Potential of polyE-323 coated capillaries for capillary electrophoresis of lipids

Journal of Chromatography A, 1317 (2013) 193–198

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Potential of polyE-323 coated capillaries for capillary electrophoresis of lipids Kert Martma 1 , Petrus W. Lindenburg 1 , Kaia-Liisa Habicht, Kaspar Vulla, Kristiin Resik, Gunnar Kuut, Ruth Shimmo ∗ Department of Natural Sciences, Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt. 25, 10120 Tallinn, Estonia

a r t i c l e

i n f o

Article history: Received 27 April 2013 Received in revised form 14 August 2013 Accepted 14 August 2013 Available online 21 August 2013 Keywords: CE Polye-323 Lipids Coating stability

a b s t r a c t In this note the feasibility of a polyamine-based capillary coating, polyE-323, for capillary electrophoresis (CE) of lipids is explored. PolyE-323 has previously been demonstrated to be suitable to suppress analyte-wall interaction of proteins in CE. However, the full applicability range of polyE-323 has not been exploited yet and it might be useful in the analysis of hydrophobic analytes, such as lipids. In this study, the stability of polyE-323 when using highly organic background electrolytes (BGEs), which are needed to solubilize the lipid analytes, was studied. For this, we used three different lipid samples: sphingomyelin, cardiolipin and a lipid extract from a cell culture. The highly organic BGEs that were used in this study consisted of 94.5% of organic solvents and 5.5% of an aqueous buffer. First, the influence of pure acetonitrile, methanol, propylene carbonate, isopropanol and chloroform on the polyE-323 coating was investigated. Then BGEs were developed and tested, using sphingomyelin and cardiolipin as test analytes in CE–UV experiments. After establishing the best BGEs (in terms of analysis time and repeatability) by CE–UV, sphingomyelin was used as a test analyte to demonstrate that method was also suitable for CE with mass-spectrometry detection (CE–MS). The LOD of sphingomyelin was estimated to be 100 nM and its migration time repeatability was 1.3%. The CE–MS analysis was further applied on a lipid extract obtained from human glioblastoma cells, which resulted in the separation and detection of a multitude of putative lipids. The results of our feasibility study indicate that CE systems based on polyE-323 coated capillaries and highly organic BGEs are promising for fast electromigration-based analysis of lipids. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Capillary electrophoresis (CE) is an electroseparation method that often complements to liquid chromatographic separation. CE has high separation power, needs only minute amounts of sample and solvents, is very versatile and often enables fast separation. A frequent drawback of CE is the tendency of molecules to interact with the inner wall of the fused silica capillary, which causes low separation efficiency and low migration time repeatability and reproducibility. To overcome this drawback, numerous capillary coatings have been developed [1,2] including various polymeric coatings [3–5]. However, the often-used polymeric coatings, such as polyacrylamide [6,7] are designed for aqueous BGEs and not much is known about their stability under highly organic conditions. This poses a problem for the analysis of very hydrophobic compounds, which have a tendency to interact with bare fused

∗ Corresponding author. Tel.: +372 6409400. E-mail address: [email protected] (R. Shimmo). 1 Contributed equally. 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.08.054

silica, but also require highly organic BGEs as they are poorly soluble in aqueous BGEs. PolyE-323 was first synthesized in the group of Bergquist and Markides in Uppsala University several years ago. It is a positively charged polyamine, containing amino-, chloride- and hydroxylgroups, resulting in an anodic electro-osmotic flow (EOF) [8]. It interacts strongly with the silanol groups on fused silica capillary walls and has been successfully used as a capillary coating in protein analysis, both with UV detection [8] and MS detection [9]. Since it was developed, polyE-323 has been reported as coating in several studies, employing microfluidic devices [10–12] and capillaries [13–15]. All these studies [10–15] were focused on the analysis of peptides and/or proteins, using aqueous BGEs such as ammonium acetate. It was demonstrated that the coating is stable in a pH range of 4–8 [8] and that up to 50% methanol or acetonitrile in the BGE does not affect the separation performance significantly [9]. Nevertheless, polyE-323 coated capillaries have not been used with more apolar BGEs. The aim of this work was to study the possibilities of polyE323 for the analysis of lipids, which require highly organic BGEs. Lipids cover a wide variety of hydrophobic metabolites that are

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important in many physiological processes. In general, lipid analysis is carried out with LC–MS, GC–MS and thin layer chromatography (TLC) [16,17] but it has also been shown that CE is suited for the separation of lipids [18]. The analysis of lipids with CE–MS could be attractive, as CE–MS offers speed and very high separation efficiency and sensitivity in comparison with spectrophotometric detection such as UV and light-induced fluorescence (LIF). Moreover, CE–MS analysis can possibly result in complementary data with respect to LC–MS and GC–MS. However, only a few works based on CE–MS of lipids have been reported yet, for example, CE–MS analysis of acylcarnitines [19] and phospholipids [20]. In the current work the goal has been to study the applicability of polyE-323 as a capillary coating in a highly organic (94.5% organic solvents) BGE environment. The best working organic BGEs have been tested on selected lipids: on sphingomyelin, which is a well-known representative of cell membrane lipids participating in intra- and extracellular signalling [21]; on cardiolipin, which is the most abundant lipid in the mitochondrial inner membrane and believed to be a key factor for many pathological conditions [22,23]; and on a mixture of lipids extracted from U-87MG cell membranes. First, the stability of the coating with several organic solvents, i.e. acetonitrile, methanol, isopropanol, propylene carbonate and chloroform, was studied. Based on the outcome of these experiments, solvents that performed best were selected to develop BGEs for hydrophobic analytes, using the moderately hydrophobic lipid sphingomyelin and highly hydrophobic lipid cardiolipin as test analytes in CE–UV experiments. Finally, a suitable buffer for CE–MS was selected and, after the CE–MS system was evaluated with sphingomyelin, a cell culture lipid extract was analyzed to study the potential of polyE-323 coated capillaries for CE–MS of a biological lipid mixture. 2. Experimental 2.1. Reagents and samples Chloroform and TRIS were purchased from AppliChem (Darmstadt, Germany). Methanol, acetonitrile and isopropanol were from Rathburn (Scotland). Sodium hydroxide, ammonium hydroxide and hydrochloric acid were from Lach-Ner (Czech Republic). Propylene carbonate, ammonium acetate, dimethyl sulfoxide (DMSO), 1,2-bis(3-aminopropylamino)ethane, epichlorohydrine, cardiolipin (sodium salt from a bovine heart), diacylglycerophosphocholine (PC 18:1/16:0) and sphingomyelin (sphingosylphosphorylcholine (SM C16:0)) were from Sigma–Aldrich (St. Louis, MO, USA) and the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate from (Sigma–Aldrich, Germany). Penicillin, Dulbecco’s modified Eagle’s Medium, streptomycin and fetal bovine serum were from PAA The Cell Culture Company (Pasching, Austria). Bidestilled water was used for all the solutions. 2.2. Instrumental Fused silica capillaries of 75 ␮m i.d., total length of 40 cm, length to detector 31.5 cm were from Composite Metal Services (Worchestershire, UK). An Agilent 3DCE system (Agilent, Waldbronn, Germany) was used for the CE experiments. An ultrasonic bath from J.P. Selecta Ultrasons Medi-II (Barcelona, Spain) was used for sample preparation. Karl Hecht GmbH & Co. KG vortexer (Sondheim/Rhön, Germany) was used to accelerate sample dissolving. An Agilent TOF 6230 mass spectrometer was used for all mass measurements. Dry gas was set to 5 L/min at 325 ◦ C, nebulizer pressure was set to 7 psig. All MS voltage parameters had same absolute values in both negative and positive mode: (1) MS heated

capillary 5000 V (2) skimmer 65 V (3) fragmentor 140 (4) RF hexapole 800 V. CE was coupled to MS using the Agilent CE–MS sheath liquid assisted interface (part number G1607-60001). The sheath liquid consisted of 50%/50% (v/v) H2 O/MeOH with 10 mM ammonium acetate and was delivered with a flow rate of 1 ␮L/min. 2.3. Procedures 2.3.1. Preparation of polyE-323 The polyE-323 coating solution was prepared according to the uncomplicated procedure described by Hardenborg et al. [8]. In a 250 mL Erlenmeyer flask, 17.65 g (0.10 mol) of 1,2bis(3-aminopropylamino)ethane was slowly mixed with 20 mL bidistilled water and 9.3 g (0.10 mol) epichlorohydrine during intensive magnetic stirring. The flask was sealed and the mixture was continuously stirred at room temperature for 48 h while the reaction mixture was thickened. Then, it was cooled down to 5 ◦ C and an additional 100 mL bidistilled water was added. Finally the equilibration reaction was allowed to continue for 1 week. The polymer solution was stored at 5 ◦ C and used without further purification. Prior to the capillary coating procedure, the polymer mix was diluted with bidistilled water to 7.5% (v/v). All the experimental work described in this paper was carried out with in-lab synthesized polyE-323. The latter had been compared to a polyE-323 solution received as a gift from Prof. Bergquist group and proved to yield similar analytical performance as the coatings described in literature [8,9].

2.3.2. Coating procedures A fused silica capillary (40 cm) was flushed for 30 min with 1 M NaOH and 1 M HCl, then for 15 min with water. After that the capillary was rinsed with 7.5% polyE-323 coating solution for 45 min (pressure 50 mbar). Then the capillary was flushed for 10 min with BGE solution. All coating procedures were performed at 25 ◦ C (set as cassette temperature). The regeneration procedure was adapted from [8] and is in essence a short version of the capillary coating procedure, meaning that an already polyE-323 coated capillary was flushed for 10 min with 1 M NaOH, for 5 min with water, for 5 min with 1 M HCl and for 5 min again with water. Then the capillary was recoated with 7.5% polyE-323 solution (10 min flush). After recoating, the capillary was flushed for 5 min with BGE. For MS, a longer capillary was used and the coating and regeneration times were adapted proportionally.

2.3.3. Preparation of samples Cardiolipin, diacylglycerophosphocholine (PC 18:1/16:0) and sphingomyelin were dissolved in MeOH, to result a 1 mM stock solution. 0.1% DMSO in BGE was used as EOF marker in CE–UV experiments. A Mettler Toledo MP225 pH Meter was used for pH adjustments in BGEs, which were done in the aqueous component of the BGEs, prior to mixing with organic solvents. The development of BGEs is described in detail in Section 3.2. The lipid extract was obtained from a human primary glioblastoma cell line (U-87 MG), which were grown in T75 cell culture flasks with Dulbecco’s modified Eagle’s Medium, supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 ␮g/mL). The cells were maintained at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Cells were sub-cultivated 1:5 once a week and medium was changed 1–2 days after sub-cultivation. To obtain a lipid extract, first, a cell membrane-enriched suspension without nucleus and mitochondria was prepared according to Lodish et al. [24], which resulted in a membrane-containing supernatant. From this fraction, lipids were extracted using the Bligh and Dyer

K. Martma et al. / J. Chromatogr. A 1317 (2013) 193–198

procedure [25], resulting in the lipid extract (in chloroform), which was kept at −18 ◦ C until used. 3. Results and discussions 3.1. Stability of polyE-323 coating under the influence of pure organic solvents As a first step of the work the effect of five well-known organic solvents (acetonitrile, methanol, isopropanol, propylene carbonate and chloroform) on the stability of the polyE-323 coating was studied. In order to correctly evaluate the effect of the organic solvent, the initial performance of every fresh (untreated) polyE-323 coating was studied by a number of successive EOF measurements in aqueous media. As the next step the polyE-323 coating was exposed to a chosen solvent for a fixed time. This was carried out by rinsing the coated capillary for 1 h periods with the solvent under study. After each hour exposure period the EOF was determined using DMSO as an EOF marker (n = 5). Fig. 1 shows how exposure to a chosen organic solvent affected the EOF mobility. It can be observed that all organic solvents affect the coating to some extent, which explains the decreasing EOF. Yet the influence of acetonitrile, propylene carbonate, methanol and isopropanol on the coating was markedly less pronounced than that of chloroform, and strong EOF was still present after 3 h of exposure. The RSDs of the EOF mobility over 15 runs during 3 h exposure to methanol and isopropanol were 14% and 11% respectively, while after exposure to acetonitrile and propylene carbonate the lowest RSD values of EOF mobility were obtained, namely less than 3%. In other words, the polyE-323 coating was most stable with acetonitrile and propylene carbonate. The exposure to chloroform resulted in big variations in EOF mobility; the overall RSD of the EOF mobility was 33%. The EOF mobility was dramatically decreasing, indicating a fast deterioration of the coating. As a consequence, chloroform was discarded. Next, various combinations of methanol. acetonitrile, isopropanol and propylene carbonate were studied as possible BGEs. First, their effects on polyE-323 coating were monitored and then the best (the least affecting) mixtures were used for lipid analysis. 3.2. Development of BGEs BGEs comprising of only one organic solvent were not considered, as they might have limited possibilities for future studies, which are aimed at the analysis of complex samples, containing a wide range of lipids. Therefore, the BGEs were designed based on two organic solvents. Yet the purely organic mixtures are not conductive; therefore an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, known to be well soluble in the organic solvents studied in this paper [26], was added (30 ␮L/20 mL). Addition of this ionic liquid to non-aqueous BGEs has been reported previously [27,28]. The addition of 1-butyl-3-methylimidazolium hexafluorophosphate increased the conductivity of used solvents markedly, yielding an average current of −17 ␮A at −20 kV. However, the EOF mobility decreased dramatically; the DMSO migration time was up to 45 min and its peak shape was poor (data not shown). This is in accordance with what is described in [28], were 1-butyl-3-methylimidazolium hexafluorophosphate is shown to decrease the EOF migration time, which is hypothesized to be due to interactions of the ionic liquid with the fused silica wall. However, in our work, addition of a small portion (5.5%) of aqueous buffer (20 mM TRIS or ammonium acetate) to the organic solvent system restored the EOF mobility. The amount of the aqueous component of the BGE was varied but the increase in the percentage of aqueous component did not have marked influence on the EOF (data

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not shown). As the analytes are hydrophobic, however, the BGE should be mainly organic and therefore the 5.5% addition of aqueous buffer was used throughout further studies. All the tested BGEs are listed in Table 1. Since acetonitrile and propylene carbonate showed the best performance in the previous section (Fig. 1), they were chosen as major component of the BGEs (56.5%). Methanol and isopropanol demonstrated somewhat poorer performance but as up to 50% of methanol has successfully been used in polyE323 coated capillaries [8] it was presumed relevant to check both methanol and isopropanol as possible components of BGEs. In Fig. 2, the results obtained with the BGEs in Table 1 are depicted. The results demonstrate that the BGEs containing propylene carbonate as the major component (BGE 3 and 4) result in very high repeatability of EOF mobility but also in long analysis times (propylene carbonate has a high viscosity). As the fast analysis is often favoured acetonitrile (BGE 1 and 2) appears to be the preferred choice. Yet propylene carbonate could be used as an alternative. Closer evaluation of the BGEs shows that acetonitrile (BGEs 1 and 2) and propylene carbonate (BGEs 3 and 4) influence the analysis time and repeatability much stronger than the secondary components (methanol and isopropanol) meaning that if needed they can easily be replaced with one another. However, as isopropanol is often a preferred choice with hydrophobic analytes BGE 3 was chosen as the propylene carbonate based BGE for further studies with lipids. To test a CE–MS compatible BGE, TRIS was replaced with ammonium acetate. The results show that the presence of ammonium acetate instead of TRIS (BGE 7 and 8 versus BGE 5 and 6) has no pronounced effect on EOF repeatability or on the overall performance of the capillary. In order to develop well MS-compatible BGE systems, the BGEs without the addition of the ionic liquid were also studied (BGE 5–8). The results shown on Fig. 2 indicate that without the addition of ionic liquid the peaks were migrating slower but the analysis repeatability was the same as with the addition of ionic liquid. All these results indicate that the polyE-323 coating is versatile in its compatibility with common BGE components and offers good potential for the development of suitable BGEs for lipid analysis. In the next section, BGEs 1–3 and 8 are tested with sphingomyelin and cardiolipin standards. 3.3. Lipid analysis with a polyE-323 coated capillary in the presence of organic solvents Sphingolipids are structural components of the cell and participate in intra- and extracellular signalling. They share the common structural feature that all are comprised of backbones called “longchain” or “sphingoid” bases, which are represented by sphingosine, (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol, and are believed to be important in a multitude of metabolic pathways [21]. CE analysis of sphingolipids that is reported in the literature involves analyte-dye interactions and LIF detection [29,30]. Cardiolipin is a phospholipid rich in unsaturated fatty acids and localized mainly within the mitochondrial inner membrane. Changes in overall cardiolipin concentration are closely related to many pathological conditions (e.g. Barth syndrome) [22,23]. CE analysis of cardiolipin is a challenging task due to the hydrophobicity of the analyte, which causes severe interactions with the capillary wall. Moreover, cardiolipin is difficult to dissolve in aqueous media. The few works that describe cardiolipin analysis by CE have been dealing with LIF detection, using BGE additives to facilitate detection [31–35]. Initial experiments carried out in uncoated capillaries, using BGE 2 from Table 1, resulted in very long analysis times and at times no peaks appeared at all (data not shown). This supports our hypothesis that an uncoated fused silica capillary is a bad choice for lipid analysis even when a nearly non-aqueous BGE is used.

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Fig. 1. The influence of exposure to different pure organic solvents on the stability of the polyE-323 coating, assessed with the measurement of EOF mobility after the exposure. Exposure time was 3 × 1 h, and after each hour EOF was measured during 5 successive experiments. Abbreviations: ACN – acetonitrile, PropCarb – propylene carbonate, MeOH – methanol, IPA – isopropanol, CHL – chloroform. The running BGE for EOF determination was TRIS (pH 7.4, ionic strength 20 mM) and the running contitions: voltage −20 kV, injection 8 s at 50 mbar, detection 210.4 nm, cassette temperature 25 ◦ C, capillary lenght 31.5/40 cm, i.d. 75 ␮m. Table 1 Composition of all tested BGEs. In BGEs 1–5 the pH 7.4 of the aqueous part was set with NaOH, in BGEs 7–8 with ammonium hydroxide. 30 ␮L/20 mL of the ionic liquid was added. BGE number

Acetonitrile (%)

1 2 3 4 5 6 7 8

56.5 56.5

Propylene carbonate (%)

Isopropanol (%)

Water (%)

Electrolyte

Ionic liquid

38

5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5

TRIS TRIS TRIS TRIS TRIS TRIS Ammonium acetate Ammonium acetate

Yes Yes Yes Yes No No No No

38 56.5 56.5

56.5 56.5 56.5 56.5

Methanol (%)

38 38 38 38 38 38

As a next step the analysis of sphingomyelin and cardiolipin was carried out with selected organic BGEs: BGEs 1–3 and BGE 8 (as described in Table 1). The results are presented on Fig. 3 which demonstrates that sphingomyelin and cardiolipin are separated from each other with all the BGEs but the separation is clearly better with TRIS containing BGEs. The repeatability of the analysis was good for both lipids and the

analysis time is in accordance with the solvents viscosity. Cardiolipin was anionic with the selected BGEs and migrated in front of neutral sphingomyelin. In order to clarify the influence of ionic liquid on the analysis performance the BGEs 1–3 were tested also without the presence of ionic liquid. The comparison demonstrated that the separation between lipids was markedly better if the ionic liquid was not

Fig. 2. EOF mobility with different organic BGE mixtures (see Table 1) with the polyE-323 coated capillary (n = 6, error bars represent standard deviation). Running conditions as in Fig. 1.

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Fig. 3. Cardiolipin mobility with different organic BGE mixtures (see Table 1) with the polyE-323 coated capillary (n = 3, error bars represent standard deviation). Running conditions as in Fig. 1. Injected cardiolipin and sphingomyelin concentration was 1 mM.

present. On the other hand, the signal intensities were dramatically lower and the analysis times were longer. The repeatability was similar for both options. The results suggested that BGEs with and without ionic liquid could be used with polyE-323 coating and the use of ionic liquid depends on the purpose of analysis. In current work the BGE 8 (without ionic liquid) was used for CE–MS (Section 3.4) to minimize the danger of contaminating the ion source of MS. 3.4. CE–MS The aim of these experiments was to demonstrate the compatibility of the CE system, based on a polyE-323 coated capillary and a highly organic BGE, with MS, using a complex sample. In all

experiments, BGE 8 (containing ammonium acetate and no ionic liquid) was used. In none of the experiments background ions that could suggest leaking of the polyE-323 coating were observed. First, the migration time repeatability was studied, using sphingomyelin, which is neutral at pH 7.4, as EOF marker. Cardiolipin was initially present in the test samples, but discarded due to detection problems, which can possibly be explained by unfavourable ionization conditions for this analyte at the used experimental conditions. A series of 9 injections of 100 ␮M sphingomyelin showed good migration time repeatability (RSD 1.3%) and peak area repeatability (RSD 6.2%). No trend of retention time shift was observed. These results indicate that the coating is very stable under these conditions. A short series of calibration samples was measured (6.25, 12.5, 25,

Fig. 4. Analysis of cell lipid extracts. Extracted ion chromatograms and examples of mass spectra in positive and negative mode. (a) Putative mass for PC 18:2/16:0; (b) putative mass for PC 18:1/16:0; (c) putative mass for PC 18:2/18:0; (d) putative mass for myristic acid residue; (e) putative mass for palmitic acid residue; (f) putative mass for stearic acid residue. All CE–MS experiments were performed with BGE 8 (Table 1). CE separation conditions: capillary length 90 cm, capillary i.d. 75 ␮m, voltage -30 kV, injection 5 s at 50 mbar followed by 10 s at 50 mbar BGE. H2 O/MeOH mixture with 10 mM ammonium acetate was used as sheath liquid at 1 ␮L/min flow rate.

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50 and 100 ␮M); the obtained curve was y = 202549x − 859783 and had an excellent linearity (R2 = 0.994). Based on the signal-to-noise ratio of the peak of the lowest concentration, the LOD for sphingomyelin was estimated to be around 100 nM. Then, a lipid extract obtained from a human primary glioblastoma cell line (U-87 MG) was analyzed, both in positive and in negative MS mode. In the positive mode, in total, 67 compounds were suggested by Agilent Masshunter ‘Find by molecular feature algorithm’ above a threshold of signal-to-noise ratio 10. Signal intensity was two orders of magnitude lower in negative mode and only 23 compounds were found above the same S/N threshold. Compounds visible in negative mode formed several well-separated signal groups, in the positive mode the majority of intense signals represented co-migrating compounds which migrated with the EOF (Fig. 4). This can possibly be explained by the fact that negatively charged compounds (which were mainly present with the pH of this CE separation) were easier detected in the negative MS mode, and not in the positive mode, leading to under-representation of positively charged lipid species. Using a standard, we could putatively identify a phosphatidylcholine 18:1/16:0. Besides phosphatidylcholine 18:1/16:0, two more phosphatidylcholines were putatively idenified from mass spectra, using exact mass information: phosphatidylcholine C18:1/C16:1 and phosphatidylcholine C 18:1/C18:1. These suggested identifications are only based on exact m/z value; fatty acid composition or location of double bonds in these phosphatidylcholines cannot be determined without MS/MS experiments. In the negative mode three different fatty acid residues were putatively identified using exact mass information: myristic, palmitic and stearic acid. Even though only a few peaks could be putatively assigned to lipid species, the majority of the observed peaks are probably lipid species as well, since the sample pretreatment, i.e. the Bligh and Dyer protocol [25], is widely used for lipid extraction and purification from biological samples [36,37]. These first results indicate that the developed CE–MS system could be promising for fast analysis of complex lipid samples. 4. Concluding remarks In this note we demonstrated the potential of the polyaminebased coating polyE-323 for CE analysis of lipids. First, the stability of the coating with various commonly used organic solvents was investigated, after which several BGEs were developed. It was found that polyE-323 is stable under highly organic conditions. Then, the method was evaluated with sphingomyelin and cardiolipin and it was found that the system had high repeatability with various BGE systems, indicating minimal analyte-wall interaction and thus that polyE-323 coated capillaries are suited for the analysis of lipids. Finally, the method was coupled to MS, using sphingomyelin as test compound for the CE–MS method performance, and an example of an analysis of a complex lipid sample is given. Further research is aimed at thorough optimization of separation and identification of the obtained lipid peaks. Also, finding the optimal detection parameters for CE–MS of lipids, such as sheath

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