrazepam 3.15 – 0.47 483 305 5.6 18.6 – –
Flurazepam – 3.19 6.5 397 305 4.2 – 10.5 5.5
Glutethimide – – – 436 401 4.8 – 6.6 6.2
Ketazolam – – 0.04 – – – – – –
Loprazolam – – – 388 – – 13.4 – –
Lorazepam – – 0.14 – – – – – –
Lormetazepam 6.32 – 0.08 487 463 6.2 – – –
HI HJ HK HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
Medazepam – – 4.44 – – – – – –
Methaqualone – – – 459 400 5.4 – 6.8 7.4
Methyprylon – – – 347 302 – – – –
Midazolam 9.75 2.1 5.9 399 306 4.2 14.9 10.2 6.3
Nitrazepam 2.96 – 1.49 448 370 4.2 16.9 6.3 6
Nordazepam – – 1.99 – – – – – –
Oxazepam 4.62 – 0.73 – – – – – –
Prazepam – – 2.19 – – – – – –
Quazepam – – – – 766 37.5 – 11.9 17.7
Temazepam 5.68 – 0.6 472 438 5.5 18.6 8.9 6.7
oxazepam – – 0.73 – – – – – –
Triazolam 4.38 – 1.83 476 390 4.2 17.4 6.4 6.7
not detected – – – – – – – – –
HI HJ HK HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
Zolpidem – – – – 291 3.2 11.9 – –
Zopiclone – – – 331 269 2.3 – 7.5 3.8
Cannabinoids
System HL
P. B. Baker et al. ,J. Analyt. Toxicol. 1980, 4, 145–152.
Column: ODS Spherisorb (250 × 4.6 mm i.d., 5 μm). Mobile phase: 0.01 M sulphuric acid:methanol:acetonitrile (7:8:9).
Cannabinoids
System HL
k
Cannabichromene 19.09
Cannabicyclol 14.78
Cannabidiol 7.47
Cannabidiolic acid 8.76
Cannabigerol 8.18
System HL
k
Cannabinol 11.77
Cannabivarin 7.47
Δ8-Tetrahydrocannabinol 14.07
Δ9-Tetrahydrocannabinol 13.35
Tetrahydrocannabinolic acid 25.83
Tetrahydrocannabivaric acid 14.64
Tetrahydrocannabivarin 8.18
Cardiac glycosides
System HM
P. H. Cobb , Analyst, Lond. 1976, 101, 768–776.(PubMed)
Column: Silica LiChrosorb SI60 (250 × 4 mm i.d., 10 μm). Mobile phase: Cyclohexane:ethanol:acetic acid (60:9:1
Cardiac glycosides
System HM
k
Digitoxigenin 2.0
Digitaxigenin bisdigitoxoside 3.9
Digitoxigenin monodigitoxoside 2.8
Digitoxin 5.4
Digoxigenin 4.5
Digoxigenin bisdigitoxoside 8.2
Digoxigenin monodigitoxoside 5.5
Digoxin 11.3
Gitaloxin 6.8
Gitoxigenin 3.7
Gitoxigenin bisdigitoxoside 6.5
Gitoxigenin monodigitoxoside 4.5
Gitoxin 8.6
System HM
k
Lanatoside A 17.9
Lanatoside B 31.8
Lanatoside C 39.5
Cardioactive drugs
The general screening systems, previously described, may be used.
HA HX HY HZ HAA
k RI RI RT RT
Ajmaline 2.8 – 277 – –
Alfuzosin – – – 2.4 10.4
Amiodarone 2.4 683 476 90.4 –
monodesethylamiodarone 1.8 – – – –
Aprindine – 433 – – 17
Bamethan 0.9 250 – – 5.9
HA HX HY HZ HAA
k RI RI RT RT
Benzthiazide – – 415 – –
Betahistine 3.1 – – – 3.2
Bretylium Tosilate 4.3 – 275 – –
Buphenine 0.9 370 – – –
Captopril – 316 283 2.1 9.7
Cilazapril – 420 – 4.5 14.4
cilazaprilate – – – 1.7 –
Clonidine 1.2 258 194 2.5 6.1
Clopamide – 377 310 – –
Debrisoquine 1.2 – 245 – –
Diltiazem – – 361 4.5 14
deacetyldiltiazem – – – – –
desmethyldiltiazem – – – – –
HA HX HY HZ HAA
k RI RI RT RT
desacetyldiltiazem – – – – –
Disopyramide 2.4 345 281 3 11.4
N-monodesisopropyldisopyramide 1.8 – – – –
Enalapril – 201 – 1.5 3.4
Encainide – 363 – – –
Felodipine – 690 – 25.8 24.4
Flecainide – 419 355 5.2 –
Hydralazine – 193 132 1.9 –
Isoxsuprine 0.8 353 301 – –
Labetalol 1.7 365 290 3 –
Lidoflazine 0.6 530 – – –
Lisinopril – 271 250 1.5 –
Lorcainide 1.8 425 – 6.6 –
HA HX HY HZ HAA
k RI RI RT RT
Methyldopa – 69 – 1.4 3
Mexiletine 1.2 329 278 – 11.5
Minoxidil – 297 – 2.4 9.8
Naftidrofuryl Oxalate – – 409 – 15.8
Nifedipine 0.2 527 464 7.2 19.5
Pargyline 0.2 – 203 – –
Pentaerithrityl Tetranitrate – 663 – – –
(pentaerithrityl) – – – – 23.1
Pentoxifylline – 355 274 2.1 11.5
Perindopril – – – 1.6 13.7
(perindoprilat) – 314 – – –
Phenoxybenzamine 0.1 396 – – –
Phentolamine 1.7 368 – 3 –
HA HX HY HZ HAA
k RI RI RT RT
Prajmalium Bitartrate 2.2 – 340 – –
Prazosin 0.8 352 – 2.5 10.6
Procainamide 1.3 208 160 1.9 –
N-acetylprocainamide 3 – – 1.8 –
Quinapril – – – 5.4 16.8
Quinidine 2.1 322 245 2.6 11
Ramipril – – – 4.2 15.7
Rescinnamine 0.6 496 407 – –
Reserpine – 467 351 – 16.4
Sotalol 1.2 226 – 2 3.8
Tocainide 1.2 247 208 2.1 –
Tolazoline 2.1 225 179 – –
Trandolapril – – – 6.1 17
HA HX HY HZ HAA
k RI RI RT RT
trandolaprilat – – – 2.1 –
Trimetazidine 3 – – – 6.1
Verapamil 2.6 447 386 7 15.4
M (nor-) 1.7 – – 6.6 –
Diuretics
System HN
R. Gill et al., unpublished data, after P. A. Tisdall et al., Clin. Chem., 1980, 26, 702–
706.
Column: ODS Hypersil (160 × 5 mm i.d., 5 μm). Mobile phase: Acetonitrile:water containing 10 mL/L acetic acid (30:70).
Diuretics
HN HX HY HAA
k RI RI RT
Acetazolamide – 268 226 6.9
Amiloride – 257 190 3.6
Bendroflumethiazide 15.35 508 – 18.6
HN HX HY HAA
k RI RI RT
Benzthiazide 9.32 – 415 –
Chlorothiazide 0.54 – 239 –
Chlortalidone 1.28 367 308 –
Clopamide 4.01 377 310 –
Clorexolone 7.26 – 391 –
Cyclopenthiazide 16.45 – 453 –
Cyclothiazide 10.78, 11.91, and 12.81 – 433 –
Etacrynic Acid – 521 497 –
Furosemide – 435 380 15.2
Hydrochlorothiazide 0.7 294 255 –
Mefruside 8.67 – 417 –
Methyclothiazide 3.82 – 364 15.4
Metolazone 4.89 – 371 –
HN HX HY HAA
k RI RI RT
Spironolactone – 592 539 20.7
Triamterene – 298 263 8.7
Trichlormethiazide 3.1 – 341 14.9
Xipamide – 488 – 18.8
Drugs of abuse
A comprehensive HPLC method for the screening of common drugs of abuse is
described in Chapter 1, Table 1.22. Furthermore, an additional eight systems (HBC,
HBD, HBE, HBF, HBG, HBI and HBJ) are provided in Chapter 2, Table 2.3.
Drugs of abuse
System
Compound HA HC HX HY HZ HAA
5-Methyltryptamine – – – – – –
Amfetamine 0.9 0.98 244 – – 3.7
Benzfetamine 1.2 0.15 – – – –
System
Compound HA HC HX HY HZ HAA
Benzoylecgonine 0.9 – – 236 1.7 9.7
Bufotenine 3.1 – – 181 – –
Cannabidiol – – 990 902 – –
Cannabinol – – 1080 1028 – –
Cocaine 2.8 – 348 289 3.3 11.9
Δ9-THC – – – – – –
Diamorphine 3 0.66 340 282 – –
Diethyltryptamine – – – – – –
Dimethyltryptamine – – – 228 – –
DOM – 1.13 340 – – –
Ketamine – – 311 262 2.4 9.6
Lysergic acid 0.8 – – 236 – –
Lysergide 0.7 – 362 – – 12
System
Compound HA HC HX HY HZ HAA
Mescaline 1.3 2.17 272 243 – –
Metamfetamine 2 2.07 262 216 2.4 8.4
Methadone 2.2 1.03 440 343 8.5 15.8
Methylenedioxyamfetamine – 0.98 266 248 2.1 8.1
Methylenedioxymethamfetamine – – 278 252 2.2 9.1
Monoacetylmorphine 3.6 0.8 – – – 7.3
Morphine 3.8 1.3 200 182 1.8 3.3
N-methyltryptamine – – – – – –
p-Methoxyamfetamine – – – – – –
Psilocin 3.1 – 240 226 – –
Psilocybine – – – 185 – –
Ergot alkaloids
System HA, previously described, may be used or System HP, below.
System HP
R. Gill et al., unpublished data, after P. J. Twitchett et al., J. Chromatogr., 1978, 150,
73–84.
Column: ODS Hypersil (100 × 5 mm i.d., 5 μm). Mobile phase: Methanol:phosphate buffer (60:40). To prepare the phosphate
buffer dissolve 3.43 g (0.022 M) sodium dihydrogen phosphate and 10.03 g (0.028 M) disodium hydrogen phosphate in sufficient water to produce 1 L.
Ergot alkaloids
HA HP
k k
Bromocriptine – 44.3
Dihydroergocristine – 18.3
Dihydroergocryptine – 15.9
Dihydroergotamine 0.6 11.4
Ergocornine 0.4 10.2
Ergocristine 0.3 17.3
Ergocryptine 0.4 15.2
Ergometrine 0.4 0.50
HA HP
k k
Ergosine 0.3 7.08
Ergosinine 0.3 17.7
Ergotamine 0.4 9.58
Iso-lysergic acid – 0.83
Iso-lysergide 2.6 0.0
Lysergamide 0.5 0.33
Lysergic acid 0.8 0.0
Lysergic acid methyl-propylamide – 1.98
Lysergide 0.7 1.83
Lysergol 1.1 0.83
Methylergometrine 0.4 0.83
Methysergide 0.4 2.33
2-Oxylysergide – 0.92
Local anaesthetics
The general screening systems, previously described may be used, as well as Systems
HQ or HR, below.
System HQ
R. Gill et al. ,J. Chromatogr. 1984, 301, 155–163.
Column: ODS Hypersil (160 × 5 mm i.d., 5 μm). Mobile phase: Methanol:water:1% v/v solution of phosphoric acid:hexylamine
(30:70:100:1.4).
System HR
R. Gill et al. ,J. Chromatogr. 1984, 301, 155–163.
Column: As for System HQ, above. Mobile phase: Methanol:1% v/v solution of phosphoric acid:hexylamine
(100:100:1.4).
Local anaesthetics
HA HQ HR HX HY HZ
k k k RI RI RT
Benzocaine 0.1 20.06 1.61 404 358 4.3
Bupivacaine 0.9 7.19 0.86 366 310 4.1
Butacaine 1.2 8.97 – 392 331 –
Butanilicaine – 4.42 – – 280 –
Chloroprocaine – 0.24 – – 250 –
HA HQ HR HX HY HZ
k k k RI RI RT
Cinchocaine 1.9 – 5.51 – 371 –
Cocaine 2.8 2.68 – 348 289 3.3
benzoylecgonine 0.9 5.68 – – – –
ecgonine 1.1 – – – – –
Cyclomethycaine – – 10.31 – 413 –
Dyclonine – – 2.78 – 347 –
Etomidate – – 475 417 –
Ketamine – – – 311 262 2.4
Lidocaine 0.6 0.79 – 288 258 2.6
M (monoethylglycinexylidide) 1.2 – – – – –
Mepivacaine 0.9 1.09 – 296 260 2.6
Methohexital – – – 503 484 –
Oxybuprocaine – 16.25 0.86 405 – –
HA HQ HR HX HY HZ
k k k RI RI RT
Piperocaine – 4.59 – 357 312 –
Pramocaine 0.6 – 2.48 415 – 6.5
Prilocaine 1 1.38 – – – 2.7
Procaine 1.9 – – 264 225 –
Propofol – – – – – 35
Proxymetacaine 2.1 1.38 – – 269 –
Quinisocaine 2.2 – 11.24 – – –
Tetracaine 2 16.25 1.33 389 321 4.4
Narcotic analgesics
Systems HA or HC, previously described, may be used or System HS, below.
System HS
P. B. Baker and T. A.Gough,J. Chromatogr. Sci. 1981, 19, 483–489.
Column: Amino-propyl bonded silica Spherisorb S5NH2 (250 × 4 mm i.d., 5 μm).
Mobile phase: Acetonitrile:tetrabutylammonium phosphate, 0.005 M, pH 7.5 (85:15).
Narcotic analgesics and narcotic antagonists
HA HC HS HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
Alphaprodine 2.8 – – 363 317 – – – –
Bezitramide 0.2 – – 564 – 22.5 – – –
Buprenorphine 0.4 0.05 – 397 339 5 14 – –
Codeine 4.8 1.21 1.9 266 237 1.9 5 6.1 3.4
morphine 3.8 1.3 5.16 – – – – – –
M (nor-) 3.1 3.51 – – – – – – –
Cyclazocine 2.1 – – – 289 – – – –
Dextromoramide 0.7 0.09 – 440 390 – 15.8 – –
Dextropropoxyphene 1.9 0.19 – – 374 7.6 15.8 – –
norpropoxyphene 1.3 – – – – – – – –
Diamorphine 3 0.66 0.35 340 282 – – 7.9 4.1
6-monoacetylmorphine 3.6 0.8 1 – – – – – –
morphine 3.8 1.3 5.16 – – – – – –
HA HC HS HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
Dihydrocodeine 7.2 2.5 – 261 208 2 4.7 – –
Dihydromorphine 5.7 2.75 – 237 156 – – – –
Dipipanone 2.2 1.61 – 500 363 – – – –
Ethoheptazine 3.3 1.55 – 359 – – – – –
Ethylmorphine 3.7 1.06 1.45 291 244 – – 6.7 3.6
Fentanyl 0.8 1.11 – 373 299 – 14.2 11.4 6
Hydromorphone 7.9 – – 240 187 – – 5.8 3.4
Ketobemidone 2.8 – – 294 245 – – – –
Levallorphan 1.9 1.46 – 356 291 – – – –
Levorphanol 4.4 3.2 – – 265 – – – –
Meptazinol 3.1 – – – 269 – – – –
Methadone 2.2 1.03 – 440 343 8.5 15.8 16.5 8.4
M (EDDP) 2.8 – – – – – – – –
HA HC HS HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
M (EMDP) 0.2 – – – – – – – –
Morphine 3.8 1.30 5.16 200 182 1.8 3.3 5.6 3.2
morphine-3-
glucuronide
– 1.56 – – – – – – –
N-oxide 3.2 – – – – – – – –
Nalorphine 1 0.29 – 260 237 – 4.8 – –
Naloxone 1.4 0.17 – – 238 2 14 – –
Norcodeine 3.1 3.51 – – 235 – – – –
Normethadone – 0.53 – – 366 – – – –
Normorphine 2.9 3.92 – – 133 – – – –
Norpipanone – 0.35 – 466 – – – – –
Oxycodone 6.9 0.85 – 277 246 – – 6.5 5.8
oxymorphone 6.7 – – – – – – – –
HA HC HS HX HY HZ HAA HAX HAY
k k k RI RI RT RT RT RT
Oxymorphone 6.7 – – 217 184 – – – –
Pentazocine 1.8 0.67 – 372 288 3.8 12.5 9.9 5.5
Pethidine 2.8 0.55 – 345 281 3.2 11.8 9.2 4.8
M (nor-) 1.7 2.04 – – – – – – –
pethidinic acid 2.8 – – – – – – – –
Phenazocine 1.3 0.3 – 409 299 – – – –
Phenoperidine 0.8 0.1 – 434 – – – – –
norpethidine 1.7 2.04 – – – – – – –
pethidine 2.8 0.55 – – – – – – –
Piritramide 0.6 0.1 – 377 343 – – – –
Thebacon 3.7 0.85 – 333 – – – – –
Tramadol – – – 328 267 2.9 – – –
Oral hypoglycemics and antidiabetics
The general screening systems, previously described, may be used.
HX HY HZ HAA
RI RI RT RT
Carbutamide – 321 – 14.5
Chlorpropamide 450 411 and 413 5 17.7
Glibenclamide 637 571 14.4 22
Gliclazide 536 483 8.8 20.5
Glipizide 478 423 4.5 17.6
Metformin 60 – 1.7 2.8
Tolazamide 452 445 6.8 –
Tolbutamide 477 424 5.9 –
Pesticides
System HAO
M. D. Osselton and R. D.Snelling,J. Chromatogr. 1986, 368, 265–271.
Column: ODS Hypersil (160 × 5 mm i.d., 5 μm), stainless steel. Mobile phase: Acetonitrile:water (60:40). Flow rate: 2 mL/min. Detection: UV diode-array (range: 200 to 450 nm).
System HAP
M. D. Osselton and R. D.Snelling,J. Chromatogr. 1986, 368, 265–271.
Column: Silica Spherisorb S5W (250 × 5 mm i.d.). Mobile phase: Dichloromethane:isoctane (60:40). Flow rate: 2 mL/min. Detection: UV diode-array (range: 200 to 450 nm).
For more information on screening pesticides, see Chapter 14, Table 14.1.
Phenothiazines and other tranquilisers
The general screening systems, previously described, may be used.
HA HX HY HZ HAA HAX HAY HAZ
k RI RI RT RT RT RT k
Acepromazine 4.1 – 350 – 10.8 – – –
Azacyclonol 1.2 – – – – 8.7 4.5 –
Benzoctamine 1.7 380 322 – – – – –
Butaperazine 3.4 464 406 – – – – –
Captodiame – 561 – – 20.2 – – –
Chlordiazepoxide – 363 285 3.2 15.2 6.9 5.3 1.68
Chlormezanone – – 334 – 15.5 6 5.3 –
Chlorpromazine 4.1 456 350 9.1 16 17 BASE 2.64
HA HX HY HZ HAA HAX HAY HAZ
k RI RI RT RT RT RT k
M (nor-) 2.2 – – – – – – –
M (sulfoxide) – – – – – 8.4 4.3 0.62
Chlorprothixene 3 459 353 10.1 – 17.6 8.3 –
Clopenthixol – 448 411 – – – – –
Clorazepic Acid – 475 388 5.6 – – – –
clorazepate – – – – 18.4 – – –
Fluanisone – 423 349 – – – – –
Flupentixol 1.2 475 435 10.7 17.4 13.7 7.5 –
sulfoxide 1.3 – – – – – – –
Fluphenazine 1.2 462 471 10.1 17.4 13.6 7.2 –
Fluspirilene – 538 – – – 18.3 9.8 –
Haloperidol 1.2 421 316 5.8 14.4 11.1 6.2 0.72
Levomepromazine 3.2 435 381 7.5 – 15.2 7.2 1.82
HA HX HY HZ HAA HAX HAY HAZ
k RI RI RT RT RT RT k
Loxapine 1.1 407 336 – 14.6 – – –
Mesoridazine 5 – 337 3.4 – 10.1 5 –
Oxypertine 0.7 402 – – – – – –
Pecazine 3.9 443 382 – – 15.3 7 –
Penfluridol – 659 656 43.4 20.2 – – –
Perazine – 403 371 6.3 – – – –
Pericyazine 1.3 410 356 4.4 – 10.2 5.1 –
Perphenazine 1.9 428 395 7.2 16 13.1 6.3 3.28
Pimozide 0.7 504 – 11.9 17.2 – – –
Pipamperone – 299 241 2.7 10.9 – – –
Pipotiazine – 431 – – 14.7 – – –
Prochlorperazine 3.9 450 323 10.4 – – – –
Promazine 5.9 407 326 5.9 – – – –
HA HX HY HZ HAA HAX HAY HAZ
k RI RI RT RT RT RT k
Prothipendyl 4.4 388 – – – – – –
Sulforidazine – 421 – 4.8 – – – –
Sulpiride – 259 235 2 3.9 – – 0.02
Thiopropazate 1 483 – – – – – –
Thioproperazine 4.1 427 305 15.4 15.2 – – –
Thioridazine 5.2 490 427 13.5 17.2 – 9.8 3.88
mesoridazine 5 – – – – – – –
Tiotixene 3.8 442 374 6.8 – – – –
Triflupromazine 2.7 484 454 12.3 – 17.3 8.9 –
Steroids
System HATa
M. J. Walters et al. ,J. Assoc. Off. Analyt. Chem. 1990, 73, 904–926.
Column: ODS Zorbax (250 × 4.6 mm i.d., 5 μm), stainless steel. Eluent: (A) methanol. Isocratic elution: (100).
Flow rate: 1.5 mL/min. Detection: UV (λ=240, 210 and 280 nm).
System HATb
M. J. Walters et al. ,J. Assoc. Off. Analyt. Chem. 1990, 73, 904–926.
Column: ODS Zorbax (250 × 4.6 mm i.d., 5 μm), stainless steel. Eluent: (A:B) methanol:water. Isocratic elution: (75:25). Flow rate: 1.5 mL/min. Detection: UV (λ=240, 210 and 280 nm).
System HAR
I. Lurie et al. ,J. Forens. Sci. 1994, 39, 74–85.
Column: ODS Zorbax (250 × 4.6 mm i.d., 5 μm). Mobile phase: (A:B) Water:methanol. Gradient elution: (30:70) to (0:100) over 15 min with 15 min hold. Flow rate: 1.0 mL/min. Detection: UV diode-array.
System HT
J. Q. Rose and W. J.Jusko, J. Chromatogr. 1979, 162; Biomed. Appl., 4, 273–280.
Column: Silica Zorbax SIL (250 × 4.6 mm i.d., 5 μm). Mobile phase: Methylene chloride:methanol (97:3).
Steroids
HT HX HY HZ HAA HAR HATa HATb
k RI RI RT RT RRT RRT RRT
Beclometasone 4.2 444 – – – – – –
dipropionate – – 711 – – – – –
Betamethasone – – – 14.2 13.3 – – –
betamethasone valerate – – 584 – – – – –
Boldenone – – – – – 0.74 – 0.76
HT HX HY HZ HAA HAR HATa HATb
k RI RI RT RT RRT RRT RRT
undecylenate – – – – – – 1.94 –
Cortisone 2.4 – 372 – – – – –
Dexamethasone 4.8 – 381 3.4 13.1 – – –
Fluoxymesterone – – 427 – – 0.78 – 0.7
Hydrocortisone 5.8 403 349 – 17.7 – – –
Hydroxyprogesterone – 1054 – – – – – –
Metenolone – – – – – – – –
acetate – – – – – – 1.26 3.54
enantate – – – – – – 1.87 –
Methandienone – – – – – 0.86 – 0.87
Methandriol – – – – – 1.25 – 1.29
dipropionate – – – – – – – 2.75
Methylprednisolone 7.5 426 390 – 18.9 – – –
Methyltestosterone – – 587 – – 1.17 – 1.27
Nandrolone – – – – – 0.84 – 0.92
Norethisterone – 536 676 – 24 – – –
Prednisolone 8.4 401 361 2.5 14.1 – – –
Prednisone 3.4 250 340 2.6 14.2 – – –
Progesterone – 672 698 – 23.8 – – –
HT HX HY HZ HAA HAR HATa HATb
k RI RI RT RT RRT RRT RRT
Testosterone – 534 508 – – – – –
acetate – – 894 – – 1.76 – 2.59
propionate – – 1003 – – 2.01 1.31 4.06
methyltestosterone – – – – – 1.17 – 1.27
isobutyrate – – – – – 2.17 – –
cipionate. – – – – – 2.63 – –
enantate – – – – – 2.6 1.8 –
undecanoate – – – – – 3.18 – –
phenylpropionate – – – – – – 1.48 –
isocaproate – – – – – – 1.62 –
cipionate – – – – – – 2.05 –
undecenoate – – – – – – 2.53 –
decanoate – – – – – – 2.78 –
undecylate – – – – – – 3.27 –
Triamcinolone – 438 312 – – – – –
acetonide 2.5 – – – – – – –
Trenbelone – – – – – – – –
hexahydrobenzylcarbonate – – – – – – 1.65 –
acetate – – – – – – – 1.71
Sulfonamides
System HU
P. H. Cobb and G. T.Hill,J. Chromatogr. 1976, 123, 444–447.
Column: Silica Spherisorb (250 × 4 mm i.d., 5 μm). Mobile phase: Cyclohexane:ethanol:acetic acid(85.7:11.4:2.9).
Sulfonamides
HU
k
Phthalylsulfathiazole 14.0
Succinylsulfathiazole 16.8
Sulfadoxine 4.4
Sulfamerazine 8.1
Sulfaquinoxaline 4.8
Sulfacetamide 7.7
Sulfachlorpyridazine 3.3
Sulfadiazine 8.7
HU
k
Sulfadimidine 7.1
Sulfafurazole 6.0
Sulfamethoxazole 4.8
Sulfamethoxydiazine 8.2
Sulfamethoxypyridazine 7.5
Sulfamoxole 12.6
Sulfanilamide 8.9
Sulfapyridine 3.8
Sulfathiazole 13.4
Xanthine stimulants
The general screening systems, previously described, may be used.
HA HX HY HZ HAA
k RI RI RT RT
Caffeine 0.2 305 259 1.9 6.7
Diprophylline – 275 227 – 3.6
Fenetylline – 336 277 – –
Proxyphylline 0.1 293 – – –
Theobromine 0.1 262 201 1.6 3.8
Theophylline 0.1 276 249 1.7 4.9
Additional systems
System HAD
G. Aymard et al. ,J. Chromatogr. Biomed. Sci. Appl. 2000, 744, 227–240.
Column: C18 Symmetry Shield (250 × 4.6 mm i.d., 5 μm) protected by 2 μm Upchurch filter.
Column temperature: 30°. Mobile phase: (A:B) M/15 potassium dihydrogen phosphate with 1% (v/v)
octane sulfonic acid:acetonitrile. Mobile phase (MP) 1: (95:5) at flow rate 1 mL/min; MP 2: (80:20) at flow rate 1 mL/min; MP 3: (30:70) at flow rate 1.2 mL/min.
Eluent switching programme: At injection, MP1 to the column. From time 12 to 30 min, MP2 to the column. From time 30 min, MP3 to the column to rinse it. From time 35 to 40 min, equilibration with MP1.
Detection: UV diode-array (λ=260 nm).
k Compound
2.7 Lamivudine
3.2 Didanosine
3.8 Stavudine
6.6 Zidovudine
8.1 Abacavir
11.1 Nevirapine
System HAF
E. Tanaka et al. ,J. Chromatogr. B. Biomed. Sci. Appl. 1996, 682, 173–178.
Column: ODS TSK-gel Super (100 × 4.6 mm i.d., 2 μm). Mobile phase: (A:B) Acetonitrile:sodium dihydrogen phosphate (5 mM, pH 6). Isocratic elution: (45:55). Flow rate: 0.65 mL/min. Detection: UV (λ=254 nm).
Retention time (min) Compound
5.3 Clonazepam
6.6 Bromazepam
9.1 Nitrazepam
13.7 Triazolam
15.0 Lorazepam
18.4 Etizolam
21.0 Chlordiazepoxide
29.8 Diazepam
32.2 Flutazolam
System HAV
D. R. Rutledge et al. ,J. Pharm. Biomed. Analysis, 1994, 12, 135–140.
Column: RP-short alkyl chain, silanol deactivated (SCD 100) (250 × 4.6 mm i.d.), stainless steel.
Mobile phase: (A:B) Methanol:dibasic potassium phosphate (0.04 M, pH 5.5). Isocratic elution: (50:50). Flow rate: 1 mL/min. Detection: UV (λ=237 nm).
k Compound
2.2 Celiprolol
2.3 Propranolol
3.6 Diltiazem deacetyldiltiazem
5.1 Diltiazem desmethyldiltiazem
6.1 Diltiazem
6.4 Imipramine
8.2 Verapamil
System HBA
J. Sastre-Toraño and H.-J.Guchelaar,J. Chromatogr. B Biomed. Sci. Appl. 1998, 720,
89–97.
Column: C18 base-deactivated silica (125 × 4.6 mm i.d., 5 μm) with base-deactivated C18 pre-column (20 × 4.6 mm i.d., 5 μm).
Eluent: (A:B) Acetonitrile:potassium dihydrogen phosphate (50 mM, pH 7.5, containing 500 μL triethylamine).
Isocratic elution: (60:40). Flow rate: 2 mL/min. Detection: Fluorescence (λex=255 nm, λem=315 nm).
Retention time (min) Compound
8.8 Erythromycin
15.7 Clarithromycin
17.1 Roxithromycin
20.7 Azithromycin
System HBB
C. Taninaka et al. ,J. Chromatogr. B Biomed. Sci. Appl. 2000, 738, 405–411.
Column: C18 (250 × 6.0 mm i.d., 5 μm). Eluent: (A:B) Acetonitrile:phosphate buffer (50 mM, pH 7.2). Isocratic elution: (43:57). Flow rate: 1.7 mL/min. Detection: Electrochemical (working electrode: glassy carbon, reference
electrode: Ag/AgCl).
Retention time (min) Compound
6.8 Clarithromycin
6.8 Erythromycin
9.6 Azithromycin
16.3 Roxithromycin
System HAE
V. Proust et al. ,J. Chromatogr. B Biomed. Sci. Appl. 2000, 742, 453–458.
Column: C18 (Lichrospher, 100 RP-18, 5 μm) with C18 pre-column (Lichrospher RP-18, 5 μm).
Mobile phase: (A:B) acetonitrile:sodium phosphate (25 mM) modified with diethylamine (0.9%) and tetrahydrofuran (1%), pH 3.0.
Isocratic elution: (44.8:55.2). Flow rate: 0.5 mL/min. Detection: UV (λ=260 nm).
Retention time (min) Compound
6.3 Delavirdine
7.0 Saquinavir
8.0 Nelfinavir
9.4 Amprenavir
22.2 Ritonavir
28.6 Efavirenz
System HAK
C. Le Guellec et al. ,J. Chromatogr. Sci. Appl. 1998, 719, 227–233.
Column: C18 Symmetry (250 × 4.6 mm i.d., 5 μm) with C18 pre-column Symmetry sentry.
Mobile phase: (A:B) Acetonitrile:potassium dihydrogen phosphate (20 mM). Elution programme: (50:50) to (70:30) in 15 min. Flow rate: 1 mL/min. Detection: UV (λ=313 nm).
Retention time (min) Compound
4.7 Carbamazepine
6.2 Clonazepam
7.6 Nordazepam
9.3 Clobazamm
not detected Phenobarbital
not detected Phenytoin
System HAL
A. Boukhabza et al. ,J. Chromatgr. 1990, 529, 210–216.
Column: C18 Novapak (150 × 4.6 mm i.d., 5 μm). Mobile phase: (A:B:C) Acetonitrile:methanol:phosphate buffer (6 mM), pH 5.7. Isocratic elution: (30:10:60). Flow rate: 1.3 mL/min.
Detection: UV diode-array (λ=242 nm). Note: The phosphate buffer stock solution is prepared using 94 mL 0.2 M
sodium dihydrogen phosphate added to 6 mL 0.2 M disodium phosphate heptahydrate.
Retention time (min) Compound
1.4 Barbital
1.45 Clonazepam 7-acetamidoclonazepam
1.55 Clonazepam 7-aminoclonazepam
2.0 Aprobarbital
2.4 Hexobarbital
3.7 Flunitrazepam M (nor)
4.4 Nordazepam oxazepam
4.4 Oxazepam
4.6 Nitrazepam
4.33 Clonazepam
5.1 Lorazepam
6.2 Flunitrazepam
6.3 Alprazolam
6.6 Triazolam
7.7 Chlordiazepoxide
7.8 Clobazam
7.9 Nordazepam
8.1 Bromazepam
8.2 Medazepam
13.2 Diazepam
System HAM
D. de Carvalho and V. L.Lanchote,Ther. Drug Monit. 1991, 13, 55–63.
Column: C18 (150 × 4.0 mm i.d., 3 μm) with C18 pre-column (40 × 4.0 mm i.d., 3 μm).
Mobile phase: (A:B) water:acetonitrile. Isocratic elution: (50:50). Flow rate: 0.7 mL/min. Detection: UV (λ=313 nm).
Retention time (min) Compound
1.8 Theophylline
1.98 Caffeine
2.0 Paracetamol
2.2 Primidone
2.7 Sulfamethoxazole
2.8 Phenobarbital
3.1 Chlordiazepoxide
3.4 Diazepam
3.4, 4.4 Oxazepam
3.5 Phenytoin
4.2 Lorazepam
4.3 Clonazepam
4.5 Nitrazepam
9.0 Imipramine
9.1 Desipramine
10.3 Diazepam
not detected Alprazolam
not detected Bromazepam
not detected Clobazam
not detected Codeine
not detected Ephedrine
not detected Levomepromazine
not detected Lidocaine
not detected Medazepam
not detected Nortriptyline
not detected Propranolol
not detected Thioridazine
not detected Triazolam
References
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Further reading
1. D. Armstrong and B.Zhang, Chiral stationary phases for high performance liquid chromatography, Anal. Chem. 2001, 73, 557A–561A.
2. J. Ayrton et al. , Use of generic fast gradient liquid chromatography–tandem mass spectroscopy in quantitative bioanalysis, J. Chromatogr. B, 1998, 709, 243–254.(PubMed)
3. S. C. Bobzin et al. , LC–NMR: a new tool to expedite the dereplication and identification of natural products, J. Ind. Microbiol. Biotechnol. 2000, 25, 342–345.(PubMed)
4. T. Fornstedt and G.Guiochon, Nonlinear effects in LC and chiral LC, Anal. Chem. 2001, 73, 609A–617A.(PubMed)
5. V. C. X. Gao et al. , Column switching in high performance liquid chromatography with tandem mass spectrometric detection for high–throughput preclinical pharmacokinetic studies, J. Chromatogr. A, 1998, 828, 141–148.(PubMed)
6. R. J. Hamilton and P.Sewell, Introduction to High Performance Liquid Chromatography, Second Edn, London, Chapman & Hall, 1977.
7. K. Heinig and F.Bucheli, Application of column–switching liquid chromatography–tandem mass spectrometry for the determination of pharmaceutical compounds in tissue samples, J. Chromatogr. B, 2002, 769, 9–26.(PubMed)
8. J. Henion et al. , Sample preparation for LC–MS–MS: Analyzing biological and environmental samples, Anal. Chem. 1998, 70, 650A–656A.(PubMed)
9. R. P. Hicks , Recent advances in NMR: expanding its role in rational drug design, Curr. Med. Chem, 2001, 8, 627–650.(PubMed)
10. Johns , Resolving isomers on HPLC columns with chiral stationary phases, Am. Lab. 1987, Jan., 72–76.
11. H. T. Karnes and M. A.Sarkar, Enantiomeric resolution of drug compounds by liquid chromatography, Pharm. Res. 1987, 4, 285–292.(PubMed)
12. G. Lunn and N. R.Schmitt, HPLC Methods for Pharmaceutical Analysis, New York, John Wiley & Sons, Vol. 1, 1997; Vols 2–4, 2000.
13. R. E. Majors , New chromatography columns and accessories at the 1997 Pittsburgh Conference Part 1. LC–GC, 1997, 15, 220–237.
14. R. E. Majors , New chromatography columns and accessories at the 1998 Pittsburgh Conference Part 1. LC–GC, 1998, 16, 228–244.
15. R. E. Majors , New chromatography columns and accessories at the 1999 Pittsburgh Conference Part 1. LC–GC, 1999, 17, 212–220.
16. R. E. Majors , New chromatography columns and accessories at the 2000 Pittsburgh Conference Part 1. LC–GC, 2000, 18, 262–285.
17. V. R. Meyer , Practical High Performance Liquid Chromatography, Second Edn, New York, Wiley Publishers, 1979.
18. S. X. Peng et al. , Direct determination of stability of protease inhibitors in plasma by HPLC with automated column–switching, J. Pharm. Biomed. Anal. 1999, 25, 343–349.
19. R. S. Plumb et al. , The application of fast gradient capillary liquid chromatography–mass spectrometry to the analysis of pharmaceuticals in biofluids, Rapid Comm. Mass Spectrom. 1999, 13, 865–872.(PubMed)
20. C. Schüfer et al., . HPLC columns: The next great leap forward- Part 1. Am. Lab., 2001, Feb., 40–41.
21. C. Schüfer et al., HPLC columns: The next great leap forward, Part 2. Am. Lab., 2001, April, 25–26.
22. C. F. Simpson , Practical High Performance Liquid Chromatography, London, Heyden and Son Ltd, 1976.
23. L. R. Snyder , HPLC past and present, Anal. Chem. 2000, 72, 412A–420A.(PubMed) 24. N. Tanaka et al. , Monolithic LC columns, Anal. Chem. 2001, 72, 420A–429A. 25. T. Wehr , Configuring HPLC systems for LC–MS, LC–GC, 2000, 18, 406–416. 26. I. Wilson et al. , 2000. Analytical chemistry: Advancing hyphenated
chromatographic systems, Anal. Chem. 2000, 71, 534A–542A. 27. J. L. Wolfender et al. , The potential of LC–NMR in phytochemical analysis,
Phytochem. Anal. 2001, 12, 2–22.(PubMed) 28. L. Y. Yang et al. , Applications of new liquid chromatography–tandem mass
spectrometry technologies for drug development support, J. Chromatogr. A, 2001, 926, 43–55.(PubMed)