electrokinetic supercharging-electrospray ionisation-mass spectrometry for separation and on-line...

Research Article

Electrokinetic supercharging-electrosprayionisation-mass spectrometry forseparation and on-line preconcentrationof hypolipidaemic drugs in water samples

Electrokinetic supercharging, a powerful on-line preconcentration technique in CE, was

for the first time hyphenated with ESI-MS for the on-line concentration and separation of

five hypolipidaemic drugs. The electrophoretic separation was performed in a co-EOF

mode using the EOF reversal agent, hexadimethrine bromide, in ammonium bicarbo-

nate electrolyte, pH 9.00. The ionic strength and the amount of methanol in the buffer

were optimised in a multivariate manner using artificial neural networks, with the

optimal conditions being 60 mM ammonium bicarbonate containing 60% methanol,

providing baseline resolution of the five hypolipidaemics within 20 min. Using electro-

kinetic supercharging, the sensitivity of the method was improved 1000-fold over a

conventional injection under field-amplified sample stacking conditions with LODs of

180 ng/L. This is the first report of the separation of hypolipidaemics by CE. The

developed method was validated and then applied to the determination of the target

drugs in water samples from Hobart city.

Keywords:

CE / ESI-MS / Electrokinetic supercharging / Hypolipidaemic drugs / Watersamples DOI 10.1002/elps.200900420

1 Introduction

Hypolipidaemic drugs have been prescribed widely for

several years for the treatment of different forms of

hyperlipidaemia. Among the different classes of hypolipi-

daemic drugs, statins and fibrates are most commonly used

as they possess high effectiveness in reducing cholesterol

and triglyceride levels in human plasma, thereby reducing

mortality associated with coronary heart disease [1]. The

daily dose of these drugs ranges between 10 and 1200 mg,

depending on the severity of the coronary heart disease and

the type of drug prescribed. Because of their acidic nature as

well as their physicochemical properties, hypolipidaemic

drugs are not fully eliminated in municipal sewage

treatment plants (STPs) and as such, small amounts are

discharged as contaminants into the receiving waters [2].

They have been detected in the aquatic environment at

concentrations ranging from ng/L to mg/L in STP effluent

and surface waters [3, 4]. Regular and continuous input of

these drugs into the environment has led to various adverse

effects on the aquatic and terrestrial organisms, for example

exposure to gemfibrozil adversely affects the immune

function of the bivalve mollusc, Mytilus galloprovincialis [5],

and it also reduces testosterone in the goldfish Carassiusauratus [6]. Atorvastatin elicits phytotoxic effects on the

aquatic macrophytes Lemna gibbaand Myriophyllum sibiri-cum [7]. The development of sensitive, rapid, and simple

methods for the analysis of such drugs in water samples is

therefore of great importance from the chemical, environ-

mental and toxicological points of view.

The hyphenation of CE with MS is nowadays considered

as a competitive approach to classical MS-hyphenated

separation techniques such as LC-MS and GC-MS [8]. The

on-line combination of CE and MS is a powerful analytical

technique that separates analytes according to the differ-

ences in their electrophoretic mobilities, and provides

information on molecular masses and/or fragmentation of

the analysed substances [9]. Despite the fact that CE-MS has

been applied routinely in drug analysis [10], proteomic

analysis [11], metabolomics [12], organic food contaminants

[13], and analysis of carbohydrates [14], only a few CE-MS

Mohamed DawodMichael C. BreadmoreRosanne M. GuijtPaul R. Haddad

Australian Centre for Research onSeparation Science (ACROSS),School of Chemistry, Universityof Tasmania, Hobart, Tasmania,Australia

Received July 12, 2009Revised August 4, 2009Accepted August 9, 2009

Abbreviations: ANN, artificial neutral network; CF-EKS, EKSand counter-flow; DCM, dichloromethane; EKS,

electrokinetic supercharging; FASS, field-amplified samplestacking; HDMB, hexadimethrine bromide; LE, leadingelectrolyte; LLE, liquid–liquid extraction; STP, sewagetreatment plant; TE, terminating electrolyte

Correspondence: Dr. Michael C. Breadmore, Australian Centrefor Research on Separation Science (ACROSS), School ofChemistry, University of Tasmania, Hobart, Tasmania, AustraliaE-mail: [email protected]: 161-3-6226-2858

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2010, 31, 1184–11931184

methods have been reported for the analysis of pharma-

ceuticals in environmental samples [15]. Ahrer et al. [16]

applied CE-ESI-MS for the determination of drug residues

in surface water samples and obtained LODs between 27

and 93 mg/L, which were decreased to 4.8 and 19 ng/L after

combination of SPE and liquid-liquid extraction (LLE).

Ahrer and Buchberger [17] also investigated the use of

aqueous and non-aqueous CE-ESI-MS for the analysis of the

same drugs in river water samples and LODs of 5–19 ng/L

were obtained. Macia et al. [18] used both aqueous and non-

aqueous BGEs in CE-ESI-MS for the analysis of acidic drugs

in surface and STP effluent water samples. The capillary

was coated with hexadimethrine bromide (HDMB) to

permanently reverse the EOF. LODs down to 100 ng/L were

achieved after SPE when the method was applied to surface

water samples, and when the method was applied for STP

water, LLE had to be included before the SPE to

preconcentrate and clean the water samples even further.

CE-ESI-TOF-MS has been reported for the determination of

antidepressant drugs in surface waters and STP effluents.

Again, SPE was used to obtain an enrichment factor of up to

1000 to obtain the necessary detection limits [19]. Sulfona-

mides in ground water samples were determined by CE-MS

and CE-MS/MS, LODs down to 5 mg/L were obtained [20].

The major drawback of CE is its high concentration

LOD, arising predominantly because of the very small inner

diameter (typically 50–100 mm) which limits the volume of

sample that can be injected and hence the absolute amount

of analyte that can be detected. The severity of this drawback

is manifested strongly when dealing with difficult samples

as in the case of analysis of pharmaceuticals in environ-

mental samples. Such samples bring further obstacles to

any CE method for two main reasons. First, pharmaceutical

compounds are typically present at very low concentrations.

In order to detect these low concentrations, injection of a

large volume of sample into the capillary is required. This is

not feasible in CE, because the amount of sample that could

be introduced into the capillary is rather limited (typically

o1 mL). Second, the matrix is not easily handled and usually

requires the use of off-line chromatographic sample clean-

up, such as SPE and/or LLE, as evidenced in all of the

currently published CE-MS methods discussed above, which

brings an additional effort to the method. These two

limitations account for the obvious lack of CE-MS methods

for analysis of pharmaceuticals in environmental samples.

In order to overcome the first limitation, the amount of

analytes injected into the capillary needs to be increased.

However, this often results in inferior separation due to the

reduction in efficiency, unless the analytes are maintained

as sharp bands as the injected volume is increased, a process

called stacking [21–23]. The most elegant solution to over-

come the second limitation is to use a very selective stacking

approach, such that only the analytes of interest are

concentrated while other components of the sample matrix

are not; this phenomenon is a characteristic outcome for

certain on-line preconcentration approaches, such as ITP

[21]. The recently developed method of electrokinetic

supercharging (EKS) is capable of achieving the above goals

and therefore presents an interesting possibility for the

enrichment of trace levels of pharmaceuticals in environ-

mental samples. EKS is considered as one of the most

powerful on-line preconcentration approaches, as it

combines the highly selective nature of ITP with the high

enrichment levels attainable by field-amplified sample

stacking. The general steps for any EKS system are as

follows: (i) filling the capillary with the BGE; (ii) introduc-

tion of a plug of leading electrolyte (LE) by hydrodynamic

injection; (iii) electrokinetic injection of the sample solution;

(iv) hydrodynamic injection of a plug of terminating elec-

trolyte (TE); (v) application of separation voltage, and start-

ing separation by ITP-CZE.

In the current work, we report the first hyphenation of

EKS with ESI-MS as a powerful combination for the

separation, on-line preconcentration and detection of hypo-

lipidaemic drugs. The electrophoretic separation was

performed in a co-EOF mode using an EOF reversal agent,

and the separation was optimised by variation of the ionic

strength of the separation buffer as well as the percentage of

the organic modifier added to the buffer. After optimisation

of separation parameters, the developed EKS-ESI-MS

method was validated, and then applied for the separation

and online preconcentration of five hypolipidaemic drugs in

drinking water and wastewater samples from Hobart city.

The presented work shows the first method for electro-

phoretic separation and on-line preconcentration of a

mixture of hypolipidaemic drugs.

2 Materials and methods

2.1 Chemicals, reagents and standards

The compounds studied, atorvastatin, fluvastatin, gemfibro-

zil, pravastatin and rosuvastatin (all 498%) were purchased

from Sequoia (Oxford, UK). 3-(Cyclohexylamino)1-propane-

sulphonic acid (CAPS) (99%), dichloromethane (DCM)

(HPLC-Grade), HDMB, and fuming hydrochloric acid

(37%) were from Sigma–Aldrich (St. Louis, MO, USA).

Ammonium hydrogen carbonate (98%) was from Ajax

Chemicals (Sydney, Australia). Sodium hydroxide (98%)

was from BDH (Kilsyth, Australia). Ammonium hydroxide

solution (28%) was from Fluka (Buchs, Switzerland).

Methanol (HPLC-Grade) was from Ajax Finechem (Seven

Hills, Australia). Water was treated with a Millipore (North

Ryde, Australia) Milli-Q water purification system. A stock

standard solution of 1 mg/mL of each drug was prepared in

methanol. A mixed standard solution of the five hypolipi-

daemic drugs was prepared at a concentration of 0.1 mg/mL

in methanol. The working standard solutions were prepared

daily by diluting the stock standard solution with Milli-Q

water. All solutions were stored in dark containers at 41C.

The working BGE solution had a concentration of 60 mM of

ammonium hydrogen carbonate (pH 9.00) containing 60%

(v/v) methanol unless otherwise stated. The BGE solutions

Electrophoresis 2010, 31, 1184–1193 CE and CEC 1185


were prepared freshly daily, sonicated for 5 min and filtered

through a 0.45 mm membrane filter.

2.2 Instrumentation

Electrophoretic separations were performed using an

Agilent 3D capillary electrophoresis system and Agilent

ChemStation software (Waldbronn, Germany). Electrophor-

esis and preconcentration experiments were carried out

using fused silica capillaries 50 mm id and 375 mm od

obtained from Polymicro Technologies (Phoenix, AZ, USA)

of 88 cm total length (88 cm effective length). The capillary

temperature was set at 251C. New capillaries were flushed

with 1 M NaOH for 120 min, with Milli-Q water for 10 min,

with HDMB (1% w/v) for 120 min, and with the BGE for

10 min.

ESI-MS detection was performed with an Agilent 6320

Ion Trap LC/MS, 6300 Ion Trap Control software, and

Agilent Pump 1200 Series.

2.3 Field-amplified sample stacking (FASS)

The hypolipidaemic drugs dissolved in Milli-Q water were

injected by applying hydrodynamic pressure at 50 mbar for

40 s; this sample plug represents 2.26% of capillary volume.

As shown in Fig. 1, all the drugs are weak acids. They have

pKa values ranging between 4.25 and 4.75 [24], and under

the experimental conditions used, they are negatively

charged and migrate towards the anode by means of a

combination of their own electrophoretic migration in

addition to the reversed EOF. Table 1 shows the pKa values

and the electrophoretic mobilities of the studied analytes, as

well as those of the major components of the BGE.

2.4 EKS

The capillary was filled with the BGE, then a short plug of

Milli-Q water was introduced by hydrodynamic injection at

40 mbar for 5 s, sample was then injected electrokinetically

by a negative voltage (�10 kV) for 170 s, and finally a small

volume of TE (1 mM CAPS) was injected hydrodynamically

at 50 mbar for 10 s. A separation voltage of �25 kV was

applied to re-stack the injected analytes and for their

separation. A positive pressure of 5 mbar was applied

during the separation process.

2.5 Preparation of water samples

Wastewater was obtained from the effluent of Self Point

STP (Hobart, Australia), while drinking water was obtained

from the tap in Hobart, Australia. Prior to analysis, the

samples were filtered through a 0.45 mm nylon membrane

syringe filter (Phenomenex, Australia) in order to eliminate

particulate matter. The samples were stored in dark glass

containers and kept in the refrigerator at �41C. The

conductivity of all the solutions used was measured using

a benchtop conductivity meter (lab CHEM-c) from TPS Pty.

(Springwater, Australia).

2.6 LLE

One millilitre of wastewater sample spiked with the

hypolipidaemic drugs (50 ppb) was acidified with 0.1 mL

hydrochloric acid, shaken, and then 1 mL of DCM was

added. The sample was then centrifuged (Eppendorf 5425

centrifuge, Hamburg, Germany) at 3000 rpm for 3 min. The

DCM layer was then separated and evaporated to dryness

under a gentle stream of air, and the dry residue was

reconstituted with 1 mL of Milli-Q water and used directly

for EKS-ESI-MS.

2.7 ESI-MS

For online hyphenation with EKS, different MS parameters

were optimised; these include sheath liquid composition,

sheath liquid flow rate, nebuliser pressure, drying gas

temperature and flow rate, and electrospray voltage. The

sheath liquid was 2 mM ammonium hydroxide in metha-

nol/Milli-Q water (50:50 v/v). A good trade-off between

electrospray stability and MS sensitivity was obtained with a

flow rate of 0.45 ml/min for the sheath liquid. Nebuliser

pressure was set to a minimum (7.00 psi) to provide stable

electrospray operation and to maximise the separation

efficiency. Additional spray chamber parameters were set

as follows: dry gas flow rate 5.00 L/min, drying gas

temperature 2501C, electrospray voltage 13.00 kV (end plate

voltage �3.00 kV). Total ion scan was performed at 200–650

m/z.

2.8 Validation study

BGE and standard solutions were freshly prepared daily for

assessing the reproducibility of the method. Precision was

expressed as percentage relative standard deviation and was

calculated by dividing the standard deviation by the mean

value of five replicate determinations for the within-day and

between-day measurements.

3 Results and discussion

The lack of CE-MS methods for the determination of

pharmaceuticals in environmental samples has inspired us

to develop a method for the determination of hypolipidae-

mic drugs in environmental samples. In previous studies

[25, 26], we reported the use of EKS and counter-flow

(CF-EKS) for the determination of non-steroidal

Electrophoresis 2010, 31, 1184–11931186 M. Dawod et al.


anti-inflammatory drugs in water samples. We obtained

sensitivity enhancements of 2400-fold with EKS, and while

the LODs with CF-EKS were even lower, the system we

developed cannot be transferred to the Agilent CE-MS

instrument as it is not possible to apply a negative pressure

on the inlet to establish a suitable counter-flow. Never-

theless, the enhancement with EKS alone was substantial

and when combined with MS this approach could be the

most appropriate solution for analysis of pharmaceuticals in

environmental samples.

3.1 Optimisation of separation selectivity

Highly efficient and rapid electrophoretic separations are

typically obtained when analytes migrate in the same

direction as the EOF (i.e. co-EOF separation). In previous

studies [25, 26], we reversed the EOF using the cationic

polymer HDMB, and found stable and reproducible results

when a small concentration of the reversing agent (0.1%

w/v) was added to the BGE. In the case of hyphenation

with ESI-MS, the addition of the surfactant to the BGE

is not desirable, because it will impair analyte detection as a

result of serious suppression of ionisation [27]. In the

present work, our experiments showed that EOF reversal

could be obtained without addition of HDMB to the running

buffer, as regular flushing with HDMB between runs

produced a reproducible reversed EOF. After establishing

a stable system with a reversed EOF, the possibility of

Figure 1. Chemical structures and molecular masses of the studied hypolipidaemic drugs.

Table 1. Electrophoretic mobility and pKa values for the

analytes and ion species included in the EKS-ESI-MS

study

Compound Electrophoretic

mobility (� 10�9 m2 v�1 s�1)

pKa value

Atorvastatin �7.90 4.20

Fluvastatin �9.60 4.10

Gemfibrozil �10.90 4.75

Pravastatin �8.40 4.60

Rosuvastatin �9.00 4.40

HCO3- �46.10 (�39.96)a) 6.35

CAPS �25.00 (�0.91)a) 10.40

a) Values between brackets are the effective mobility values for

the LE and TE calculated from PeakMaster 5.2 software (http://

www.natur.cuni.cz/�gas/pm52.exe).



separating the hypolipidaemics in a MS-compatible BGE

was examined. We tested the separation of the drugs with

ammonium bicarbonate 30 mM, pH 9.00. In this BGE, the

separation was accomplished in less than 15 min, but

baseline resolution was not obtained for all analytes. In

order to improve resolution, we undertook a multivariate

optimisation of the ionic strength and methanol, as

both are well known to have a significant influence on

resolution and both are also MS-compatible [28]. The

experimental space was defined between 10–70 mM ammo-

nium bicarbonate and 0–70% v/v methanol. Higher

concentrations of the buffer increased electric current, and

this was undesirable both from the loss in efficiency due to

Joule heating effect [29] as well as the current limitations

due to ESI hyphenation (the system current limit is set to

50 mA by Agilent software). Higher percentages of methanol

caused a substantial reduction in the magnitude of the

reversed EOF, and resulted in severe prolongation of the

separation time. Over this experimental space, separations

were performed at nine training points using the four

corner points, the centre point, and four mid points.

Additional separations were performed at four random

points selected within the space, with two used in

the verification points of the artificial neutral network

(ANN) training, with the remaining two being used to

check the accuracy of the trained ANN. From these data an

ANN with an (2-11-3) architecture was trained with

very good correlation between the observed and predicted

values for separation time, current, and minimum resolu-

tion (r2 values of 0.9977, 0.9983, and 0.9998, respectively).

Figure 2 shows the response surface for minimum

resolution from the trained ANN. It is clear that the optimal

conditions based on the highest minimum resolution

showed that the best results were obtained with 60 mM of

the buffer and 60% v/v of methanol. This combination gives

a reasonable separation time and the current profile was

within the system limit for CE-ESI. A representative

separation obtained under these conditions is shown in

Fig. 3. The five hypolipidaemic drugs were successfully

separated in less than 20 min. It is noteworthy that we tested

the same ANN experiments but using ammonium acetate as

a BGE. Similar results were obtained, yet inclusion of

methanol (450% v/v) with acetate buffer gave a highly

intense peak of HDMB with m/z of 265, which suppressed

the ionisation of the drugs.

3.2 EKS-ESI

One of the online preconcentration strategies that has not

been hyphenated with MS is EKS. This technique was first

described by the group of Hirokawa [30, 31] for the analysis

of rare-earth ions. Urbanek et al.[32] applied EKS for

determination of sub-ppb levels of Fe(II), Co(II), and Ni(II)

with LODs at the low ng/L level for standards, however, the

LODs were ten times higher for real samples due to a lower

loading rate during the field amplified stacking injection

step. Recently, Busnel and co-workers [33] applied EKS for

peptide analysis with 1000–10 000-fold improvement in

sensitivity. Interestingly a buffer system that is compatible

with MS was used, yet coupling their method with MS was

not applied.

It is well known that there is a limitation in the BGEs

that can be used in any on-line CE-MS analysis. This limited

choice of electrolytes brings considerable restrictions not

only to the CE separation itself but also to the potential

sample stacking [9]. For EKS, this has implications on the

choice of the BGE as well as the LE and the TE. This

limitation leads to further difficulties for the EKS-ESI-MS

hyphenation.

In our system, ammonium bicarbonate was used as a

separation buffer, as the high electrophoretic mobility of

bicarbonate allows it to also act as a LE for these drugs. The

choice of the TE is not straightforward. The terminator

should ideally have an electrophoretic mobility lower than

that of the analytes of interest, but for MS detection, it is also

important that it does not foul the MS. In our previous

systems [25, 26] we used CHES as a TE. Initial experiments

using CHES as the TE gave poor results as CHES co-

migrated with atorvastatin (as shown in Fig. 4A) and also

caused contamination of the electrospray, requiring tens of

minutes for the signal to reach baseline again. CAPS was

investigated as an alternative TE because it has a higher pKa

value than CHES and therefore will have a lower mobility,

thereby minimising the chance of co-migration with any of

the analytes. When this was tried, surprisingly CAPS was

also more compatible with ESI-MS than CHES as it did not

contaminate the electrospray kit, and the baseline returned

to its normal value after detection of CAPS peak, as shown

in Fig. 4B.

The optimum conditions involved filling the capillary

with the BGE, after which a short plug of Milli-Q water was

Figure 2. Response surface estimated from the minimumresolution of hypolipidaemic drugs with concentration ofammonium bicarbonate and percentage of methanol.



introduced at a pressure of 50 mbar for 6 s. This plug has

been reported to be important for improvement of sensi-

tivity and reproducibility before the field amplified stacking

injection process [34]. In our system, there was no further

improvement in sensitivity with the water plug, yet it was

very important for stabilisation of the electric current during

electrokinetic injection of the sample. The sample was then

introduced electrokinetically by application of �10 kV for

170 s. Systematic attempts to increase the injection time

and/or injection voltage resulted in inferior reproducibility

of the method due to instability of electric current; this is

due to the introduction of large amount of the low

conductivity sample matrix. Moreover, breakage of the

capillary approximately 2.5 cm from the capillary inlet was

observed after 20–30 runs. This is most likely a result of a

high localised electric field at the interface between the

Figure 3. Base peak and extracted ionchromatograms of five hypolipidaemicdrugs 10 ppm in Milli-Q water. CEconditions: fused silica capillary,88 cm�50 mm id; BGE 60 mM ammo-nium bicarbonate containing 60%methanol, pH 9.00. Hydrodynamic injec-tion of sample at 50 mbar for 40 s.Separation voltage: �25 kV. MS condi-tions as in the text.



low conductivity sample matrix and the high conductivity

BGE zone [35]. After injection, the TE (CAPS, 1 mM) was

introduced hydrodynamically by injection at 50 mbar for

10 s. This volume of TE was just sufficient for ITP step to

take place and longer injection plugs were judged to leave

smaller room for the capillary for subsequent separation of

the sample after destacking of the ITP step. Finally, the

separation voltage (�25 kV) was set for the ITP-CZE step.

Figure 4B shows the separation obtained with EKS-ESI-MS

using CAPS as TE.

Under optimum conditions, the EKS-ESI-MS method

yielded a 1000-fold improvement in detection sensitivity of

the hypolipidaemic drugs when compared with conven-

tional FASS. The LODs (at three times signal to noise) were

560, 180, 230, 200, and 260 ng/L for atorvastatin, fluvastatin,

gemfibrozil, pravastatin, and rosuvastatin, respectively.

Figure 4. Base peak chroma-tograms of five hypolipidae-mic drugs 50 ppb in Milli-Qwater. CE conditions: fusedsilica capillary, 88 cm� 50 mmid; BGE 60 mM ammoniumbicarbonate containing 60%methanol, pH 9.00. EKI ofsample at �10 kV for 170 s.Hydrodynamic injection of1 mM TE at 50 mbar for 10 s.(A) CHES and (B) CAPS.Separation voltage: �25 kV.MS conditions as in the text.

Table 2. Within-day and between-day reproducibilities (RSD%) of migration times, peak areas, and peak heights for EKS-ESI-MS

Compound Within-day RSD% (n 5 5) Between-day RSD% (n 5 5)

Migration time Peak area Peak height Migration time Peak area Peak height

Atorvastatin 0.75 2.90 8.12 6.38 7.96 9.36

Fluvastatin 0.41 2.89 3.44 4.49 4.06 6.32

Gemfibrozil 0.26 2.02 4.82 3.96 .65 5.70

Pravastatin 0.32 2.46 6.02 6.46 7.88 7.11

Rosuvastatin 0.21 2.04 2.88 3.24 4.51 5.50



Figure 5. Base peak chroma-togram obtained from EKS-ESI-MS of (A) drinking waterspiked with 50 mg/L of thedrugs and (B) drinking watersample spiked with 50 mg/L ofthe drugs, then diluted withMilli-Q water 1:4 and condi-tions as in Fig. 4B.

Figure 6. Base peak chroma-togram obtained from EKS-ESI-MS of (A) wastewaterspiked with 50 mg/L of thedrugs and (B) wastewatersample spiked with 50 mg/L ofthe hypolipidaemic drugsafter LLE. Conditions as inFig. 4B.



3.3 Method validation

The reproducibility of the method was investigated by

determining the within-day and between-day precision values.

Within-day precision was evaluated by performing five

replicate separations of the five hypolipidaemic drugs at a

concentration of 50 mg/L. Between-day precision was evalu-

ated by performing the same separations for five different

days. Precision (expressed as percentage relative standard

deviation (RSD%)) was calculated for migration times, peak

areas, and peak heights. Table 2 shows the RSD% values for

within-day and between-day precision for the above para-

meters. These precision evaluation studies were performed on

the same capillary. The RSD% studies were conducted on

different capillary sections from the same batch as well as

different capillaries from different batches, and there was no

significant difference in RSD% values obtained.

3.4 Application to water samples

To demonstrate the potential of the developed EKS-ESI-MS

method for the analysis of hypolipidaemic drugs in different

water samples, the proposed method was applied for the

analysis of two water samples. Drinking water was collected

from the tap, while wastewater was collected from the

effluent of Self Point STP (Hobart, Australia).

Figure 5 shows the electropherograms of drinking water

spiked with 50 mg/L of the hypolipidaemic drugs and drinking

water spiked with 50 mg/L of each of the drugs after dilution of

1:4 with Milli-Q water. It is well known that the conductivity of

the sample plays an important role in any EKS system, as

matrix ions will be injected in the EKI step, and thereby

decrease the amounts of analytes injected. Measuring the

conductivity of the drinking water, it was found to be 51

mS/cm, as opposed to that of the standards which was 3.8 mS/

cm. A 1:4 dilution would reduce the conductivity of the water

sample to approximately 12 mS/cm and bring the conductivity

of drinking water close to the standard solutions and subse-

quently facilitate the applicability of EKS. An experimental

study confirmed that a 1:4 dilution was the lowest dilution

factor that decreases the conductivity of the sample enabling

online preconcentration by EKS.

When unspiked wastewater was analysed, none of the

drugs was detected, however, an unknown negative ion peak

was obtained with m/z of 315. Upon spiking the sample

with the drugs, the LODs were found to be 20 times higher

than the standard solutions due to the higher conductivity of

the wastewater sample compared to standard solutions of

the studied drugs [26]. The measured conductivity

of wastewater was 521 mS/cm, which requires several folds

of dilution, which dramatically increases the LODs and

hinders the application of EKS. For this reason, we devel-

oped a simple LLE method to extract the target analytes and

to eliminate ions present in the high conductivity sample

matrix. Figure 6 shows the electropherogram of a waste-

water sample spiked with 50 mg/L of each of the hypolipi-

daemic drugs before and after LLE with DCM. LLE of spiked

wastewater samples showed values for recovery %7SD

(n 5 3) of 44.6472.98, 88.6373.21, 92.3373.89,

49.6672.17 and 88.1272.09 for atorvastatin, fluvastatin,

gemfibrozil, pravastatin and rosuvastatin, respectively.

LODs were 1260, 203, 250, 403 and 294 ng/L for atorvasta-

tin, fluvastatin, gemfibrozil, pravastatin and rosuvastatin,

respectively. Although the LODs for wastewater samples are

higher than those obtained for the standard solutions of the

drugs, they are lower than the levels reported for some of

these analytes in wastewater samples in some cities in

Europe and North America. Gemfibrozil was detected at

concentrations up to 478 ng/L in STP effluents (Vancouver,

Canada) [36], 497 ng/L in STP effluent (Near Ebro river,

North East Spain) [37] and 340 ng/L in ground water

(Ontario, Canada) [38]. It is also important to report that the

EKS-ESI-MS method presented is suitable for the analysis of

the target analytes in low conductivity water samples with-

out the need for sample pretreatment.

4 Concluding remarks

The current work demonstrates the development of a new,

powerful CE-MS hyphenation. The simple and sensitive

method was applied for the separation and on-line preconcen-

tration of hypolipidaemic drugs in water samples. The method

uses a combination of EKS as an on-line preconcentration

technique in CE with the highly precise ESI-MS detection

system. The proposed method allowed the separation of five

hypolipidaemic drugs in less than 20 min. To the best of our

knowledge, this is the first separation and/or on-line

preconcentration system for these drugs. The enhancement

in detection sensitivity was 1000-fold over FASS and allows

LODs to get down to 180 ng/L. When the method was applied

to drinking and wastewater samples, the LODs achieved with

drinking water spiked were about five times higher than

standards, while for wastewaters, the high conductivity of the

samples required LLE to obtain sufficiently low LODs with

EKS. Although no further improvement in preconcentration

was obtained with LLE, the extraction method could be

modified in the future to obtain additional enhancement in

sensitivity. The proposed method could be applied directly

without sample pretreatment for analysis of some of these

analytes in some European and North American cities, where

the expected concentrations of such drugs are slightly higher

than the LODs achieved with the proposed method.

M. D. thanks the Egyptian ministry for higher education forprovision of a scholarship. This work was supported by theAustralian Research Council through the award of an ARC QEIIFellowship to M. C. B. (DP0984745), ARC postdoctoral Fellowshipto R. M. G. (DP0557083) and a Federation Fellowship to P. R. H.(FF0668673). The authors also thank Mrs. Francis Smith fromHobart city council for supplying the STP effluent samples.

The authors have declared no conflict of interest.



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electrokinetic supercharging-electrospray ionisation-mass spectrometry for separation and on-line...

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