Analysis of low-molecular-mass organic acids using capillary zone electrophoresis-electrospray ionization mass spectrometry

A capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) method was developed to facilitate identification and determination of eleven low-molecular-mass (LMW) organic acids (i.e. oxalic, lactic, malonic, maleic, citric, tartaric, adipic, glutaric, gluconic, isosaccharinic and succinic acid) in different sample matrices. This CZE method was adapted to suit MS conditions. Sheath liquid, sheath flow and MS parameters were optimized to achieve high mass spectrometric sensitivity. The CZE-ESI-MS procedure showed good sensitivity (limit of detection of < 0.05-0.7 mg/l for all acids), linearity (r2 = 0.9925-0.9998) and reproducibility (2.09-5.34% RSD). The applicability of the CZE-ESI-MS was demonstrated on LMW organic acids in an ale sample. In addition the (here presented) method also provided quantification of fumaric, galacturonic and 2-ketoglutaric acid with high sensitivity.     

Selectivity in microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) is a most promising separation technique providing good selectivity and high separation efficiency of anionic, cationic as well as neutral solutes. In MEEKC lipophilic organic solvents dispersed as tiny droplets in an aqueous buffer by the use of surfactants provide a pseudo-stationary phase to which the solutes may have an affinity either to the surface or they may even partition into the droplets. When the droplets are charged, typically negatively, they will migrate opposite to the electroosmotic flow and hence separation of neutral solutes may take place. In the present paper focus has been set on how to change selectivity in MEEKC. Changes in the nature of surfactant as well as in pH have been shown to be powerful tools in changing the selectivity. The type of lipophilic organic phase is of less importance for the separation of fairly lipophilic solutes. Also changes in the temperature surrounding the capillary may alter the selectivity.     

Application of microemulsion electrokinetic chromatography to the analysis of a wide range of pharmaceuticals and excipients

Microemulsion electrokinetic chromatography (MEEKC) is a capillary electrophoresis (CE) technique in which solutes partition with moving oil droplets present in a microemulsion buffer. Ionised species will also separate by electrophoresis. In this paper MEEKC is shown to give highly efficient and relatively rapid separations for a wide range of pharmaceuticals, vitamins and excipients. A single set of operating conditions was used to resolve both water-soluble and insoluble compounds. The method was also used to separate both ionic and neutral compounds. The method was especially useful in the analysis of water-insoluble neutral compounds such as steroids and lecithin, which are difficult to analyse by CE. The method was found to be both quantitative and highly repeatable. The quality of the separation was found to be dependent upon the sample diluent used if large injection volumes are employed. The use of MEEKC for the determination of complex mixtures such as multi-ingredient formulations and drug-related impurities was successfully demonstrated. MEEKC offers significant advantages over many forms of CE and capillary electrochromatography (CEC) and should be considered as an extremely useful option in pharmaceutical analysis.     

Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Comparison of atmospheric pressure photoionization and ESI for CZE-MS of drugs

The performance of atmospheric pressure photoionization (APPI) and ESI for CZE was compared using a set of seven drugs (basic amines, quaternary amines and steroids) and four different BGEs. The influence of volatile and nonvolatile BGEs of acidic and neutral pH on the MS responses of test compounds was evaluated by infusion of test solutions into the respective ion sources, and by actual CZE-MS experiments. The infusion experiments indicate that sodium phosphate buffers cause ionization suppression in ESI-MS, although for the amines the suppression was modest (25-60% signal reduction). By contrast, APPI-MS responses were not affected by nonvolatile BGEs. With phosphate buffers, ESI-MS responses for the basic amines were still a factor 3-13 higher than the APPI-MS signals, whereas the steroids yielded similar responses in ESI-MS and APPI-MS. The quaternary amines could readily be detected in ESI-MS, but detection in APPI-MS required specific interface conditions. Using typical CZE-APPI-MS settings, quaternary amines remained undetected. Remarkably, the S/Ns observed in CZE-ESI-MS for the test compounds, were generally similar when using volatile and nonvolatile BGEs. For basic compounds, the S/Ns obtained in CZE-ESI-MS were a factor 2-5 higher than in CZE-APPI-MS, whereas steroids yielded equal S/Ns in both methods. Overall, it is concluded that when using relatively low BGE concentrations, the sensitivity of ESI-MS detection in CZE is more favorable than APPI-MS detection, even when nonvolatile BGEs are employed.     

Overview on advances in capillary electrophoresis-mass spectrometry of carbohydrates: a tabulated review

The increasing interest for carbohydrates as holder of essential bioinformations has boosted their full characterization through analytical techniques. The intent of this review is to summarize the recent trends regarding on-line and off-line CE-MS coupling for carbohydrate analysis. A statistical survey on the articles that use derivatizing agents as well as on the analyzer and type of instrument coupling (i.e. on- or off-line) is depicted. From a general overview it can be concluded that, whereas derivatization might be useful for the detection of neutral carbohydrates improving separation selectivity with volatile buffers and increasing sensitivity of the MS detection, relatively few works with derivatized carbohydrates were found; this was noticed in particular for glycosides and saccharides carrying ionizable groups, which are normally analyzed without any chemical modification. The most applied coupling is the on-line sheath-liquid interface; for on-line applications, ESI is the sole source used, whilst the most common analyzer is the IT. MS(n) is often exploited, as fragmentation increases the achieved structural information. CE-MS turned out to be mainly used for the analysis of carbohydrates in drug development (i.e. study of oligosaccharides from pathogens, carbohydrate-based drugs and drug metabolites), in nutrition and for characterization of glycans from glycoproteins. The reader will find elucidating tables regarding these recent CE-MS applications, including the main information on the analysis conditions. Comments are meant to help the immediate focus on the usefulness of the analytical technique and predict the difficulties found during analysis and, in case, their overcoming.     

Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis-electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 μg mL(-1) for carnosine, 0.17 μg mL(-1) for anserine and 0.64 μg mL(-1) for buserelin in water and 0.19 μg mL(-1) for carnosine, 0.50 μg mL(-1) for anserine and 0.74 μg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.     

Twenty‐one years of microemulsion electrokinetic chromatography (1991–2012): A powerful analytical tool

Microemulsion electrokinetic chromatography (MEEKC) is a CE separation technique, which utilizes buffered microemulsions as the separation media. In the past two decades, MEEKC has blossomed into a powerful separation technique for the analysis of a wide range of compounds. Pseudostationary phase composition is so critical to successful resolution in EKC, and several variables could be optimized including surfactant/co‐surfactant/oil type and concentration, buffer content, and pH value. Additionally, MEEKC coupled with online sample preconcentration approaches could significantly improve the detection sensitivity. This review comprehensively describes the development of MEEKC from the period 1991 to 2012. Areas covered include basic theory, microemulsion composition, improving resolution and enhancing sensitivity methods, detection techniques, and applications of MEEKC.     

Comparison of microemulsion electrokinetic chromatography and micellar electrokinetic chromatography methods for the analysis of phenolic compounds

In this study, microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatography (MEKC) were compared for their abilities to separate and detect thirteen phenolic compounds (syringic acid, p-coumaric acid, vanillic acid, caffeic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin, and (-)-gallocatechin), and two other ingredients (caffeine and theophylline) in teas and grapes. Separation of phenolic compounds was improved by changing the SDS concentration for MEEKC, but the SDS concentration rarely affected the resolution for MEKC. Organic modifier (acetonitrile or methanol) was found to markedly influence the resolution and selectivity for both MEEKC and MEKC systems. In addition, a higher voltage and a higher column temperature improved the separation efficiency without any noticeable reduction in resolution for MEEKC whereas they caused a poor resolution for the MEKC system. Although separations with baseline resolution were achieved by the optimized MEEKC and MEKC methods, the separation selectivity resulting from the proposed MEEKC method was completely different from that of MEKC.     

微乳电动色谱法的研究进展

微乳电动色谱(MEEKC)是一种以微乳为背景电解质,以电压为驱动力的分离技术。本文对MEEKC中的各调节参数进行了概述,重点归纳了MEEKC法在正辛醇-水分配系数测定、中药和手性化合物分离方面的应用,以及在操作技术上的新进展,如多路进样技术以及在线联用质谱检测技术等,并通过对相关文献的分析,对一直存有争议的MEEKC与胶束电动色谱(MEKC)的联系与区别进行了讨论,最后提出了MEEKC法未来的发展空间。     

Identification and quantitation of cis-ketoconazole impurity by capillary zone electrophoresis-mass spectrometry

trans-Ketoconazole was identified and quantified as impurity of cis-ketoconazole, an antifungal compound, by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS). The chirality of this impurity was demonstrated separating their enantiomers by adding heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin to the separation buffer in capillary electrophoresis (CE) with UV detection. However, MS detection was hyphenated to the CE instrument for its identification. As both compounds are diastereomers, they have the same m/z values and are needed to be separated prior to the MS identification. A 0.4M ammonium formate separation buffer at pH 3.0 enabled the separation of the impurity from cis-ketoconazole. Under these conditions, the optimization of ESI-MS parameters (composition and flow of the sheath-liquid, drying temperature, drying gas flow, and capillary potential) was carried out to obtain the best MS sensitivity. CZE-ESI-MS optimized conditions enabled the identification of trans-ketoconazole as impurity of cis-ketoconazole. In addition, the quantitation of this impurity was achieved in different samples: cis-ketoconazole standard and three different pharmaceutical formulations (two tablets and one syrup) containing this standard. In all cases, percentages higher than 2.0 were determined for the impurity. According to ICH guidelines, these values required the identification and quantitation of any impurity in drug substances and products.     

Cover Picture: Electrophoresis 2'2011

Issue no. 2 is a regular issue assembled of 16 solid and original research articles distributed over 3 distinct parts. Part I is on novel trends in fundamentals and methodologies including theoretical models for selectivity of charged solutes in MEKC, system peaks in indirect detection, measuring epimerization constants by MEEKC, bundled CE using micro‐structured fibers, 2‐D separations by coupling CIEF and CEC, high speed DNA CE, MCE of N‐glycans and mucin expression in a microfluidic gradient device. Part II is concerned with detection, sensitivity enhancement, on‐column preconcentration and microdialysis sampling involving the design of continuous full filling CEC‐ESI‐MS using nanoparticles, CE‐fluorescence using tapered optical fiber, CZE separation of pesticide residues in water samples with acid‐assisted on‐column preconcentration and CE‐LIF to detect neurotransmitter amino acids and carbamathione in brain microdialysis samples. Novel methods for the separation and profiling of various proteins and large nucleic fragments are described in 4 consecutive papers grouped in part III. Featured articles include: Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography (( 10.1002/elps.201000405 )) Bundled capillary electrophoresis using microstructured fibres ( 10.1002/elps.201000442 )) Two‐dimensional separation system by on‐line hyphenation of capillary isoelectric focusing with pressurized capillary electrochromatography for peptide and protein mapping ( 10.1002/elps.201000419 )) Microchip electrophoresis of N‐glycans on serpentine separation channels with asymmetrically tapered turns ( 10.1002/elps.201000461 ))     

Anion-selective exhaustive injection-sweeping microemulsion electrokinetic chromatography

In this study, anion-selective exhaustive injection-sweeping (ASEI-sweeping) technique, which is a selective on-line sample concentration technique, was first proposed in microemulsion electrokinetic chromatography (MEEKC) for analyses of eight acidic phenolic compounds. In contrast to a capillary that is typically filled with nonmicellar background solution in conventional ASEI-sweeping MEKC method, in the proposed ASEI-sweeping MEEKC method, a capillary is filled with a low pH microemulsion solution (pH 2.0), and then with a short acid plug (pH 2.0, 1.9 cm) before field-amplified sample injection. This proposed design has two functions. First, the microemulsion solution that is present at the front of capillary column is able to avoid phase separation of microemulsion solution during MEEKC separation. Second, the presence of the short acid plug would effectively limit the partition behavior of acid analytes with the oil droplets in the microemulsion during field-amplified sample injection; otherwise, the stacking effect of acid analytes would be markedly reduced. This optimal ASEI-sweeping MEEKC method afforded about 96,000-fold to 238,000-fold increases in detection sensitivity in terms of peak areas without any separation efficiency loss when compared to normal MEEKC separation. Furthermore, trace levels (about 3 ng/g) of gallic acid and catechin in foods were also detected successfully by the proposed ASEI-sweeping MEEKC technique.     

Background theory and applications of microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) is an electrodriven separation technique. Separations are achieved using microemulsions which are nanometre-sized oil droplets suspended in aqueous buffer. The surface tension between the oil and water components is reduced by covered the oil droplet with an anionic surfactant such as sodium dodecyl sulphate and a co-surfactant such as a short-chain alcohol. This review summarises the various microemulsion types and compositions that have been used in MEEKC. The effects of key operating variables such as pH and temperature are also described. The application areas of MEEKC are also described in some detail. MEEKC has been applied to a wide range of water-soluble and insoluble both charged and neutral compounds. Examples are described which include analysis of derivatised sugars, proteins, pesticides and a wide range of pharmaceuticals. At present there are only a limited number of publications describing the use of MEEKC but it is anticipated that this number will increase rapidly in the near future as more awareness of the separation possibilities that MEEKC presents increases.     

On-line concentration of neutral analytes by complexation and acetonitrile sweeping in nonionic microemulsion electrokinetic chromatography with direct ultraviolet detection

To separate and detect neutral solutes in nonionic microemulsion electrokinetic chromatography (MEEKC), a novel method was developed, combining complex formation and acetonitrile (ACN) sweeping. In this report, dynamic borate complexation and on-line sweeping occurred simultaneously during a run. The operating parameters which affected the performance of analyte sweeping in nonionic MEEKC were examined in terms of borate complexation, ACN content, Brij-35 concentration and sample plug length. In addition, the validation of the method included tests of the limit of detection, reproducibility and sensitivity enhancement. 60–110-Fold of magnitude improvement in detection sensitivity for model compounds (ginsenoside Rf, ginsenoside Rb2, ginsenoside Re) using Brij-35 microemulsion was demonstrated. Furthermore, the method was applied to the determination of glucosides in the plant extract.     

Analytical atomic spectroscopy using ion trap devices

Investigations using ion trap devices for analytical atomic spectroscopy purposes have focused on the use of an inductively coupled plasma (ICP) ion source with ion trap mass spectrometric (ITMS) detection. Initial studies were conducted with an instrument assembled by simply appending an ion trap as the detector to a fairly conventional ICP/MS instrument, i.e. leaving an intermediate linear quadrupole between the plasma source and the ion trap. The principal advantages found with this system include the destruction of nearly all problematic and typical ICP/MS polyatomic ions (e.g., ArH(+), ArO(+), ArCl(+), Ar(2)(+), etc) and a dramatic reduction of the primary plasma source ion, Ar(+). These results prompted the development of a second-generation plasma source ion trap instrument in which direct coupling of the ICP and ion trap has been effected (i.e. no intermediate linear quadrupole); the same performance benefits have been largely preserved. Initial operation of this instrument is described, characterized, and compared to the originally described ICP/ITMS and conventional ICP/MS systems. In addition, experiments aimed at improving ICP/ITMS sensitivity and selectivity using broadband resonance excitation techniques are described. Finally, the potential for laser optical detection of trapped ions for analytical purposes is speculated upon.     

Comparison of oil-in-water and water-in-oil microemulsion electrokinetic chromatography as methods for the analysis of eight phenolic acids and five diterpenoids

Oil-in-water (O/W) and water-in-oil (W/O) MEEKC were compared for their abilities to separate and detect eight phenolic acids and five diterpenoids in Radix et Rhizoma Salviae Miltiorrhizae (RRSM). The effects of oil type and concentration, organic modifier, SDS, and buffer concentration on separation were examined in order to optimize the two methods. Oil contents and organic modifier were found to markedly influence the separation selectivity for both O/W and W/O systems. SDS concentration rarely affected separation resolution for O/W MEEKC, and separation of eight phenolic acids and five diterpenoids could be improved by changing the buffer concentration for W/O MEEKC. A highly efficient O/W MEEKC separation method, where the 13 compounds were separated with baseline resolution, was achieved by using a microemulsion solution of pH 8.0 containing 0.6% cyclohexane, 3.0% SDS, 6.0% 1-butanol, and 3.0% ACN. The W/O MEEKC was unable to resolve all the components. In addition, the analytic time in O/W MEEKC was shorter than that in W/O MEEKC. Finally, the developed O/W MEEKC method was successfully applied to determine analytic compounds in RRSM samples.     

磷脂微乳电动色谱的定量结构保留关系研究

以大豆磷脂为主要的表面活性剂,制备适合毛细管电动色谱使用的不同构成比的微乳体系, 应用溶剂化参数模型研究了中性溶质在其中的定量结构保留关系.使用动态涂层毛细管, 以二甲基亚砜和十二烷基苯分别作为电渗流和微乳液滴迁移的标记物, 测定了26个具有不同结构小分子中性化合物在17种微乳电动色谱体系下的保留因子, 建立了线性溶剂化能量关系(LSER)方程.通过比较两体系的LSER方程系数比较体系相似性.结果表明, 本研究建立的磷脂微乳电动色谱体系在线性溶剂化特征上和其它构成的微乳电动色谱体系相似.对溶质保留贡献较大的是溶质体积和有效氢键碱度, 油相种类及浓度对溶质的保留选择性无明显影响.     

No-discharge atmospheric pressure chemical ionization: evaluation and application to the analysis of animal drug residues in complex matrices

Alternative ionization methods are increasingly being utilized to increase the versatility and selectivity of liquid chromatography/mass spectrometry (LC/MS). One such technique is the practice of using commercially available atmospheric pressure chemical ionization (APCI) sources with the corona discharge turned off, a process termed no-discharge APCI (ND-APCI). The relative LC/MS responses for several different classes of veterinary drugs were obtained by using ND-APCI, electrospray ionization (ESI), and APCI. While the ND-APCI-MS and -MSn spectra for these compounds were comparable with ESI, ND-APCI provided advantages in sensitivity and selectivity for some compounds. Drugs that were charged in solution as cations or sodium adducts responded particularly well with this technique. Instrumental parameters such as temperatures, gas and liquid flow rates, and source design were investigated to determine their effect on the process of ND-APCI. This paper explores advantages of using ND-APCI for the determination and confirmation of drug residues that might be found in food matrices, including malachite green residues in fish tissue and avermectin residues in milk.     

Screening of octanol-water partition coefficients for pharmaceuticals by pressure-assisted microemulsion electrokinetic chromatography

A rapid screening assay for the determination of octanol-water partition coefficients (log P(OW)) of pharmaceuticals was developed by using pressure-assisted microemulsion electrokinetic chromatography (MEEKC). The microemulsion system contains 50 mM sodium dodecyl sulfate, 0.87 M l-butanol, 82 mM heptane, and 50 mM borate-phosphate (2:3) at pH 10. Ten standard compounds with known log P(OW) values from -0.26 to 4.88 were used for constructing the calibration curve of log P(OW) against the MEEKC retention factor, log k. The log P(OW) values of the compounds were calculated based on the log k values measured by MEEKC and the slope and intercept of the calibration curve. For 13 literature and 32 Roche compounds, about 90% of the log P(OW) values measured by MEEKC are within 0.5 log units of the values from the literature and potentiometric titration. The throughput is about 2 samples/h using +20 kV voltage plus 5 mbar air pressure for separation. This MEEKC method is applicable for log P(OW) screening of weakly basic, weakly acidic, and neutral pharmaceuticals with log P(OW) = 0-5 and pKa < or = 10.