Journal of Chromatography B (v.826, #1-2)

A simple, sensitive and reliable HPLC ion-pairing method with fluorescence detection, was developed for penciclovir determination in plasma and aqueous humor, with a Zorbax SB-aq C18 (100 mm × 2.1 mm) column. Plasma samples were treated by solid-phase extraction with Oasis® MCX (30 mg) cartridges. Ganciclovir, an antiviral drug structurally related to penciclovir, was used as internal standard (I.S.). Aqueous humor samples were directly injected into the chromatographic system. Separation was performed by a gradient elution with a mobile phase consisting of a mixture of acetonitrile and phosphate buffer 50 mM containing 5 mM of sodium octanesulfonate, pH 2.0, at a flow rate of 0.3 ml/min.The method was validated and showed good performances in terms of linearity, sensitivity, precision and trueness. Quantification limit was obtained at 0.05 μg/ml for aqueous humor and at 0.1 μg/ml for plasma. Finally, the proposed analytical method was used to measure penciclovir in clinical samples for a pharmacokinetic study, after oral administration of famciclovir.
Keywords: Penciclovir; Liquid chromatograpy–fluorescence detection; Plasma; Aqueous humor; Validation;

Fast “hyperlayer” separation development in sedimentation field flow fractionation by James R. Kassab; Philippe J.P. Cardot; Richard A. Zahoransky; Serge Battu (8-16).
Specific prototypes of sedimentation field flow fractionation devices (SdFFF) have been developed with relative success for cell sorting. However, no data are available to compare these apparatus with commercial ones. In order to compare with other devices mainly used for non-biological species, biocompatible systems were used for standard particle (latex: 3–10 μm of different size dispersities) separation development. In order to enhance size dependent separations, channels of reduced thickness were used (80 and 100 μm) and channel/carrier-phase equilibration procedures were necessary. For sample injection, the use of inlet tubing linked to the FFF accumulation wall, common for cell sorting, can be extended to latex species when they are eluted in the Steric Hyperlayer elution mode. It avoids any primary relaxation steps (stop flow injection procedure) simplifying series of elution processing. Mixtures composed of four different monodispersed latex beads can be eluted in 6 min with 100 μm channel thickness.
Keywords: Sedimentation field flow fractionation; Latex particles; Steric Hyperlayer;

A sensitive GC/CI/MS/MS method was developed for the simultaneous determination of cocaine (COC), anhydroecgonine methylester (cocaine pyrolysis product, AEME), ecgonine methylester (cocaine enzymatic hydrolysis product, EME) and cocaethylene (cocaine with ethanol trans-esterification product, COET) in human hair samples. After acid hydrolysis, hair samples were extracted with an automated solid phase extraction (SPE). The analysis of cocaine and its three metabolites was performed using an ion-trap spectrometer in positive chemical ionization with isobutane as gas reagent. The procedure was validated. Weighted linear regression was found appropriate in a concentration range of 0.10–5.00 ng/mg for AEME, 0.05–5.00 ng/mg for COC, EME and COET. The limit of detection was estimated at 0.005 ng/mg for COC and COET, at 0.025 ng/mg for EME, and at 0.050 ng/mg for AEME. Method performance was evaluated in terms of trueness and precision using quality control (QC) samples over the investigated ranges. Method selectivity and robustness were also demonstrated.
Keywords: Chemical ionization: CI; Cocaine; GC/MS/MS; Hair; Ion-trap; Validation;

Tanshinone IIA (TS) and cryptotanshinone (CT) are the major active constituents contained in Radix salvia miltiorrhiza. This paper described a rapid, sensitive and specific assay for the simultaneous quantitative determination of TS and CT in rat plasma. After a single step of liquid–liquid extraction, plasma samples were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) using a reversed-phase C18 column (150 mm × 2.0 mm, 5 μm, Shim-pack VP-ODS column). The assay was linear in the concentration range of 2–200 ng/ml. The lower limits of quantification of TS and CT were 1 and 0.2 ng/ml, respectively. Recoveries of TS and CT were greater than 80%. The precisions and accuracies determined from 5 days were all within 12%. The assay was applied to a pharmacokinetic study in rats after an oral administration of total tanshinones with a dose of 150 mg/kg (containing 12% of TS and CT). Results showed that this simple and rapid method was sensitive enough to follow the plasma levels of TS and CT in rats, even though the concentration maximums of both were below 20 ng/ml after an oral administration of total tanshinones.
Keywords: Tanshinone IIA; Cryptotanshinone; Liquid chromatography–mass spectrometry; Pharmacokinetics;

A validated LC/MS/MS method for the quantification of pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies by Gabriella Szekely-Klepser; Kimberly Wade; Dayna Woolson; Richard Brown; Scott Fountain; Erick Kindt (31-40).
A novel skin tissue extraction method coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies. The analyte is extracted from the matrix (2 mm skin punch biopsies) using a simple oxidative degradation procedure. The extract supernatants are evaporated, reconstituted in mobile phase solvent, and injected into the LC/MS/MS system without further derivatization. The chromatographic separation is achieved on a reverse phase high performance liquid chromatography (HPLC) column. The accuracy and precision of the method was determined over the concentration range of 1–1000 ng/mL PTCA from human skin extracts in three validation batch runs. Inter-assay precision (%CV) and accuracy (%R.E.) of the quality control samples were ≤18.5% (at lower limit quality control, LLQC) and ≤5.25%, respectively. The sensitivity and throughput of this assay is significantly improved relative to previously published methods resulting in much smaller tissue requirements and analysis time. This procedure could potentially be used in the investigation of therapies associated with skin pigmentation.
Keywords: Pyrrole-2,3,5-tricarboxylic acid; PTCA; Mass spectrometry; LC/MS/MS; Human skin; Pigmentation; Eumelanin; Melanin; Biomarker;

Determination of atorvastatin in human serum by reversed-phase high-performance liquid chromatography with UV detection by Gholamreza Bahrami; Bahareh Mohammadi; Shahla Mirzaeei; Amir Kiani (41-45).
A rapid and sensitive high-performance liquid chromatographic method was validated and described for determination of atorvastatin in human serum. Following liquid–liquid extraction of the drug and an internal standard (sodium diclofenac), chromatographic separation was accomplished using C18 analytical column with a mobile phase consisting of sodium phosphate buffer (0.05 M, pH 4.0) and methanol (33:67, v/v). Atorvastatin and the internal standard were detected by ultraviolet absorbance at 247 nm. The average recoveries of the drug and internal standard were 95 and 80%, respectively. The lower limits of detection and quantification were 1 and 4 ng/ml, respectively, and the calibration curves were linear over a concentration range of 4–256 ng/ml of atorvastatin in human serum. The analysis performance was studied and the method was applied in a randomized cross-over bioequivalence study of two different atorvastatin preparations in 12 healthy volunteers.
Keywords: Chromatography; HPLC; Atorvastatin; Serum; Bioequivalence study; Statins;

A simultaneous HPLC separation of the six major kavapyrones and the flavokavins A–C in an ethanolic extract of Piper methysticum was carried out on a Symmetry C18 column. For quantitative determinations of the flavokavins, calibration curves with correlation coefficients between 0.9986 and 0.9998 were established. Detection limit for each flavokavin of 0.5 ng per injection was measured at 355 nm. The precision of the HPLC analysis was verified by six determinations of the content of flavokavins in the kava extract. Flavokavins A–C contents of 0.62 ± 0.01 mg/100 mg, 0.34 ± 0.01 mg/100 mg and 0.14 ± 0.003 mg/100 mg ethanolic kava extract was found, respectively. From the corresponding relative standard deviation of 1.53, 1.99 and 2.30% the confidential interval (P  = 95) of the mean value was calculated for each flavokavin. The accuracy of the method was proven by recoveries between 99.2 ± 0.3% and 101.1 ± 0.4% for the flavokavins A–C.
Keywords: HPLC; Kava; Flavokavins; Kavapyrones;

Pathogenic antibody removal using magnetically stabilized fluidized bed by Mehmet Odabaşı; Nihal Özkayar; Serpil Özkara; Serhat Ünal; Adil Denizli (50-57).
Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads were used in the removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma in a magnetically stabilized fluidized bed. mPHEMA beads, in the size range of 80–120 μm, were produced by suspension technique. Then, DNA was immobilized onto mPHEMA beads by carbodiimide activation. Magnetic beads were contacted with blood in in vitro systems. Loss of blood cells and clotting times were followed. mPHEMA beads were characterized by scanning electron microscopy (SEM). Important results obtained in this study are as follows: the mPHEMA beads have a spherical shape and porous structure. Loss of cells in the blood contacting with mPHEMA/DNA was negligible. The anti-dsDNA adsorption capacity decreased significantly with the increase of the flow-rate. With increasing anti-dsDNA antibody concentration, the amount of antibody adsorbed per unit mass increased, then reached saturation. Maximum anti-dsDNA antibody adsorption capacity was found to be 97.8 mg/g. Pathogenic antibody molecules could be repeatedly adsorbed and desorbed with these magnetic beads without noticeable loss in their antibody adsorption capacity. Because of the good blood-compatibility, mPHEMA is hopeful for the treatment of SLE by magnetically stabilized fluidized bed systems in the future.
Keywords: Antibody removal; Magnetic adsorbents; Affinity beads; DNA; SLE;

An ionic liquid, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl)/salt aqueous two-phase systems (ATPS) was presented as a simple, rapid and effective sample pretreatment technique coupled with high-performance liquid chromatography (HPLC) for analysis of the major opium alkaloids in Pericarpium papaveris. To find optimal conditions, the partition behaviors of codeine and papaverine in ionic liquid/salt aqueous two-phase systems were investigated. Various factors were considered systematically, and the results indicated that both the pH value and the salting-out ability of salt had great influence on phase separation. The recoveries of codeine and papaverine were 90.0–100.2% and 99.3–102.0%, respectively, from aqueous samples of P. papaveris by the proposed method.
Keywords: Aqueous two-phase systems; Ionic liquid; Extraction; Opium alkaloids;

Quantification of pralidoxime methylsulfate (Contrathion®) in human urine by capillary zone electrophoresis by Pascal Houzé; Hafedh Thabet; Alexandre Delfour; Lucile Larrouy; Thierry Le Bricon; Frédéric J. Baud (63-68).
Pralidoxime methylsulfate (Contrathion®) is widely used to treat organophosphate poisoning. For the first time, we developed a specific assay for urinary pralidoxime using capillary zone electrophoresis (CZE) in the following conditions: fused-silica capillary (length: 47 cm, internal diameter: 75 μm), electrolyte solution: 25 mM sodium borate (pH 9.1), voltage: 15 kV, temperature: 25 °C, injection time: 1 or 2 s, on-line UV detection: 280 nm. Sample preparation did not require a deproteinization step (1:5 dilution in water). The method was linear between 0.125 and 2 mg mL−1 of pralidoxime (quantification limit: 0.10 mg mL−1). Coefficients of variation for intra- and inter-assay precision were below 10% for all three control levels (0.15–1.15 mg mL−1). This assay was successfully applied to urine specimens from organophosphate poisoned patients treated by Contrathion® (n  = 10). This CZE method allows the measure of pralidoxime in urine within 15 min with excellent precision, selectivity, and sensitivity. It is simple (no pretreatment) and convenient, thus suitable for the monitoring of Contrathion® therapy in organophosphate poisoned patients.
Keywords: Pralidoxime; Capillary zone electrophoresis; Urine;

In this work, solid phase microextraction-gas chromatograph (SPME-GC) was applied to analyze alkanes and aromatic hydrocarbons in human breath, providing a potential non-invasive method to screen lung cancer. This method has been optimized and evaluated. It provided quantification limits ranging from 0.04 to 4.2 ng/mL, linear correlations ranging from 0.9845 to 0.9966 and R.S.D. values less than 9.8%. Total 30 breath samples, from 15 lung cancer patients and 15 healthy persons, were analyzed, and the alkanes and aromatic hydrocarbons were detected in 73.3% lung cancer patients and in 13.3% healthy persons by this method. Above all, It was demonstrated that this SPME-GC method provided a sensitive and non-invasive measure means to analyze alkanes and aromatic hydrocarbons in human breath, and brought forward a potential application for screening lung cancer.
Keywords: Solid phase microextraction; Human breath; Lung cancer; Volatile organic compounds; Alkanes and aromatic hydrocarbons;

A new high-performance liquid chromatographic method for determination of warfarin enantiomers by Abdimajid Osman; Kerstin Arbring; Tomas L. Lindahl (75-80).
Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cm × 4.6 mm I.D., 5 μm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08–10 μg/mL. In this range, warfarin showed to be linear (r 2  = 0.9997 for S-warfarin and r 2  = 0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10 × LOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r  = 0.23, y  = 0.3044x  + 0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.
Keywords: S- and R-warfarin; Oxybenzone; INR;

As the epidemiological and physiological investigation of isoflavones and lignans expands, the need for sensitive methods for analyzing large numbers of samples intensifies. We have developed a method using high-performance liquid chromatography (HPLC) equipped with a coulometric electrode array detector for separation and sensitive detection of daidzein (Da), equol (Eq), genistein (Ge) and enterolactone (Enl) in dried blood spots (DBS). Detection limits ranged from 4.5 pg or 0.09 ng/mL (Eq) to 19 pg or 0.38 ng/mL (Ge) on column. Signal linearities ranged from detection limits to 200 ng/mL (Eq, Enl) and 600 ng/mL (Da, Ge) sample concentration. Correlations between DBS and serum concentrations were 0.66 (Enl), 0.88 (Eq), 0.98 (Ge) and 0.99 (Da). Intra-assay coefficients of variation (CVs) were less than 8% and inter-assay CVs ranged from 2.4 to 20.2% for Da, Eq and Ge for three levels of controls. Enl intra-assay CV was 13.6% for the low pooled control. Analytic recovery ranged from 87% (inter-assay Ge) to 98% (inter-assay Enl). DBS concentrations of Da, Ge and Eq were stable for at least 8 weeks at 4 and 25 °C, and at 37 °C for at least 5 weeks, with Enl showing greater variability at all temperatures but relative stability for 7 weeks. Measurement of samples from 135 perimenopausal Japanese women consuming habitual diets in Kyoto and Fukushima prefectures showed the former to have the expected lower concentrations of Da and Eq (416 and 87 nM) as well as Enl (49 nM) compared to the latter locale (566, 145 and 72 nM, respectively). This method could be useful in large epidemiological research or detailed physiological studies.
Keywords: Multi-channel coulometric electrode array detection; Isoflavones; Phytoestrogens; Dried blood spots;

N-Terminal isotope tagging (NIT) is an important proteomic tool for quantifying proteins in complex mixtures. Here we describe a modified version of the isotope-coded propionylation procedure of Zhang et al. [Zhang et al., Rapid Commun. Mass Spectom. 16 (2002) 2325], which uses ‘light’ D0 and ‘heavy’ D10-propionic anhydride. The method has been extensively modified to improve both the kinetics and overall yield of propionylation. Using albumin as a model protein, the overall variation in quantification yields, calculated using several tryptic peptides, was within ±10% (S.D. ±0.2) error. The efficacy of the method is demonstrated by the quantitative differences obtained for vimentin in cell lysates of C2C12 myoblasts upon their myogensis to myotubules.
Keywords: Quantitative proteomics; N-terminal isotope tagging (NIT); Propionylation; nanoLC ESI/MS/MS; Myogenesis;

A selective chiral high performance liquid chromatographic (HPLC) method coupled with achiral column was developed and validated to separate and quantify tetrahydropalmatine (THP) enantiomers in dog plasma. Chromatography was accomplished by two steps: (1) racemic THP was separated from biological matrix and collected on a Kromasil C18 column (150 mm × 4.6 mm, 5 μm) with the mobile phase acetonitrile-0.1% phosphoric acid solution, adjusted with triethylamine to pH 6.15 (47:53); (2) enantiomeric separation was performed on a Chiralcel OJ-H column (250 mm × 4.6 mm, 5 μm) with the mobile phase anhydrous ethanol. The detection wavelength was set at 230 nm. (+)-THP and (−)-THP were separated with a resolution factor (Rs) of at least 1.6 and a separation factor (α) greater than 1.29. Linear calibration curves were obtained over the range of 0.025–4 μg/ml in plasma for each of (+)-THP and (−)-THP (R 2  > 0.999) with a limit of detection (LOD) of 0.005 μg/ml and the recovery was greater than 88% for each enantiomer. The relative standard deviation (R.S.D.) and relative error values were less than 10% at upper and lower concentrations. The method was used to determine the pharmacokinetics of THP enantiomers after oral administration of racemic THP. The results presented herein showed the stereoselective disposition kinetics of THP in dogs and were a further contribution to the understanding of the kinetic behavior of THP analogues.
Keywords: Enantiomer separation; Tetrahydropalmatine; Pharmacokinetics;

Purification and partial characterization of recombinant Cu, Zn containing superoxide dismutase of Cordyceps militaris in E. coli by Zunsheng Wang; Zhuojing He; Qiong Shen; Yuxiang Gu; Suxia Li; Qinsheng Yuan (114-121).
The cDNA of Cu, Zn containing superoxide dismutase from the Cordyceps militaris SH (cm-SOD) was overexpressed in Escherichia coli BL 21 (DE3) using the pET-21a expression vector. The recombinant cell overexpressed the protein corresponding to 35 ± 3% of total bacterial protein in cytosol. The purification was performed through three steps: DEAE-FF, CM-52, and G-100. After this purification procedure, a specific activity of 27272.7 U/mg of protein was reached, corresponding to 6.1-fold purification with a yield of 85.0%. The purity was homogeneous by SDS–PAGE analysis and 94.2 ± 1.0% by CZE analysis. A subunit molecular mass of the recombinant enzyme was 15704 Da with a Cu and Zn element. In addition, the dimeric and polymeric structures were observed on MALDI-TOF-MS. Isoelectric point value of 7.0 was obtained for the recombinant enzyme that was sensitive to H2O2 and KCN. The recombinant enzyme remained 80 ± 2% residual activity at pH 7.8, at 50 °C for 4 h incubation. The properties: N-terminal amino acid sequence (the first 12 amino acid residues), pI, subunit molecular mass, thermo-stability of the purified recombinant SOD are similar to that of the native Cu, Zn-SOD from C. militaris (N-cm-SOD).
Keywords: Cordyceps militaris; Recombinant Cu, Zn containing superoxide dismutase; Purification; Molecular properties;

Two-dimensional liquid chromatography (2D-LC) coupled on-line with electrospray ionization tandem mass spectrometry (2D-LC-ESI-MS/MS) is a new platform for analysis and identification of proteome. Peptides are separated by 2D-LC and then performed MS/MS analysis by tandem MS/MS. The MS/MS data are searched against database for protein identification. In one 2D-LC-ESI-MS/MS run, we obtained not only the structural information of peptides directly from MS/MS, but also the retention time of peptides eluted from LC. Information on the chromatographic behavior of peptides can assist protein identification in the new platform for proteomics. The retention time of the matching peptides of the identified protein was predicted by the hydrophobic contribute of each amino acid on reversed-phase liquid chromatography (RPLC). By using this strategy proteins were identified by four types of information: peptide mass fingerprinting (PMF), sequence query, and MS/MS ions searched and the predicted retention time. This additional information obtained from LC could assist protein identification with no extra experimental cost.
Keywords: Proteomics; Protein identification; Retention time; Prediction; Tandem mass spectrometry;

N-acylaziridines as potential proinsecticides of carboxylic acids by B. Merelli; S. Hamm; A. Carlin-Sinclair; J.-C. Cherton (129-138).
To determine the reversible masking potential of carboxylic acids afforded by the N-acyl structure in a proinsecticide perspective, the hydrolysis of monosubstituted N-acylaziridines and unsubstituted N-acylpyrrolidine was studied by reversed-phase high-performance liquid chromatography (HPLC) during in vitro assays conducted in the presence of insect tissues or of α-chymotrypsin. Chromatographic analysis of unextracted biological samples so-called “the direct injection approach” was simpler and more accurate than the “extraction approach” because it avoids problems associated with extraction. Thus, periodical injections of samples of biological insect tissues or of α-chymotrypsin incubated with N-acyl substrates were performed on packings allowing direct injection: a wide-pore column or a monolithic column. Moreover, to allow the simultaneous monitoring of the carboxylic acids and of the parent substrates, ion-pairing was used. In these conditions, it was shown that N-acylpyrrolidine is not hydrolyzed whatever the enzymatic conditions or the pH. On the other hand, the unmasking of the carboxylic acid is the preponderant mode of hydrolysis of N-acylaziridines in the presence of α-chymotrypsine and the exclusive one in the presence of locust fat-body, which establishes the convenience of this structure in our proinsecticide perspective. Due to the enzymatic character of the unmasking of the carboxylic acid during biological hydrolysis of N-acylaziridines, the research of possible chiral recognitions was undertaken. Thus, the enantiomeric composition of these substrates was analysed at the stage of their approximative half hydrolysis using a chiral α-AGP column. It appeared that locust fat-body hydrolyses preferentially the (R)-isomers of N-acylaziridines while the reverse is observed when α-chymotrypsine is used.
Keywords: N-acylaziridine; Proinsecticide; Direct injection; Metabolism; Chirality;

A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150 μl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01 M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5 min for the elution of milk proteins and then 0.01 M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01 M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265 nm. The calibration graphs were linear in the concentration ranges of 25–800 ng/ml and 50–400 ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX–TMP combination.
Keywords: Restricted access media (RAM); Column-switching; Direct injection; Sulfonamide; Sulfamethoxazole; Trimethoprim; Bovine milk;

Determination of quantitative changes in the pattern of serum bile acids is important for the monitoring of diseases affecting bile acid metabolism. A sensitive and specific high-performance liquid chromatography (HPLC)–MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)-MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%. The developed method was applied to a group of 21 healthy volunteers and preliminary reference intervals in serum were established. In patients with drug-induced cholestasis, an elevation of primary bile acids has been shown.
Keywords: Bile acids; High-performance liquid chromatography–tandem mass spectrometry; Electrospray ionization; Serum;

N-nitroso compounds (NOC) are potent carcinogens. Reliable methods for the analysis of volatile carcinogenic NOC are well established; however selective and sensitive methods for routine analysis of thermally unstable, ionic or non-volatile NOC are still needed. For this purpose, a method based on micellar electrokinetic chromatography (MEKC) with laser induced fluorescence (LIF) detection is described for the simultaneous determination of a broad range of N-nitroso compounds. In this procedure, the nitroso group is photolytically cleaved from the NOC to yield the corresponding amine. The amines are then derivatized with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), identified and quantified using MEKC-LIF. For the standard mixture of NOC, this method has good sensitivity and a large dynamic range. The detection limit provided by the method is 9 ppb for N-nitrosopyrrolidine.
Keywords: Nitrisamines; Laser fluorimeric detection; Carcinogen;

The determination of drug–protein binding and free drug concentration in plasma applying the equilibrium sampling through membrane (ESTM) technique has been studied using supported liquid membrane extraction in a single hollow fibre without any membrane carrier. In the extraction setup, the donor phase (plasma or buffer) was placed in the vial, into which was immersed the hollow fibre with the acceptor phase situated in the lumen. This proposed technique was applied to study the drug–protein binding of five local anaesthetics and two antidepressants as model substances, and the influence of the total drug concentration on the drug–protein binding was investigated. The brief theoretical background for determination of the drug–protein binding under equilibrium conditions is described. The developed method shows a new, improved and simple procedure for determination of free drug concentration in plasma and extent of drug–protein binding.
Keywords: Equilibrium sampling through membrane (ESTM); Hollow fibre; Drug–protein binding; Local anaesthetic; Antidepressant;

A platform for high-throughput molecular characterization of recombinant monoclonal antibodies by Mark J. Bailey; Andrew D. Hooker; Carolyn S. Adams; Shuhong Zhang; David C. James (177-187).
We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest™), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF.
Keywords: IgG2; Recombinant monoclonal antibody; Glycosylation; Glycopeptide; High-throughput characterization; Normal phase liquid chromatography; MALDI-TOF MS;

LC–MS/MS-analysis of prostaglandin E2 and D2 in microdialysis samples of rats by Ronald Schmidt; Ovidiu Coste; Gerd Geisslinger (188-197).
For the determination of prostaglandins in microdialysis samples, usually immunoassays are used. However, these assays may show cross-reactivity among various prostaglandins. To overcome this problem a specific method for the determination of prostaglandin E2 and D2 in rat microdialysis samples by using liquid chromatography–electrospay ionization-tandem mass spectrometry (LC–ESI-MS/MS) is described. Prostaglandin E2 and D2 were extracted from microdialysis samples with liquid–liquid extraction using deuterated prostaglandin D2, [2H4]-PGD2, as internal standard. Subsequently, prostaglandins were separated with a phenomenex Synergi Hydro-RP column and determined with a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface operating in negative ionization mode. The method showed a LLOQ of 25 pg/ml for prostaglandin E2 and 50 pg/ml for prostaglandin D2. The applicability of the method is shown in rat spinal cord microdialysis samples following peripheral nociceptive stimulation.
Keywords: PGE2; PGD2; Chromatography; Tandem mass spectrometry; ESI; Microdialysis;

A quantitative and selective chromatography method for determining coverages of multiple proteins on surfaces by Michela Ombelli; Russell J. Composto; Qing Cheng Meng; David M. Eckmann (198-205).
Competitive protein adsorption plays a key role in the surface hemocompatibility of biological implants. We describe a quantitative chromatography method to measure the coverage of multiple proteins physisorbed to surfaces. In this method adsorbed proteins are displaced by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and then analyzed by high performance liquid chromatography to separate and quantify the individual proteins, in this case bovine serum albumin (BSA) and bovine fibrinogen (Fg). CHAPS displaced over 95% of the adsorbed proteins and was easily removed from solution by dialysis. This method was tested by measuring the coverage of BSA, 66 kDa, and Fg, 340 kDa, simultaneously adsorbed from solutions with concentration of 20 μg/ml, on bare and dextranized silicon. Relative to silicon, the dextranized surfaces were found to strongly inhibit protein adsorption, decreasing BSA and Fg coverages by 76 and 60%, respectively.
Keywords: Multiple protein coverages; SEC-HPLC; Biocompatible surfaces; Dextran;

Determination of dihydroxynaphthalenes in human urine by gas chromatography–mass spectrometry by Renan Wu; Suramya Waidyanatha; Alistair P. Henderson; Berrin Serdar; Yuxin Zheng; Stephen M. Rappaport (206-213).
A gas chromatography–mass spectrometry (GC–MS) method was developed for measuring 1,2-dihydroxynaphthalene (1,2-DHN) and 1,4-dihydroxynaphthalene (1,4-DHN) in urine. The method involves enzymatic digestion of urinary conjugates to release the DHNs which were then analyzed as trimethylsilyl derivatives by GC–MS. For 1,2-DHN and 1,4-DHN, respectively, the assay limits of detection were 0.21 and 0.15 μg/l, the assay limits of quantitation were 0.69 and 0.44 μg/l, and the coefficients of variation were 14.7 and 10.9%. This method was successfully applied to determine urinary levels of 1,2-DHN and 1,4-DHN in coke workers (14 top workers and 13 side-bottom workers) and 21 matching control workers from the steel industry of northern China. The geometric mean (GM) levels of 1,2-DHN were approximately 100 and 30 times higher than those of 1,4-DHN in exposed and control subjects, respectively. The GM levels 1,2-DHN and 1,4-DHN were significantly higher for coke workers (1,2-DHN: top workers – 552 μg/l, side-bottom workers – 260 μg/l; 1,4-DHN: top workers – 3.42 μg/l, side-bottom workers – 3.56 μg/l) than for controls (1,2-DHN: 38.8 μg/l; 1,4-DHN: 1.21 μg/l) (p  0.0031). In each exposure category, levels of the DHNs were marginally greater in smokers than in nonsmokers (p  = 0.0646). Strong correlations were observed among 1,2-DHN and 1,4-DHN and previously measured urinary levels of naphthalene, 1-hydroxynaphthalene, and 2-hydroxynaphthalene in these subjects (r s  ≥ 0.623; p  < 0.0001). Also, levels of 1,2-DHN were significantly correlated with those of serum albumin adducts of l,2-naphthoquinone (r s  = 0.492, p  = 0.0004). These results indicate that 1,2- and 1,4-DHN are good biomarkers for assessment of naphthalene exposure in coke workers. Since the DHNs are precursors of the naphthoquinones, which have been implicated as toxic products of naphthalene metabolism, measurements of urinary DHNs may have toxicological significance.
Keywords: 1,2-Dihydroxynaphthalene; 1,4-Dihydroxynaphthalene; Gas chromatography–mass spectrometry; Urine; Coke workers; Biomarker; Naphthalene;

A rapid, sensitive and selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of piroxicam, meloxicam and tenoxicam in human plasma was developed. Piroxicam, meloxicam, tenoxicam and isoxicam (internal standard) were extracted from human plasma with ethyl acetate at acidic pH and analyzed on a Sunfire column with the mobile phase of methanol:ammonium formate (15 mM, pH 3.0) (60:40, v/v). The analytes were detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in the multiple-reaction-monitoring (MRM) mode. The standard curve was linear (r  = 1.000) over the concentration range of 0.50–200 ng/ml. The coefficient of variation (CV) and relative error (RE) for intra- and inter-assay statistics at three QC levels were 1.0–5.4% and −5.9 to 2.8%, respectively. The recoveries of piroxicam, meloxicam and tenoxicam ranged from 78.3 to 87.1%, with that of isoxicam being 59.7%. The lower limit of quantification for piroxicam, meloxicam and tenoxicam was 0.50 ng/ml using a 100 μl plasma sample. This method was successfully applied to a pharmacokinetic study of piroxicam after application of transdermal piroxicam patches to humans.
Keywords: LC–MS/MS; Piroxicam; Meloxicam; Tenoxicam; Human plasma;

Bioanalysis and pharmacokinetics of the p38 MAPkinase inhibitor SB202190 in rats by Jai Prakash; Vinay Saluja; Jan Visser; Frits Moolenaar; Dirk K.F. Meijer; Klaas Poelstra; Robbert J. Kok (220-225).
We have developed a sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for the quantification of the p38 MAPkinase inhibitor SB202190 in serum, kidney homogenates and urine samples. Liquid–liquid extraction of SB202190 from the samples was performed using diethylether after adding a derivative of SB202190 as internal standard (I.S.). Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of acetonitrile–water–trifluoroacetic acid (30:70:0.1, v/v/v; pH 2.0). Both drug and I.S. were measured at 350 nm and eluted at 5.0 and 10.6 min, respectively. Peak-height ratios of the drug and the I.S. were used for the quantification of SB202190 from the different matrixes. The limit of quantitation of SB202190 in serum, kidney and urine were 0.25 μg/ml, 1 μg/g and 1 μg/ml, respectively. The average recoveries were 74, 75 and 92% in serum, kidney and urine, respectively. The intra- and inter-day precision (% CV) and accuracy (% bias) were below 15% for all concentrations. The method was successfully applied for a pharmacokinetic study of SB202190 in rats.
Keywords: Liquid–liquid extraction; High performance liquid chromatography; Pharmacokinetic studies; Drug analysis; Renal fibrosis;

The flavonoid compound mangiferin is found in the leaves, stem bark, fruit peels and root of Mangifera indica L. and in many other herbal species with many potential pharmacological properties. We have established an analytical method of mangiferin extracted from M. indica L. bark and Mangifera persiciformis C.Y. Wu et T.L. Ming leaves utilizing CZE. An electrolytic buffer containing 0.05 M borate buffer, pH 7.4 with methanol (1:0.3, v/v) was deemed suitable for mangiferin analysis. An ideal mangiferin electropherogram with a migration time at approximately 10.50 min was obtained. Repeatability tests showed that the R.S.D.s for both intra- and inter-day migration time and peak area for all manigferin sources tested were less than 4%. The linearity range of this method was 5–1000 μg/ml. The detection limit of this method was 1.5 μg/ml. Quantitative analysis of mangiferin was also performed with this method. The accuracy of quantitation at 10, 500 and 1000 μg/ml of control mangiferin were 99.00, 99.38 and 99.14%, respectively (n  = 10). The repeatability of quantitation (R.S.D.) was below 3%. Our results demonstrated that CZE is a simple and reliable method in mangiferin analysis and more studies are needed to detect other mangiferin resources, such as clinical biological samples, in pharmacology and pharmacokinetic studies.
Keywords: Capillary electrophoresis; Flavonoid; Mangiferin; Mangifera indica; Mangifera persiciformis;

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of capecitabine, its intermediate metabolites (DFCR, DFUR) and the active metabolite 5-fluorouracil in mouse plasma, liver and human xenograft tumours. The method was also cross-validated in human plasma and human tumour for clinical application. The method has greater sensitivity than previously published methods with an equivalent accuracy and precision. It uses less biological material (plasma, tissue) and should therefore be applicable to biopsies in patients treated with capecitabine.
Keywords: Capecitabine; 5-Fluorouracil; Quantification; Plasma; Tumour; Liver;

This study describes an high-performance liquid chromatographic (HPLC)–UV method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF) and cortisol (F) in human urine and plasma. The within-day relative standard deviation of the three concentrations for both analytes was less than 6.2%. Accuracy determined at three concentrations ranged between 95 and 107%. The extraction recoveries were 64.1 ± 4.3 and 88.1 ± 2.4% at three concentrations for 6β-OHF and F in urine, respectively. The extraction recoveries were 88.7 ± 1.4% at three concentrations for F in plasma. This is the first HPLC method that can simultaneously determine 6β-OHF and F in human urine and plasma and is suitable for routine assessment of the CYP3A activity expressed as 6β-hydroxylation clearance.
Keywords: 6β-Hydroxycortisol; Cortisol; HPLC; CYP3A;

Analysis of methylene blue in human urine by capillary electrophoresis by Holger Borwitzky; Walter E. Haefeli; Jürgen Burhenne (244-251).
A capillary electrophoresis method for the determination of the dye methylene blue (tetramethylthionine, MB) in human urine depending on liquid/liquid-extraction and diode array detection has been developed, validated, and applied to samples of healthy individuals, who had been dosed with methylene blue within clinical studies. After extraction with dichloromethane and sodium hexanesulfonate, sample extracts were measured on an extended light path capillary. The dye was detected simultaneously at 292 and 592 nm using methylene violet 3 RAX as internal standard. The limit of quantification was 1.0 μg/ml. The accuracy of the method varied between −15.2 and +0.8% and the precision ranged from 2.0 to 12.0%. The method was linear at least within 1.0 and 60 μg/ml. In contrast to earlier indirect determinations no leuco methylene blue (LMB) was directly detected in urine, whereas in aqueous test solutions containing surplus amounts of ascorbic acid leuco methylene blue was well separated from MB in a single run.
Keywords: Capillary electrophoresis; Methylene blue; Leuco methylene blue; Human urine; Validation;

Quantitative analysis of pyoluteorin in anti-fungal fermentation liquor of Pseudomonas species by capillary zone electrophoresis with UV–vis detector by Qiu-Ling Wang; Xue-Hong Zhang; Liu-Yin Fan; Wei Zhang; Yu-Qian Xu; Hong-Bo Hu; Cheng-Xi Cao (252.e1-252.e6).
This paper investigated potential utility of capillary zone electrophoresis (CZE) for very succinct but robust quantitative analysis of pyoluteorin (Plt) in anti-fungal fermentation liquor of Pseudomonas species. The experimental conditions for the separation and quantification of Plt were optimized at first. The optimized conditions are: 80 mmol/L pH 8.40 Gly-NaOH buffer, 51 cm total length (42 cm effective) and 75 μm I.D. capillary, 230 nm wavelength, 25 kV, 13 mbar 10 s pressure sample injection and 24 °C air-cooling. Under the optimized conditions, the migration times of Plt and the internal standard phenobarbital are 2.09 and 2.49 min, respectively, the linear response of Plt concentration ranges from 5.0 to 1000 μg/mL with high correlation coefficient (r  = 0.99977, n  = 9), the limits of detection (LOD) and quantification (LOQ) for Plt are 0.66 and 2.2 μg/mL, the precision values (expressed as R.S.D.) of intra- and inter-day are 1.19–1.94% and 1.55–6.21%, respectively, the recoveries of Plt at three concentration levels of 750, 250 and 50 μg/mL range from 90.31% to 97.85% and to 98.96%, respectively. The developed method can be well used for the quantification of Plt in the fermentation liquor.
Keywords: Capillary zone electrophoresis; Fermentation; Pseudomonas species; Pyoluteorin;

Simultaneous determination of adenine and guanine in ruminant bacterial pellets by ion-pair HPLC by Pilar García del Moral; María Jesús Arín; José Antonio Resines; María Teresa Díez (257-260).
An ion-pair reversed-phase high-performance liquid chromatography with gradient elution and UV detection was used to measure adenine (A) and guanine (G) in lyophilized bacterial pellets from ruminants using allopurinol as internal standard. The separation was performed on a Symmetry C18 column and the detection was monitored at 280 nm. Calibration curves were found to be linear in the concentration range from 5 to 50 mg/l with correlation coefficients (r 2) > 0.999. Mean recoveries of A and G standards added to bacterial samples were 102.2 and 98.2, respectively. The method proposed yielded sharp, well-resolved peaks within 25 min and was successfully applied for the determination of A and G in bacterial pellets.
Keywords: Purine bases: Adenine and Guanine; Ion-pair reversed-phase high-performance liquid chromatography;

Sensitive and accurate analyses of free 3-nitrotyrosine in exhaled breath condensate by LC–MS/MS by Thomas Göen; Alice Müller-Lux; Petra Dewes; Anita Musiol; Thomas Kraus (261-266).
The quantitative determination of 3-nitro-l-tyrosine, a biological marker for inflammatory processes, in exhaled breath condensate (EBC) is described. The clean-up and preconcentration was performed by solid phase extraction (SPE). After liquid chromatography the specific detection was performed by tandem mass spectrometry using electron spray ionisation and selected reaction monitoring (SRM). 13C9-3-nitrotyrosine was used as an internal standard. For reliability, tests for the precision of the method, the losses during preparation, a test for nitrating artifacts and the comparibility of calibrants in EBC and buffer solution were performed. The calibration of the method was linear over a range of 10–500 pg/mL. The within-run coefficients of variation (CV) of the samples were found to be 8.4% at 25 pg/mL and 8.3% at 250 pg/mL. The day-to-day CV was found to be 11.2%. The limit of quantification was 3.9 pg/mL. The losses during preparation were 15%. The discrepancy between the calibration with EBC and buffer solution was below 10%. No artificial production of 3-nitrotyrosine was observed during the procedure. The application of the method on the EBC samples of healthy smokers (N  = 10) and non-smokers (N  = 10) showed no difference between the two groups. The concentration of 3-nitrotyrosine ranged between the limit of quantification and 184 pg/mL and was distinctly lower than data detected by an immunoassay procedure. The procedure was proven to be accurate, sensitive and in contrast to GC methods less elaborate and is recommended for the determination of 3-nitrotyrosine in exhaled breath condensate.
Keywords: Biological effect monitoring; Respiratory symptoms; Exhaled breath condensate; Oxidative stress; Nitric oxide;

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C18 column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 μg/ml for iohexol and iothalamate, 5 to 40 μg/ml for PAH and 2.5 to 40 μg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.
Keywords: Iohexol; Iothalamate; p-Aminohippuric acid (PAH); n-Acetyl-PAH; ERPF; GFR; HPLC;

Simultaneous liquid chromatographic determination of doxorubicin and its major metabolite doxorubicinol in parrot plasma by Catherine M. Gilbert; Ross P. McGeary; Lucio J. Filippich; Ross L.G. Norris; Bruce G. Charles (273-276).
A new microscale method is reported for the determination of doxorubicin and its active metabolite, doxorubicinol, in parrot plasma. Sample workup involved acetonitrile protein precipitation, ethyl acetate extraction, followed by back extraction into HCl. Separations were achieved on a phenyl-hexyl column at 30 °C using acetonitrile (17%, v/v) in 0.01 M orthophosphoric acid (83%, v/v) delivered via a linear flow program. Fluorometric detection wavelengths were 235 nm (excitation) and 550 nm (emission). Calibration plots were linear (r 2  > 0.999), and recoveries were 71–87% from 20 to 400 ng/mL. Assay imprecision was ≤8.5% and inaccuracy was ≤6.3%. The limit of quantification was 25 ng/mL.
Keywords: Doxorubicin; Doxorubicinol; Parrot; Liquid chromatography;

Contamination in HPLC quantified benzaldehyde is from polypropylene microtubes by Tomer Shemesh; Connie Karschimkus; Kevin G. Rowley (277-278).
Semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6) is a copper-containing enzyme predominantly expressed by vascular smooth muscle cells. SSAO deaminates primary amines to produce aldehydes and oxygen peroxides, and may thus play a role in vascular damage. SSAO activity can be quantified by assaying benzaldehyde production using fluorescent derivatisation and separation by HPLC. We performed the derivatisation step in polypropylene or borosilicate glass tubes over 45 min at 95 °C. High and obstructing background levels of benzaldehyde were found in one batch of polypropylene vials, as opposed to its alternatives. Treatment and handling of product shipment into the country did not account for introduction of contaminant into packaged vials nor did any reagent used in the assay. We conclude that the source of contamination was most likely due to variation in the commercial production process. Use of borosilicate vials for assays based on aldehyde production and derivatisation is recommended.
Keywords: Semicarbazide-sensitive amine oxidase; HPLC; Benzaldehyde;

Author Index (279-281).

Keyword Index (282-287).