Journal of Chromatography B (v.843, #2)

Liquid chromatography–electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood by Violeta Kontrimavičiūtė; Hélène Breton; Olivier Mathieu; Jean-Claude Mathieu-Daudé; Françoise M.M. Bressolle (131-141).
A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using Oasis®HLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 μm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2 mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89–179 μg/l for ibogaine; 1–200 μg/l for noribogaine) and to whole blood concentrations (1.78–358 μg/kg for ibogaine; 2–400 μg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89–102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were ≥94% in plasma and ≥57% in whole blood. The lower limits of quantitation were 0.89 μg/l for ibogaine and 1 μg/l for noribogaine in plasma, and 1.78 μg/kg for ibogaine and 2 μg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4 h at 4 °C and 20 °C and 2 months at −20 °C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.
Keywords: Ibogaine; Noribogaine; Plasma and whole blood; Quantitation; LC-ESI-MS; Forensic samples;

Microdialysis is an increasingly employed technique for the determination of tissue pharmacokinetics. A high-performance liquid chromatography method for the quantitative determination of caspofungin in human microdialysates with amperometric detection is described. Since microdialysis of caspofungin is performed with a 100,000 molecular mass cut-off membrane, microdialysates contain protein that was precipitated at pH 4 with acetonitrile. Addition of 1-propanol (33%, v/v) to the sample extract improved the analytical recovery to 81–89%. Caspofungin and the internal standard clarithromycin were separated isocratically on a cyanopropyl silica column using acetonitrile–0.05 M citrate (33:67, v/v), adjusted to an apparent pH of 6.9, at a flow rate of 1.0 ml/min, and amperometric detection at +950 mV oxidation potential. Within-day and between-day imprecision and inaccuracy were <11%. The lower limit of quantification was 0.07 μg/ml. The method was applied to in vitro microdialysis experiments. Ringer's solution containing 1% (w/v) human albumin was used for the perfusing and surrounding medium, respectively. Albumin did not entirely prevent adsorption of caspofungin to the surface of membrane and/or tubing. When the binding-sites were saturated with albumin plus caspofungin prior to the start of sampling, the percentage of drug appearing in the microdialysate (“recovery”) remained stable over the concentration range tested.
Keywords: Caspofungin; Echinocandin; Microdialysis; High-performance liquid chromatography; Amperometric detection;

Liquid chromatography–tandem mass spectrometric determination of tenofovir-diphosphate in human peripheral blood mononuclear cells by Tracy King; Lane Bushman; Jennifer Kiser; Peter L. Anderson; Michelle Ray; Thomas Delahunty; Courtney V. Fletcher (147-156).
To facilitate the evaluation of drug safety, virologic activity, and pharmacokinetics, an anion exchange isolation of tenofovir-diphosphate (TFV-DP) from human peripheral blood mononuclear cells (hPBMCs), coupled with dephosphorylation, desaltation, and detection by LC–MS–MS was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular tenofovir moieties was produced. TFV-DP was isolated from TFV-monophosphate (TFV-MP) and tenofovir (TFV), dephosphorylated with acid phosphatase to form TFV and then desalted and concentrated, making it possible for tandem mass spectral detection. An LC–MS–MS methodology was developed and validated for the determination of TFV concentrations, which directly correspond with the intra-hPBMC TFV-DP concentration. The assay was linear in the range of 50–10,000 fmol per sample. The lower limit of quantitation (LLOQ) of the method is 10 fmol per million cells with 5 million hPBMCs used. This paper outlines the development and validation of the determination of TFV-DP concentrations in femtomoles per million hPBMCs.
Keywords: Tenofovir-diphosphate; LC–MS–MS; Peripheral blood mononuclear cells;

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC–tandem mass spectrometry. Whole blood samples (500 μl) were subjected to liquid–liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3 → 255.3). The assay was linear from 0.2 to 25 μg/l (r 2  > 0.997, n  = 5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 μg/l) were 95.8–103.2 and <5.5%, respectively. At the lower limit of quantification (0.2 μg/l) the inter- and intra-day analytical recovery was 99.0–102.8% with imprecision of <7.6% (n  = 5). The assay had a mean relative recovery of 100.5 ± 5.8% (n  = 15). Extracted samples were stable for 16 h. FTY720 quality control samples were stable at room temperature for 16 h, at 4 °C for at least 8 days and when taken through at least three freeze–thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans.
Keywords: FTY720; HPLC; Mass spectrometry; APCI; Immunosuppressant drug;

An analytical method based on high-performance liquid chromatography (HPLC) with ultraviolet detection (269 nm) was developed for the determination of pioglitazone in human plasma. Rosiglitazone was used as an internal standard. Chromatographic separation was achieved with a reversed-phase Apollo C18 column and a mobile phase of methanol–acetonitrile-mixed phosphate buffer (pH 2.6; 10 mM) (40:12:48, v/v/v) with a flow rate of 1.2 ml/min. The calibration curve was linear over the range of 50–2000 ng/ml (r 2  > 0.9987) and the lower limit of quantification was 50 ng/ml. The method was validated with excellent sensitivity, accuracy, precision, recovery and stability. The assay has been applied successfully to a pharmacokinetic study with human volunteers.
Keywords: Pioglitazone; HPLC; Pharmacokinetics; Quantification; Validation; Solid-phase extraction; Reversed-phase chromatography;

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N G,N G-dimethyl-l-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) – 140 μM (1.0 nmol per injection) using N G-monomethyl-l-arginine (l-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82 ± 0.05 μM (n  = 4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.
Keywords: N G,N G-Dimethyl-l-arginine (ADMA); Dimethylarginine dimethylaminohydrolase (DDAH); Monolithic silica column; 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F); Fluorescence; Column-switching;

Neural stem cell separation from the embryonic avian olfactory epithelium by sedimentation field-flow fractionation by I. Comte; S. Battu; M. Mathonnet; B. Bessette; F. Lalloué; P. Cardot; C. Ayer-Le Lièvre (175-182).
The aim of the present study was to isolate neural stem cells from a complex tissue: the avian olfactory epithelium; by using sedimentation field flow fractionation (SdFFF). By using “Hyperlayer” elution mode, fraction collection and cell characterization methods, results shows that SdFFF could be a useful cell sorter to isolate an enriched, viable and sterile immature neural cell fraction from which the reconstitution of a complete epithelium was possible. In culture, SdFFF eluted cells first led to a “pseudoplacodal” epithelioid cell type from which derived “floating cells”. These cells were then able to generate neurosphere-like structures which were composed of cell having many features of immature cells: undifferentiated, self-renewable and multipotentiality. Such a population might be used as a model to improve our understanding of the mechanisms of olfactory neoneurogenesis.
Keywords: Avian embryos; Olfactory epithelium; Neural stem cells; SdFFF; Cell separation;

Characterization and quantification of eight water-soluble constituents in tubers of Pinellia ternata and in tea granules from the Chinese multiherb remedy Xiaochaihu-tang by Ping Chen; Chuan Li; Shipiao Liang; Guoqiang Song; Yan Sun; Yanhong Shi; Songlin Xu; Jiwen Zhang; Shuqun Sheng; Yiming Yang; Min Li (183-193).
In traditional Chinese medicine, multiple herbs are usually used in combination to generate the joint actions of a multiherb remedy. The recent development of LC-hyphenated techniques enables efficient and rapid profiling of the chemical constituent in extracts from multiherb remedies. Xiaochaihu-tang is a seven-herb remedy that has attracted a great deal of attention for reported ability to treat liver dysfunction. Dried tubers of Pinellia ternata (banxia in Chinese) is one of the ingredients, but its chemical contribution to Xiaochaihu-tang remains poorly understood. In the study presented here, LC–UV–MS, LC–MS–MS, and LC–NMR were used in a complementary manner to determine the nature and content of eight water-soluble constituents of banxia and their presence in various tea granules from Xiaochaihu-tang. Among the eight chemicals identified in banxia, cytidine, adenosine, tryptophan, uridine, and adenine are reported for the first time, while tyrosine, guanosine, and phenylalanine were previously described. These chemicals are also present in all of the samples of Xiaochaihu-tang granules, and the amounts of the chemicals ingested due to a daily dose of the multiherb remedies range from 0.008 to 6.3 mg.
Keywords: LC-hyphenated techniques; Xiaochaihu-tang; Pinellia ternate;

A method of pinpoint-sampling followed by on-line pre-concentration of the sample, throughout in-capillary derivatization and capillary electrophoretic separation was evaluated by demonstrating the detection of taurine, 2-aminoethanesulfonic acid at a specific location of a rat brain. The direct sampling of taurine from the rat brain was accomplished by using voltage injection associated with two kinds of driving forces, electrophoretic flow and electroosmotic flow (EOF). The capillary tube (75 μm of inner diameter × 375 μm of outer diameter) of the capillary electrophoresis (CE) apparatus was already filled with a CE run buffer, viz., 40 mM phosphate–borate buffer (pH 10) containing 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as the derivatization reagent. One end of a platinum wire (0.5 mm o.d.), used as the anode, and the inlet end of capillary tube (from which a 1.0 cm long polyimide coating was removed), were pricked down onto the surface of either the cerebrum or cerebellum of a rat brain at a location of very small dimension. When a low voltage (5 kV, 30 s) was applied, taurine began to move from the rat brain into the capillary tube, and, simultaneously, electric focusing of taurine occurred by the action of “the pH-junction effect” at the inlet end of the capillary tube. After completing the injection, both the platinum wire and capillary tube were detached from the brain and dipped into the run buffer in an anode reservoir filed with the same solution as that in the capillary tube for the CE apparatus. Then, by applying a high voltage (20 kV) between the ends of the capillary tube, taurine was automatically derivatized to yield the fluorescent derivative, separated and detected with fluorescence (E x  = 340 nm, E m  = 455 nm) during migration throughout the capillary tube. The migration profiles obtained from cerebrum and cerebellum appeared to be different, but the peak corresponding to taurine was identified on both electropherograms. The efficacy of the present method including sample on-line pre-concentration prior to throughout in-capillary derivatization CE was first verified with several preliminary experiments by using samples of taurine in water, saline and a piece of 1.5% agar–gel block, as an alternate standard for the rat brain used in this study.
Keywords: Capillary electrophoresis; On-line pre-concentration; Taurine; Throughout in-capillary derivatization; Rat brain;

A gas chromatography–electron impact ionization-mass spectrometric (GC–EI-MS) assay was developed for the determination of ethylene oxide–hemoglobin adduct (N-2-hydroxyethylvaline, HEVal). HEVal and deuterated HEVal (d 4-HEVal) were synthesized for identification and quality control. Globin samples were separated from red blood cells (RBCs) by acidic isopropanol and extracted with ethyl acetate. HEVal adduct in globin was transformed to HEVal-pentafluorophenylthiohydantoin derivative by modified Edman-degradation method, which was extracted from globin with diethylether. d 4-HEVal was used as an internal reference standard. The dried extract was derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)-NH4I (1000:4, w/w) containing 0.4 mg of dithioerthritol. The TBDMS derivative of HEVal had very good chromatographic property and offered sensitive response of GC–EI-MS. The recovery of HEVal was about 81.6% and the coefficient of variation was 5.0% at the concentration of 311 pmol/g. Low limit of detection (LOD) of the assay was 1.8 pmol/g in 0.1 g hemoglobin. The experiments have demonstrated to detect background level of HEVal adduct in human blood. HEVal adduct in globin was detected between 12 and 6573 pmol/g.
Keywords: EO–hemoglobin adduct; Hydroxyethylvaline; GC–EI-MS; Silylation;

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum by Xiaolai Ma; Zunsheng Wang; Suxia Li; Qiong Shen; Qinsheng Yuan (209-215).
Heparinase I has been purified from F. heparinum by a novel scheme with 10 mM CaCl2 added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4–7.0 and 41 °C. CaCl2 is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 °C or freezing-thawing.
Keywords: CaCl2; Activity stabilizer; Purification; Flavobacterial heparinum; Heparinase I;

In this work, we present an improved method for the determination of a wide range of intracellular metabolites from mammalian cells by anion-exchange chromatography. The analysis includes the measurement of intermediates from glycolysis and tricarboxylic acid cycle as well as several additional nucleotides and sugar nucleotides. The use of an electrolytic on-line eluent generation device made the method highly convenient, reliable and prone to errors. Due to short delay times of the eluent generator, rapid KOH gradient changes could be applied to improve separation and to speed up elution. Suppressed conductivity and UV in series was used for detection. The detection wavelength of the UV detector was switched from 220 to 260 nm during the elution for a more selective signal depending on the absorption of analytes. Standards from more than 50 metabolites of major cellular pathways were chromatographically tested and compared to chromatograms from extraction samples of Madin–Darby canine kidney (MDCK) and BHK21 cells. A validation for most substances was performed. Detection limits were below the micromolar range and the coefficient of correlation (R 2) was above 0.99 for most analytes. Working ranges were between 0.125–3.875 and 4.5–139.5 μM. Sample pH had a major impact on the quantification of several metabolites, but measurements were robust within a pH range of 6.5–9.0.
Keywords: Anion-exchange chromatography; On-line eluent generation; Metabolism; Tricarboxylic acid cycle; Glycolysis; Organic acids; Nucleotides; Sugar nucleotides; Sugar phosphates; Mammalian cells;

Simultaneous determination of fluvoxamine isomers and quetiapine in human plasma by means of high-performance liquid chromatography by Maria Addolorata Saracino; Laura Mercolini; Giuseppina Flotta; Lawrence J. Albers; Roberto Merli; Maria Augusta Raggi (227-233).
An original HPLC–UV method has been developed for the simultaneous determination of the atypical antipsychotic quetiapine and the geometric isomers of the second-generation antidepressant fluvoxamine. The analytes were separated on a reversed-phase C8 column (150 mm × 4.6 mm i.d., 5 μm) using a mobile phase composed of acetonitrile (30%) and a 10.5 mM, pH 3.5 phosphate buffer containing 0.12% triethylamine (70%). The flow rate was 1.2 mL min−1 and the detection wavelength was 245 nm. Sample pretreatment was carried out by an original solid-phase extraction procedure using mixed-mode cation exchange (DSC-MCAX) cartridges; only 300 μL of plasma were needed for one analysis. Citalopram was used as the internal standard. The method was validated in terms of linearity, extraction yield, precision and accuracy. Good linearity was obtained in plasma over the 5.0–160.0 ng mL−1 concentration range for each fluvoxamine isomer and over the 2.5–400.0 ng mL−1 concentration range for quetiapine. Extraction yield values were always higher than 93%, with precision (expressed as relative standard deviation values) better than 4.0%. The method was successfully applied to human plasma samples drawn from patients undergoing polypharmacy with the two drugs. Satisfactory accuracy values were obtained, with mean recovery higher than 94%.
Keywords: Quetiapine; Fluvoxamine isomers; Human plasma; Liquid chromatography; Solid-phase extraction;

Determination of penehyclidine by gas chromatographic–mass spectrometry and its application to pharmacokinetics in humans, rabbits and mice by Ming Xue; Shulan Yuan; Jinxiu Ruan; Jianzhong Qiao; Yanxia Xu; Zhenqin Zhang; Keliang Liu (234-239).
A sensitive and specific gas chromatographic–mass spectrometry with the extracted ion chromatograms (GC–MS/EIC) method has been developed and validated for the identification and quantification of penehyclidine (PH) in human and animal blood. The chromatography was on HP-5 capillary column (12 m × 0.2 mm × 0.3 μm). PH-d5 was selected as the internal standard (IS). Simultaneous MS detection of PH and IS was performed at m/z 315 (PH) and m/z 320 (PH-d5), and the EIC of the two compounds was at 175 and 180. PH and PH-d5 eluted at approximately 8.2 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 0.1–100 ng/ml in the blood. The lower limit of quantification (LLOQ) was reproducible at 50 pg/ml in both human and animal blood. The within-day and between-day precisions were no more than 9.1%. This method was successfully applied to quantification and pharmacokinetic studies of PH in the subjects. The concentration–time profiles of PH in humans, rabbits and mice were all fitted to first order absorption two-compartment open model after i.m. a single dose. The differences in absorption, distribution and elimination of PH among the species were found. The results provided the important information for developing a novel anti-cholinergic drug and for obtaining a more effectual remedy in clinical practice.
Keywords: Penehyclidine; Anticholinergic drug; Quantification; Pharmacokinetics; GC–MS/SIM;

Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A by Terrina Dickinson Laing; Armando J. Marenco; Diana M. Moore; Graham J. Moore; David C.W. Mah; William E. Lee (240-246).
Botulinum neurotoxin serotype A (BoNT/A) is a proteolytic enzyme that induces muscle paralysis. It is a cause of food poisoning, a potential bioterrorist threat and, in low doses an emerging pharmaceutical product. No effective treatment is currently available for BoNT intoxication. Previously we developed a BoNT/A light chain enzyme assay using a peptide substrate based on the SNAP-25 protein target, with HPLC separation and UV detection of assay products, and applied the method to screen combinatorial peptide libraries for inhibitory activity to BoNT/A. We now report on development of a capillary electrophoresis laser-induced fluorescence (CE-LIF) method for measuring BoNT/A activity. The enzyme assay products were labeled with CBQCA dye followed by CE separation on a bare fused silica column in a HEPES-based buffer and LIF detection. All assay products were separated in CE within 8 min compared to incomplete separation of assay products within 1 h by HPLC. The labeled products showed linear dependence of intensity versus concentration, and quantitative mole-fraction assignments. We used the CE-LIF method to screen combinatorial peptide libraries for potential modulating effects on BoNT/A peptidase activity. With some of the libraries, peptides co-migrated with assay products and interfered with quantitation. In such cases, interference was reduced by substituting sodium dodecyl sulfate (SDS) for Tween-20 in the running buffer. Separation in the capillaries then occurred by micellar electrokinetic chromatography (MEKC). The CE-LIF method is quick and lends itself to high-throughput or microfluidic formats.
Keywords: Capillary electrophoresis; Fluorescence; Botulinum neurotoxin; Micellar electrokinetic chromatography; Combinatorial peptide libraries;

A liquid chromatography–ion trap mass spectrometry method with three “time segments” has been developed to determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) in edible goldfish muscle. By using the optimized “time segments”, MG and LMG as well as the internal standard atrazine-d5 were analyzed with good sensitivity with positive ESI–MS in a single run. The homogenized fish muscle tissues were extracted with a solution of perchloric acid and acetonitrile, followed by partitioning with dichloromethane. Strata-x polymeric solid-phase extraction column was used for the clean-up process. The determination of MG and LMG was achieved by using a reversed-phase HPLC gradient program coupled with MS/MS in multiple-reaction-monitoring mode. Matrix calibration curves were linear over the ranges of 5–500 ng/ml for MG and 1–100 ng/ml for LMG. Recoveries of the fish tissue extraction at three spiked levels (2, 10 and 30 ng/g for MG as well as 0.4, 2 and 6 ng/g for LMG) were better than 71% and 89%, respectively. Relative standard derivations from six determinations were less than 8%. The method detection limits were 0.13 ng/g for MG and 0.06 ng/g for LMG.
Keywords: LC–ion trap MS; Time segments; Malachite green; Leucomalachite green; Edible goldfish;

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC–APCI–MS by P. Songsermsakul; G. Sontag; M. Cichna-Markl; J. Zentek; E. Razzazi-Fazeli (252-261).
The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1–0.5 μg/l or μg/kg, the limits of quantification from 0.5–1.0 μg/l or μg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.
Keywords: Zearalenone; Metabolites; Liquid chromatography–mass spectrometry; Urine; Plasma; Faeces; Horse;

A sensitive and selective LC–MS/MS method has been developed and validated for the quantification of abiraterone acetate and its metabolite, abiraterone (an androgen biosynthesis inhibitor) in human plasma. Analytes were extracted by SPE with cation mixed-mode polymer cartridges. Chromatography was performed on a Luna C5 5 μm, 50 mm × 2.1 mm i.d. column, using a mobile phase of 2% propan-2-ol in acetonitrile and 10 mM ammonium acetate. The assay was linear from 5 to 500 nM (r 2  = 0.998). The intra- and inter-day coefficients of variation were <13.9% for both analytes. This method will be applied to a clinical trial investigating the pharmacokinetics of abiraterone acetate and abiraterone in patients with prostate cancer.
Keywords: Abiraterone; Liquid chromatography–tandem mass spectrometry; Prostate cancer; Validation;

Development of an analytical method for organotin compounds in fortified flour samples using microwave-assisted extraction and normal-phase HPLC with UV detection by Xiupin Wang; Lan Ding; Huarong Zhang; Jianhua Cheng; Aimin Yu; Hanqi Zhang; Li Liu; Zhihong Liu; Ying Li (268-274).
The normal high-performance liquid chromatography with UV detection was applied for the determination of tributyltin chloride (TBT), triphenyltin chloride (TPhT), tetraphenyltin (TrPhT), triethyltin chloride (TET) and tetraethyltin (TrET) from flour samples. The separation was performed in the isocratic mode on cyanopropyl column with a mobile phase of hexane–acetonitrile–THF (97/1/2). Under the experimental conditions used, quantitative limit of TBT, TPhT, TrPhT, TET and TrET are 0.95, 0.46, 0.97, 0.75 and 0.96 μg/ml, respectively. Microwave-assisted extraction of organotin (OT) compounds at 100 °C with an extraction time of 3 min was described. The extraction of organotin can be finished in acetic acid–hexane (20/80) medium. The quantitative extraction of five organotin compounds was achieved with recoveries ranging from 88 to 101% R.S.D. 3–8%.
Keywords: Speciation analysis; Organotin compounds; Microwave-assisted extraction; HPLC; Flour;

Automated μ-SPE tips were successfully utilized for the determination of posaconazole in rat plasma. The bioanalytical method using μ-SPE tips was successfully qualified for routine quantitation of posaconazole over the concentration range of 10.0–10,000 ng/mL in rat EDTA plasma. Inter-assay precision, based on percent relative deviation for n  = 18 replicate quality controls, was ≤5.7%. Inter-assay accuracy based on n  = 18 replicate quality controls was ±7.7%. Complete solid phase extraction using μ-SPE tips was demonstrated on a Tomtec liquid handler where >95% recovery for posaconazole was obtained. The μ-SPE tips had sufficient capacity to extract at least 100 μL plasma fortified with 10 μg/mL of posaconazole and the analyte could be efficiently eluted with as little as 60 μL of methanol. Of particular note is the unique ability of these μ-SPE tips to perform exhaustive solid phase extraction more commonly performed when using liquid/liquid extraction.
Keywords: Solid-phase extraction; SPE; Extraction methods; LC–MS/MS; Preclinical studies; Posaconazole; OMIX;

Micellar electrokinetic chromatography–electrospray ionization mass spectrometry for the identification of drug impurities by Roelof Mol; Else Kragt; Ilias Jimidar; Gerhardus J. de Jong; Govert W. Somsen (283-288).
Previously, we have presented a system hyphenating continuous micellar electrokinetic chromatography (MEKC) with electrospray ionization mass spectrometry (ESI-MS). Here we evaluate this technique for its applicability in impurity profiling of drugs using galantamine and ipratropium as test samples. A background electrolyte (BGE) of 10 mM sodium phosphate (pH 7.5), 12.5–15% acetonitrile and 20 mM sodium dodecylsulfate (SDS) was used for the MEKC–MS analysis of a galantamine sample containing a number of related impurities, and a heat-treated solution of ipratropium containing a number of unknown degradation products. MEKC provided efficient separation of all sample constituents. Despite the presence of non-volatile BGEs, all impurities in the galantamine sample could be detected by ESI-MS in their respective extracted ion traces (XICs) with a detection sensitivity in the sub-μg/ml range (full-scan mode). MS/MS detection provided useful product spectra allowing the structural characterization of the respective galantamine impurities. With the MEKC–MS/MS system, two degradation products could be revealed and identified in the heat-stressed ipratropium sample. The presented method shows good potential for the detection and structure elucidation of minor impurities in drug substances.
Keywords: Micellar electrokinetic chromatography (MEKC); Mass spectrometry (MS); Impurity profiling;

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm × 4.6 mm i.d., 5 μm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n  = 8) in the range of 0.2–25 μg/ml, the limit of detection and quantitation were 0.10 and 0.20 μg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.
Keywords: Norgestrel; Enantiomeric separation; Hydroxypropyl-beta-cyclodextrin; HPLC;

A HPLC method with improved sensitivity for the determination of ochratoxins (OT) A, B and α in plasma and milk was developed. Plasma analysis involved a simple liquid–liquid extraction with chloroform; while for milk, an additional immunoaffinity clean-up step was necessary. The method showed a good linearity (r 2  > 0.999). The limit of quantification (LOQ) of OTA was 5 and 200 ng/l for milk and plasma, respectively. Average recovery was 89% in both matrices, except for OTα in milk that was only 18% due to poor immunoaffinity binding. OT remained stable in −20 °C stored samples; OTA concentration in plasma and milk did not change after 8 and 18 months of storage, respectively. The developed method has been applied to contaminated plasma and milk samples obtained from dairy ewes fed with ochratoxin-contaminated feed.
Keywords: Ochratoxins; Milk; Plasma;

Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography by Benjamin J. Davies; Megan K. Herbert; Janet K. Coller; Andrew A. Somogyi; Robert W. Milne; Benedetta C. Sallustio (302-309).
The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias <15%, except at the lowest limit of quantification (<20%). The inter-assay coefficients of variation and bias were <15%. The application of this method to plasma and urine samples of five CYP2D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (±S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58 ± 0.35 and 0.16 ± 0.06 l/h, respectively. The mean (±S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6 ± 11.6%.
Keywords: HPLC; Liver microsomes; Metabolites; Perhexiline; Pharmacokinetics;

Silica-immobilized enzyme reactors; application to cholinesterase-inhibition studies by Heather R. Luckarift; Glenn R. Johnson; Jim C. Spain (310-316).
A rapid and economical method is reported for the preparation of an immobilized enzyme reactor (IMER) using silica-encapsulated equine butyrylcholinesterase (BuChE) as a model system. Peptide-mediated silica formation was used to encapsulate BuChE, directly immobilizing the enzyme within a commercial pre-packed column. The silica/enzyme nanocomposites form and attach simultaneously to the metal affinity column via a histidine-tag on the silica-precipitating peptide. BuChE–IMER columns were integrated to a liquid chromatography system and used as a rapid and reproducible screening method for determining the potency of cholinesterase inhibitors. The IMER preparation method reported herein produces an inert silica-encapsulation matrix with advantages over alternative systems, including ease of preparation, high immobilization efficiency (70–100%) and complete retention of activity during continuous use.
Keywords: Immobilized enzyme reactor; Butyrylcholinesterase; Silica-encapsulation; Cholinesterase inhibitors;

Colloidal gas aphrons (CGA), which are surfactant stabilised microbubbles, have been previously applied for the recovery of proteins from model mixtures and a few studies have demonstrated the potential of these dispersions for the selective recovery of proteins from complex mixtures. However there is a lack of understanding of the mechanism of separation and forces governing the selectivity of the separation. In this paper a mechanistic study is carried out to determine the main factors and forces influencing the selectivity of separation of whey proteins with CGA generated from ionic surfactants. Two different separation strategies were followed: (i) separation of lactoferrin and lactoperoxidase by anionic CGA generated from a solution of sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT); (ii) separation of β-lactoglobulin by cationic CGA generated from a solution of cetyltrimethylammonium bromide (CTAB). Separation results indicate that electrostatic interactions are the main forces determining the selectivity however these could not completely explain the selectivities obtained following both strategies. Protein–surfactant interactions were studied by measuring the zeta potential changes on individual proteins upon addition of surfactant and at varying pH. Interestingly strongest electrostatic interactions were measured at those pH and surfactant to protein mass ratios which were optimum for protein separation. Effect of surfactant on protein conformation was determined by measuring the change in fluorescence intensity upon addition of surfactant at varying pH. Differences in the fluorescence patterns were detected among proteins which were correlated to differences in their conformational features which could in turn explain their different separation behaviour. The effect of conformation on selectivity was further proven by experiments in which conformational changes were induced by pre-treatment of whey (heating) and by storage at 4 °C. Overall it can be concluded that separation of proteins by ionic CGA is driven mainly by electrostatic interactions however conformational features will finally determine the selectivity of the separation with competitive adsorption having also an effect.
Keywords: Colloidal gas aphrons; Whey proteins; Ionic surfactants; z-Potential; Fluorescence; Protein–surfactant interactions; Competitive adsorption;

A liquid chromatography–electrospray ionization tandem mass spectrometric (LC–MS/MS) method was developed for the simultaneous qualitative and quantitative determination of sphingolipid metabolites such as ceramides, sphingisine, sphinganine, sphingomyelins, and ceramide 1-phosphates in the extracts of human promyelocytic leukemia cells (HL-60). The assay uses C4 ceramide as an internal standard; simple liquid extraction; a short XTerra MS C18 (3 μm, 50 mm × 2.0 mm) column; a gradient mobile phase of 5 mM ammonium formate (pH 4.0)/methanol/tetrahydrofuran (5/2/3 → 1/2/7); mass spectrometric detection using electrospray ionization. This LC–MS/MS method allowed the identification of 22 sphingolipid derivatives at pmol levels. In addition, this technique was successfully applied to analyze the changes of the sphingolipids profiles in cancer cells treated with apoptosis inducing agents, C2 ceramide and H2O2.
Keywords: Sphingolipid; Ceramides; LC–MS/MS; HL-60 cells;

A replaceable capillary gel electrophoretic (replaceable CGE) method was developed for the separation of two sets of model compounds of single-stranded oligodeoxynucleotide mixtures (18–20 mers), phosphodiester oligodeoxynucleotides (PO-ODNs) and their phosphorothioate modifications (PS-ODNs), with equal sequences differing in a single base. Polyethylene glycol (PEG) 35000 was chosen as the sieving matrix. It was confirmed that PEG polymer solution less influenced resolutions of the PS-ODNs compared with those of the PO-ODNs, while acetonitrile used as an additive in the system improved the separation significantly. It was also noticed that the effect of temperature on separation was much larger than that of denaturant urea.
Keywords: Single-based resolution; Phosphodiester oligodeoxynucleotide; Phosphorothioate modified oligodeoxynucleotide; Replaceable capillary gel electrophoresis;

A simple and rapid liquid chromatography method for simultaneous determination of zidovudine and nevirapine in plasma by Geetha Ramachandran; A.K. Hemanthkumar; V. Kumaraswami; Soumya Swaminathan (339-344).
We describe a simple, fast, isocratic, reversed-phase high performance liquid chromatographic method for simultaneous determination of plasma zidovudine and nevirapine with UV detection at 260 nm. The method involves liquid–liquid extraction with ethyl acetate and using 3-isobutyl 1-methyl xanthine as internal standard. The system requires a C18 column (150 mm × 4.6 mm I.D.) and a mobile phase composed of potassium dihydrogen phosphate (15 mM; pH 7.5) and acetonitrile in the ratio of 80:20 (v/v). The assay was linear from 0.025 to 10.0 μg/ml for zidovudine and 0.05 to 10.0 μg/ml for nevirapine. The intra- and inter-day variations were less than 10% for both the drugs. The method was specific and sensitive enough to allow quantification of zidovudine and nevirapine in concentrations observed clinically. The average recoveries of zidovudine and nevirapine from plasma were 95 and 94%, respectively. The method was applied to a pharmacokinetic study in HIV-infected patients who were receiving antiretroviral treatment with zidovudine and nevirapine containing regimens. The method spans the blood concentration range of clinical interest. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring in patients taking a combination treatment of zidovudine and nevirapine.
Keywords: Zidovudine; Nevirapine; Plasma; HPLC;