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A literature assessment of sample pretreatments and limits of detection for capillary electrophoresis of drugs in biological fluids and practical investigation with some antimalarials in plasma.

A literature survey on published reports of the determination of drugs in biological fluids shows that all methods of sample pretreatment have been used and that the limits of detection achieved vary widely, ranging from low ngcm(-3) to microgcm(-3). The most widely used injection method was hydrodynamic and, in the majority of cases, whenever low detection limits were achieved, this was a result of preconcentration during the sample pretreatment. Only a small proportion of the reported methods employed electrokinetic injection and utilised the field amplified sample injection (FASI) techniques. An experimental investigation of the alternative hydrodynamic and electrokinetic injection methods for a small set of antimalarial drugs is reported. It was found that electrokinetic injection with FASI from an acetonitrile-water matrix produced dramatic improvements in detection limits. This improvement could not, however, be achieved when the drugs were in plasma using protein precipitation, liquid-liquid extraction or solid phase extraction pretreatment methods. This highlights the importance of sample pretreatment in utilising the potential sensitivity of capillary electrophoresis with electrokinetic injection.

Antimalarials↗

Determination of atropine in biological fluids by micellar electrokinetic capillary chromatography in the presence of strychnine and tetracaine.

The identification and quantitation of atropine, in whole blood and gastric contents in the presence of strychnine and tetracaine is described. This method uses liquid-liquid extraction and micellar electrokinetic chromatography (MECC). Separations are made using a 50 cm long capillary and a borate/phosphate buffer at pH 9.2 with 50 mM sodium dodecyl sulfate (SDS). Linearity was established for the three compounds between 1.0 and 100 microg/mL, using scopolamine as internal standard. The limit of detection for atropine was estimated at 0.06 microg/mL and the limit of quantitation at 0.2 microg/mL. The run time is less than 30 min. Alternate parameters are proposed to reduce the run time to under 10 min. The method was applied to a forensic post-mortem case.

Anesthetics, Local↗

Quantitation of talinolol and other beta-blockers by capillary electrophoresis for in vitro drug absorption studies.

A capillary zone electrophoresis method is described for the enantioseparation of talinolol using heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin (HDAS-beta-CD) as a chiral selector. After liquid-liquid extraction of talinolol from physiological solution, electrokinetic injection was employed to improve the sensitivity. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A baseline separation of the talinolol enantiomers was achieved in less than 10 min using 100 mM phosphate solution as background electrolyte and pH 3.5, at the presence of 3.0 mM HDAS-beta-CD and at 20 degrees C. In addition, this analytical condition proved to be useful for the enantioseparation of a number of other beta-blocking agents such as alprenolol, atenolol, bisoprolol, celiprolol, metipranolol, oxprenolol, and sotalol. For determining talinolol, the method could be validated in terms of precision, accuracy and linearity, and was found to be suitable in determination of talinolol enantiomers in highly diluted samples obtained from in vitro experiments.

Absorption↗

Effect of NaOH on large-volume sample stacking of haloacetic acids in capillary zone electrophoresis with a low-pH buffer.

Large-volume sample stacking (LVSS) is an effective on-capillary sample concentration method in capillary zone electrophoresis, which can be applied to the sample in a low-conductivity matrix. NaOH solution is commonly used to back-extract acidic compounds from organic solvent in sample pretreatment. The effect of NaOH as sample matrix on LVSS of haloacetic acids was investigated in this study. It was found that the presence of NaOH in sample did not compromise, but rather help the sample stacking performance if a low pH background electrolyte (BGE) was used. The sensitivity enhancement factor was higher than the case when sample was dissolved in pure water or diluted BGE. Compared with conventional injection (0.4% capillary volume), 97-120-fold sensitivity enhancement in terms of peak height was obtained without deterioration of separation with an injection amount equal to 20% of the capillary volume. This method was applied to determine haloacetic acids in tap water by combination with liquid-liquid extraction and back-extraction into NaOH solution. Limits of detection at sub-ppb levels were obtained for real samples with direct UV detection.

Acetates↗

Study on thioridazine 5-sulfoxide epimerization and degradation by capillary electrophoresis.

We present a method for the stereoselective analysis of thioridazine 5-sulfoxide (THD 5-SO) in human plasma based on liquid-liquid extraction with diethyl ether and chiral resolution of the stereoisomers by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin and sulfated beta-cyclodextrin as chiral selectors. The method showed recovery rates of 85.5% for both THD 5-SO (slow-eluting, SE) enantiomers. The coefficients of variation observed in the precision studies, as well as the accuracy values, were below 10%. After validation, the method was used to study the stability and configurational changes of this THD metabolite. Our results showed that both enantiomers of THD 5-SO (SE) were stable under conditions of variation of temperatures (38 degrees C, 4 degrees C and -20 degrees C), pH (5.0, 7.0 and 8.5) and ionic strengths (0.2, 0.5 and 1.0 mol/L). The influence of light on the stability of the THD 5-SO (SE) stereoisomers was also studied using standard solutions prepared in methanol and an inversion in configuration was observed under UV light (254 and 366 nm).

Cyclodextrins↗

Determination of B2 and B6 vitamers in serum by capillary electrophoresis-molecular fluorescence-charge coupled detector.

A method for the separation by capillary zone electrophoresis and determination of vitamers of two important water-soluble vitamins, i.e., B2 and B6, is proposed here. The working conditions for optimal separation were obtained by a multivariate methodology in order to succeed in the best resolution in the shortest analysis time. The optimization of the buffer composition together with other variables, such as analysis and injection voltage, temperature of the capillary cassette, and injection time, resulted in a solution of 30 mM KH2PO4 adjusted to pH 8.5 with formic acid. Concerning the detection step, the target analytes were quantified by molecular fluorescence, for which two different detectors, a photomultiplier tube (PMT) and a charge-coupled detector (CCD), were compared in terms of resolution, sensitivity, and precision. A Xe-Hg lamp was used as an irradiation source in the two cases. The best option was the CCD, which provides three-dimensional electropherograms and enables to solve the overlapped peaks. Besides, the sensitivity of the CCD was similar to that of the PMT, due to the treatment data, obtaining limits of detection and quantification from 1.16 to 27.1 ng/g and from 3.83 to 89.4 ng/g, respectively. The method was applied to the serum samples for which a prior liquid-liquid extraction using ethanol in an acid medium was mandatory for eliminating the interferences and concentrating the analytes by a factor of 5. The rapidity of the analysis (13 min for the electrophoretic separation) and the excellent precision (repeatability and within-laboratory reproducibility between 2.86 and 4.11% and 7.03 and 8.45%, respectively, both expressed as relative standard deviation) demonstrated the capability of the proposed method for a clinical routine analysis.

Electrophoresis, Capillary↗

Enantioselective analysis of primaquine and its metabolite carboxyprimaquine by capillary electrophoresis.

An enantioselective capillary electrophoresis method for the simultaneous determination of primaquine (PQ) and carboxyprimaquine (CPQ) in rat liver mitochondrial fraction, suitable for in vitro metabolism studies is presented. The drug and metabolite were extracted by liquid-liquid extraction using ethyl ether. The enantiomers were resolved in a fused-silica capillary, 50 microm inside diameter (ID) and 24 cm of effective length, using an electrolyte solution consisting of a 20 mmol/L sodium phosphate solution, pH 3.0, and 10% w/v maltodextrin. Hydrodynamic sample injection was used with a 10 s injection time at 50 mbar pressure. The applied voltage was 22 kV and the capillary temperature was controlled at 20 degrees C. Detection was carried out at 264 nm. Under these conditions, the enantiomeric fractions of the drug and of its metabolite were analyzed within 6 min. The extraction procedure was efficient in removing endogenous interferents and low values (<10%) for the coefficients of variation and deviation from theoretical values were demonstrated for both within-day and between-day assays. The method described allows the determination of PQ and CPQ enantiomers as low as 100 and 40 ng/mL, respectively. After validation, the method was used for an in vitro metabolism study of PQ. The results showed that the enantiomer (-)-PQ was preferentially metabolized to (-)-CPQ.

Animals↗

Determination of some acidic drugs in surface and sewage treatment plant waters by capillary electrophoresis-electrospray ionization-mass spectrometry.

We describe an analytical method involving solid-phase extraction (SPE) and capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) for determining some pharmaceutical compounds - naproxen, clofibric acid and bezafibrate - in real water samples. The electrospray parameters were optimized to maximize sensitivity. When a mixed aqueous-organic solvent and CZE-ESI-MS were used to analyze these drugs in water samples, the capillary was coated with hexadimethrin bromide (HDB) to permanently reverse the EOF. The method was developed from off-line SPE-CZE-MS and was validated with surface water. The detection limits were 100 ng.L(-1) for all analytes. The method was applied to analyze water samples from the influent and effluent of a sewage treatment plant. A liquid-liquid extraction step was required before SPE, and the compounds studied were found, some of them between detection and quantification limits.

Acids↗

Characterization of the stereoselective biotransformation of ketamine to norketamine via determination of their enantiomers in equine plasma by capillary electrophoresis.

A robust CE method for the simultaneous determination of the enantiomers of ketamine and norketamine in equine plasma is described. It is based upon liquid-liquid extraction of ketamine and norketamine at alkaline pH from 1 mL plasma followed by analysis of the reconstituted extract by CE in the presence of a pH 2.5 Tris-phosphate buffer containing 10 mg/mL highly sulfated beta-CD as chiral selector. Enantiomer plasma levels between 0.04 and 2.5 microg/mL are shown to provide linear calibration graphs. Intraday and interday precisions evaluated from peak area ratios (n = 5) at the lowest calibrator concentration are < 8 and < 14%, respectively. The LOD for all enantiomers is 0.01 microg/mL. After i.v. bolus administration of 2.2 mg/kg racemic ketamine, the assay is demonstrated to provide reliable data for plasma samples of ponies under isoflurane anesthesia, of ponies premedicated with xylazine, and of one horse that received romifidine, L-methadone, guaifenisine, and isoflurane. In animals not premedicated with xylazine, the ketamine N-demethylation is demonstrated to be enantioselective. The concentrations of the two ketamine enantiomers in plasma are equal whereas S-norketamine is found in a larger amount than R-norketamine. In the group receiving xylazine, data obtained do not reveal this stereoselectivity.

Anesthetics↗

Rapid stereoselective separations of amphetamine derivatives with highly sulfated gamma-cyclodextrin.

The highly sulfated gamma-CD (HS-gamma-CD) is a chiral selector widely used in CE for the enantioseparation of pharmaceutical compounds. This paper investigated different approaches to reduce the stereoselective analysis time of amphetamine (AT) derivatives according to the chiral selector concentration in the BGE. With high HS-gamma-CD concentration, tested analytes were separated in 3.5 min as anionic complexes with short-end injection technique in reversed polarity mode. However, this procedure presented some limitations in terms of efficiency and resolution, excessive Joule heating and poor compatibility with MS detection. With low HS-gamma-CD concentration, compounds were separated as cations. Conventional approaches to reduce CE analysis time demonstrated critical resolution between some analytes. Therefore, the use of the partial-filling technique compatible with MS detection was carried out. Under optimized conditions, the analysis time for the chiral separation of seven AT like compounds was reduced to 6 min. Moreover, sensitivity of CE-MS was sufficient for the determination of ATs in plasma following a simple liquid-liquid extraction.

Amphetamines↗

Electrophoretic identification and quantitation of compounds in the polyphenolic fraction of extra-virgin olive oil.

A capillary zone electrophoresis method has been carried out to determine and quantitate some compounds of the polyphenolic fraction of virgin olive oil which have never previously been determined before using capillary electrophoresis, such as elenolic acid, ligstroside aglycon, oleuropein aglycon, and (+)-pinoresinol. The compounds were identified using standards obtained by semipreparative high-performance liquid chromatography (HPLC). A detailed method optimization was performed to separate the phenolic compounds present in olive oil using a methanol-water extract of Picual extra-virgin olive oil, and different extraction systems were compared (C18-solid phase extraction (SPE), Diol-SPE, Sax-SPE and liquid-liquid extraction). The optimized parameters were 30 mM sodium tetraborate buffer (pH 9.3) at 25 kV with 8 s hydrodynamic injection, and the quantitation was carried out by the use of two reference compounds at two different wavelengths.

Chromatography, High Pressure Liquid↗

Sensitive chiral analysis by capillary electrophoresis.

In this review, an updated view of the different strategies used up to now to enhance the sensitivity of detection in chiral analysis by CE will be provided to the readers. With this aim, it will include a brief description of the fundamentals and most of the recent applications performed in sensitive chiral analysis by CE using offline and online sample treatment techniques (SPE, liquid-liquid extraction, microdialysis, etc.), on-column preconcentration techniques based on electrophoretic principles (ITP, stacking, and sweeping), and alternative detection systems (spectroscopic, spectrometric, and electrochemical) to the widely used UV-Vis absorption detection.

Electrophoresis, Capillary↗

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples.

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.

Anti-Arrhythmia Agents↗

Capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenates.

A rapid, selective, and low-cost chiral capillary electrophoretic method was developed for the simultaneous analysis of hydroxychloroquine (HCQ) and its three chiral metabolites: desethylchloroquine (DCQ), desethylhydroxychloroquine (DHCQ), and bisdesethylchloroquine (BDCQ) in the microsomal fraction of liver homogenates. After liquid-liquid extraction using toluene as extracting solvent, the drug and metabolites were resolved on a fused-silica capillary (50 microm ID, 50 cm total length, and 42 cm effective length), using 100 mmol/L of Tris/phosphate buffer, pH 9.0 containing 1% w/v sulfated-beta-CD and 30 mg/mL hydroxypropyl-beta-CD. Detection was carried out at 220 nm. The extraction procedure was efficient in removing endogenous interferents, and low values (<or=15%) for CVs and deviation from theoretical values were demonstrated for both within-day and between-day assays. The quantitation limit was 125 ng/mL with linear response over the 125-2000 ng/mL of concentration range for all metabolites. After validation, the method was used for an in vitro metabolism study of HCQ. The major HCQ metabolite formed by microsomal enzymes was (-)-(R)-DHCQ.

Animals↗

Enhanced method performances for conventional and chiral CE-ESI/MS analyses in plasma.

Due to its high efficiency, selectivity, and sensitivity, CE-ESI/MS has evolved as an efficient technique for the drugs and metabolites analysis in biological matrices. However, a sample preparation is mandatory prior to CE-ESI/MS analysis. To achieve fast and simplified sample preparation of plasma samples, protein precipitation (PP) and liquid-liquid extraction (LLE) were used with two injection techniques: hydrodynamic (HD) and electrokinetic (EK) injection. CE-ESI/MS analyses of pharmaceutical compounds and amphetamine derivatives were developed. Detection limits of 1 ppm were reached with PP and HD injection whereas 1 ppb was detected when samples were prepared with LLE and injected by EK. Same experiments were performed for stereoselective determinations in partial-filling mode and detection limits achieved were equivalent to conventional analysis (0.5 ppb per enantiomer). When complex matrices are analyzed, MS signal suppression or enhancement effects are generally not reproducible and could compromise results with ESI. Therefore, matrix effect was investigated in CE-ESI/MS with a commercially available coaxial sheath-liquid ESI interface used as postcapillary infusion system to determine MS signal alterations. Matrix effects were differentially evidenced according to the selected sample preparation. With PP, signal suppression was observed out of the analyses migration window, while for LLE no relevant matrix effect occurred in all experiments.

Amphetamines↗

Influence of P-glycoprotein inhibition on the distribution of the tricyclic antidepressant nortriptyline over the blood-brain barrier.

The distribution of the antidepressant drug nortriptyline (NT) and its main metabolite E-10-hydroxy-nortriptyline (E-10-OH-NT) across the blood-brain barrier was considered in relation to inhibition of the multidrug transporter P-glycoprotein (P-gp). Rats received NT in doses of 25 mg/kg orally, 10 mg/kg i.p. or 25 mg/kg i.p. Half the rats were treated with the P-glycoprotein inhibitor cyclosporine A (CsA) (200 mg/kg) 2 h prior to NT administration, and the other half served as a control group. NT and the metabolite were extracted from brain and serum by liquid-liquid extraction and analysed by HPLC with UV-detection. The brain to serum ratio of NT was increased in the CsA treated groups (22.3-26.8) compared with the control groups (16.5-22.7), the difference being statistically significant in two of the three experiments (p<0.05). Increased brain-serum ratios were also found for E-10-OH-NT, but the differences were not statistically significant. These results suggest that inhibition of P-gp by CsA increases the accumulation of NT in the brain. Administration of the antipsychotic drug risperidone (0.5 mg/kg s.c.), which is a P-gp substrate, instead of CsA did not exert any measurable influence on the blood-brain ratio of NT concentrations. In conclusion, the results show that drug-drug interaction at P-gp may influence the intracerebral NT concentration, but apparently, a major inhibition of P-gp is necessary to attain a measurable effect.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

High-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry for the determination of flocoumafen and brodifacoum in whole blood.

A high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) assay was developed and validated to determine quantitatively flocoumafen and brodifacoum in whole blood using warfarin as an internal standard (IS). Liquid-liquid extraction, using ethyl acetate, was used to isolate flocoumafen, brodifacoum and the IS from the biological matrix. Detection was performed on a mass spectrometer by negative electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curves were linear (r(2) > 0.998) in the concentration range of 0.1-100.0 ng ml(-1) with a lower limit of quantification of 0.05 ng ml(-1) for flocoumafen, and 0.1 ng ml(-1) for brodifacoum in whole blood. Intra-day and inter-day relative standard deviations (RSDs) were less than 8.0% and 10.8%, respectively. Recoveries of flocoumafen and brodifacoum ranged from 78.0% to 83.7%. This assay can be used to determine trace flocoumafen and brodifacoum in whole blood to investigate suspected poisoning of human and animals.

4-Hydroxycoumarins↗

A generalized extraction-TLC procedure for identification of drugs.

A combination of liquid-liquid extraction and thin-layer chromatography permits rapid identification of a large variety of drugs in pharmaceutical or street dosage forms, or in biological materials. The system utilizes selective pH extraction combined with internal standards. Limits of sensitivity are in the submicrogram level. The procedure is rapid and requires minimal equipment and training of personnel. It can be supplied in toxicology involving both clinical and forensic demands.

Chromatography, Thin Layer↗