Journal of Chromatography B (v.823, #2)

A liquid chromatography–electrospray ionization tandem mass spectrometric (LC/ESI–MS/MS) method for the determination of 2β-(3-hydroxypropoxy)-1α,25-dihydroxy vitamin D3 (ED-71) in human serum has been developed. ED-71 in human serum was extracted using two solid-phase extraction steps on Bond Elut C18 and NH2 cartridge. The separation of ED-71 and preED-71 isomer was attained by LC using 2 mmol/L ammonium acetate–methanol (15:85, v/v) as a mobile phase on a Symmetry C18 column (5 μm, 150 mm × 2.1 mm i.d.). ESI–MS/MS analysis was operated using selected reaction monitoring (SRM) in positive ion mode. The method achieved a lower limit of quantitation of 25 pg/mL. The calibration curve (25–3200 pg/mL) gave acceptable linearity (r  > 0.9964). Intra-assay precision ranged from 2.3 to 9.7%. Inter-assay precision ranged from 1.0 to 3.4%. The accuracy was within 90.8–107.0%. This highly sensitive and reproducible method is able to determine only biologically active ED-71 by separating it from preED-71, which is considered to be applicable for the determination of serum samples from pharmacokinetic studies in human.
Keywords: 2β-(3-Hydroxypropoxy)-1α,25-dihydroxy vitamin D3 (ED-71); preED-71; LC/ESI–MS/MS; Human serum;

Analytical method development and validation of mianserin hydrochloride and its metabolite in human plasma by LC–MS by Bhupendrasinh Chauhan; Shubha Rani; Swati Guttikar; Anjali Zope; Nimisha Jadon; Harish Padh (69-74).
Mianserin is a tetracyclic antidepressant drug and administered as racemate of R (−) and S (+) mianserin hydrochloride in a dose of 30–90 mg/day in divided doses. Liquid chromatography-mass spectroscopy (LC–MS) is a tool, which is widely used for determination of drug and their metabolites in biological fluids because of its high sensitivity and precision. Here we describe a liquid chromatography mass spectroscopy method for simultaneous determination of mianserin and its metabolite, N-desmethylmianserin, from human plasma using a liquid–liquid extraction with hexane:isoamylalcohol (98:2) and back extraction with 0.005 M formic acid solution. This method is specific and linear over the concentration range of 1.00–60.00 ng/ml for mianserin and 0.50–14.00 ng/ml for N-desmethylmianserin in human plasma. The lowest limits of quantification (LLQ) is 1.00 ng/ml for mianserin and 0.50 ng/ml for N-desmethylmianserin. Intraday and interday precision (%C.V.) is <10% for both mianserin and N-desmethylmianserin. The accuracy ranges from 94.44 to 112.33% for mianserin and 91.85–100.13% for N-desmethylmianserin. The stability studies showed that mianserin and N-desmethylmianserin in human plasma are stable during short-term period for sample preparation and analysis. The method was used to assay mianserin and its metabolite, N-desmethylmianserin, in human plasma samples obtained from subjects who had been given an oral tablet of 30 mg of mianserin.
Keywords: Mianserin; N-desmethylmianserin; Human plasma; Single ionization mode (SIM);

Downstream processing of MDCK cell-derived equine influenza virus by Deba Prasad Nayak; Sylvia Lehmann; Udo Reichl (75-81).
A microcarrier-based process was used to produce equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93) in Madin Darby Canine kidney (MDCK) cells. The virus was purified in a sequence of downstream processing steps comprising of depth filtration, inactivation, ultrafiltration (UF) and gel filtration. In the ultrafiltration step, the hemagglutinin (HA) was recovered to 100%. A high increase of neuraminidase (NA) activity indicated the removal of some inhibitory compounds during this step. At the same time, the level of contaminating proteins and DNA was reduced by more than 88%. In the subsequent size exclusion chromatography (Sepharose CL 2B), the recovery of HA and NA in the “virus peak” was 37.8 and 59.8%, respectively compared to the concentrated feed material. Inconsistencies in the overall mass balance for HA and NA (70.0 and 69.2%) during gel filtration indicated non-specific interactions of the inactivated virus to the gel matrix which is supported by a HA recovery of about 50% in shake flask experiments performed as a control. Overall 35.8% of HA and 291.6% of NA were recovered. More than 95.7% of the host cell proteins and 98.7% of the host cell DNA were removed during downstream processing.
Keywords: Chromatography; Downstream processing; Influenza; Virus; MDCK cell and vaccine;

Two-dimensional electrophoresis database of fluorescence-labeled proteins of colon cancer cells by Yasuharu Mori; Tadashi Kondo; Tesshi Yamada; Akihiko Tsuchida; Tetsuya Aoki; Setsuo Hirohashi (82-97).
We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.
Keywords: Proteome; Two-dimensional gel electrophoresis; 2D database; 2D-DIGE; colon cancer;

An accurate and improved HPLC method was set up to measure both dihydrouracil (UH2) and uracil (U) in plasma, and to assess their ratio.Analytes retention time, separation and peak purity were greatly optimized with a Hypercarb column and a diode array detector. U and UH2 limits of quantification were 1.25 and 0.625 ng/mL. U and UH2 within-day precisions were 0.9–2.3% and 0.7–5.6%. Between-day precisions were 1.3–5.3% and 1.3–7.1%. Accuracy was 0.1–6.1%.UH2/U ratio between-day variability was low, but ratio decreased from 02:00 p.m.This method is now used in practice to detect patients at risk of fluoropyrimidine toxicity and to individually adapt the dosage.
Keywords: Fluoropyrimidines; Uracil; Dihydrouracil; HPLC;

Determination of puerarin in human plasma by high performance liquid chromatography by Zhongze Ma; Qingli Wu; David Y.W. Lee; Michael Tracy; Scott E. Lukas (108-114).
Puerarin, an isoflavone C-glycoside, has been identified as the major active component isolated from Pueraria lobata (Kudzu) responsible for suppression of alcohol drinking. In order to conduct clinical studies of Kudzu's efficacy, a method for measuring its bioavailability and pharmacokinetic profile is needed. We have developed a gradient reversed-phase HPLC system for pharmacokinetic study of puerarin in human plasma. Solid-phase extraction was performed on an abselut Nexus cartridge (60 mg/3 ml) possessing adsorbent function with a recovery of >97% and 4-hydroxybenzoic acid was used as an internal standard. The HPLC assay was performed on a YMC ODS-A column (150 mm × 4.6 mm i.d., 5 μm particle size). The HPLC mobile phase consisted of methanol/0.5% acetic acid with 20–35% methanol gradient at a flow-rate of 0.8 ml/min. The UV wavelength was set at 254 nm. Calibration of the overall analytical procedure gave a linear signal (r  > 0.999) over a puerarin concentration range of 5–500 ng/ml in human plasma. The lower limit of quantification was ca. at 8 ng/ml of puerarin in plasma. The detection limit (defined as signal-to-noise ratio of about 3) was approximately 3 ng/ml. The preliminary pharmacokinetic study after oral administration of the Kudzu capsules containing 400 mg of puerarin to a healthy volunteer confirmed that the present method was suitable for determining puerarin in human plasma.
Keywords: Puerarin; Kudzu; Human plasma; Reversed-phase HPLC;

HPLC assay for bupropion and its major metabolites in human plasma by Katarzyna K. Loboz; Annette S. Gross; John Ray; Andrew J. McLachlan (115-121).
Bupropion is used clinically as an antidepressant and in smoking cessation. As it is metabolised to hydroxybupropion specifically by CYP2B6, bupropion has also been used as a probe to assess CYP2B6 activity. A specific and reproducible HPLC assay has been developed to simultaneously quantify bupropion and its major metabolites hydroxybupropion, threohydrobupropion and erythrohydrobupropion in human plasma. The analysis was performed on an Aqua C18 HPLC column, with a mobile phase consisting of 45:55 of methanol:0.05 M phosphate buffer (pH 5.5) and simultaneous UV detection at 214 nm (bupropion metabolites) and 254 nm (bupropion, internal standard timolol maleate). The assay showed a linear response for bupropion (2.5–250 ng/mL), threohydrobupropion (5–250 ng/mL), erythrohydrobupropion (10–250 ng/mL) and hydroxybupropion (10–1000 ng/mL). Extraction recovery was reproducible and greater than 55% for each analyte. The inter- and intra-day assay variability (measured as percent coefficient of variation; %CV) was less than 15% for all analytes. Limit of quantification was 2.5 ng/mL for bupropion, 5 ng/mL for threohydrobupropion and 10 ng/mL for hydroxybupropion and erythrohydrobupropion. This assay is more sensitive than currently published methods using HPLC with UV detection for the simultaneous quantitation of bupropion and metabolites and can be used for assessing CYP2B6 activity in vivo following a single dose of bupropion.
Keywords: HPLC; Bupropion; Hydroxybupropion; Threohydrobupropion; Erythrohydrobupropion; CYP2B6 phenotype;

Enzymatic diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by detecting 2-octenoyl-CoA production using high-performance liquid chromatography: A practical confirmatory test for tandem mass spectrometry newborn screening in Japan by Go Tajima; Nobuo Sakura; Hiroko Yofune; Yutaka Nishimura; Hiroaki Ono; Yuki Hasegawa; Ikue Hata; Masahiko Kimura; Seiji Yamaguchi; Yosuke Shigematsu; Masao Kobayashi (122-130).
Many of the previously described enzymatic assay methods for the diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency have been dependent upon the measurement of radioisotope-labeled co-products or reduction of electron acceptors. We have developed a direct assay method to detect 2-enoyl-CoA production using high-performance liquid chromatography (HPLC). Crude cell lysate prepared from lymphocytes were incubated with n-octanoyl-CoA and ferrocenium hexafluorophosphate. The detection of 2-octenoyl-CoA was significantly reproducible. We applied the assay to samples from four infants suspected to have MCAD deficiency by tandem mass spectrometry (MS/MS) newborn screening conducted in the Hiroshima area of Japan. Three of them were proved to have pathologically reduced residual enzyme activities, although they were associated with various clinical and biochemical phenotypes. In addition, another symptomatic Japanese patient and her presymptomatic sibling who were detected by MS/MS selective screening were successfully diagnosed by our enzymatic assay. These results indicate that the method can be a useful confirmatory test for MS/MS screening of MCAD deficiency.
Keywords: Medium-chain acyl-CoA dehydrogenase; Enzymatic assay; 2-Octenoyl-CoA; High-performance liquid chromatography; Tandem mass spectrometry; Newborn screening; Japanese;

A systematic method for the sensitive, precise and accurate determination of hair lipids, including trace amounts of intrinsic endogenous cholesterol (CH), ceramide/N-palmitoyl-dl-dihydrosphingosine (CER/PDS), cholesterol sulfate (CS) and chemically bound 18-methyl eicosanoic acid (18-MEA), has been developed in combination with TLC/FID (flame ionization detection), LC/MS and GC/MS. TLC/FID was used for the simultaneous determination of squalene (SQ), wax esters (WEs), triglycerides (TGs) and free fatty acids (FFAs). Optimal conditions for LC/MS to determine CS and 18-MEA were developed using selected ion monitoring (SIM) under the negative ion mode of electrospray ionization. An alternative procedure for the determination of 18-MEA was also established using commercially available heneicosanoic acid (HEA). In GC/MS, the optimal selection of ions for SIM of trimethylsilylated CH and CER/PDS, and the use of on-column injection has enabled their simultaneous detection. This newly developed method has been used to characterize the hair lipid composition from the proximal root end to the distal tip of chemically untreated hair fibers from two different females, and specific changes of hair lipids probably due to its origin and individuals have been demonstrated for the first time. This method may be useful for clarifying the important roles of intrinsic endogenous 18-MEA, CS, CH and CERs in the function of the cell membrane complex of hair fibers.
Keywords: Hair; Lipids; TLC/FID; GC/MS; LC/MS;

We developed a sensitive method for determination of 23 flavonoids and phenolic acids, which represent phenolic acids and five subclasses of flavonoids. Plasma samples were extracted with selective solid-phase-extraction columns and separated by RP-high-performance liquid chromatography (HPLC). For detection an electrochemical detector was used. Identification and test of purity were carried out via retention times and spectra analyses. Limits of detection varied from 1.45 to 22.27 nmol/l. Recovery varied from 81% to 106%. Reproducibility for all analytes was below 10% (coefficient of variation, CV (%)) and ranged between 3.1% and 9.8%. This method can be applied to samples from interventional studies as well as observational studies.
Keywords: Flavonoids; Phenolic acids; Polyphenols; HPLC; Biomarker; Diet;

High-performance thin-layer chromatographic determination of lamotrigine in serum by Kuldeep M. Patil; Subhash L. Bodhankar (152-157).
A simple and rapid high-performance thin-layer chromatographic (HPTLC) determination of lamotrigine (LTG) in serum is reported. The method involves extraction of the drug by ethyl acetate followed by separation on TLC silica plates using a mixture of toluene-acetone-ammonia (7:3:0.5), as eluting solvent. Densitometric analysis was carried out at 312 nm with lamotrigine being detected at R f of 0.54. The analytical method has excellent linearity (r  = 0.998) in the range of 20–300 ng/spot. This assay range is adequate for analyzing human serum, as it corresponds to lamotrigine concentrations measured in human serum from epileptic patients. The method was validated for sensitivity, selectivity, extraction efficiency, accuracy and intra and inter-day reproducibility. The limit of detection and limit of quantification were found to be 6.4 and 10.2 ng, respectively. Good accuracy is reported in the range of 92.06–97.12% and high precision with %CV in range of 0.53–2.59. The method was applied for determination of serum lamotrigine levels in epileptic patients and in pharmacokinetic study of lamotrigine administered orally to rabbits.
Keywords: Lamotrigine; Serum; High-performance thin-layer chromatography;

The microsomal fraction of rat liver containing uridine diphospho-glucuronosyltransferase (UDPGT; EC 2.4.1.17) has been covalently immobilized on a high performance chromatographic support. In this study Nucleosil Si-500 silica was converted into diol-bonded silica and subsequently converted into an aldehyde form through oxidation with sodium periodate. The microsomal fraction was immobilized via Schiff base formation followed by reduction with sodium cyanoborohydride. The resulting immobilized enzyme reactor (IMER) was placed in a multi-dimensional chromatographic system which utilized a mixed mode (C18 and anion exchange) column to trap the parent compound and glucuronide and a C18 column to separate the substrate and product. The IMER system was used for the online glucuronidation of 4-methylumbelliferone (4Me7OHC) and acetaminophen (APAP). The Michaelis-Menten kinetic parameters (K m and V max) associated with the formation of 4Me7OHC and APAP glucuronides demonstrated that the immobilization had not significantly affected the enzymatic activity of the UDPGT relative to the non-immobilized enzyme. The IMER retained enzymatic activity for more than 6 weeks. The results of this study demonstrate an easy and convenient way to identify compounds which may be glucuronidated and to synthesize and characterize the resulting products.
Keywords: Glucuronides; Immobilized enzyme reactors; Rat liver microsomes;

Simultaneous determination of clobazam and its major metabolite in human plasma by a rapid HPLC method by Mohammadreza Rouini; Yalda H. Ardakani; Lida Hakemi; Maryam Mokhberi; Gheise Badri (167-171).
A rapid and specific HPLC method has been developed and validated for simultaneous determination of clobazam, the anticonvulsant agent, and its major metabolite in human plasma. The sample preparation was a liquid–liquid extraction with tuloene yielding almost near 100% recoveries of two compounds. Chromatographic separation was achieved with a Chromolith™ Performance RP-18e 100 mm × 4.6 mm column, using a mixture of a phosphate buffer (pH 3.5; 10 mM)–acetonitrile (70:30, v/v), in isocratic mode at 2 ml/min at a detection wave-length of 228 nm. The calibration curves were linear (r 2  > 0.998) in the concentration range of 5–450 ng ml−1. The lower limit of quantification was 5 ng ml−1 for two compounds studied. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 0.89–9.1% and 2.1–10.1% R.S.D., respectively. The developed procedure was applied to assess the pharmacokinetics of clobazam and its major metabolite following administration of a single 10 mg oral dose of clobazam to healthy volunteers.
Keywords: Clobazam; N-Desmethylclobazam; Plasma; Monolithic; Pharmacokinetics;

A validated new, simple and highly sensitive reversed-phase HPLC method is developed for studying the pharmacokinetics of germacrone after intravenous administration of zedoary turmeric oil (ZTO) oil-in-water microemulsion. The method did not require a complex and expensive equipment. A high extraction recovery (>80%) of germacrone was obtained. Linear calibration curves obtained with the peak-area ratio (y) of germacrone to internal standard (tanshinoneIIA) versus drug concentration (x) were found to be linear between 8.08 and 808 ng/ml. The limit of quantitation was 8.08 ng/ml.The monitored compounds were completely separated from others in ZTO and from endogenous species in plasma by HPLC. Pharmacokinetic investigations were performed on 18 male rabbits after intravenous administration of ZTO microemulsion via the ear vein at germacrone doses of 3.2, 6.4 and 9.6 mg/kg. The plasma concentration–time data fit to a two-compartment intravenous model with a weight of 1/C 2 (C: germacrone concentration in plasma). Germacrone exhibited linear pharmacokinetics after intravenous administration of ZTO microemulsion to rabbits over the germacrone dose range 3.2–9.6 mg/ml.
Keywords: Zedoary turmeric oil; Germacrone; HPLC; Protein precipitation; Solvent extraction; Pharmacokinetic;

Validated HPLC assay for iron determination in biological matrices based on ferrioxamine formation by Angelo Tesoro; Jasmina Novakovic; Jake J. Thiessen; Michael Spino (177-183).
A simple, robust and reproducible HPLC method has been developed and validated for iron determination in biological matrices. It is based on chelation with desferrioxamine (DFO) and the measurement of the chelate ferrioxamine (FO). The method was developed to permit monitoring of iron bio-kinetics and estimation of iron status in experimental animals. The chromatography was performed on a stainless steel XTerra MS C18 column (Waters; 250 mm × 4.6 mm i.d., 5 μm) using a gradient of Tris–HCl buffer (10 mM, pH 5) and acetonitrile. The method was validated in terms of selectivity, linearity (0.3–80 nmol on-column), limit of detection (0.2 nmol on-column), low limit of quantification (0.3 nmol on-column), recovery (91–102%), intra- and inter-day reproducibility, stability, and robustness. The method's universal applicability was illustrated by monitoring plasma and heart iron kinetic profiles in rats after a single intraperitoneal (i.p.) injection of 200 mg/kg iron dextran.
Keywords: Iron; RP–HPLC; Ferrioxamine; Iron bio-kinetics;

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography by Andrew J. Mannes; Dorothy Cimino Brown; Jason Keller; Lauren Cordes; Roderic G. Eckenhoff; Robert M. Caudle; Michael J. Iadarola; Qing C. Meng (184-188).
A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.

High-performance liquid chromatography method for the quantification of entacapone in human plasma by N.V.S. Ramakrishna; K.N. Vishwottam; S. Wishu; M. Koteshwara; J. Chidambara (189-194).
A simple, sensitive and selective HPLC method with UV detection (315 nm) was developed and validated for quantitation of entacapone in human plasma, the newest addition to the group of antiparkinsonian agents. Following a single-step liquid–liquid extraction (LLE) with ethyl acetate/n-hexane (30/70, v/v), the analyte and internal standard (rofecoxib) were separated using an isocratic mobile phase of 30 mM phosphate buffer (pH 2.75)/acetonitrile (62/38, v/v) on a reverse phase C18 column. The lower limit of quantitation was 25 ng/mL, with a relative standard deviation of less than 8%. A linear range of 25–2500 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 2.2–4.2% and 1.7–7.8%, respectively. The between-batch and within-batch accuracy was 98.7–107.5% and 97.5–106.0%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of entacapone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.
Keywords: Entacapone; HPLC; Human plasma; Pharmacokinetic study;

Stereoselective analysis of metoprolol and its metabolites in rat plasma with application to oxidative metabolism by Vanessa Bergamin Boralli; Eduardo Barbosa Coelho; Paula Macedo Cerqueira; Vera Lucia Lanchote (195-202).
We investigated the stereoselective kinetic disposition and metabolism of metoprolol (MET) in rats. The racemic MET (15 mg/kg) was given by oral gavage and blood samples were collected from 0 to 10 h (n  = 6 at each time point). The enantiomeric concentrations of MET and its metabolites α-hydroxymetoprolol (α-OHM) and O-demethylmetoprolol (ODM) were determined by HPLC using a Chiralpak® AD chiral column and fluorescence detection. The pharmacokinetic parameters of unchanged MET and the formation of ODM did not show to be stereoselective. In contrast, the AUC (ng h/mL) of α-hydroxymetoprolol isomers were higher to I′R [638.2(525.2–706.2) for 1′R2R and 659.6(580.4–698.1) for 1′R,2S, mean, (95%CI)] than to I′S products [58.3(47.4–66.1) for 1′S,2R and 57.1(44.7–67.9) for 1′S,2S, mean, (95%CI)]. We conclude that the kinetic disposition of unchanged MET and the formation of ODM are not enantioselective in rats but the metabolism of α-OHM yields predominantly the 1′R-product.
Keywords: Metoprolol; Enantiomers; Metabolism; Rats; Pharmacokinetics; CYP2D6;

Determination of d- and l-enantiomers of methionine and [2H3]methionine in plasma by gas chromatography–mass spectrometry by Hiroshi Hasegawa; Yoshihiko Shinohara; Kenji Akahane; Takao Hashimoto (203-208).
A method for the stereoselective determination of d- and l-enantiomers of both methionine and [2H3]methionine in rat plasma was developed using gas chromatography–mass spectrometry with selected-ion monitoring (GC–MS-SIM). dl-[2H7]Methionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The amino acids were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. Endogenous l-methionine concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0 and 6.3%, respectively. The intra- and inter-day reproducibility in the amounts of d- and l-[2H3]methionine determined were in good agreement with actual amount added.
Keywords: d-Methionine; GC–MS; Stable isotope; Chiral inversion;

Membrane proteins were obtained from the mitochondrial fraction of HL-60 cells by solubilization with octyl glucoside and bound to heparin-gels. Bound proteins were successively eluted with solutions containing increasing concentrations of Mg2+ in the first and increasing concentrations of Ca2+ in the second chromatography. After SDS-PAGE and subsequent N-terminal amino acid analysis of proteins on each band, 13 proteins were identified. Fifteen out of the 37 proteins analysed were modified at their N-termini. These results show that this two-step affinity chromatography method using divalent cations as eluents can be applied to a variety of membranes for the isolation of specific proteins.
Keywords: Heparin binding proteins; Membrane proteins; Affinity chromatography; Divalent cations;

A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid – liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm × 6 mm i.d.) which was packed with 5 μm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5–2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively.
Keywords: Reverse phase chromatography; HPLC; Lamivudine; Serum; Bioequivalence Study;

Use of chlorite to improve HPLC detection of pyridoxal 5′-phosphate by Karen L. Ericson; J. Dennis Mahuren; Yvonne M. Zubovic; Stephen P. Coburn (218-220).
The sensitivity of fluorescent detection of the biologically active form of Vitamin B-6, pyridoxal 5′-phosphate (PLP), in biological samples has been improved approximately four-fold by adopting chlorite as a post-column derivatization reagent (instead of bisulfite) in high-performance liquid chromatography (HPLC) separation. Chlorite oxidizes PLP to the more fluorescent 4-pyridoxic acid 5′-phosphate, and avoids the toxicity and heating of the cyanide procedure. Detection of another major metabolite, 4-pyridoxic acid (4-PA), is not effected. Detection of pyridoxal (PL) is slightly lowered due to eluting at a lower pH.
Keywords: Pyridoxal phosphate; Vitamin B-6;

Author Index (221-223).

Keyword Index (224-228).