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Journal of Chromatography A, 1130 (2006) 259–264

Short communication

Separation of phospholipids by capillary zone electrophoresiswith indirect ultraviolet detection

Fei Gao a,b, Juan Dong a, Wei Li a, Tao Wang b,∗, Jie Liao c, Yiping Liao a, Huwei Liu a,∗a Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemistry,

Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, Chinab Institute of Nephrol, First Hospital, Peking University, Beijing 100034, China

c Medical Experiment and Analysis Center, Chinese PLA General Hospital, Beijing 100853, China

Available online 18 April 2006

bstract

A simple method for separation of different anionic and zwitterionic phospholipid classes by capillary zone electrophoresis (CZE), using indirect

V detection with adenosine monophosphate (AMP) as background electrolyte and the UV-absorbing additive, was successfully developed in

his study. The separation conditions including apparent pH (pH*) of running buffer, concentration of AMP, organic solvent, applied voltage andapillary temperature were systematically optimized. The application of this method to human blood sample was also briefly examined.

2006 Elsevier B.V. All rights reserved.

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eywords: Capillary zone electrophoresis; Phospholipids; Separation; Indirect

. Introduction

Phospholipids are not only essential structural constituentsf biological membranes but also important substances in sig-al transduction pathways [1]. In addition, phospholipids aremportant sources of arachidonic acid, which can be metabo-ized by cyclooxygenase or lipoxygenase pathways to produceiologically active prostaglandins or leukotrienes [2,3]. Gener-lly, the phospholipids are divided into several classes based onifferences in their polar head groups, and their distributions arereatly different in different organs [4].

Traditionally, thin-layer chromatography (TLC) has been theimplest technique in lipid analyses [5], but quantification bycanning of the stained chromatograms lacked precision becausef problems with nonuniformity of staining of both the phos-holipids and the background. Normal-phase high-performanceiquid chromatography (NP-HPLC) has been used for the sepa-ation of phospholipid classes in recent years [6–9]. The obviousisadvantage of most HPLC methods in the analysis of phospho-

ipids is the high cost for magnitude of mobile phase and time-onsuming, which would not meet the needs for high throughputnalysis.

∗ Corresponding authors. Tel.: +86 10 62754976; fax: +86 10 62751708.E-mail address: hwliu@pku.edu.cn (H. Liu).

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021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2006.03.070

etection

Capillary electrophoresis (CE) provides an alternative toPLC in the analysis of phospholipid classes for its high separa-

ion efficiency, application versatility, and instrument simplicity.hile the methodologies of CE are well established for ana-

yzing a variety of substances, its application for phospholipidnalysis is limited because of the poor aqueous solubility andow UV absorbance of phospholipids, and only a few papers onhe separation of phospholipids have been published [10–16],hich include micellar electrokinetic capillary chromatographic

MEKC) methods with UV detection [10–11] or laser-induceduorescence (LIF) detection [12]. Although LIF can give highensitivity for phospholipids, the derivatization process of theample is complex and nonstoichiometric. Raith et al. [13] sepa-ated phospholipids by CE on-line coupled to mass spectrometryCE–MS) using nonaqueous buffer. The separation for mostf phospholipids was acceptable, but an extra pressure muste applied in the inlet of capillary to guarantee a sufficientiquid flow between CE capillary and the electrospray ioniza-ion (ESI) needle because of the very low electrophoretic flowEOF) generated by the capillary they used. Guo et al. [14] sep-rated and determined phospholipids in plant seeds using theonaqueous capillary electrophoresis (NACE) with UV detec-

or, but the detection sensitivity was not satisfactory. On thether hand, indirect UV detection exhibits greater sensitivityor phospholipids using an appropriate chromophore additive.addadian et al. [15] and Chen et al. [16] reported CZE with

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ndirect UV detection for the determination of phospholipids andyso-phospholipids, respectively. However, only anionic phos-holipids were separated and the buffer systems they utilizedere relatively complex.In this study, by using adenosine monophosphate (AMP) as

GE and chromophore simultaneously, separation of differentnionic and zwitterionic phospholipid classes is obtained by aimple CZE method with indirect UV detection within 15 min.he optimal CE conditions were investigated and the methodas also validated. This method has been used to separate phos-holipids in human blood samples.

. Materials and methods

.1. Apparatus and conditions

All experiments were performed with an HP3D CE systemith air-cooling and a diode-array detector (Agilent Technolo-ies, Palo Alto, CA, USA), and an uncoated fused-silica capil-ary, 50 �m I.D. × 375 �m O.D. (Yongnian Optical Fiber Fac-ory, Hebei, PR China), with a total length of 48.5 cm (40.0 cm toetection window) was utilized, and maintained at 25 ◦C unlesstherwise specified. On-line indirect UV detection was set at59 nm. Samples were introduced into the capillary by pres-ure injection at 50 mbar for 5 s. Separation were carried outnder applied potential of 30 kV. The capillary was washed with.0 mol L−1 sodium hydroxide (1 min), water (2 min), methanol2 min) and buffer (5 min) between different buffers. Betweenonsecutive analysis, the capillary was rinsed with methanol2 min) and running buffer (5 min) to guarantee good repro-ucibility.

.2. Chemicals and reagents

All phospholipid standards were obtained from SigmaSt. Louis, MO, USA), including 1, 2-diacyl-sn-glycero-3-hosphoenthanolamine from egg yolk (PE), l-�-phosphatidyl--serine from soybean (PS), l-�-phosphatidylinositol,mmonium salt from soybean (PI), 1-palmitoyl-2-oleoyl-sn-lycero-3-phospho-rac-(1-glycerol) ammonium salt (PG) and-sn-phosphatidic acid sodium salt from egg yolk lecithin (PA).he nominal purity of the standard compounds was no lower

han 98%.Human blood sample was kindly provided by the First

ospital of Peking University (Beijing). Adenosine 5′-mono-hosphate monohydrate (99%) (AMP) was purchased fromhanghai Chemical Reagent Co. (Shanghai, China). HPLCrade methanol and sodium hydroxide (analytical-reagentrade) were from Dima Technology (USA) and Beijing Chemi-al Factory (Beijing, China), respectively. Redistilled water wassed for the preparation of solutions.

.3. Buffer and standard solution preparation

A stock solution of 150 mmol L−1 AMP was prepared ineionized water, and then diluted to appropriate concentrationsith the organic solvents. These buffers were adjusted to the

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A 1130 (2006) 259–264

esired pHs with 1.0 mol L−1 NaOH. Because the pH in aque-us buffer is not the same as in organic solvents like methanol, weall it apparent pH (pH*). In our experiments, the standard phos-holipid samples (100 �g mL−1) were dissolved in methanolnd then diluted to obtain tested standard solutions for calibra-ion at the concentration range from 1.0 to 100 �g mL−1. Both ofhe buffer and standard solutions were filtered through a 0.45 �mylon membrane filter and degassed by ultrasonication beforenjection.

.4. Blood sample preparation

The procedure for extraction of blood plasma lipids devel-ped by Uran et al. [17] was used with modifications. Briefly,.7 mL of water was added to 300 �L of the blood sample, thenmL of methanol and 10 mL of chloroform was added and the

olution was sonicated for 60 s both before and after adding chlo-oform. Finally, 5 mL of water was added and mixed for 5 s andhen centrifuged at 3024 × g for 10 min under temperature 4 ◦C.he lower chloroform phase was sampled and dried by evapora-

ion under nitrogen; the samples were stored dry at −70 ◦C. Prioro analysis, the extracted samples were redissolved in 1 mL of

ethanol and the solution was filtered through a 0.45 �m nylonembrane filter.

.5. Peak identification

The peaks on the electropherogram of the mixed phospho-ipids were identified by comparing the migration time with thatf standard phospholipids and by comparison of the peak heightefore and after standard addition in the mixed solution. Theolecular species in PA class were not identified due to the lack

f the corresponding standard phospholipids.

. Results and discussion

.1. Optimization of CZE conditions

In indirect UV detection, a chromophore is necessarilyncluded in background electrolyte (BGE) to generate highackground absorption. For our work, we tried several UV-bsorbing additives such as p-toluenesufonic acid sodium salt,aphthalenesulfonic acid sodium salt and AMP. AMP was cho-en because of its high molar absorptivity, large ratio of back-round absorbance to background noise, and favorable transferatio [18], as well as closely matched mobility with phospho-ipids. As shown in Fig. 1, the BGE (a 150-fold dilution ofeparation buffer) gave two maximum absorption peaks at 207nd 259 nm, and the maximum difference of absorption betweenhe phospholipids and BGE was at 259 nm. Therefore, 259 nmas chosen for the detection in the subsequent experiments.A series of buffers at pH* 8.5–10.0 were investigated by

sing a 5 mmol L−1 AMP in methanol/water (9:1, v/v). The pH*ffected not only the degrees of dissociation of AMP and phos-holipids, but also the EOF. As shown in Fig. 2, the migrationrder was PE > PI > PG > PS > PA based on the charge-to-mass

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ig. 1. The UV spectrum of BGE containing 33 �mol L AMP at pH* 9.5 inethanol–water (9:1, v/v), compared with the spectrum of 25 �g L−1 standard

hospholipid sample.

atio of analytes. According to the proton binding constant ofifferent phospholipid classes [19], in the range of tested pH*,cidic PA carries two negative charges, so it migrated at last,hile PI and PG bear one negative charges owing to the depro-

onation of the phosphate. It is interesting to note the migrationehaviors of PE and PS at varying pH*. As the pH* valuencreased from 8.5 to 10.0, the status of zwitterionic PE changed

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ig. 2. Effect of pH* on the separation of the phospholipids. Conditions: 5 mmol L−1

ndirect UV at 259 nm; injection, 50 mbar, 5 s; (A) pH* 8.5, (B) pH* 9.0, (C) pH* 9.5A with a variety of fatty acid groups, which were not identified.

A 1130 (2006) 259–264 261

rom almost neutral (migrated close to EOF marker) to one neg-tively charged, while the negative charge of PS from one toartial two due to the further deprotonation of serine residue.herefore, at lower pH* value, i.e. pH* 9.0, PS migrated close

o PG, whereas at higher pH* value, i.e. pH* 10.0, PS was par-ially overlapped with PA due to the change in molecular chargetate. Thus, the pH* 9.5 was chosen for further optimization.

The effect of AMP concentration on the separation was stud-ed in the range of 5–15 mmol L−1 at pH* 9.5. The results inig. 3 show that, with the increasing concentration of AMP, theOF was decreased, leading to long migration time of analytes.owever, the increase in AMP concentration can improve the

esolution between phosphosplipid classes, as well as molec-lar species of PA (shown in Fig. 3B), indicating that thisethod might be applied to the separation of molecular species

f PA in clinical diagnosis [20]. When the AMP concentrationncreased further, the resolution between different species of PAas obviously decreased (Fig. 3C) and the background noise

lso increased. Taking the resolution and migration time into

eparation.The problems associated with the separation of phospho-

ipids in more content of water are illustrated in Fig. 4. In higher

AMP in methanol/water (9:1, v/v); applied voltage, 30 kV; temperature, 25 ◦C;and (D) pH* 10.0. Peaks (a–d) were referred to different molecular species of

262 F. Gao et al. / J. Chromatogr. A 1130 (2006) 259–264

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ig. 3. Effect of AMP concentration on the separation of the phospholipids. Coethanol/water (9:1, v/v); applied voltage, 30 kV; temperature, 25 ◦C; indirect

ontent of water, the baseline drifted drastically; the peak tail-ng was observed and for some phospholipids, peaks were lost

ompletely due to hydrophobic adsorption of these phospho-ipids on the capillary wall and/or ion–ion interactions that causerecipitation of the longer chain phospholipids [21]. However,

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ig. 4. Separation of phospholipids in (A) methanol/water (7:3, v/v), (B) methanol/watMP in, applied voltage, 30 kV; temperature, 25 ◦C; indirect UV at 259 nm; injection

ns: pH* 9.5; (A) 5 mmol L−1, (B) 10 mmol L−1 and (C) 15 mmol L−1 AMP in259 nm; injection, 50 mbar, 5 s.

/10 water in the buffer is necessary for the solubility of AMPnd the use of 90% methanol is essential for the solubility and

electivity of phospholipids [15,16], where good peak shapes forll phospholipids were obtained and the baseline became moretable.

er (8:2, v/v) and (C) methanol/water (9:1, v/v). Conditions: pH* 9.5; 5 mmol L−1

, 50 mbar, 5 s.

F. Gao et al. / J. Chromatogr. A 1130 (2006) 259–264 263

Fig. 5. The electropherogram of human serum sample. The 5 mmol L−1 AMP in mindirect UV at 259 nm; injection, 50 mbar, 5 s.

Table 1The calibration equations, correlation coefficient (r), limit of detection (LOD)and repeatability (RSD) of migration time (MT) and peak area (A) for the sepa-rated phospholipids (n = 5)

Phospholipids Calibrationequations

r LOD(�g/mL)

RSD (%)

MT A

PE y = 3.02x + 10.1 0.9966 3.3 0.31 0.72PI y = 2.01x + 7.97 0.9976 3.3 0.42 2.00PG y = 5.18x + 24.0 0.9956 2.0 0.40 1.00PS y = 3.60x + 15.0 0.9966 5.0 1.00 1.10PA y = 4.69x + 50.9 0.9960 5.0 0.48 0.48

Separation conditions: 5 mmol L−1 AMP in methanol/water (9:1, v/v) at pH 9.5;a5b

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pplied voltage, 30 kV; temperature, 25 ◦C; indirect UV at 259 nm; injection,0 mbar, 5 s. The concentration of standard phospholipid samples for repeata-ility test was 20 �g mL−1.

The effects of applied voltage in the range of 10–30 kV andhe capillary temperature on the separation were studied. Theoltage of 30 kV and temperature 25 ◦C were chosen for oureparation system.

.2. Method validation

The repeatability of migration time and peak area for eachhospholipid under the optimized conditions are acceptable,ummarized in Table 1, where the relative standard deviationRSD) of migration time and peak area was ≤1.0 and ≤2.0%,espectively. The calibration curves of the peak area (A) versusoncentration of phospholipids (c) are also established with aorrelation coefficient (r) greater than 0.99. The limits of detec-ion (LODs) for all detected phospholipids were varied from 2.0o 5 �g mL−1. The results obtained showed that the sensitivityf this CZE method with indirect UV detection is not lower thanhat of HPLC method with evaporative light-scattering detec-ion (ELSD), where the detection limit is ranged from 1.5 to�g mL−1 [7].

.3. Application to human blood sample

Human blood serum was prepared by liquid–liquid extrac-ion according to the procedure described in Section 2.4. The

ample was then analyzed by this CZE method, and the resultsre showed in Fig. 5. Although the baseline drifts slightly dueo the matrix effect, a separation of the main phospholipids wasbtained. For the sensitivity of the method presented, it was not

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ethanol/water (9:1, v/v), pH* 9.5; applied voltage, 30 kV; temperature, 25 ◦C;

igh enough for determination of phospholipids in the blooderum sample. In the future work we will concentrate the PLractions to improve the CE traces or use some on-line precon-entration techniques for quantitative determination [22,23].

. Concluding remarks

In this work, a CZE-indirect UV method using 5 mmol L−1

denosine monophosphate as BGE and UV-absorbing additiven methanol–water (9:1, v/v) under 25 ◦C temperature and 30 kVpplied voltage was established for the separation of anionic andwitterionic phospholipid classes with different polarity. Phos-holipids in human blood serum were profiled and tested. Theethod shows great potential for determination of phospholipids

n biological samples.

cknowledgements

The authors are grateful to Ms. C.C. Li for her suggestionsegarding to the manuscript preparation. This study is financiallyupported by the National Science Foundation of China (NSFC),rant Nos. 20575003 and 90209056.

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