Liquid Chromatographic Liquid Chromatographic Methods for the Extraction of Methods for the Extraction of Pharmaceutical Compounds Pharmaceutical Compounds

From Biological FluidsFrom Biological Fluids

Nick GrayNick Graysanofisanofi--aventisaventis

BackgroundBackgroundQuantitative bioanalysis is performed in the Quantitative bioanalysis is performed in the pharmaceutical industry to support the pharmaceutical industry to support the development of new pharmaceutical compounds. development of new pharmaceutical compounds.

Analysis is predominantly in blood plasma, or Analysis is predominantly in blood plasma, or urine, but also serum, milk, inurine, but also serum, milk, in--vitro incubation vitro incubation media.media.

Blood samples are taken during a defined time Blood samples are taken during a defined time profile following dose administration and profile following dose administration and concentration time profile is constructed.concentration time profile is constructed.

These data determine the key pharmacokinetic These data determine the key pharmacokinetic parameters to characterise the exposure, parameters to characterise the exposure, distribution and clearance of the drug and relate distribution and clearance of the drug and relate these to safety and efficacy.these to safety and efficacy.

BackgroundBackground

Pharmaceutical development is highly Pharmaceutical development is highly regulated environment. regulated environment.

Providing Providing ‘‘onon--lineline’’ pharmacokinetic data pharmacokinetic data for First in Man studies provides a safe for First in Man studies provides a safe framework for dose escalation studies. framework for dose escalation studies.

BackgroundBackgroundBioanalytical methodology must be:Bioanalytical methodology must be:•• Very sensitive (Lower Limits of Quantification in the Very sensitive (Lower Limits of Quantification in the

pg/mL to ng/mL range; pg/mL to ng/mL range; femtogrammefemtogramme scale injections)scale injections)

•• High throughput; typically 200 samples per period for an High throughput; typically 200 samples per period for an ‘‘onon--lineline’’ study study –– ideally achievable overnightideally achievable overnight

•• Very robust; multiple development studies running Very robust; multiple development studies running simultaneously.simultaneously.

•• High quality; to comply with strict regulatory High quality; to comply with strict regulatory requirementsrequirements

Samples are Samples are ‘‘dirtydirty’’•• Proteins, salts, lipids, endogenous and exogenous Proteins, salts, lipids, endogenous and exogenous

compoundscompounds

BackgroundBackground

Technique is LCTechnique is LC--MS/MSMS/MS

•• Typically ESI or APCITypically ESI or APCI

•• Internal standard calibration with stable Internal standard calibration with stable isotope labelled analyte.isotope labelled analyte.

GC and CE separations are also used but GC and CE separations are also used but LC is predominant technique in industry.LC is predominant technique in industry.

•• This presentation relates to preparation of This presentation relates to preparation of samples for HPLC analysis.samples for HPLC analysis.

Trend in LCTrend in LC--MS analysisMS analysis

LLOQ

Throughput

Assay Sensitivity

Run time

Productivity

Method Development

LCLC--MS/MSMS/MSOperation in SRM mode (LCOperation in SRM mode (LC--MS/MS) gives MS/MS) gives the method superior selectivitythe method superior selectivity•• Only molecules of the analyte mass are Only molecules of the analyte mass are

detecteddetected•• M+ M+ (M(M--n)n)++Stable isotope internal standardisation Stable isotope internal standardisation means that the differences between means that the differences between injections are minimised.injections are minimised.

Refine the sample a bit and inject ?Refine the sample a bit and inject ?•• Protein precipitation/dilute and shoot ?Protein precipitation/dilute and shoot ?

LCLC--MS ChromatogramMS Chromatogram

Time ->

Sign

al R

espo

nse

->

All components

Precursor ion --> Product ion

Detected InterferenceDetected Interference

Undetected coUndetected co--eluentseluents SuppressionSuppression

InterferenceInterference

Contaminant with Close Mass Contaminant with Close Mass ‘‘hiddenhidden’’

Molecular isotopes with same mass Molecular isotopes with same mass ‘‘visiblevisible’’

Matrix effectMatrix effect

Loss of signal responseLoss of signal response

Loss of precisionLoss of precision

Deviation from linearityDeviation from linearity

Selective peak detection follows the iceberg Selective peak detection follows the iceberg phenomenonphenomenon

ExtractionExtractionSolid Phase ExtractionSolid Phase Extraction

Off lineOff line

In lineIn line

Direct Injection TechniquesDirect Injection Techniques

Restricted Access MediaRestricted Access Media•• StericSteric restrictionrestriction

Internal SurfaceInternal Surface

External SurfaceExternal Surface

•• Dynamic restrictionDynamic restrictionTurbulent Flow ChromatographyTurbulent Flow Chromatography

Multiplexed analysisMultiplexed analysis

Solid Phase ExtractionSolid Phase ExtractionAlkyl modified silica stationary

phase in a disposable cartridge.

‘Digital’ Chromatography– On/Off mechanism

– 100% retention / 0% retention

– ‘Washed’ to remove

unretained components

– Elution in organic solvent• evaporation/reconstitution

• direct analysis

Waters OasisTM

Solid Phase ExtractionSolid Phase Extraction

PolypropylenePolypropylenecartridgecartridge

SorbentSorbentAlkylAlkyl--silica,silica,Reverse phaseReverse phasepolymerpolymer

1. Condition1. ConditionAddition of Addition of solventsolvent

2. Add sample2. Add sampleDispense to the Dispense to the cartridge, allow cartridge, allow sample to sample to adhere to adhere to sorbentsorbent

3. Wash3. WashWith aqueous or With aqueous or organic solvents organic solvents elute to wasteelute to waste

4. Elute4. EluteWith eluting With eluting solventsolvent

InjectInject

Solid Phase Extraction Solid Phase Extraction AutomationAutomation

SPE is well automated– Robotics– Microtitre plate

technology

Can be integrated with HPLC autoinjector.Resource intensive– Cartridges single use– Other consumables

requiredGilson ASPEC

•• Examples; Waters Oasis, Examples; Waters Oasis, BiotageBiotage EvoluteEvolute

Direct injection techniquesDirect injection techniquesThe extraction and analysis of the mixture are The extraction and analysis of the mixture are incorporated into a single process.incorporated into a single process.

Analyte molecules interact with a Analyte molecules interact with a chromatographic stationary phase, while chromatographic stationary phase, while macromolecules are restricted from macromolecules are restricted from interaction.interaction.

Manual extraction steps are unnecessary and Manual extraction steps are unnecessary and samples in plasma can be injected directly samples in plasma can be injected directly onto the HPLC/MS/MS.onto the HPLC/MS/MS.

Elution gradient is applied, and retained Elution gradient is applied, and retained analyte molecules are eluted to the detection analyte molecules are eluted to the detection system or through a second analytical column.system or through a second analytical column.

Entire test aliquot is injected Entire test aliquot is injected -- sample sample conserved and possibility of 100% recovery conserved and possibility of 100% recovery from injected sample.from injected sample.

External Surface Restricted External Surface Restricted Access MaterialAccess Material

• Standard chromatographic particle.• Surface modified by an external polymeric cage

• Small analyte molecules can permeate surface.• Macromolecules are too large to fit within the mesh and are eluted

unretained.• Analytes are retained while macromolecules pass directly through

column.• Analytes are then eluted through the column.• Example, Pinkerton SPM

Silica Particle core

Inert meshwork over particle surface

Internal Surface Restricted Internal Surface Restricted Access MaterialAccess Material

• Porous spherical silica particle with inert biocompatible outer surface, and alkylated hydrophobic inner surface.

• Small analyte molecules have access to stationary phase on innersurface.

• Macromolecules are too large to fit within the pores, and only contact with the inert biocompatible outer surface.

• Analytes are retained while macromolecules pass directly throughcolumn.

• Example, BAS Biotrap, Merck AGP, Pinkerton ISRP

Contents transferred to Extraction column

Analyte molecules with access to stationary phase are retained

on head of column

Analyte pre-concentration

Direction of flow reversed through Extraction column, Analytical column brought online with Extraction column.

Analyte molecules transferred to analytical column and ‘Injected’ for chromatographic analysis

Sample Injected

Macromolecules not retained by column

Waste

On Line Extraction By Direct Injection TechniquesOn Line Extraction By Direct Injection Techniques

Waste

Pump

Pump INJECTOR

1

2

3

4

5

6

DetectionSystem

Analytical Mobile Phase

Column Switching : Load PositionColumn Switching : Load Position

ExtractionMobile Phase

Extraction Column

Analytical Column

Plasma

Waste

Pump

Pump INJECTOR

1

2

3

4

5

6

DetectionSystem

Analytical Mobile Phase

ExtractionMobile Phase

Extraction Column

Analytical Column

Column Switching : Inject PositionColumn Switching : Inject Position

• Trace enrichment from large volumes of plasma• Qualitative analysis to determine metabolic profiles.

• Loading up to 10 mL of plasma in a single run.• Column lifetime up to 150 mL of plasma.• Flow rates up to 5 mL/min

• Large volumes of aqueous environmental samples.

Restricted Access ChromatographyRestricted Access Chromatography

Turbulent flow chromatographyTurbulent flow chromatography

• Unmodified alkylated silica particle• Large particle size (50 µm) • High linear velocity (5 mL/min through a 1.0mm i.d.

column.)• This has two important characteristics :

• Turbulent flow characteristics• Flat flow profile• Efficient mass transfer

• High linear velocity prevents large macromolecules from interacting with the stationary phase.

• Laminar flow : Frictional drag on the column walls gives rise to differing rates of flow across the column diameter.

• Turbulent Flow : Rapid movement of the liquid evens out the rate at which different molecules travel through the column.

• Turbulent flow gives rise to narrow, efficient peaks.

Turbulent flow : The Analytical system.Turbulent flow : The Analytical system.

Turbulent flow : The Analytical system.Turbulent flow : The Analytical system.Efficient separations at high linear velocity

According to the van Deemter relationship that isn’t possible.

h = a + b/v + cv

A Term

C Term

Flow velocity

h

• Turbulent flow is the region beyond laminar flow.

• As flow rate is increased beyond the laminar region, efficiency increases with flow rate.

• Turbulent flow is recognised in open tube chromatography. In a packed bed, ‘tubes’ are formed in the spaces between packing particles

• Because the flow rate is high, speed of analysis increased and therefore there is a significant improvement in productivity.

Turbulent flow : The Analytical system.Turbulent flow : The Analytical system.

LipophilicLipophilic PhasePhase

Silica ParticleSilica Particle

Plasma-drugComplex

Plasma

Drug

High linear velocity prevents large molecules from interacting with the stationary phaseDiffusion is driven by a concentration gradient.Analyte molecules can be retained on the chromatographic stationary phase inside the pores

Plasma proteins are eluted in the void volume.Analyte molecules can be eluted by increasing the organic gradient through the column.

Turbulent flow : The Extraction system.Turbulent flow : The Extraction system.

Analysis of pharmaceutical compound Analysis of pharmaceutical compound in plasma using Turboflowin plasma using Turboflow

• Extraction (load)• pH 3.0, 20 mM ammonium formate • 5.0 mL/min• Column : Turboflow® C8 50µm 50 x 1.0mm • loading time : 0.75 mins

• Elution (inject)• pH 3.0, 20 mM ammonium formate / Methanol (20:80)• 1.0 mL/min• Column : Phenomenex Luna C18 (2) 5µm 30 x 2.0mm• 45 °C• Retention time of pharmaceutical compound : 1 min• Stop time : 3 mins

Schematic Showing a Typical Schematic Showing a Typical TurboflowTurboflow--LCLC--MS MethodMS Method

Inefficiency of MS Acquisition During Inefficiency of MS Acquisition During InjectionInjection

Schematic Showing a Multiplexed Schematic Showing a Multiplexed TurboflowTurboflow--LCLC--MS MethodMS Method

Multiplex analysisMultiplex analysis

Twin autosampler, two extraction pumps, two analytical pumps

ConclusionsConclusionsThere are a variety of techniques and materials available to chromatographically separate analytes from interfering endogenous molecules.Chromatographic extractions can be selective, reliable and well automated. Direct injection techniques contribute to improving the efficiency of extractions in bioanalysis.– Efficient sample recovery, higher sensitivity,

lower LOQThese techniques have been shown to demonstrate a high degree of precision and accuracy.

AcknowledgementsAcknowledgementsStuart McDougall, Head of Bioanalysis, sanofi-aventisBioanalysis and Mass Spectrometry teams, sanofi-aventis, Alnwick, UK