Journal of Chromatography B (v.857, #2)

Investigation of the lipophilic behaviour of some thiazolidinediones by Costas Giaginis; Stamatios Theocharis; Anna Tsantili-Kakoulidou (181-187).
Various lipophilicity aspects of five well-known PPAR-γ ligands, belonging to the thiazolidinedione (TZD) class, ciglitazone (CSZ), troglitazone (TGZ), netoglitazone (NGZ) and the ampholytic pioglitazone (PGZ) and rosiglitazone (RGZ), have been explored. The compounds were found to be highly lipophilic as assessed by direct octanol–water partitioning experiments and further confirmed by reversed phase HPLC measurements under different conditions. Immobilised artificial membrane (IAM) chromatographic indices were also determined as an alternative expression of lipophilicity. They were found to show less diversity forming two clusters. Experimental log  D/log  P values were compared to those predicted by three widely used calculation systems. For the two ampholytic TZDs, the lipophilicity and retention/pH profiles were established over a broad pH range and compared to the corresponding calculated profiles. Lipophilicity indices derived under the different conditions were further compared to biological activity, concerning in vitro transactivation (pEC50) and binding affinity (pK i) data, taken from literature. The most active TZD (RGZ) in both transactivation and binding assay proved to be the less lipophilic analogue. An equation relating pEC50 data to experimental log  D 7.4 or reversed-phase log  k w values could be established, while pK i data did not lead to satisfactory correlation.
Keywords: Thiazolidinediones; PPAR-γ ligands; Lipophilicity; n-Octanol/water partitioning; RP-HPLC; IAM chromatography;

Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method by Milan Blanusa; Iva Perovic; Milica Popovic; Natalija Polovic; Lidija Burazer; Mina Milovanovic; Marija Gavrovic-Jankulovic; Ratko Jankov; Tanja Cirkovic Velickovic (188-194).
A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study were theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use.
Keywords: Actinidin; Act c 1; Allergen; Art v 1; Ion-exchange; HPLC; Quantification; Kiwi fruit; Mugwort; Pollen;

We have developed and validated a method for the quantification of fentanyl, a synthetic opioid, in dog plasma by on-line SPE with a hydrophilic column coupled to tandem mass spectrometry in positive electrospray mode. A column-switching instrument with 10-port valve and two HPLC pumping systems were employed. Deuterated fentanyl served as the internal standard. A Waters Oasis HLB extraction column and a Waters Atlantis HILIC Silica analytical column in a column-switching set-up with gradient elution were utilized. Both fentanyl (analyte) and the internal standard (fentanyl-d5) were determined via multiple reaction monitoring (MRM) and the MS/MS ion transitions monitored were m/z 337.0/188.0 and 342.0/188.0, respectively. Each plasma sample was chromatographed within 5 min. The calibration curves were linear over a widely range of 0.01–50 ng/mL using weighted linear regression analysis (1/x). The low limit of quantitation was 0.01 ng/mL. The intra- and inter-day accuracy ranged from 102 to 112% and the overall precision was less than 3%. The recoveries ranged from 90 to 105% in plasma at the concentrations of 0.04, 0.4, 4 and 40 ng/mL. No influence of freeze/thaw and long-term stability were observed. This validated method has been successfully applied to analyze the dog plasma samples of a pharmacokinetics study.
Keywords: Fentanyl; On-line SPE; HILIC; LC/MS/MS; Bioanalysis; Pharmacokinetics;

The major active biological constituents in Citrus herbs are flavonoids, especially hesperidin, naringin and alkaloids, mainly synephrine, with beneficial medical effects on human health. They are used as the markers to control the quality of Citrus herbs. In this paper, a new ion pairing chromatographic method was developed to exclude the most polar solute (synephrine) from the viod volume and to maintain selectivity between the two other solutes (hesperidin and naringin). Perfluorinated carboxylic acids, which are appropriate for MS detection due to their volatility, were used as ion-pairing agents. The problems of the synephrine separation, such as band tailing and low retention, were solved successfully by using perfluorinated carboxylic acids. The effect of heptafluorobutyric acid (HFBA) was the best in the three investigated perfluorinated carboxylic acids. For the flavanone glycosides, the influence of the perfluorinated acids on retention time was rather weak. The two different kinds of the analytes were separated satisfactorily in one run using an isocratic eluent and the total analysis time takes less than 10 min. The abundance of pseudomolecular ions was recorded using selected ion monitoring (SIM) mode of m/z 135.1, 273.1 and 303.1 for synephrine, naringin and hesperidin, respectively. The contents of hesperidin, naringin and synephrine in several Citrus herbs were simultaneously determined by the proposed method.
Keywords: HPLC-DAD-ESI/MS; Perfluorinated carboxylic acids; Hesperidin; Naringin; Synephrine; Citrus herbs;

Purification and analysis of a 5 kDa component of enamel matrix derivative by Alexandra Mumulidu; Bergisa Hildebrand; Beata Fabi; Lars Hammarström; David L. Cochran; Michel Dard; Stephanie Lemoult (210-218).
High performance liquid chromatography (HPLC) methods were used to analyse a 5 kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain®, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm × 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5 kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5 kDa component. From 1200 mg of EMD (of which 9% is the 5 kDa component), approximately 65 mg of lyophilised 5 kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.
Keywords: Size-exclusion HPLC; Reversed-phase HPLC; Protein purification; Qualitative analysis; Quantitative analysis; Method validation; Fractionation; Enamel matrix derivative; Emdogain;

Estimation of carvedilol in human plasma by using HPLC-fluorescence detector and its application to pharmacokinetic study by Rajeshwari Rathod; L. Poorna Chandra Prasad; Shubha Rani; Manish Nivsarkar; Harish Padh (219-223).
A simple, precise and sensitive high performance liquid chromatography procedure has been developed for determination of carvedilol in human plasma. The method was developed on Lichrosphere R CN column using a mobile phase of acetonitrile/20 mM ammonium acetate buffer with 0.1% triethylamine (pH adjusted to 4.5) (40/60, v/v). The peaks were detected by using fluorescence detector (excitation wavelength 282 nm and emission wavelength 340 nm). Carvedilol and domperidone (internal standard) were extracted by liquid–liquid extraction procedure using dichloromethane. This method was specific and had a linearity range of 1–128 ng/ml with intra- and inter-day precision (%C.V.) less than 15%. The accuracy ranges from 87.3 to 100.88% and the recovery of carvedilol was 69.90%. The stability studies showed that carvedilol in human plasma was stable during short-term period for sample preparation and analysis. This method was used to assay the carvedilol in human plasma samples obtained from subjects who had been given an oral tablet of 12.5 mg carvedilol.
Keywords: Carvedilol; HPLC-fluorescence detector; Liquid–liquid extraction; Pharmacokinetics;

A chiral selective, accurate and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for direct separation of individual enantiomers of torcetrapib (TTB) [(+)-TTB and (−)-TTB]. TTB enantiomers and IS were extracted from a small aliquot of plasma (100 μL) by simple liquid–liquid extraction using acetonitrile as extraction solvent. The enantiomers were resolved on Chiralpak AD-H® (250 mm × 4.6 mm, 5 μm) with the mobile phase consisting of n-hexane:isopropyl alcohol (IPA) in the ratio of 95:5 (v/v). The eluate was monitored using an UV detector set at 254 nm. Baseline separation of the TTB enantiomers and the internal standard (IS, DRL-17859), free from endogenous interferences was achieved. The resolution factor between the enantiomers was optimized and found to be not less than five. During method development, the IPA content in the mobile phase was optimized for separation of peaks of interest. Additionally, both flow rate and column temperature were optimized for an improved baseline separation of the enantiomers. Ratio of peak area of each enantiomer to IS was used for quantification of plasma samples. Nominal retention times of (+)-TTB, (−)-TTB and IS were 9.4, 13.8 and 17.5 min, respectively. The standard curves for TTB enantiomers were linear (r 2  > 0.999) in the concentration range 0.1–10 μg/mL for each enantiomer. Absolute recovery, when compared to neat standards, was 88.7–90.0% for TTB enantiomers and 100% for IS from the hamster plasma. The lower limit of quantification (LLOQ) for each enantiomer of TTB was 0.1 μg/mL. The inter-day precisions were in the range of 4.57–6.32 and 5.66–11.0% for (+)-TTB and (−)-TTB, respectively. The intra-day precisions were in the range of 1.60–7.36 and 2.76–13.6% for (+)-TTB and (−)-TTB, respectively. Accuracy in the measurement of quality control (QC) samples was in the range of 95.6–109% and 92.7–108% for (+)-TTB and (−)-TTB, respectively. Both enantiomers were stable in a series of stability studies, viz. bench-top (up to 12 h), auto-sampler (up to 24 h) and freeze/thaw cycles (n  = 3). Stability of TTB enantiomers was established in hamster plasma for 15 days at −80 °C. The application of the assay to a pharmacokinetic study of (−)-TTB in hamsters is described.
Keywords: Torcetrapib; Enantioselective; Quantitation; Chiralpak AD-H®; Chirality; HPLC–UV; Hamsters; Pharmacokinetics;

Purification and characterization of RGD tumor-homing peptide conjugated human tumor necrosis factor α over-expressed in Escherichia coli by Dingyuan Ma; Yuan Chen; Lei Fang; Guanghui Jin; Bin Zhou; Lin Cao; Jianqiang Ye; Zichun Hua (231-239).
A number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor α (TNFα) to permit its systemic use in cancer therapy. Because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of TNFα, we prepared RGD-hTNF consisting of human TNF fused with the ACDCRGDCFCG peptide, a ligand of αvβ3 and αvβ5 integrins. Recombinant RGD-hTNF was produced in Escherichia coli as a polyhistidine fusion protein. Between polyhistidine tag and RGD-hTNF, a tobacco etch virus (TEV) protease cleavage site (ENLYFQG) was introduced to ensure the release of intact RGD-hTNF. The purification strategy consisted of the target protein capture step by immobilized metal affinity chromatography (IMAC), TEV protease cleavage of fusion protein, the subtractive depletion of removed His-tag by IMAC and the final gel filtration step. As a result, about 18 mg of intact RGD-hTNF was obtained from 1 l of bacteria culture. The purified RGD-hTNF was characterized by SDS-PAGE, Western blot, mass spectroscopy and gel filtration. Since the RGD-hTNF molecule retained the cytotoxic activity of the TNF moiety and the integrin binding ability of the RGD moiety, the purification method provided material for assessing its anti-tumor activity in animal model.
Keywords: Tumor necrosis factor α; RGD peptide; Fusion protein; Immobilized metal affinity chromatography; Enterokinase; TEV protease;

On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides by Gushinder Kaur-Atwal; Daniel J. Weston; Philip S. Green; Susan Crosland; Philip L.R. Bonner; Colin S. Creaser (240-245).
Capillary column immobilised metal affinity chromatography (IMAC) has been combined on-line with electrospray ionisation/quadrupole time-of-flight mass spectrometry for the fractionation of histidine-containing peptides. IMAC beads (Poros 20MC, 20 μm) containing imidodiacetate chelating groups on a cross-linked poly(styrene-divinylbenzene) support were packed into a fused silica column (250 μm i.d.), which was interfaced to the electrospray ion source of the spectrometer. A Cu(II) activated column was used to isolate histidine-containing peptides from tryptic and other peptide mixtures with an average breakthrough of 9.1%, to reduce the complexity of the mass spectral analysis. The analysis cycle time was reduced to less than 15 min, at an optimum flow rate of 7.5 μL/min, without sacrificing peptide selectivity. Direct coupling of capillary IMAC with MS allows on-line separation, using MS compatible loading and elution buffers, and detection in a high-throughput fashion when compared to off-line strategies.
Keywords: Capillary LC–MS; Immobilised metal affinity chromatography; Electrospray ionisation mass spectrometry; Copper(II); Histidine-containing peptides;

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method with ultraviolet detector (UV) has been developed for the determination of bifendate in 100 μl plasma of rats. Sample preparation was carried out by deproteinization with 100 μl of acetonitrile. A 20 μl of supernatant was directly injected into the HPLC system with methanol–double distilled water (65/35, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed with a μBondapak C18 column at 30 °C. The peak was detected at 278 nm. The calibration curve was linear (r 2  = 0.9989) in the concentration range of 0.028–2.80 μg/ml in plasma. The intra- and inter-day variation coefficients were not more than 6.55% and 6.07%, respectively. The limit of detection was 5 ng/ml. The mean recoveries of bifendate were ranged from 94.53% to 99.36% in plasma. The present method has been successfully applied to the pharmacokinetic study of bifendate liposome in rats.
Keywords: Bifendate;

Acylcarnitine profiles have been used to diagnose specific inherited metabolic diseases. For some acylcarnitines, however, the detailed structure of their acyl group remains a question. One such incompletely characterized acylcarnitine is cis-3,4-methylene-heptanoylcarnitine. To investigate this problem, we isolated the “C8:1” acylcarnitine from human urine, transesterified it to form its acyl picolinyl ester, and characterized it by GC/EI-MS. These results were compared to GC/EI-MS results from authentic standards we synthesized (cis-3,4-methylene-heptanoylcarnitine, trans-2-octenoylcarnitine, 3-octenoylcarnitine, cis-4-octenoylcarnitine, and trans-4-octenoylcarnitine). Only cis-3,4-methylene-heptanoylcarnitine matched the urinary “C8:1” acylcarnitine. The standards were then spiked in human urine, converted to pentafluorophenacyl esters, and detected by HPLC/MS. cis-3,4-Methylene-heptanoylcarnitine exactly matched the “C8:1” acylcarnitine in urine, whereas none of the other C8:1 acylcarnitine standards matched. Based on the data from GC/EI-MS and HPLC/MS, the “C8:1” acylcarnitine in human urine is shown to be cis-3,4-methylene-heptanoylcarnitine.
Keywords: Acylcarnitines; cis-3,4-Methylene-heptanoylcarnitine; GC/MS; HPLC/MS;

A selective and sensitive screening method for the detection of prohibited narcotic and stimulating agents in doping control is described and validated. This method is suitable for the detection of all narcotic agents mentioned on the World Anti-Doping Agency (WADA) doping list in addition to numerous stimulants. The analytes are extracted from urine by a combined extraction procedure using CH2Cl2/MeOH (9/1, v/v) and t-butylmethyl ether as extraction solvents at pH 9.5 and 14, respectively. Prior to GC–MS analysis the obtained residues are combined and derivatised with MSTFA. The mass spectrometer is operated in the full scan mode in the range between m/z 40 and 550. The obtained limits of detection (LOD) for all components included in this extensive screening method are in the range 20–500 ng/ml, which is in compliance with the requirements set by WADA. Besides narcotic and stimulating agents, this method is also capable of detecting several agents with anti-estrogenic activity and some beta-agonists. As an example, a positive identification of hydroxyl-methoxy-tamoxyfen is shown.
Keywords: GC–MS; Doping; Narcotic agents; Stimulants; Derivatisation;

In vitro phase I metabolism of BYZX, a novel central-acting cholinesterase inhibitor for the treatment of the symptoms of Alzheimer's disease, was studied in human liver microsomes (HLM) and the metabolite formation pathways were investigated by chemical inhibition experiments and correlation analysis. The residual concentration of substrate and the metabolite formed in incubate were determined by HPLC method. The calibration curves of BYZX were linear over the concentration range from 5.07 μM to 200.74 μM. The relative standard deviations of within day and between day were less than 5% (n  = 5). The limit of detection (LOD) was 0.18 μg/mL (S/N = 3) and the limit of quantification (LOQ) was 0.55 μg/mL (R.S.D. = 5.2%, n  = 5). The determination recoveries of BYZX were in the range of 98.2–104.8%. The apparent K m of BYZX in HLM was 53.25 ± 17.2 μM, the V max was 0.94 ± 0.77 μM/min/mg protein, and the intrinsic clearance value (Clint) was 0.018 ± 0.02 mL/min/mg protein. Ketoconazole and cyclosporin A were the most potent inhibitors on BYZX metabolism in HLM with IC50 being 0.89 μM and 18.17 μM, respectively. And the inhibition constant (K i) of ketoconazole was 0.42 μM. The metabolite of BYZX was N-des-ethyl-BYZX elucidated by LC–MS-MS. The results demonstrated that the developed HPLC method was reliability, simple technique, and was applicable to be used for the researches of in vitro metabolism of BYZX. CYP3A4 was the major isozyme responsible for BYZX metabolism; N-dealkylation was the major metabolic pathway of BYZX. The predominant metabolite of BYZX was N-des-ethyl-BYZX detected in vitro phase I metabolism in HLM.
Keywords: BYZX; In vitro metabolism; HPLC–MS-MS; Human liver microsomes (HLM);

Fast urinary screening for imipramine and desipramine using on-line solid-phase extraction and selective derivatization by Marta Cruz-Vera; Rafael Lucena; Soledad Cárdenas; Miguel Valcárcel (275-280).
A continuous-flow configuration based on sequential solid-phase extraction and derivatization is proposed for the screening of urine samples for imipramine and related metabolites. For the first time, a 50/50 (v/v) methanol/nitric acid mixture is used as both eluent and derivatizing reagent. Sample aliquots are injected into the flow manifold and driven by a water stream to an RP-C18 column where the drugs are quantitatively retained. Following clean-up step with 40/60 (v/v) methanol/water, the eluent/derivatizing reagent is injected and passed through the sorbent column, eluted drugs reacting with nitric acid to form a blue dye that is monitored at 600 nm. The global signal thus obtained for the antidepressants can be used to estimate their total concentration in the samples without the need to individually quantify the analytes. This total index can be used for timely decision-making in case of overdosage. The proposed method is sensitive and selective; thus, typical interferents such as endogenous and diet compounds have no substantial effect on the analytical signal. This allows imipramine and its metabolites to be determined at therapeutic levels in urine samples.
Keywords: Imipramine; Desipramine; Tricyclic antidepressants; Urine samples; Flow analyser; Solid-phase extraction; Derivatization;

Identification of amygdalin and its major metabolites in rat urine by LC–MS/MS by B.Y. Ge; H.X. Chen; F.M. Han; Y. Chen (281-286).
Amygdalin and its metabolites in rat urine were identified using liquid chromatography–electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (ΔM), retention times and MS2 spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.
Keywords: Amygdalin; LC–MS/MS; Metabolite; Rat urine;

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-15N2] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M−H) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08–0.18% and between-set CVs of 0.02–0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.
Keywords: Uric acid; Serum; LC/ID-MS; Candidate reference method;

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C18 bonded silica column with a deionized water–methanol–acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 μg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1–20 μg/kg were in the range of 71.4–99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 μg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 μg/kg were in the range of 6.2–13.9% and 4.0–8.7%, respectively. The intra-day precision (n  = 5) for nitroimidazoles residues in chicken spiked at 20 μg/kg is 6.9%, and the inter-day precision for 5 days (n  = 25) is 11%. The method is capable of identifying nitroimidazole residues at ≥0.7 μg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.
Keywords: Solid-phase extraction; High-performance liquid chromatography; Nitroimidazoles; Multi-residue analysis; Meat sample;

Enantioselective quantification of omeprazole and its main metabolites in human serum by chiral HPLC–atmospheric pressure photoionization tandem mass spectrometry by Jens Martens-Lobenhoffer; Ines Reiche; Uwe Tröger; Klaus Mönkemüller; Peter Malfertheiner; Stefanie M. Bode-Böger (301-307).
Omeprazole is a proton pump inhibitor drug in widespread use for the reduction of gastric acid production. It is also proposed as a test substance for the phenotyping of cytochrome CYP3A4 and CYP2C19 enzyme activities. For this purpose, it is necessary to quantify, additionally to omeprazole, the two main metabolites 5-hydroxyomeprazole and omeprazole-sulfon in human plasma. Since omeprazole is a racemic mixture of two enantiomers and its enzymatic decomposition depends in part on its chiral configuration, full information about its metabolic breakdown can only be gained by enantioselective quantification of the drug and its metabolites. We introduce a new LC–MS/MS method that is capable to simultaneously quantify omeprazole and its two main metabolites enantioselectively in human serum. The method features solid-phase extraction, normal phase chiral HPLC separation and atmospheric pressure photoionization tandem mass spectrometry. As internal standards serve stable isotope labeled omeprazole and 5-hydroxyomeprazole. The calibration functions are linear in the range of 5–750 ng/ml for the omeprazole enantiomers and omeprazole-sulfon, and 2.5–375 ng/ml for the 5-hydroxyomeprazole enantiomers, respectively. Intra- and inter-day relative standard deviations are <7% for omeprazole and 5-hydroxyomeprazole enantiomers, and <9% for omeprazole-sulfon, respectively.
Keywords: Omeprazole; Omeprazole-sulfon; 5-Hydroxyomeprazole; Chiral separation; APPI; LC–MS/MS;

The combination of two sensitive, selective and reproducible reversed phase liquid chromatographic (RP-HPLC) methods was developed for the determination of artesunate (AS), its active metabolite dihydroartemisinin (DHA) and mefloquine (MQ) in human plasma. Solid phase extraction (SPE) of the plasma samples was carried out on Supelclean LC-18 extraction cartridges. Chromatographic separation of AS, DHA and the internal standard, artemisinin (QHS) was obtained on a Hypersil C4 column with mobile phase consisting of acetonitrile–0.05 M acetic acid adjusted to pH 5.2 with 1.0 M NaOH (42:58, v/v) at the flow rate of 1.50 ml/min. The analytes were detected using an electrochemical detector operating in the reductive mode. Chromatography of MQ and the internal standard, chlorpromazine hydrochloride (CPM) was carried out on an Inertsil C8-3 column using methanol–acetonitrile–0.05 M potassium dihydrogen phosphate adjusted to pH 3.9 with 0.5% orthophosphoric acid (50:8:42, v/v/v) at a flow rate of 1.00 ml/min with ultraviolet detection at 284 nm. The mean recoveries of AS and DHA over a concentration range of 30–750 ng/0.5 ml plasma and MQ over a concentration of 75–1500 ng/0.5 ml plasma were above 80% and the accuracy ranged from 91.1 to 103.5%. The within-day coefficients of variation were 1.0–1.4% for AS, 0.4–3.4% for DHA and 0.7–1.5% for MQ. The day-to-day coefficients of variation were 1.3–7.6%, 1.8–7.8% and 2.0–3.4%, respectively. Both the lower limit of quantifications for AS and DHA were at 10 ng/0.5 ml and the lower limit of quantification for MQ was at 25 ng/0.5 ml. The limit of detections were 4 ng/0.5 ml for AS and DHA and 15 ng/0.5 ml for MQ. The method was found to be suitable for use in clinical pharmacological studies.
Keywords: Artesunate; Dihydroartemisinin; Mefloquine; SPE; RP-HPLC-EC; RP-HPLC-UV; Pharmacological studies;

This report describes a procedure for purification of large conductance calcium-activated potassium (BK, maxi-K) channels using immobilised metal affinity chromatography (IMAC) under non-denaturing conditions. An amino-terminal histidine fusion tag was added to hSlo, the human BK channel, and expressed in Sf9 insect cells. Following IMAC purification and production of proteoliposomes, protein function was assessed electrophysiologically in planar bilayer lipid membranes. Single channel openings had conductances of 250–300 pS and were inhibited by paxilline, demonstrating that the BK channels remained functional following IMAC purification. This method to obtain functional human ion channels will be useful in assays to screen potential pharmaceuticals.
Keywords: IMAC; His-tag; Ion channel; Sf9 insect cell; Purification; Planar lipid bilayer;

A sensitive and rapid high-performance liquid chromatographic method for the analysis of fluvoxamine, a selective serotonin reuptake inhibitor in human serum, is described using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent. The drug and an internal standard (fluoxetine) were extracted from 0.25 mL of serum using ethyl acetate as extracting solvent and subjected to pre-column derivatization by the reagent. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.8) containing 1 mL/L triethylamine (72:28 v/v) was used and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm × 4.6 mm) column. The fluorescence derivatives of the drugs were monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. The calibration curve was linear over the concentration range of 0.5–240 ng/mL with a limit of quantification (LOQ) of 0.5 ng/mL using 0.25 mL serum sample. The method validation was performed for its selectivity, specificity, sensitivity, precision and accuracy. In this method, which was applied in a randomized cross-over bioequivalence study of two different fluvoxamine preparations in 24 healthy volunteers, the sensitivity and run time of analysis were significantly improved.
Keywords: HPLC; Fluvoxamine; Serum; 4-Chloro-7-nitrobenzofurazan; NBD-Cl; Derivatization;

Quantification of darunavir (TMC114) in human plasma by high-performance liquid chromatography with ultra-violet detection by Lauriane Goldwirt; Stéphanie Chhun; Elisabeth Rey; Odile Launay; Jean-Paul Viard; Gérard Pons; Vincent Jullien (327-331).
A precise and accurate high-performance liquid chromatography (HPLC) method with UV detection has been developed and validated for darunavir, a peptidic protease inhibitor. An internal standard, methylclonazepam, was added to 100 μL of plasma before a solid-phase extraction on C18 Bond Elut column. The eluted solutions were evaporated to dryness and reconstitued with 100 μL of mobile phase before being injected in the chromatographic system. The separation was performed on a C8 column using an acetonitrile and ultrapure water mixture (40:60, v/v) as mobile phase. All compounds were detected at a wavelength of 266 nm. The method was linear and validated over a concentration range of 0.25–20 mg/L. The within-day precision, ranged from 3.0 to 7.9%, while the within-day accuracy ranged from −11.4 to 0.5%. The between day precision and accuracy were respectively less than 13.7 and −11.4%. The mean recovery was 75.7% for darunavir and 66.7% for methylclonazepam. This method provides a useful tool for therapeutic drug monitoring in HIV patients.
Keywords: TMC114; Darunavir; Liquid chromatography; UV detection;

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for determination of scopoletin in rat plasma using psoralen as internal standard. Chromatographic separation was achieved on a C18 column using methanol and distilled water (49:51, v/v) containing 0.05% (v/v) phosphoric acid as mobile phase. The UV detector was set at 345 nm. The calibration curve was linear over the range of 0.165–9.90 μg/ml with a correlation coefficient of 0.9994. The recovery for plasma samples of 0.165, 1.32 and 6.60 μg/ml was 93.2%, 95.9% and 95.5%, respectively. The RSD of intra- and inter-day assay variations was less than 6.7%. This HPLC assay is a precise and reliable method for the analysis of scopoletin in pharmacokinetic studies.
Keywords: Scopoletin; HPLC; Plasma; Pharmacokinetics;

An overpressured layer chromatography (OPLC) method was evaluated for broad-scale screening of basic drugs in 5 g autopsy liver samples using two parallel OPLC systems. Sample preparation included enzymatic digestion with trypsin and liquid–liquid extraction with butyl chloride. Chromatographic separation was performed as dual-plate analysis, with mobile phases composed of trichloroethylene–methylethylketone–n-butanol–acetic acid–water (17:8:25:6:4, v/v) (OPLC1), and butyl acetate–ethanol (96.1%)–tripropylamine–water (85:9.25:5:0.75, v/v). Identification was based on automated comparison of corrected R f values (hRfc) and in situ UV spectra with library values by dedicated software. The identification limit was determined for 25 basic drugs in liver ranging from 0.5 to 10 mg/kg. The OPLC method proved to be well suited for routine screening analysis of basic drugs in post-mortem samples of varying quality, combining the benefit from moderately high separation power with the ease of disposable plates.
Keywords: Drug screening; Tissue; Planar chromatography; OPLC;

Improved RP-HPLC method to determine neferine in dog plasma and its application to pharmacokinetics by Libo Zhao; Xiaomin Wang; Jianhong Wu; Yongsheng Jia; Wenquan Wang; Shaohui Zhang; Jialing Wang (341-346).
Existing methods to determine neferine, a bisbenzylisoquinline alkaloid, either have no internal standard or lack selectivity, or take longer time. Here an improved reverse-phase high-performance liquid chromatographic (RP-HPLC) method was established in biological samples. The extraction recovery was 90.9% for neferine at concentration level of 0.2 μg/ml and 77.7% for dauricine (the internal standard) at 5 μg/ml in dog plasma, respectively. The linear quantification range of the method was 25–2 000 ng/ml in dog plasma, with linear correlation coefficients greater than 0.999. The intra-day and inter-day relative standard deviations (R.S.D.s) for nerferine at 50, 200 and 1 000 ng/ml levels in dog plasma fell in the range of 3.0–5.4% and 4.3–9.5%, respectively. The RP-HPLC method was successfully applied to a pharmacokinetics study, in which experimental dogs received a single dose of neferine (5 mg/kg i.v. or 10 mg/kg p.o.). The pharmacokinetic result was presented.
Keywords: Improved; RP-HPLC; Neferine; Dog plasma; Pharmacokinetics;

Indirect determination of pyruvic acid by capillary electrophoresis with amperometric detection by Xin Lu; Wei-Hua Huang; Feng Ai; Zong-Li Wang; Jie-Ke Cheng (347-351).
A method of indirectly measuring pyruvic acid (PA) by capillary electrophoresis with amperometric detection is proposed for the first time. It is based on the oximation reaction between PA and hydroxylamine (NH2OH), and the quantification of PA was performed by direct and sensitive amperometric detection of excessive NH2OH after the oximation reaction. This method displayed a good sensitivity, and the detection limits of NH2OH and PA are 1.76 × 10−7 and 3.88 × 10−7  mol/L, respectively at S/N = 3. The linear relationship between the peak current and PA concentration is exhibited over the range from 4 × 10−6 to 1 × 10−4  mol/L. This method has been applied to determine PA in rat plasma with satisfactory results.
Keywords: Capillary electrophoresis; Amperometric detection; Carbon fiber microelectrode; Hydroxylamine; Pyruvic acid;

Determination of vancomycin in serum by liquid chromatography–high resolution full scan mass spectrometry by T. Zhang; D.G. Watson; C. Azike; J.N.A. Tettey; A.T. Stearns; A.R. Binning; C.J. Payne (352-356).
A liquid chromatography–mass spectrometry (LC–MS) method was developed for the analysis of vancomycin (VCM) in human serum. The method was based on full scan data with extracted ions for the accurate masses of VCM and the atenolol internal standard obtained by Fourier transform MS. VCM was extracted from serum using strong cation exchange (SCX) solid phase extraction (SPE). The method was found to be linear in the range 0.05–10 μg/ml, which was adequate for quantification of VCM in serum samples, with a limit of quantification (LOQ) of 0.005 μg/ml and a limit of detection (LOD) of 0.001 μg/ml. Intra-day precision (n  = 5) was ±3.5%, ±2.5%, ±0.7% at 0.05, 0.5 and 5 μg/ml, respectively. Inter-day precision (n  = 5) was ±7.6%, ±6.4%, ±3.9% at 0.05, 0.5 and 5 μg/ml, respectively. The process efficiency for VCM was in the range 89.2–98.1% with the recovery for the atenolol internal standard (IS) being 97.3%. The method was used to determine VCM levels in patients during peri-operative infusion of the drug, which was found to result in drug levels within the required therapeutic window.
Keywords: Vancomycin; Serum; Fourier transform mass spectrometry; Ion suppression;