Journal of Chromatography B (v.792, #2)
Editorial Board (OFC).
News Section (N1-N3).
Fingerprint analysis of Flos Carthami by capillary electrophoresis by Yi Sun; Tao Guo; Yin Sui; Famei Li (147-152).
Capillary electrophoresis (CE) was employed in fingerprint analysis of Flos Carthami. A standardized procedure was used to develop the CE fingerprint. An electrophoretic profile of genuine Flos Carthami from Fengqiu, He’nan, China, was first established as the characteristic fingerprint. This profile was then used to identify and assess the consistency of the herb. A study with a limited number of samples from nine sources showed a fair consistency in their CE fingerprints with that of the genuine sample. Flos Carthami was well distinguished from Stigma Croci, a possible substitute in traditional Chinese medicine, and Flos Hemerocallis, a commercial adulterant, by comparing the fingerprints of each herb.
Synthesis, characterization, and use of 2-[(2H9)butoxy]acetic acid and 2-(3-methylbutoxy)acetic acid as an internal standard and an instrument performance surrogate, respectively, for the gas chromatographic–mass spectrometric determination of 2-butoxyacetic acid, a human metabolite of 2-butoxyethanol by Kenneth K. Brown; Kenneth L. Cheever; Mary Ann Butler; Peter B. Shaw; Jeffery L. McLaurin (153-166).
2-[(2H9)Butoxy]acetic acid and 2-(3-methylbutoxy)acetic acid were synthesized, mixed with 2-butoxyacetic acid, and separated by capillary gas chromatography on a fused-silica column with a length of 50 m, inside diameter of 0.200 mm, and a “free fatty acid phase” wall coating of 0.3 μm film. 2-[(2H9)Butoxy]acetic acid, 2-butoxyacetic acid, and 2-(3-methylbutoxy)acetic acid were baseline resolved at retention times of 13.55, 13.78, and 15.20 min; 2-(3-methylbutoxy)acetic acid having a peak efficiency of 360 000. Mass spectrometric detection using selected ion monitoring at m/z 66, 57, and 71 showed linear analytical responses from 0.04 ng to at least 200 ng with a limit of detection of 0.04 ng for 2-butoxyacetic acid.
Keywords: 2-Butoxyacetic acid;
Application of stable isotope dilution assays based on liquid chromatography–tandem mass spectrometry for the assessment of folate bioavailability by Michael Rychlik; Michael Netzel; Inga Pfannebecker; Thomas Frank; Irmgard Bitsch (167-176).
A pilot study was performed to prove the suitability of stable isotope dilution assays for assessing the bioavailability of endogenous folates in foods. By using [2H4]folic acid, [2H4]tetrahydrofolate, [2H4]5-methyltetrahydrofolate, [2H4]5-formyltetrahydrofolate and [2H4]10-formylfolic acid as internal standards, folates in spinach, apple juice and blood plasma were quantified by liquid chromatography coupled to tandem mass spectrometry. To liberate the pteroyl monoglutamates, sample extracts of foods were treated by rat plasma. Sample clean-up was achieved by solid-phase extraction on anion-exchange cartridges, which proved to be sufficient to obtain mass chromatograms devoid of matrix interferences. The bioavailability study was designed as a short-time protocol with three meals, the first consisting of 600 g spinach (meal A), the second consisting of 600 g apple sauce with additionally 400 μg synthetic folic acid (meal B) and the third consisting solely of 600 g apple sauce (meal C). Prior to the meals, the participating volunteer’s tissue was saturated with folates to achieve a significant response of plasma folate to the meals. After consumption of meals A and B a significant rise in folate plasma level compared to meal C (mean level at 28 μg/ml) was observed. The relative bioavailability of folate following meal A exceeded significantly the suggested value of 50% for food folates by taking the dose-normalized area under the curve (AUC) following ingestion of meal B as reference.
Novel sulfamethazine ligand used for one-step purification of immunoglobulin G from human plasma by Yang Liu; Rui Zhao; Dihua Shangguan; Hongwu Zhang; Guoquan Liu (177-185).
To replace conventional affinity ligand like protein A or protein G, a pseudobioaffinity ligand seems to be an alternative for the purification of immunoglobulin G (IgG). In this study, sulfamethazine (SMZ) was chosen as novel affinity ligand for investigating its affinity to human IgG. Monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) (PGMA) beads were employed as the support for high-performance affinity chromatography. SMZ was immobilized on PGMA beads using bisoxirane (ethanediol diglycigyl ether) as spacer. The resultant affinity media presented minimal non-specific interaction with other proteins. Results of high-performance frontal analysis indicated that the media showed specific affinity to human IgG with a dissociation constant on the order of 10−6 M. The SMZ affinity column proved useful for a very convenient one-step purification of IgG from human plasma. Antibody purity after a one-step purification was higher than 90%, as determined by densitometric scanninng of sodium dodecyl sulfate–polyacrylamide gel electrophoresis of purified fraction under reducing condition. The results obtained indicate that SMZ is a valuable affinity ligand for purification of human IgG.
Keywords: Immunoglobulins; Sulfamethazine;
Immunoaffinity extraction and tandem mass spectrometric analysis of human chorionic gonadotropin in doping analysis by Lay-Harn Gam; Sock-Ying Tham; Aishah Latiff (187-196).
A confirmatory and quantitative HPLC–tandem mass spectrometry (MS–MS) method for human chorionic gonadotropin hormone (hCG) at concentrations as low as 5 IU/l following immunoaffinity extraction of the glycoprotein from urine was developed. The extraction method involved retention of urinary hCG in the immunoaffinity column via specific antigen–antibody interaction. A variety of eluents were then used to quantitatively elute hCG from the immunoaffinity column. Qualitative and quantitative analysis of hCG were undertaken using MS–MS by identifying the amino acid sequence of the marker peptide βT5 obtained from hCG by tryptic digestion and the peak areas of three product ions b6 +, b9 + and y11 +, respectively.
Keywords: Chorionic gonadotropin; Glycoproteins;
Liquid chromatography–tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone by B. Starcevic; E. DiStefano; C. Wang; Don H. Catlin (197-204).
A liquid chromatography tandem mass spectrometry assay for serum testosterone (T) and trideuterated testosterone (d3T) was developed in order to support clinical research studies that determine the pharmacokinetics, production rate, and clearance of testosterone by administration of trideuterated testosterone. After adding 19-nortestosterone as the internal standard (I.S.), sodium acetate buffer, and ether, to a serum aliquot, the mixture was shaken and centrifuged, and the ether was dried. The extract was reconstituted in methanol and 15 μl was injected into a liquid chromatograph equipped with an autosampler and Applied Biosystems-Sciex API 300 triple quadrupole mass spectrometer operated in the positive ion mode. T, d3T, and I.S. were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0–20 ng/ml for T and 0–2.0 ng/ml for d3T. The LOQs for T and d3T were 0.5 ng/ml and 0.05 ng/ml. The recoveries for T and d3T were 91.5 and 96.4%. For T at 1.25 ng/ml and 4.0 ng/ml, the intra-day precision (RSD, %) was 3.9 and 4.3% and intra-day accuracy 0.01 and 4.5%, respectively. The inter-day precision at these levels was 5.3 and 5.4% and inter-day accuracy was 1.9 and 0.3%. For d3T at 0.125 ng/ml and 0.4 ng/ml, the intra-day precision (RSD, %) was 2.8 and 8.3% and intra-day accuracy was 1.8 and 5.6%. The inter-day precision at these levels was 10.0 and 7.6% and inter-day accuracy was 5.7 and 3.4%. The concentrations of T in the 38 healthy subjects ranged from 2.5 to 14.0 ng/ml (mean 6.2 ng/ml).
Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography by Miao-Fang Lin; Mabel Royal; Kirk Hayenga; Greg Conn (205-215).
We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu2+/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 μM with LOD/LOQ values below 2.0 μM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH4SO4, and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 μM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.
Keywords: Ethylenediaminetetraacetic acid; Recombinant proteins;
Determination of the platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) in human urine by liquid chromatography–tandem mass spectrometry by Tomoyuki Oe; Ye Tian; Peter J. O’Dwyer; David W. Roberts; Christopher J. Bailey; Ian A. Blair (217-227).
A validated stable isotope dilution liquid chromatography–tandem mass spectrometry assay for the novel platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) (ZD0473) in human urine has been developed. This method uses selected reaction monitoring on the transition of m/z 393 [M+NH4]+ to m/z 304 [M+NH4−NH3−2×H35Cl]+ for ZD0473, and m/z 400 [M+NH4]+ to m/z 310 [M+NH4−NH3−H35Cl−2H35Cl]+ for the internal standard [2H7]ZD0473. Standard curves were prepared over the range from 0.15 to 50 μg/ml. The lower limit of quantitation was 0.2 μg/ml for 100 μl of urine. This simple, rapid, reliable, and sensitive method of quantitation displayed acceptable accuracy and precision over the 3 days of assay validation. A novel platinum adduct was formed during the storage of ZD0473 in human urine. The adduct did not correspond to any of the typical sulfhydryl adducts that have been identified previously for platinum drugs. Formation of the adduct was prevented by the addition of 50% (w/v) sodium chloride to the urine. The assay can be used to quantify intact ZD0473 in the urine of subjects dosed with this new platinum drug.
Keywords: cis-Amminedichloro(2-methylpyridine)platinum(II); Platinum adduct;
Validation of a sensitive and automated 96-well solid-phase extraction liquid chromatography–tandem mass spectrometry method for the determination of desloratadine and 3-hydroxydesloratadine in human plasma by Liyu Yang; Robert P Clement; Bhavna Kantesaria; Larisa Reyderman; Francis Beaudry; Charles Grandmaison; Lorella Di Donato; Robert Masse; Patrick J Rudewicz (229-240).
To support clinical development, a liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed and validated for the determination of desloratadine (descarboethoxyloratadine) and 3-OH desloratadine (3-hydroxydescarboethoxyloratadine) concentrations in human plasma. The method consisted of automated 96-well solid-phase extraction for sample preparation and liquid chromatography/turbo ionspray tandem mass spectrometry for analysis. [2H4]Desloratadine and [2H4]3-OH desloratadine were used as internal standards (I.S.). A quadratic regression (weighted 1/concentration2) gave the best fit for calibration curves over the concentration range of 25–10 000 pg/ml for both desloratadine and 3-OH desloratadine. There was no interference from endogenous components in the blank plasma tested. The accuracy (%bias) at the lower limit of quantitation (LLOQ) was −12.8 and +3.4% for desloratadine and 3-OH desloratadine, respectively. The precision (%CV) for samples at the LLOQ was 15.1 and 10.9% for desloratadine and 3-OH desloratadine, respectively. For quality control samples at 75, 1000 and 7500 pg/ml, the between run %CV was ≤7.5% for desloratadine and ≤6.3% for 3-OH desloratadine. Between run %bias ranged from 4.1 to 8.0% for desloratadine and −11.5 to −4.8% for 3-OH desloratadine. Desloratadine and 3-OH desloratadine were stable in human plasma for 401 days at −22 °C, after five freeze/thaw cycles, up to 24 h at room temperature, and in reconstituted sample extracts (up to 185 h at 5 °C). This LC–MS–MS method for the determination of desloratadine and 3-OH desloratadine in human plasma met regulatory requirements for selectivity, sensitivity, goodness of fit, precision, accuracy and stability.
Keywords: Desloratadine; 3-Hydroxydesloratadine;
Confirmation of amphetamine, methamphetamine, MDA and MDMA in urine samples using disk solid-phase extraction and gas chromatography–mass spectrometry after immunoassay screening by Zengping Huang; Shaoyu Zhang (241-247).
A method using mixed phase disk solid-phase extraction (SPE) and gas chromatography–mass spectrometry (GC–MS) was developed for confirmation of amphetamine (AMP), methamphetamine (MET), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples after immunoassay screening. Disk SPE provided hydrophobic (C18) and strong cation-exchange (SCX) interactions. The analytes were retained on SCX functional groups in the disk and eluted with ammoniated ethyl acetate after washed with methanol. Confirmation and quantitation was exercised by selected ion monitoring using nikethamide as chromatographic standard. Recoveries of the amphetamines were between 73.0 and 104.6% with RSDs in range of 2.1–6.4% (n=3). The limits of detection were 2 ng/ml for AMP, MET and MDMA, and 4 ng/ml for MDA. Five real urine samples were tested with the method after immunoassay screening, and the results were comparable to those of traditional liquid–liquid extraction (LLE). The method was solvent-saved, simple, rapid and reliable, and the extract was cleaner than that of LLE.
Keywords: Amphetamines; Methamphetamine; 3,4-Methylenedioxyamphetamine; 3,4-Methylenedioxymethamphetamine;
Application of a C-peptide electrospray ionization-isotope dilution–liquid chromatography–tandem mass spectrometry measurement procedure for the evaluation of five C-peptide immunoassays for urine by Colette Fierens; Dietmar Stöckl; Dimitri Baetens; André P. De Leenheer; Linda M. Thienpont (249-259).
This study applied electrospray ionization-isotope dilution–liquid chromatography–tandem mass spectrometry for the evaluation of five urinary C-peptide immunoassays via split-sample measurements. The immunoassays measured in duplicate in the same run, the comparison method in triplicate over different runs. From the data, the within-run imprecision and the method comparison total RSDs were calculated. Regression analysis revealed on the one hand systematic differences, on the other, an excellent correlation between the test and comparison methods. From the spread of the data around the regression line in comparison with the 95% prediction intervals from the total RSD, sample-related effects and/or specificity problems were apparent and investigated.
Diagnosis of maple syrup urine disease by determination of l-valine, l-isoleucine, l-leucine and l-phenylalanine in neonatal blood spots by gas chromatography–mass spectrometry by Chunhui Deng; Yonghui Deng (261-268).
A novel method was developed for the diagnosis of maple syrup urine disease (MSUD) by the determination of l-valine, l-leucine, l-isoleucine and l-phenylalanine in dried blood spots of newborns by gas chromatography–mass spectrometry (GC–MS). The four amino acids were extracted from blood samples by methanol and derivatized by n-butanol and trifluroacetic anhydride under optimum reaction conditions. The corresponding single derivatives of the four amino acids were obtained under the optimum conditions. Their contents in blood samples were analyzed quantitatively by measurement of their derivatives by GC–MS in selected ion monitoring mode. MSUD can be diagnosed on the basis of the ratio of the total content of l-leucine and l-isoleucine to that of l-phenylalanine. The present method only took a short time to perform and required minimal sample preparation, which provided low detection limits and a relative standard deviation of less than 5.0%. The derivatization reactions of the four amino acids, l-valine, l-isoleucine, l-leucine and l-phenylalanine, with n-butanol and trifluroacetic anhydride were investigated and the optimum reaction conditions, including reaction time and temperature, were obtained and used for the determination of the amino acids in plasma samples.
Keywords: Valine; Isoleucine; Leucine; Phenylalanine;
Simultaneous enantioselective analysis of chiral urinary metabolites in patients with Zellweger syndrome by Alexandra Muth; Jochen Jung; Steffi Bilke; Annette Scharrer; Armin Mosandl; Adrian C Sewell; Hans Böhles (269-277).
Enantio-MDGC–MS analysis with heptakis-(2,3-di-O-methyl-6-O-tert.-butyl-dimethylsilyl)-β-cyclodextrin as the chiral main column is a powerful tool for the separation of chiral compounds. This paper reports on the simultaneous stereodifferentiation of 2-hydroxyisocaproic acid (HICA), 3-methyladipic acid (3-MA), 2-hydroxyglutaric acid (2-HG), 3-(4-hydroxyphenyl)-lactic acid (HPLA), 2-hydroxysebacic acid (2-HS) and 3-hydroxysebacic acid (3-HS) in a single chromatographic run. These chiral urinary metabolites are useful in the diagnosis of peroxisomal diseases such as Zellweger syndrome (ZS). In this investigation, urine samples from nine patients with ZS were analysed in order to reveal the enantiomeric ratio of these chiral metabolites. The stereodifferentiation of the analysed chiral compounds may provide important information on their biochemical origin.
Keywords: Chiral metabolites;
Stationary phase with specific surface properties for the separation of estradiol diastereoisomers by B Buszewski; M Jezierska-Świtała; S Kowalska (279-286).
The aim of this study was to develop a procedure that enabled the separation of estradiol diastereoisomers. For this purpose a series of stationary phases with different surface properties has been utilized. Two of them contain various interaction sites, such as: cholesterol, n-acylamide, amine and silanols localised in the organic layer bonded to the surface of silica gel (SG-CHOL and SG-CHOL/AP). The other one contains mainly alkylamide ligands and also residual aminopropyl and silanol groups (SG-AP), as well as the last one consisting of hydrocarbonaceous material (SG-C18). In order to select the best type of stationary phase for this analysis, after chromatographic separation of 17-α-estradiol and 17-β-estradiol, selectivity and resolution of the analytes were compared. The best separation of hormones was obtained for SG-CHOL packing, as a consequence of the structure and the properties of this stationary phase. To better understand the retention mechanism and the properties of the stationary phases, used in the separation of steroid compounds, the functional group contributions (τ) were compared with Hansch substituent constants (π).
Keywords: Estradiol; Steroids;
Identification and characterisation of clonal incomplete T-cell-receptor Vδ2–Dδ3/Dδ2-Dδ3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection: implications for minimal residual disease monitoring in childhood acute lymphoblastic leukemia by Udo zur Stadt; Conny Eckert; Johannes Rischewski; Katharina Michael; Steffi Golta; Manuela Müller; Reinhard Schneppenheim; Hartmut Kabisch (287-298).
Incomplete T-cell-receptor δ (TCR-δ) rearrangements are widely used for detection of minimal residual disease in childhood acute lymphoblastic leukemia. In a substantial number of cases both alleles are rearranged and polymerase chain reaction (PCR) products have either to be cloned or excised and reamplified from acrylamide gels. Here we describe a novel HPLC-based method for identification and characterization of clonal TCR-δ targets. Clonality of PCR amplified TCR-δ products was examined on a high-resolution micropellicular DNASep matrix (WAVE Nucleic Acid Fragment Analysis System, Transgenomic) and subsequently classified into clonal, biclonal or negative samples. Vδ2-Dδ3 and Dδ2-Dδ3 rearrangements were analyzed by denaturing high-performance liquid chromatography (DHPLC), using triethylammonium acetate as an ion-pairing reagent, with a linear acetonitrile gradient at 50 °C. Biclonal elution profiles consisted of two individual homoduplex peaks and one heteroduplex peak unique for each patient sample. For characterization of biclonal rearrangements DHPLC separated samples were subjected to a second run and individual clonal peaks were collected. A software-controlled fragment collector was arranged in tandem with the HPLC system for this purpose and purified PCR products were collected in a time-dependent manner. Fractions were air dried and subsequently sequenced directly. The specificity of the observed patient specific sequences was tested via real time quantitative PCR on a LightCycler system.
Direct screening of lyophilised biological fluids for bile acids using an evaporative light scattering detector by Andrés Criado; Soledad Cárdenas; Mercedes Gallego; Miguel Valcárcel (299-305).
The usefulness of lyophilisation for the direct screening of biological fluids for bile acids was investigated. Human serum and urine were lyophilised without losses of the target compounds and further extracted with n-hexane in acidic medium under magnetic stirring. An integrated flow injection-liquid chromatographic system coupled to an evaporative light scattering detector (ELSD) was used for automated screening/confirmation. The continuous module allows sequential filtration of the organic phase, solvent changeover and solid-phase extraction for clean-up and preconcentration purposes. Retained bile acids were eluted with an acetonitrile–methanol (65:35, v/v) stream. For screening purposes, the effluent was directly introduced in the ELSD detector and the total bile acid content of the sample determined. For confirmatory analysis, another aliquot of the sample was processed in the screening module but the effluent was directed to the chromatographic columns, which provided the free bile acid profile of the sample. Fasting serum and urine samples obtained from healthy individuals were lyophilised and processed. Good agreement was obtained in the analysis of the two matrices assayed following the screening and confirmatory methods.
Keywords: Bile acids;
Determination of phloroglucinol in human plasma by high-performance liquid chromatography–mass spectrometry by Hohyun Kim; Hyeongjin Roh; Hee Joo Lee; Soo Youn Chung; Sun Ok Choi; Kyung Ryul Lee; Sang Beom Han (307-312).
A sensitive and selective liquid chromatographic method coupled with mass spectrometry (LC–MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconstituted samples were injected into a C18 XTerra MS column (2.1×100 mm) with 3.5-μm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with flow-rate at 200 μl/min. The mass spectrometer was operated in negative ion mode with selective ion monitoring (SIM). Phloroglucinol was detected without severe interferences from plasma matrix when used negative ion mode. Phloroglucinol produced a parent molecule ([M–H]−) at m/z 125 in negative ion mode. Detection of phloroglucinol in human plasma was accurate and precise, with quantification limit at 5 ng/ml. This method has been successfully applied to a study of phloroglucinol in human specimens.
Liquid chromatographic analysis of the cis(Z)- and trans(E)-isomers of clopenthixol in human plasma using a novel solid phase extraction procedure by Vincenzo Pucci; Francesca Bugamelli; Roberto Mandrioli; Claudio Bartoletti; Nicola Rossi; Maria Augusta Raggi (313-321).
A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in human plasma has been developed. The chromatographic analysis was carried out isocratically on a reversed-phase column (C8 150×4.6 mm I.D., 5 μm) using a mixture of 25 mM phosphate buffer and acetonitrile (65:35 v/v, pH* 3.0) as the mobile phase, and ultraviolet detection at 230 nm. Plasma sample pretreatment was accomplished by means of an original solid-phase extraction (SPE) procedure carried out on cyanopropyl cartridges, with a high extraction yield and good selectivity. Under the optimum conditions, calibration graphs of spiked human plasma samples were obtained over the concentration ranges 1–300 ng ml−1 for cis(Z)-clopenthixol and 1–200 ng ml−1 for trans(E)-clopenthixol. The limit of detection (LOD) was 0.3 ng ml−1 for both cis(Z)- and trans(E)-isomers of clopenthixol. The method was successfully applied to the determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in plasma samples of schizophrenic patients undergoing therapy with zuclopenthixol.
Isolation of an unknown metabolite of capecitabine, an oral 5-fluorouracil prodrug, and its identification by nuclear magnetic resonance and liquid chromatography–tandem mass spectrometry as a glucuroconjugate of 5′-deoxy-5-fluorocytidine, namely 2′-(β-d-glucuronic acid)–5′-deoxy-5-fluorocytidine by Franck Desmoulin; Véronique Gilard; Robert Martino; Myriam Malet-Martino (323-332).
A new metabolite of capecitabine, a prodrug of 5-fluorouracil, was detected by 19F NMR in bile and liver of rats treated with this anticancer drug. Crude bile and perchloric acid extract of liver was subjected to liquid–liquid separation followed by a pre-purification step on a preparative octadecyl silane column (C18). The compound was purified by HPLC optimised to allow the detection of the unknown metabolite and its assumed precursor 5′-deoxy-5-fluorocytidine (5′-DFCR). Treatment with β-glucuronidase from three sources showed that it was a glucuroconjugate of 5′-DFCR. HPLC–TIS-MS–MS and 1H NMR allowed identification of the unknown metabolite as 2′-(β-d-glucuronic acid)–5′-deoxy-5-fluorocytidine.
Keywords: Capecitabine; 5′-Deoxy-5-fluorocytidine; 5-Fluorouracil;
Optimisation and validation of a sensitive high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma and red cells using pre-column semicarbazide derivatisation by Dinesh Talwar; Tara Quasim; Donald C. McMillan; John Kinsella; Cathy Williamson; Denis St.J. O’Reilly (333-343).
There are few studies in which direct measurement of vitamin B6 status in both plasma and red cells has been assessed. The aims of the present study were to evaluate the use of a simple, robust HPLC method of direct pyridoxal 5′-phosphate (PLP) measurement in plasma and red cells and to assess its use in establishing reference ranges in a healthy population. A reverse phase HPLC method with pre-column derivatisation using semicarbazide for the simultaneous measurement of PLP, its degradation product, 4-pyridoxic acid (PA) and pyridoxal (PL) in plasma and red cells was developed. Pre-column derivatisation, reverse phase chromatography and detection procedures were optimised. The recovery, precision, linearity and sensitivity of the assay for plasma and red cell PLP, PA and PL was established. The recovery of PLP was greater than 95% for both plasma and red cell samples. The Intra and Inter batch imprecision for PLP was less than 6% and 7%, respectively. The method for PLP was linear up to at least 1000 nmol/l and the detection limit was 2.1 nmol/l (limit of quantification; 5.8 nmol/l). Accuracy of PLP measurements in plasma were acceptable, showing a mean bias of 4.5% from the mean value of laboratories (N=34) participating in an external quality assurance scheme. Geometric mean (95% reference intervals) for plasma and red cell PLP in the healthy subjects (N=126) were 56 (21–138) nmol/l and 410 (250–680) pmol/g Hb, respectively. There was a strong positive correlation (r 2=0.81) between plasma and red cell PLP levels in the reference population. The HPLC method described was found to be suitable for the routine measurement of PLP in both plasma and red cells.
Keywords: Vitamins; Pyridoxal 5-phosphate;
Artifact production in the assay of anhydroecgonine methyl ester in serum using gas chromatography–mass spectrometry by Stefan W. Toennes; Anabel S. Fandiño; Franz-Josef Hesse; Gerold F. Kauert (345-351).
The detection of the pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) after cocaine smoking using gas chromatography–mass spectrometry is hampered by the artifactual production of AEME. The amount of AEME increases with the amount of cocaine used producing false positive values in authentic samples. A method for the correction of quantitative values was established using calibration of pyrolysis and estimation of the artifactual AEME. Authentic AEME in serum was differentiated from the artifact above 3.5 μg/l, 99% prediction limits of the quantitation were ±3.1 μg/l. In 16 serum samples and five postmortem blood samples, cocaine and AEME were detected, but after application of the correction method only ten were truly positive for AEME.
Keywords: Anhydroecgonine methyl ester; Cocaine;
Simple and rapid method for the simultaneous determination of the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma using liquid chromatography by Bregt S Kappelhoff; Hilde Rosing; Alwin D.R Huitema; Jos H Beijnen (353-362).
Efavirenz and nevirapine are non-nucleoside reverse transcriptase inhibitors for the treatment of HIV-1-infected individuals. A simple and rapid high-performance liquid chromatographic method for the simultaneous quantification of efavirenz and nevirapine in human plasma suitable for therapeutic drug monitoring is described. Sample pre-treatment consisted of protein precipitation with acetonitrile and subsequently dilution with distilled water. The drugs were separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 275 nm. The method was validated over the therapeutically relevant concentration range of 0.05–15.0 mg l−1 and 0.25–15.0 mg l−1 for efavirenz and nevirapine, respectively, using a volume of 100 μl of plasma. The calibration curves were linear over this concentration range. Carbamazepine was used as internal standard. The assay proved to be accurate (accuracies varied between −12.7 and 8.5%) and precise (intra- and inter-assay precisions were less then 5.9%). The tested batches of control human plasma and frequently co-administered drugs did not interfere with the described methodology. Efavirenz and nevirapine were stable under various relevant storage conditions. This validated assay is suited for use in pharmacokinetic studies with efavirenz and nevirapine and can readily be implemented in the setting of a hospital laboratory for the monitoring of efavirenz and nevirapine concentrations.
Keywords: Efavirenz; Nevirapine;
Capillary electrophoretic separation and quantification of flavone-O- and C-glycosides in Achillea setacea W. et K. by Elke Marchart; Brigitte Kopp (363-368).
A simple method for the rapid separation and quantification of flavone-O- and C-glycosides in A. setacea W. et K. by capillary zone electrophoresis (CZE) with UV detection is described. Using 25 mM sodium borate with 20% (v/v) of methanol (pH 9.3) as running buffer sufficient separation of the analytes was achieved within 19 min. For the quantitative determination isorhamnetin-3-O-rutinoside was used as internal standard. The method was successfully applied to a rapid characterisation of the flavonoid complex and a precise quantification of the single and total amount of the flavonoids in different samples of A. setacea.
Keywords: Flavone glycosides;
Determination of tezosentan, a parenteral endothelin receptor antagonist, in human plasma by liquid chromatography–tandem mass spectrometry by Paul L.M. van Giersbergen; Peter Wipfli; Jasper Dingemanse (369-373).
An analytical method was developed for the quantification of tezosentan in human plasma obtained in clinical studies. The method was linear in the range 1 to 512 ng/ml. After liquid–liquid extraction, the samples were analyzed by reversed-phase HPLC with tandem mass spectrometry. The limit of quantification was 1 ng/ml and the extraction recovery was at least 88.2%. Intra- and inter-assay coefficients of variation were below 10%. Stability tests revealed that tezosentan is stable under the different conditions tested.
Extraction of oxytocin and arginine–vasopressin from serum and plasma for radioimmunoassay and surface-enhanced laser desorption–ionization time-of-flight mass spectrometry by David R. Cool; David DeBrosse (375-380).
Oxytocin and arginine–vasopressin (AVP) are secreted into the blood in low concentrations. To analyze these peptides, we investigated two common extraction procedures, acetone–ether precipitation and C18-SepPak columns. Recovery from both procedures approached 70–80% of the spiked amount, though the SepPak columns were more efficient. C18-SepPak columns were used to sequentially separate oxytocin from AVP by eluting oxytocin first with 98% acetone followed by elution of AVP with 80% acetonitrile. Surface-enhanced laser desorption–ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to analyze oxytocin and AVP extracted with C18-SepPak columns from an autistic patient’s plasma sample. We conclude that C18-SepPaks provide more consistent and efficient peptide extraction from serum or plasma that augments both quantitative and qualitative analysis by radioimmunoassay and SELDI-TOF MS.
Keywords: Oxytocin; Arginine–vasopressin;
Determination of dopamine in single rat pheochromocytoma cell by capillary electrophoresis with amperometric detection by Liyao Zhang; Sanfu Qv; Zongli Wang; Jieke Cheng (381-385).
A method is described for the direct identification of dopamine in single cultured rat pheochromocytoma cell by capillary electrophoresis (CE) with amperometric detection. The separation and detection conditions were optimized. The dopamine in single cell analysis was identified based on the migration time of standard dopamine and internal standard (epinephrine). The amount of dopamine in a single cell ranged from 0.29 to 1.28 fmol.
Author Index to Vol. 792 (387-390).
Compound Index to Vol. 792 (391-394).