Journal of Chromatography B (v.822, #1-2)

Snake venoms contain several trypsin-like enzymes with equivalent physicochemical characteristics and similar inhibition profiles. These are rather difficult to separate by classical purification procedures and therefore constitute a good model for affinity chromatography analysis. Some of these trypsin homologues present fibrinogenase activity, mimicking one or more features of the central mammalian coagulation enzyme, thrombin. It was previously demonstrated that a number of amidine derivatives are able to interact specifically with some of these serine proteases. To understand the enzyme–sorbent interactions we have investigated the ability of two commercially available benzamidine affinity matrices to purify thrombin-like serine proteases (TLSP) with similar biological properties from two snake venoms (Bothrops jararacussu and Lachesis muta rhombeata). Curiously, each sorbent retained a single but distinct TLSP from each venom with high yield. Molecular modeling analysis suggested that hydrophobic interactions within a specific region on the surface of these enzymes could be generated to explain this exquisite specificity. In addition, it was demonstrated that a specific tandem alignment of the two benzamidine sorbents enables the purification of three other enzymes from B. jararacussu venom.
Keywords: p-Aminobenzamidine; Thrombin-like; Bothrops jararacussu; Lachesis muta rhombeata; Affinity chromatography; Serine proteinase; Molecular modelling; Enzymatic specificity;

Direct liquid chromatography determination of the reactive imine SJG-136 (NSC 694501) by Andrew Cheung; Elaine Struble; Jingyi He; Chun Yang; Euphemia Wang; David E. Thurston; Paul Liu (10-20).
SJG-136 (NSC 694501), 8,8′-[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one], which is being developed as a DNA-interactive antitumor agent, contains highly reactive imines in the diazepinone portions of the molecule. Water or alcohol adds readily to the imino moiety to form the corresponding carbinolamine or its alkyl ether, respectively. This sensitivity to protic substances poses a formidable challenge to the formulation and HPLC assay development for the compound. After studying the solution chemistry of SJG-136 and its potential interaction with various stationary phases, two reversed-phase liquid chromatographic assays for the compound have been developed. A direct assay that separates SJG-136 from its water or methanol adducts and an indirect assay that quantifies SJG-136 as its dihydrate adduct are reported. The latter method, which is more practical for drug development, has been validated. It is reproducible (R.S.D. < 2%), linear (r 2  = 0.9999) and accurate (within 98–102% recovery), with a lower detection limit of 2.5 ng.
Keywords: SJG-136; NSC 694501; Benzodiazepine; Water adducts; NMR; HPLC assay; Development; Validation;

HPLC analysis of PAMAM dendrimer based multifunctional devices by Mohammad T. Islam; Istvan J. Majoros; James R. Baker (21-26).
Comprehensive high-performance liquid chromatography (HPLC) analyses were performed on poly(amidoamine) (PAMAM) dendrimer based multifunctional devices. The nanometer-size devices were synthesized by conjugating partially acetylated (Ac) poly(amidoamine) dendrimers of generation 5 (G5) with fluorescein isothiocyanate (FITC), folic acid (FA) and methotrexate (MTX). The devices are intended for targeted intracellular drug delivery to tumor cells through the folate receptor. Methods were developed for detection and separation of various surface functionalized dendrimer conjugates and small molecules (FITC, FA, MTX) using a common gradient. Results indicate that the HPLC technique can be used as a quality control tool for determining purity of the G5 carrier, its acetylated form, and mono-, bi- and tri-functional nanodevices. More importantly, the chromatograms of these novel nanodevices, reported for the first time, provide information on critical properties such as polydispersity, surface heterogeneity and solubility. The benchmark data can be used to optimize the physicochemical properties of the conjugates to improve drug delivery to cancer cells.
Keywords: HPLC; PAMAM dendrimer; Multifunctional device; Dendrimer–drug conjugate; Drug delivery;

A highly sensitive LC–MS–MS assay for analysis of midazolam and its major metabolite in human plasma: Applications to drug metabolism by Valquiria A.P. Jabor; Eduardo B. Coelho; Neife A.G. dos Santos; Pierina S. Bonato; Vera L. Lanchote (27-32).
The present report describes a rapid, selective and a highly sensitive assay for midazolam (MDZ) and its major metabolite 1-hydroxymidazolam (1-OH-MDZ) in human plasma employing liquid chromatography–tandem mass spectrometry (LC–MS–MS) detection. The method involves liquid–liquid extraction sample clean-up, separation on a Purospher RP 18-e column and detection with an electrospray interface in the positive ion mode. The overall recoveries were about 100% and 80% for midazolam and 1-hydroxymidazolam, respectively. Accuracy, precision and linearity were acceptable for biological samples with quantitation limits of 0.1–100 ng mL−1 plasma for both analytes. The validated method was successfully applied to quantify plasma concentration of midazolam and 1-hydroxymidazolam in authentic samples from a healthy volunteer following a single 15 mg oral dose of midazolam (apparent total clearance: 3.47 L h−1  kg−1 and AUC0 − αIOH-MDZ/MDZ: 0.338).
Keywords: Midazolam; LC–MS–MS; Plasma; Pharmacokinetics; CYP3A4;

Monitoring in vivo oxidative stress implicates the evaluation of damage and defence parameters by well-established, validated methods. We report two optimized and validated HPLC methods for measurement of malondialdehyde (MDA) and fat-soluble vitamins in rat plasma. For the MDA method, optimization experiments of the thiobarbituric acid test resulted in the addition of 1% butylhydroxytoluene to the reaction mixture and in a heating time reduction to 40 min, ensuring inhibition of further lipid peroxidation during the test. Validation experiments showed good linearity, precision and recovery. The use of HPLC with coulometric array detection technology permits simultaneous and sensitive analysis of different fat-soluble vitamins and related compounds (tocopherols, retinoids, carotenoids and coenzyme Q10), which are identified by both retention time and electrochemical characteristics. Furthermore, this method is extended to the analysis of coenzyme Q9, the predominant homologue in rats. Validation experiments with rat plasma gave good results.
Keywords: Oxidative stress; Lipid peroxidation; Fat-soluble vitamins;

Effects of pure n-alkanes and crude oil on bacterial phospholipid classes and molecular species determined by electrospray ionization mass spectrometry by Nicolas Mazzella; Josiane Molinet; Agung Dhamar Syakti; Alexandre Barriol; Alain Dodi; Jean-Claude Bertrand; Pierre Doumenq (40-53).
Phospholipids are major components of bacterial membrane. Furthermore, the growth in vitro on xenobiotics such as n-alkanes, aromatic compounds or alkanols bring about to a bacterial membrane adaptive response. Concerning this work, we studied the membrane lipid composition of a hydrocarbon-degrading gram-positive bacterium (Corynebacterium sp.) on a soluble substrate and we detected four different phospholipid classes: phosphatidylglycerol, phosphatidylinositol, cardiolipin and acyl phosphatidylglycerol. In addition, a study of the lipid composition was performed after an in vitro culture on either pure n-alkane or crude oil. The growths on such hydrophobic substrates showed major qualitative and quantitative modifications. In the case of a growth on either heneicosane or crude oil, an increase of odd-numbered fatty acids was observed. Furthermore, the phospholipid polar head group composition was highly influenced by the crude oil addition. These modifications were, respectively, interpreted as the consequence of hydrocarbon assimilation and membrane fluidity adaptation. Finally, Corynebacterium sp. was taken back on the initial ammonium acetate substrate in order to determine its restoration abilities after a petroleum contamination.
Keywords: RP-HPLC/ESI/MS; Tandem ESI/MS/MS; Petroleum; Hydrophobic substrates; Gram-positive bacteria; Corynebacterium sp;

Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells by Chen-Li Chiang; Ching-Shan Sung; Ting-Feng Wu; Chuh-Yean Chen; Chiung-Yuen Hsu (54-60).
The aim of this study was to develop a simple and rapid method for purification of ultrapure supercoiled plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe3O4) were prepared by chemical precipitation method using Fe2+, Fe3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe3O4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, Fourier transformation infrared spectroscopy and superconducting quantum interference device magnetometer. The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 35 μg of high-purity (A260/A280 ratio = 1.87) plasmid DNA was isolated from 3 ml of overnight bacterial culture. EGFP expression was detected by fluorescent microscopy in the transformed E. coli cells, indicating the biological activities of DNA fragments were retained after purified from magnetic nanobeads. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmids takes less than 10 min. Thus, the separation and purification qualities of PEI-modified magnetic nanobeads as well as its ease of use surpass those of conventional anion-exchange resins.
Keywords: Plasmid DNA; Purification; Superparamagnetic nanoparticles; PEI;

Capillary electrophoresis of glutathione to monitor oxidative stress and response to antioxidant treatments in an animal model by N. Maeso; D. García-Martínez; F.J. Rupérez; A. Cifuentes; C. Barbas (61-69).
Glutathione plays a central role in metabolism and antioxidant defence. Several factors can influence the analytical efficiency and rapidity of the quantitative determination of glutathione. Procedures in sample pre-treatment have been compared in order to minimize analytical errors. Capillary electrophoresis has been chosen as a more adequate technique for obtaining a rapid and simple method for glutathione and glutathione disulfide determination in the blood and liver of the rat. The methods, once optimised, have been validated and applied for monitoring the oxidative stress in an animal model, such as the rat made diabetic by streptozotocin injection, when the animals are treated with antioxidants and compared with the corresponding controls.
Keywords: Glutathione; Antioxidants; Oxidative stress; Rat; Blood; Liver; In vivo assay;

A sensitive and efficient method was developed for the determination of carvedilol and its metabolites in human urine by gas chromatography–mass spectrometry (GC–MS). Urine samples were hydrolyzed with β-glucuronidase/arylsulfatase (from Helix pomatia) and the target compounds were extracted with liquid–liquid extraction. The extracts were completely derivatized with MSTFA and MBTFA and analyzed by GC–MS using an Ultra-2 column. The linearity of the assay ranges were 0.75–75 ng mL−1 for carvedilol and o-desmethyl carvedilol (o-DMC), and 3.0–75 ng mL−1 for 4-hydroxyphenyl carvedilol (4-HPC) and 5-hydroxyphenyl carvedilol (5-HPC). The absolute recovery of carvedilol and its metabolites added to a blank urine sample was 80.1–97.8%. The limits of detection (LOD) and quantitation (LOQ) of carvedilol and o-DMC were 0.30 and 0.75 ng mL−1, and its of 4-HPC and 5-HPC were 0.75 and 3.0 ng mL−1, respectively. The reproducibilities were 1.86–11.5% for the intra-day assay, and 0.70–1.71% for the inter-day assay precision and the degree of inaccuracy was −3.0 to 3.9% at the concentration of 75 ng mL−1. The proposed GC–MS method was effective for the determination of carvedilol and its three metabolites in human urine.
Keywords: Carvedilol; GC–MS; Metabolites;

Analysis of mitochondrial DNA in microfluidic systems by Patricia Taylor; Dammika P. Manage; Karmon E. Helmle; Yao Zheng; D. Moira Glerum; Christopher J. Backhouse (78-84).
Abnormalities in mitochondrial function play a major role in many human diseases. It is often of critical importance to ascertain what proportion of the mitochondria within a cell, or cells, bear a given mutation (the mitochondrial “demographics”). In this work, a rapid, novel, on-chip procedure was used, in which a restriction enzyme was employed to excise a mitochondrial DNA (mtDNA) sequence from plasmid DNA that acted as a prototypical mitochondrial genome. The DNA was then denatured, reassembled to form duplexes, fluorescently labelled and analysed. This method was able to differentiate between a homogeneous population and a heterogeneous population. Using a microfluidic chip, the method could be performed in about 45 min, even without robotics or multiplexed operation, whereas conventional methods of analysis require days to perform. This method may ultimately form the basis for a means of characterizing the mitochondrial demographics of a single cell.
Keywords: Microfluidic; Lab-on-a-chip; Electrophoresis; Heteroduplex analysis; Restriction enzyme digest;

New method of qualitative and quantitative analysis of nucleotides in human cerebrospinal fluid (CSF), based on the combination of extraction of purines and pyrimidines to the solid phase (SPE) and high-performance liquid chromatography (HPLC), was proposed. Use of SPE and lyophilization of samples allowed for the first time to detect the presence of di- and triphosphonucleotides in human CSF. Concentration of those compounds varied from 0.003 to 5.0 μM. Differences in the nucleotide mixture composition in human CSF detected with the new method are coupled with the neurological disorders and might be a basis for an efficient diagnostic tool.
Keywords: Human cerebrospinal fluid; SPE; HPLC; Nucleotides; Neurological diseases;

Immobilization of lipase onto micron-size magnetic beads by Xianqiao Liu; Yueping Guan; Rui Shen; Huizhou Liu (91-97).
A novel and economical magnetic poly(methacrylate-divinylbenzene) microsphere (less than 8 μm in diameter) was synthesized by the modified suspension polymerization of methacrylate and cross-linker divinylbenzene in the presence of magnetic fluid. Then, surface aminolysis was employed to obtain a high content of surface amino groups (0.40–0.55 mmol g−1 supports). The morphology and properties of these magnetic supports were characterized with scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and a vibrating sample magnetometer. These magnetic supports exhibited superparamagnetism with a high specific saturation magnetization (σ s) of 14.6 emu g−1. Candida cylindracea lipase was covalently immobilized on the amino-functionalized magnetic supports with the activity recovery up to 72.4% and enzyme loading of 34.0 mg g−1 support, remarkably higher than the previous studies. The factors involved in the activity recovery and enzymatic properties of the immobilized lipase prepared were studied in comparison with free lipase, for which olive oil was chosen as the substrate. The results show that the immobilized lipase has good stability and reusability after recovery by magnetic separation within 20 s.
Keywords: Magnetic microsphere; Synthesis; Suspension polymerization; Candida cylindracea lipase; Immobilization;

Two-dimensional (2D) gel electrophoresis is the most common protein separation method in proteomics research. It can provide high resolution and high sensitivity. However, 2D gel methods have several limitations, such as labor-intensive procedures, poor reproducibility, and limited dynamic range of detection. In fact, many investigators have returned to couple the one-dimensional (1D) SDS-PAGE with mass spectrometry for protein identification. The limitation of this approach is the increased protein complexity in each one-dimensional gel band. To overcome this problem and provide reproducible quantitative information, we describe here a 2D method for protein mixture separation using a combination of high performance liquid chromatography (HPLC) and 1D SDS-PAGE. The study shows that the step-gradient fractionation method we have applied provides excellent reproducibility. In addition, high mass accuracy of LC–FTICR-MS can allow more confident protein identifications by high resolution and ultra-high mass measurement accuracy. This approach was applied to comparative proteomics since protein abundance level changes can be easily visualized with side-by-side vertical comparison in one gel. Furthermore, separation of multi-samples in the same gel significantly reduces run-to-run variation, as is shown with differential image gel electrophoresis (DIGE). Finally, this approach readily incorporates immunological methods to normalize relative abundances of multiple samples within a single gel. This paper presents the results of our developments and our initial application of this strategy for mapping protease function of beta amyloid cleaving enzyme (BACE) in biological systems.
Keywords: Multidimensional protein profiling; SAX; SDS-PAGE; BACE; Mass spectrometry;

Preliminary investigation of using volatile organic compounds from human expired air, blood and urine for locating entrapped people in earthquakes by M. Statheropoulos; E. Sianos; A. Agapiou; A. Georgiadou; A. Pappa; N. Tzamtzis; H. Giotaki; C. Papageorgiou; D. Kolostoumbis (112-117).
A preliminary investigation on the possibility of using volatile organic compounds (VOCs) determination of expired air, blood and urine, for the early location of entrapped people in earthquakes, has been carried out. A group of 15 healthy subjects has been sampled. The identification of a common “core” of substances might provide indications of human presence that can be used for the development of a real time field analytical method for the on site detection of entrapped people. Expired air samples have been analyzed by thermal desorption GC/MS and VOCs from blood and urine by headspace SPME–GC/MS. Acetone was the only compound found common in all three matrices. Isoprene was found in both expired air and blood samples. Acetone and isoprene along with a number of saturated hydrocarbons were among the major constituents identified in expired air analysis. Various ketones (2-pentanone, 4-heptanone, 2-butanone) were also determined over urine specimens. Using the techniques and methods of field analytical chemistry and technology appears to be the proper approach for applying the results of the present study in real situations.
Keywords: Blood; Earthquakes; Entrapped people; Expired air; SPME; Thermal desorption; Urine; VOCs;

Differentiation between Candida species isolated from diabetic foot by fatty acid methyl ester analysis using gas chromatography by Emilija Mlinaric Missoni; Desanka Rade; Sandra Nederal; Smilja Kalenic; Josipa Kern; Verica Vazic Babic (118-123).
Gas chromatography (GC) was used to differentiate 100 isolates of Candida species (Candida parapsilosis, Candida albicans, Candida tropicalis, Candida famata and Candida glabrata) from 22 of 509 diabetic patients in whom the same species had been isolated from ulcer and interdigital spaces of the same and/or the other foot. All clinical isolates were identified by quantitative differences in the composition of six cell fatty acids (CFA). The values of the coefficients of variability (CV) of CFA show that the isolates from foot ulcers and interdigital spaces of the same diabetic patient probably belong to different chemotypes of the same Candida species.
Keywords: Candida species; Diabetic foot ulcers; Gas chromatography; Cell fatty acids;

A sensitive internal standard method for the analysis of a DNA-adduct of N,N-dimethylformamide (N 4-methylcarbamoylcytosine, NMC-C) in human urine has been developed. A sample pre-treatment involving an acidic hydrolysis is followed by the sample clean-up performed with solid-phase extraction (SPE) technique using a cation-exchange resin. A two-dimensional liquid chromatography is used to separate the target analyte from the matrix using first a C18 reversed phase column with incorporated hydrophilic moieties and then a C8 bonded reversed phase column for the final separation. Quantification is carried out by positive electrospray ionisation and mass spectrometry detection of the transitions from molecule ions to product ions (169 → 112 and 172 → 115) for the analyte and the labelled internal standard, respectively. The detection limit in urine reaches down to 8 ng/L (48 pmol/L). In the general population NMC-C could not be detected. In 10 out of 32 urine samples of occupationally to DMF exposed subjects NMC-C could be detected. The concentrations ranged up to 172 ng/L (1023 pmol/L) with a 95th percentile of 121 ng/L (720 pmol/L).
Keywords: N,N-Dimethylformamide; DNA-adduct; Biochemical effect monitoring; Urine;

Determination of telithromycin in human plasma and microdialysates by high-performance liquid chromatography by Friederike Traunmüller; Rainer Gattringer; Markus A. Zeitlinger; Wolfgang Graninger; Markus Müller; Christian Joukhadar (133-136).
A high-performance liquid chromatography method for the quantitative determination of telithromycin in biological fluids is described. The method is suitable for plasma and microdialysates from the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue. Plasma samples were deproteinised with trichloroacetic acid and neutralised with sodium hydroxide. Microdialysates were analysed without further preparation step. Telithromycin was separated isocratically on a reverse-phase column using acetonitrile–0.03 M ammonium acetate, pH 5.2 (43:57, v/v) at a flow rate of 0.8 ml min−1, and fluorescence detection (excitation 263 nm, emission 460 nm). The calibration curve was linear from 0.01 to 5 μg ml−1. Within- and between-day imprecision and inaccuracy was ≤10%. The limits of quantification were 0.02 and 0.015 μg ml−1 for plasma and microdialysates, respectively. Since telithromycin is decomposed in aqueous solution at ambient temperature, it is strongly recommended to store samples frozen at −80 °C, to maintain the temperature at 4 °C during all preparation steps, and to analyse samples within 120 min after thawing.
Keywords: Telithromycin; Ketolide; Microdialysis; High-performance liquid chromatography;

Opiates are some of the most widely prescribed drugs in America and are often abused. Demonstrating the presence or absence of opiate compounds in postmortem fluids and/or tissues derived from fatal civil aviation accidents can have serious legal consequences and may help determine the cause of impairment and/or death. However, the consumption of poppy seed products can result in a positive opiate drug test. We have developed a simple method for the simultaneous determination of eight opiate compounds from one extraction. These compounds are hydrocodone, dihydrocodeine, codeine, oxycodone, hydromorphone, 6-monoacetylmorphine, morphine, and thebaine. The inclusion of thebaine is notable as it is an indicator of poppy seed consumption and may help explain morphine/codeine positives in cases where no opiate use was indicated. This method incorporates a Zymark® RapidTrace™ automated solid-phase extraction system, gas chromatography/mass spectrometry, and trimethyl silane (TMS) and oxime-TMS derivatives. The limits of detection ranged from 0.78 to 12.5 ng/mL. The linear dynamic range for most analytes was 6.25–1600 ng/mL. The extraction efficiencies ranged from 70 to 103%. We applied this method to eight separate aviation fatalities where opiate compounds had previously been detected.
Keywords: Opiate; Thebaine; Postmortem; Zymark®; Gas chromatography;

Measurement of adenosine deaminase (ADA) activity using spectrophotometric method presents problem, regarding the quantitative estimation of the substrate degradation and product formation, due to the closely apposed λ max of the substrates, product and the inhibitor. The feasibility of applying reverse-phase HPLC technique, for studying adenosine deaminase-catalyzed reaction product and inhibition study was examined. We have drawn a comparison between the HPLC-based method over the corresponding spectrophotometric method. A gradient elution pattern was used to separate substrate (adenosine and deoxyadenosine), product (inosine and deoxyinosine) and standard adenosine deaminase inhibitor (erythro-9-(3-nonyl-ρ-aminobenzyl)-adenine) in the HPLC method. The product formation was quantitated by monitoring the absorbance at 260 nm with the progress of time. The limit of detection as well as the limit of quantification of the respective enzymatic product were found to be in nano molar (nM) range in the HPLC method. This study was also extended to monitor adenosine deaminase activity in different cancer cells of hematological origin. The HPLC-based method is found to be suitable for the quantitative estimation of adenosine deaminase-catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC-based method has specific advantages over the spectrophotometric method. Moreover, the concentration of different nucleotides in cell lysate and body fluid can be measured using this HPLC method.
Keywords: Reverse-phase HPLC; Spectrophotometric-enzyme kinetics; Adenosine deaminase; Adenosine; Inosine; EHNA;

Efficient HPLC method for the determination of nicarbazin, as dinitrocarbanilide in broiler liver by Emiliana Capurro; Martin Danaher; Aniello Anastasio; Maria Luisa Cortesi; Michael O’Keeffe (154-159).
A simple, fast and reliable HPLC-UV method has been developed for the determination of dinitrocarbanilide residues in broiler liver. Liver samples (2 g) were extracted with two portions of acetonitrile (10 and 5 ml), defatted with hexane and evaporated to dryness under nitrogen. Extracts were reconstituted in acetonitrile–water (70/30, v/v, 500 μl), loaded onto C18 solid phase (SPE) cartridges and eluted with acetonitrile–water (70/30, v/v, 2.5 ml) into clean test-tubes. Extracts were evaporated to dryness and reconstituted in acetonitrile–water (80/20, v/v, 500 μl). An aliquot of the extract was assayed by high performance liquid chromatography (HPLC) with UV detection at 350 nm. The method was validated according to EU guidelines using liver tissues fortified at levels of 100, 200 and 300 μg/kg, with dinitrocarbanilide. The decision limit (CCα) and the detection capability (CCβ) were calculated from the within laboratory repeatability data to be 228 and 266 μg/kg, respectively. The mean recovery was typically >70% and the limits of quantitation was 12.5 μg/kg (based on the lowest standard on the calibration curve).
Keywords: Dinitrocarbanilide; Nicarbazin; Broiler liver; C18 SPE; HPLC-UV;

A liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) method for the quantification of major chlorpyrifos (CP) metabolites, i.e. diethyl thiophosphate (DETP), diethyl phosphate (DEP), and 3,5,6-trichloro-2-pyridinol (TCP), in human urine was developed. Simultaneous separation of the parent compound and its primary biotransformation products was achieved within 20 min in gradient elution mode employing a mixed-mode reversed-phase/weak anion exchange (RP/WAX) separation principle. The analytical method was developed for a toxicokinetic study of an acute poisoning incidence with a CP containing pesticide formulation. An initial mass spectrometric screening performed with unprocessed urine samples revealed that CP is not excreted unchanged by the kidney. Hence, the quantitative assay was validated for DETP (quantifier transition: m/z 169 → 95, qualifier transition: m/z 169 → 141), DEP (m/z 153 → 79, 153 → 125), and TCP (m/z 196 → 35, 198 → 35) taking dibutyl phosphate (DBP) (m/z 209 → 79, 209 → 153) as internal standard. Clean-up of urine samples prior to LC–ESI–MS/MS analysis was carried out by a liquid–liquid extraction step with a mixture of ethylacetate and acetonitrile (70:30; v/v). Linearity was observed between 0.25 and 75 mg L−1, and the signal-to-noise ratio at 0.25 mg L−1 was better than six for the individual analytes. Recoveries, precision, and accuracies were all adequate across the validated range of 1–75 mg L−1 for the present toxicological case study.
Keywords: Chlorpyrifos; Metabolites; Reversed-phase/weak anion exchange stationary phase; Liquid chromatography–tandem mass spectrometry; Human poisoning; Urine;

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 (5 μm, 150 mm × 2.0 mm) column. Acetonitrile:water containing 5 μM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R 2  > 0.9999) was observed throughout the range of 10–5000 ng/ml in 0.5 ml rat plasma and 5–5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R 2  > 0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93–110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.
Keywords: Astragaloside IV; HPLC; LC/MS/MS; Pharmacokinetics;

A validated method for the quantification of enterodiol and enterolactone in plasma using isotope dilution liquid chromatography with tandem mass spectrometry by Anneleen Kuijsten; Michel N.C.P. Buijsman; Ilja. C.W. Arts; Patrick P.J. Mulder; Peter C.H. Hollman (178-184).
Enterolactone and enterodiol are phytoestrogens with structural similarity to endogenous estrogens. Because of their biological activities, they may affect the development of several diseases. To quantify enterodiol and enterolactone in plasma, we developed and validated a liquid chromatography–tandem mass spectrometry method with electrospray ionization using 13C3 labeled isotopes. The method consists of a simple enzymatic hydrolysis and ether extraction followed by a rapid LC separation (run-time of 11 min). Detection limits as low as 0.15 nM for enterodiol and 0.55 nM for enterolactone were achieved. The within-run R.S.D. ranges from 3 to 6% and the between-run R.S.D. ranges from 10 to 14% for both enterolignans. This method allows simple, rapid, and sensitive quantification, and is suitable for measuring large numbers of samples.
Keywords: Lignans; Enterodiol; Enterolactone; Plasma; Liquid chromatography; Mass spectrometry;

The fluorescence emission of the fluoroquinolones enoxacin (ENO), ciprofloxacin (CIPRO), norfloxacin (NOR) and ofloxacin (OFLO) notably increased by UV irradiation during few minutes, in ethanolic–water medium. An HPLC method has been developed, for the determination of these fluoroquinolones, based in the separation of the formed irradiation photoproducts. Optimization of the analytical wavelengths has been carried out by fast multiemission scanning fluorescence detection. The highest sensitivity has been found when measuring at emission wavelengths of 407 and 490 nm, for ENO and OFLO, respectively, and at 444 nm for both NOR and CIPRO (exciting at 277 nm). According to the criterium of Clayton, using 0.05 as false positive and false negative error assurance probabilities, detection limits of 7.3, 6.0, 6.3 and 14.5 ng/mL, for ENO, NOR, CIPRO and OFLO, respectively, have been found. Urine and serum samples have been successfully analyzed, with recovery values ranging among 99–97% and 98–103%, for urine and serum, respectively.
Keywords: Photoinduced fluorescence (PIF); Fluoroquinolones; HPLC; Serum; Urine;

Solid phase microextraction gas chromatographic analysis of organophosphorus pesticides in biological samples by Heleni Tsoukali; Georgios Theodoridis; Nikolaos Raikos; Ifigeneia Grigoratou (194-200).
Headspace-solid phase microextraction (HS-SPME) was studied and optimised for the determination of four common organophosphorus pesticides (OPPs) in biological samples. Various parameters controlling SPME were studied: choice of SPME fiber, type and content of salt added, preheating and extraction time, desorption time, extraction temperature. Capillary gas chromatographic analysis with nitrogen phosphorus detection (GC–NPD) facilitates sensitive and selective detection of the OPPs: malathion, parathion, methyl parathion and diazinon. Fenitrothion was used as the internal standard. The method was applied to the determination of the pesticides in human biological specimens: whole blood, blood plasma, urine, cerebrospinal fluid, liver and kidney. Limits of detection ranged from 2 to 55 ng/ml depending on pesticide and type of specimen. The developed methodology overcomes limitations and obstacles of conventional methods such as the use of organic solvents, the formation of emulsions and the tedious-cumbersome procedures. The proposed protocol is seen as an attractive alternative to be used in routine toxicological analysis.
Keywords: SPME; Organophosphorus pesticides; Biological samples; Sample preparation;

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of emtricitabine and tenofovir in human blood plasma is described. Using 200 μL of plasma and BOND ELUT-C18 Varian columns, the solid phase extraction (SPE) method results in a clean baseline and high extraction efficiencies (100% for emtricitabine and 98.6% for tenofovir). An Atlantis™ dC-18 analytical column is used along with an 18 min linear gradient elution of phosphate buffer (pH 5.7) and methanol to provide sharp peaks for emtricitabine at 280 nm, tenofovir at 259 nm, and the internal standard 2′,3′didoxyuridine (DDU) at 262 nm. The method was validated over the range of 10–10,000 ng/mL for both analytes, and is accurate (average accuracies of three different concentrations ranged from 98 to 105% for emtricitabine and 97 to 103% for tenofovir) and precise (within- and between-day precision ranged from 1.7 to 3.7% and 3.7 to 5.2%, respectively). This method is suitable for use in clinical pharmacokinetic studies and is nimble enough for therapeutic drug monitoring.
Keywords: HIV; Antiretroviral; NRTI; NtRTI; HPLC; Chromatography;

Effect of hippuric acid on the gaschromatographic retention of S-phenylmercapturic acid by G. Marrubini; E. Terulla; G. Brusotti; G. Massolini (209-220).
S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 μg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health.In this study a GC–MS method was adopted for measuring urinary PMA, which involved liquid–liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m × 0.25 mm i.d., 0.25 μm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.
Keywords: Benzene; Toluene; Biological monitoring; S-phenylmercapturic acid; Hippuric acid; Phase soaking;

We developed a sensitive method to detect several classes of pesticides and their metabolites in maternal and cord whole blood using electron-impact gas chromatography–mass spectrometry (GC–MS). The method can detect parent and metabolite compounds at levels of <0.10 and 0.20 μg/mL, respectively, with high accuracy and recovery. Analysis of blood from mother–infant dyads from an area of high pesticide use in the Philippines showed detectable levels of propoxur, 3-phenoxybenzoic acid (3-PBA), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE) in maternal and umbilical cord blood. GC–MS analysis of several classes of parent pesticides and their metabolites in maternal and cord blood provides a sensitive and specific method to detect pesticide exposure during pregnancy.
Keywords: Pyrethroids; Pesticides; Whole blood; Parental pesticide exposure; Cord blood; GC–MS; Environmental pollutants; Carbamates; Organophosphates; Herbicide;

A rapid and sensitive assay for quantification of nalbuphine, butorphanol and morphine in blood (50 μL) and brain microdialysate (∼40 μL) samples was developed. Blood samples were extracted with ethyl acetate. Analysis was performed with high-performance liquid chromatography (HPLC) coupled to an electrochemical detector. The mobile phase was a mixture of 0.1 M sodium phosphate buffer, methanol and octane-sulfonic acid with ratio and pH depending on compound and matrix. The limits of quantification in blood samples were 25, 50 and 25 ng/mL for nalbuphine, butorphanol and morphine, respectively and 0.5 ng/mL for morphine in microdialysate samples. Based on sample volume, sensitivity and reproducibility, these assays are particularly suitable for pharmacokinetic/pharmacodynamic studies in rodents.
Keywords: Pharmacokinetic/pharmacodynamic studies; Opioids; HPLC; Blood; Microdialysate;

This paper describes a simple, fast and sensitive liquid chromatography–mass spectrometry method for quantification of an anti-thrombocythemic agent, anagrelide in human plasma. The samples were subjected to a liquid–liquid extraction after addition of a buffer and an internal standard. Chromatography was performed on an Inertsil ODS2 column and the extract was injected onto a HPLC system coupled with mass spectrometric detection. Linear responses for standards were observed from 50 to 7500 pg/ml. The accuracy of intra-assay and inter-assay were in the ranges 4.3–4.4% and 4.8–5.6%, respectively. The method is simple and reproducible with a run time of less than 2 min.
Keywords: HPLC; Anagrelide; Human;

An efficient and noninvasive method consisting of an original sampling device, solid phase microextraction (SPME) and gas chromatography–mass spectrometry (GC–MS) was developed to analyze volatile organic emanations from the skin of human arms. The emanations were sampled by SPME connected with the active sampling device for 30 min and transferred into GC–MS immediately for the consequent analysis. The sampling projects for 15 candidates were scheduled in both winter and spring with the same optimized conditions. Thirty-five compounds were finally identified according to various degrees of certainty. Different emission behaviors specified with principal component analysis (PCA) and similar fingerprint characteristics were observed clearly by comparisons of chromatograms of different seasons. Top ten emanations contributing to characteristics in different seasons were attempted to be described using comparisons based on common model strategy. The large amounts of experimental data were all handled by the corresponding chemometrics strategies with the homemade chromatographic data processing system. The results suggest that the analysis based on fingerprint characteristics of human skin emanations could provide useful and important clues to reveal biomarkers among the mixture of human skin emanations.
Keywords: Human emanations; Fingerprint characteristics; Original sampling system; SPME;

Quantification of carvedilol in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry by Ney Carter do Carmo Borges; Gustavo Duarte Mendes; Diogo de Oliveira Silva; Vinicius Marcondes Rezende; Rafael Eliseo Barrientos-Astigarraga; Gilberto De Nucci (253-262).
A rapid, sensitive and specific method to quantify carvedilol in human plasma using metoprolol as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid–liquid extraction using a diethyl-ether solvent. After removed and dried the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile–water (50/50; v/v). The extracts were analyzed by a high performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC–MS/MS). Chromatography was performed isocratically on Alltech Prevail C18 5 μm analytical column, (150 mm × 4.6 mm i.d.). The method had a chromatographic run time of 3.5 min and a linear calibration curve over the range 0.1–200 ng ml−1 (r 2  > 0.997992). The limit of quantification was 0.1 ng ml−1. This HPLC–MS/MS procedure was used to assess the bioequivalence of two carvedilol 25 mg tablet formulations (carvedilol test formulation from Laboratórios Biosintética Ltda and Coreg® from Roche Químicos e Farmacêuticos S.A standard reference formulation). A single 25 mg dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 2-week wash-out interval. Since the 90% CI for C max and AUCs ratios were all inside the 80–125% interval proposed by the US Food and Drug Administration Agency, it was concluded that carvedilol formulation elaborated by Laboratórios Biosintética Ltda is bioequivalent to Coreg® formulation for both the rate and the extent of absorption.
Keywords: Healthy volunteer; Plasma; Pharmacokinetics; Carvedilol; LC–MS/MS; Bioequivalence;

Separation and quantification of a novel two-component vaccine adjuvant by Constantia E. Kritsch; Agnes Berger; Christa Heinrich-Cseh; Agnes Bugajska-Schretter; Wolfgang Zauner (263-270).
Two reversed-phase HPLC methods were developed for the quantitative determination of the two components of the novel vaccine adjuvant IC31. The adjuvant consists of a mixture of a synthetic oligodeoxynucleotide (ODN) and an 11-mer cationic peptide. The negatively charged oligodeoxynucleotide and the positively charged peptide form a complex that has to be quantitatively dissociated for analysis. Dissociation of the complex was achieved with a basic heparin solution (1000 IU/ml) when analyzing the ODN, whereas 30% acetic acid was used for the determination of the peptide. Both methods are suitable for identification and quantification but also for stability indicating investigations.
Keywords: Cationic peptide; Complex dissociation; Heparin; ODN; Reversed-phase HPLC;

N,N-Diethyl-m-toluamide (DEET) and oxybenzone are two essential active ingredients in insect repellent and sunscreen preparations. We developed and validated a simple, sensitive, and selective HPLC assay to simultaneously measure DEET, oxybenzone and five primary metabolites of DEET and oxybenzone in biological samples including plasma, urine and skin strips. The compounds were separated on a reversed-phase C18 column using three-stage gradient steps with methanol and water. DEET and two relevant metabolites were detected at 254 nm, while oxybenzone and three relevant metabolites were detected at 289 nm. The limit of detection was 0.6 ng for DEET and 0.5 ng for oxybenzone, respectively. The developed method was further applied to analyze various biological samples from an in vivo animal study that evaluated concurrent use of commercially available insect repellent and sunscreen preparations.
Keywords: Reversed-phase HPLC with UV detection; Concurrent application; Repellent DEET; Sunscreen oxybenzone; Relevant metabolites;

Tadalafil is a potent reversible phosphodiesterase-5 inhibitor used for the treatment of erectile dysfunction. This study describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the determination of tadalafil in 50 μl of rat plasma. Tadalafil and the internal standard lamotrigine were extracted with 0.5 ml of tert-butyl methyl ether, after the samples alkalinized with 20 μl of sodium hydroxide solution (1N). Chromatographic separation was achieved on a C18 column with the mobile phase of acetonitrile–water containing 20 mM phosphate buffer (pH 7) (35/65, v/v), at a flow rate of 1 ml/min. The eluant was detected at 290 nm. The retention time was about 4.5 min for lamotrigine and 15 min for tadalafil. No endogenous substances were found to interfere. Calibration curves were linear from 10 to 2000 ng/ml. The recovery of tadalafil from plasma was greater than 77%. The limit of quantitation was 10 ng/ml. The intra- and inter-day imprecision (expressed as coefficient of variation, C.V.) did not exceed 10.7%, and the accuracy was within 5.9% deviation of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring tadalafil concentration.
Keywords: Tadalafil; Erectile dysfunction; HPLC; Pharmacokinetics;

Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection by Violeta Kontrimavičiūtė; Michel Larroque; Vitalis Briedis; Delphine Margout; Françoise Bressolle (285-293).
A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm × 4.6 mm i.d., 3 μm particle size). The excitation wavelength was set at 230 nm for the first 15.8 min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0–12.5% and accuracy was 95.4–104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was ≤17% and accuracy was 95–105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.
Keywords: Ibogaine; 12-Hydroxyibogaine; Plasma; High-performance liquid chromatography; Fluorimetric detection; Validation;

An accurate, sensitive, reproducible, and selective liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for determination of aripiprazole and its main metabolite, OPC-14857, in human plasma was developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 7.5 min. The calibration curve, ranging from 0.1 to 100 ng/ml, was fitted to a 1/y 2-weighted linear regression model. The assay showed no significant interference. Lower limit of quantitation (LLOQ) for both analytes was 0.1 ng/ml using 0.4 ml of plasma. Intra- and inter-assay precision and accuracy values for aripiprazole and OPC-14857 were within regulatory limits.
Keywords: Aripiprazole; OPC-14857; LC–MS/MS;

HPLC determination of pirenzepine dihydrochloride in rabbit aqueous humor by Jiasheng Tu; Pengmei Li; Xiaoyan Yang; Hui Pang (300-303).
Pirenzepine was considered as a pharmacologic agent of preventing form-deprivation myopia. To assess the ocular bioavailability of pirenzepine, a HPLC method for determination of pirenzepine in rabbit aqueous humor was developed. An HPLC system was used in the reverse phase mode for the determination of pirenzepine. A Luna RP18 5 μm 4.6 mm × 150 mm column was employed at 35 °C. The mobile phase was methanol/0.02 M KH2PO4/sodium 1-pentanesulfonate (350/650/1, v/v/m, pH was adjusted to 8.0 by dropping 1 M NaOH). The flow rate was 1 ml/min. Pirenzepine was monitored at 280 nm. Sample treatment procedure consists of deproteinisation with methanol. Calibration curves fitted by plotting the peak area versus concentration were linear in the range 20–400 ng/ml. The limit of quantification (LOQ) of present method was 20 ng/ml. Within-day and inter-day coefficient of variation was lower than 10%. Analytical recoveries were determined as 92.4, 95.4 and 101.4% at concentrations of 40, 200 and 400 ng/ml. In conclusion, this HPLC method using a simple sample treatment procedure appears suitable for monitoring ocular concentration of pirenzepine.
Keywords: HPLC; Pirenzepine; Concentration in aqueous humor;

Immobilisation of oligo-peptidic probes for microarray implementation: Characterisation by FTIR, Atomic Force Microscopy and 2D fluorescence by S. Soultani-Vigneron; V. Dugas; M.H. Rouillat; J. Fédollière; M.C. Duclos; E. Vnuk; M. Phaner-Goutorbe; V. Bulone; J.R. Martin; J. Wallach; J.P. Cloarec (304-310).
Proteomic microarrays show a wide range of applications for the investigation of DNA–protein, enzyme–substrate as well as protein–protein interactions. Among many challenges to build a viable “protein microarray”, the surface chemistry that will allow to immobilised various proteins to retain their biological activity is of paramount importance. Here we report a chemical functionalisation method allowing immobilisation of oligo-peptides onto silica surface (porous silica, glass, thermal silicon dioxide). Substrates were first derivatised with a monofunctional silane allowing the elaboration of dense and uniform monolayers in highly reproducible way. Prior to the oligo-peptides grafting, this organic layer was functionalised with an amino-polyethyleneglycol. The coupling step of oligo-peptides onto functionalised supports is achieved through activation of the C-terminal function of the oligo-peptides. Chemical surface modifications were followed by FTIR spectroscopy, AFM measurements and fluorescence scanning microscopy. A systematic study of the oligo-peptide grafting conditions (time, concentration, solvent) was carried out to optimise this step. The oligo-peptides grafting strategy implemented in this work ensure a covalent and oriented grafting of the oligo-peptides. This orientation is ensured through the use of fully protected peptide except the terminal primary amine. The immobilized peptides will be then deprotected before biological recognition. This strategy is crucial to retain the biological activity of thousands of oligo-probes assessed on a microarray.
Keywords: Oligo-peptide immobilization; Microarray; Biotin; Streptavidin; FTIR; AFM; Fluorescence;

Fast analysis of pravastatin in production media by Andrej Kocijan; Rok Grahek; Andrej Bastarda; Lucija Zupančič Kralj (311-315).
High throughput methods (high performance liquid chromatography and capillary electrophoresis) were developed to determine pravastatin in production media. The analyses were performed on particle column, monolithic column and silica capillary filled with borate buffer pH 9.3 containing 20 mM SDS. All three methods successfully separate pravastatin from interfering compounds (matrix, mevastatin and 6-epi pravastatin) and runtimes are shorter than 1 min. Solvent consumptions for methods using small particle column, monolith column and MECK were 132, 510 and 1.5 mL h−1. The most sensitive was the method using particle column (LOD was about 10−5  mg mL−1), followed by the system using monolith column (LOD was 2 × 10−4  mg mL−1) and the MECK method (LOD was about 0.02 mg mL−1).
Keywords: Pravastain; Fast analysis; Pharmaceutical analysis; Monolith column; Small particle column; Fast MECK method;

In this study a protocol for the analysis of thiamin and thiamin coenzymes in whole blood was developed. Thiamin and its coenzymes are analyzed by reversed phase liquid chromatography (RPLC), precolumn derivatisation with alkaline potassium ferricyanide and fluorescence detection, all at pH 10. Under these relatively high pH conditions the detectability of the analytes and the robustness of the method were substantially improved. The use of a high pH resistant RPLC column was a crucial step in developing this analysis method. Reproducibility, linearity, recovery, detection limit and column robustness were investigated. The within-batch CV was <2.5%, the between-batch CV <4.5%. The method was linear far above the physiological relevant concentration level. Recovery was almost 100% on an average. The limit of quantification was 1 nmol/l. The robustness of the RPLC column proved to be very high. Up to 1500 injections hardly any substantial changes in retention times and efficiency were observed. In summary: Using a high pH resistant RPLC column resulted in a robust, sensitive and precise method for the analysis of total Vitamin B1 and especially of TDP.
Keywords: Thiamin; Vitamin B1; HPLC; Whole blood analysis; High pH mobile phase;

High performance liquid chromatographic determination of topiramate in human serum using UV detection by Gholamreza Bahrami; Shahla Mirzaeei; Bahareh Mohammadi; Amir Kiani (322-325).
Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid–liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05 M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 μg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992
Keywords: Reverse phase chromatography; HPLC; Topiramate; UV detection;

High-performance liquid chromatography method for the quantification of pantoprazole in human plasma by N.V.S. Ramakrishna; K.N. Vishwottam; S. Wishu; M. Koteshwara (326-329).
A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid–liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10 mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry® C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20–5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3–3.2% and 0.7–3.3%, respectively. The between-batch and within-batch bias was −0.5 to 8.2 % and −2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.
Keywords: Pantoprazole; HPLC; Quantification; Human plasma;

High throughput assay for the determination of lumefantrine in plasma by A. Annerberg; T. Singtoroj; P. Tipmanee; N.J. White; N.P.J. Day; N. Lindegårdh (330-333).
A high throughput bioanalytical assay for the determination of lumefantrine in plasma has been developed and validated extensively. The within-day precisions for lumefantrine were 5.2, 3.5 and 2.5% at 200, 2000 and 15000 ng/mL, respectively. The between-day precisions were 4.0, 2.8 and 3.1% at 200, 2000 and 15000 ng/mL, respectively. The lower limits of quantification (LLOQ) and the limits of detection (LOD) were 25 and 10 ng/mL, respectively using 0.250 mL plasma. The average recovery of lumefantrine was 85% and independent upon concentration. The use of 96-well plate format and short chromatographic run has increased the daily sample throughput four times. The assay is particularly suitable for large therapeutic drug monitoring studies using day 7 sampling.
Keywords: Antimalarial; High throughput; Liquid chromatography; Lumefantrine; Solid phase extraction; 96-Well;

A capillary zone electrophoresis method with laser induced fluorescence detection for the chiral separation of highly fluorescent enantiomeric derivatives of d/l-Serine from 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-d/l-Serine) was developed and optimized. Enantiomeric separation of NBD-d/l-Serine was accomplished by using 40 mM hydroxypropyl-β-cyclodextrin (HP-β-CD) contained in 100 mM borate buffer, pH 10.0. A 70 cm (effective length of 50 cm) uncoated fused-silica capillary at a voltage of 15 kV was used for the separation. The optimized electrophoretic conditions were subsequently applied to the analysis of d-Serine in rat brain, and satisfactory analytical results with respect to accuracy were obtained. This assay showed acceptable precision, with linearity in the d-Serine concentration range of 0.2–20.0 μM. The limit of detection for d-Serine was 3.0 × 10−7  M.
Keywords: Capillary zone electrophoresis; Enantiomeric separation; d-Serine determination;

A fast, sensitive method for the simultaneous determination of α-tocopherol and α-tocopheryl acetate in mixed micelles by Stéphane Castan; Claude Villard; Stefan Jakob; Antoine Puigserver; El Hassan Ajandouz (339-346).
This report improves analytical procedures to investigate the behaviour of the two Vitamin E forms, α-tocopherol (Tol) and α-tocopheryl acetate (Tac), in model systems mimicking the intestinal medium. We describe how to prepare mixed micelles as vehicle for Tac and Tol and the HPLC method for their quantification in the micelles. Tac and Tol were extracted using ethanol-hexane-drying procedure, whereas the separation and detection were performed in methanol and by UV method, respectively. Both compounds were eluted in less than 4 min. In the range between 1.7 μM and 54 μM of Tac or Tol in the micelles, their recovery were 89% and 81%, respectively, with correlation coefficient over 0.99 and R.S.D. of less than 7.2% in all cases. Limits of detection and quantification for Tac and Tol in mixed micelles ranged between 1 μM and 2 μM and between 3 μM and 5 μM, respectively. The behaviours of Tac and Tol were quite different during the extraction procedure and both were influenced by the vitamin concentration and the relative volume of organic solvents.
Keywords: α-Tocopherol; α-Tocopheryl acetate; Mixed micelles; Reverse phase HPLC;