Journal of Chromatography B (v.791, #1-2)
Editorial Board (iii).
News Section (N1-N2).
Phenylisothiocyanate and dansyl chloride precolumn derivatizations for the high-performance liquid chromatography analysis of the antitumoral agent ES-285 in dog plasma by C Maraschiello; E Miranda; E Millán; P Floriano; J Vilageliu (1-11).
Chromophor and fluorophor addition reactions involving phenylisothiocyanate (PITC) and dansyl chloride (DC) were optimized to adapt two high-performance liquid chromatography (HPLC) procedures designed for the accurate determination of the novel antitumoral agent ES-285 in Beagle dog plasma. ES-285 was reacted with PITC at 60 °C for 15 min in the presence of triethylamine. The dansyl derivative was obtained by reaction of ES-285 with dansyl chloride in a basic medium at 80 °C for 20 min. Both reactions also worked for ES-299, a compound structurally related to ES-285 which was used as internal standard. The treatment of ES-285 and ES-299 spiked plasma samples with a basic phosphate buffer and MeOH permitted the extraction of the drug from the matrix. The purification of the analytes was carried out by solid-phase extraction followed by precolumn derivatization with PITC and DC. The phenylisothiocyanate adducts were analyzed by isocratic HPLC with UV detection at 254 nm. The ES-285 and ES-299 derivatives were eluted from a C18 column at ∼6.9 and ∼8.1 min, respectively. The eluent ACN–water (95:5, v/v) was delivered to the column at a flow-rate of 1 ml/min and the analysis was completed in 15 min. The dansyl derivatives were analysed by a two-HPLC column system with fluorescence detection and gradient elution. The column temperature was maintained at 40 °C. The analysis lasted 55 min with the elution of ES-285 and ES-299 derivatives at ∼35.2 and ∼37.9 min, respectively. The PITC- and DC-based procedures were characterized by limits of quantification of 20 and 15 ng/ml, respectively. Both procedures were validated according to the ICH and FDA guidelines. They were selective for ES-285 and provided accurate, precise and reproducible results. ES-299 was shown to be a suitable internal standard. The HPLC procedure involving derivatization with DC was more sensitive and permitted to process plasma sample volumes as low as 100 μl. On the other hand, the PITC-based procedure characterised by a quite similar LOQ permitted a higher throughput but implied the processing of a 500-μl plasma volume.
Keywords: ES-285; Phenylisothiocyanate; Dansyl chloride;
Kinetic differentiation mode chromatography using 8-quinolinol and fluorimetric detection for sensitive determination of aluminum adhering to the gastric mucosa by Koji Kashimura; Yasuhiro Mizushima; Eiichi Hoshino; Shuzo Matsubara (13-19).
A highly sensitive method of kinetic differentiation (KD) mode high-performance liquid chromatography (HPLC) with fluorimetric detection was established using 8-quinolinol to measure aluminum adhering to the gastric mucosa. After sucralfate was hydrolyzed by 1 mol/l hydrochloric acid, an 8-quinolinolate–aluminum complex was produced by reacting aluminum with an 8-quinolinol solution. Then contaminants in the gastric mucosa and sucralfate were removed by liquid–liquid extraction with chloroform. Next, the 8-quinolinolate–aluminum complex was separated on a reversed-phase column that was specifically designed to detect aluminum (50×4.6-mm I.D.). Separation was done at a flow-rate of 0.8 ml/min, using BES buffer containing sodium dodecyl sulfate (pH 7.0) as the mobile phase. Fluorescence was detected at 370 nm (excitation) and 504 nm (emission). The sensitivity of this method was more than 1000 times greater than that of absorptiometry using 8-quinolinol. The detection and quantitation limits were 1.68 and 5.11 ng/ml, respectively. When tested with aluminum solutions of 10, 30, and 90 ng/ml, the intra-assay and inter-assay coefficients of variation were below 7.1%, with an error of less than 8.3%. Aluminum adhering to the gastric mucosa was determined by HPLC and absorptiometry after administration of sucralfate to rats. The HPLC method showed that aluminum levels were higher at sites of ulceration than in the normal mucosa at all times after sucralfate administration. When the values above zero obtained for absorptiometry were assessed, there was a significant correlation (r=0.993, P<0.0001) between the aluminum concentrations measured by the two methods. This new HPLC method could be applied to the determination of aluminum in small samples, such as human gastric mucosal biopsy specimens.
Keywords: 8-Quinolinol; Aluminum;
Analysis of pentacyclic triterpenic acids from frankincense gum resins and related phytopharmaceuticals by high-performance liquid chromatography. Identification of lupeolic acid, a novel pentacyclic triterpene by Berthold Büchele; Waltraud Zugmaier; Thomas Simmet (21-30).
An HPLC gradient method with photodiode array detection was developed for the simultaneous analysis of 12 different pentacyclic triterpenic acids in Indian and African frankincense gum resins as well as in related phytopharmaceuticals. The triterpenic acids were obtained by an exhaustive extraction procedure. Identification of the compounds was based on retention times, UV-spectra and add on technique with standards isolated from African frankincense. The method allows differentiation of frankincense of different origin and standardization of frankincense-based phytopharmaceuticals. Further, this is the first report identifying a novel pentacyclic triterpene, lupeolic acid, as a constituent of frankincense gum resins.
Keywords: Pentacyclic triterpenic acids; Lupeolic acid; Triterpenes;
Determination of chloramphenicol residues in shrimps by liquid chromatography–mass spectrometry by M Ramos; P Muñoz; A Aranda; I Rodriguez; R Diaz; J Blanca (31-38).
A liquid chromatographic method with mass spectrometric detection and identification (LC–MS) is presented for the determination of chloramphenicol (CAP) in shrimp tissues. Homogenized shrimp samples were extracted with phosphate buffer (pH 7.0). Clean-up was carried out on a C18 SPE cartridge. Chloramphenicol was determined by LC–MS-ESI in negative mode. The column used was a Symmetry Shield with a mixture of acetonitrile–water (25:75) as mobile phase. Shrimp samples were fortified at CAP levels between 0.2 and 50 ng g−1 with 5D-CAP as internal standard. At these levels, accuracies lay between 101 and 110% and between-day reproducibilities were lower than 7.1%, expressed as the variation coefficient of the mean. Limit of decision (CCα) was 0.02 ng g−1. Limit of quantitation (LOQ) was 0.2 ng g−1.
Liquid chromatographic–mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP 74588) in plasma by Robert A Parise; Ramesh K Ramanathan; Michael J Hayes; Merrill J Egorin (39-44).
Imatinib mesylate (Gleevec, Glivec, STI571) is a targeted, small molecule inhibitor of the oncogenes, BCR/ABL and c-KIT, and has striking antitumor activity in patients with chronic myelogenous leukemia or gastrointestinal stromal tumors. We have developed a liquid chromatographic–electrospray ionization mass spectrometric (LC–MS) method for quantifying imatinib and its main metabolite (CGP 74588) in plasma. The assay uses deuterated imatinib as the internal standard; acetonitrile deproteination; a Phenomenex Luna C18(2) (5 μm, 50×4.6 mm) reversed-phase analytical column; a gradient mobile phase of 0.1% formic acid in methanol and water; and mass spectrometric detection using electrospray positive mode electron ionization. The assay has a lower limit of quantitation (LLOQ) of 30 ng/ml and is linear between 30 and 10 000 ng/ml for both imatinib and CGP 74588. We demonstrated the suitability of this assay for imatinib using it to quantify the concentrations of imatinib and CGP 74588 in plasma of a patient given a 200-mg dose of imatinib orally. We believe that this LC–MS assay should be an important tool for future pharmacokinetic studies of imatinib.
Keywords: Imatinib; CGP 74588;
Determination of three major components of bitespiramycin and their major active metabolites in rat plasma by liquid chromatography-ion trap mass spectrometry by Dafang Zhong; Xiangguo Shi; Lu Sun; Xiaoyan Chen (45-53).
The rapid, selective and sensitive liquid chromatographic–ion trap mass spectrometric (LC–MS n ) method was developed and validated for determination of three major components (isovaleryspiramycins, ISV-SPMs) of a novel macrolide antibiotic bitespiramycin and their major active metabolites (spiramycins, SPMs) in rat plasma. The analytes ISV-SPMs, SPMs, internal standard roxithromycin and azithromycin were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C18 column using two mobile phase systems. Detection was carried out on an ion trap mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). Three components (ISV-SPM I, II, III or SPM I, II, III) could be simultaneously determined within 6.5 min. Linear calibration curves were obtained in the concentration ranges of 4–200 ng/ml for ISV-SPM I and SPM I, 12–600 ng/ml for ISV-SPM II and SPM II, and 18–900 ng/ml for ISV-SPM III and SPM III. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples were less than 8.8 and 10.4% for ISV-SPMs, and 9.3 and 11.2% for SPMs, respectively. The method was applied for the evaluation of the pharmacokinetics of bitespiramycin in rats following peroral/intravenous administration.
Keywords: Bitespiramycin; Isovaleryspiramycin; Spiramycin; Antibiotics;
Membrane cartridges for endotoxin removal from interferon preparations by Jinghua Li; Yingguang Shao; Zhaoan Chen; Runzi Cong; Junde Wang; Xueliang Liu (55-61).
The suitability of membrane cartridges for the removal of endotoxin from both distilled water and interferon preparations was examined. The endotoxin concentrations were reduced to 4.0 and 7.3 EU/ml, respectively, when about 4000 ml of distilled water with 20 and 28 EU/ml were passed through the deoxycholate and chitosan immobilized membrane cartridges. When 200 ml of interferon preparation with endotoxin concentration more than 80 EU/ml and pH 3.9 were applied to a deoxycholate immobilized membrane cartridge at a flow-rate of 9 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml. However, if an interferon preparation of 450 ml, with more than 80 EU/ml of endotoxin and pH 3.9 was applied to the chitosan immobilized membrane cartridge at a flow-rate of 18 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml.
Keywords: Interferon; Chitosan; Deoxycholate; Endotoxin;
Gradient liquid chromatography of leucine-enkephalin peptide and its metabolites with electrochemical detection using highly boron-doped diamond electrode by T.A Ivandini; B.V Sarada; C Terashima; T.N Rao; D.A Tryk; H Ishiguro; Y Kubota; A Fujishima (63-72).
Boron-doped diamond thin film (BDD) electrodes have been used to study the oxidation reactions and to detect leucine-enkephalinamide (LEA) and its metabolites, tyrosine (T), tyrosyl-alanine (TA), tyrosyl-alanine-glycine (TAG) and leucine-enkephalin (LE) using cyclic voltammetry (CV), flow-injection analysis (FIA), and gradient liquid chromatography (LC) with amperometric detection. At diamond electrodes, well-defined and highly reproducible cyclic voltammograms were obtained with signal-to-background (S/B) ratios 5–10 times higher than those observed for glassy carbon (GC) electrodes. The analytical peaks of LC for LEA and its metabolites were well resolved. No deactivation of BDD electrodes was found after several experiments with standard as well as plasma samples, indicating high stability of the electrode. Calibration curves were linear over a wide range from 0.06 to 30 μM with regression coefficients of 0.999 for all compounds. The limits of detection obtained based on a signal-to-noise ratio of 3:1 were 3, 2.2, 2.7, 20 and 11 nM for T, TA, TAG, LE and LEA, respectively. These values were at least one order lower than those obtained at GC electrodes, which has given limits of detection of 22.88, 20.64, 89.57, 116.04 and 75.67 for T, TA, TAG, LE and LEA, respectively. Application of this method to real samples was demonstrated and validated using rabbit serum samples. This work shows the promising use of conducting diamond as an amperometric detector in gradient LC, especially for the analysis of enkephalinamide and its metabolites.
Keywords: Leucine-enkaphalin; Peptides; Enkephalins;
Determination of haemoglobin A1c by liquid chromatography using a new cation-exchange column by Maria Beatriz de la Calle Guntiñas; René Wissiack; Guy Bordin; Adela Rosa Rodrı́guez (73-83).
The use of a new cation-exchange column, ProPac SCX-10, for the determination of haemoglobin A1c (HbA1c) by high-performance liquid chromatography is described. After optimization of the analytical method for the separation of the various isoforms of haemoglobin with the ProPac SCX-10 column, the method was applied to the determination of HbA1c in blood from 59 volunteers. Three of the 59 had previously been diagnosed as diabetics. Interference studies for carbamylation, acetylation and pre-HbA1c were carried out via “in-vitro” experiments. No interference due to carbamylation was observed at the urea values normally found in uremic patients undergoing dialysis. No interference from pre-HbA1c was detected either. The method is able to separate haemoglobin A (α2β2), haemoglobin S (haemoglobin from sickle cell anaemia patients) and haemoglobin A2 (α2δ2) without interference. The method of Hampel was applied to detect outliers. A value of 3.29±0.44% (2σ) for HbA1c was obtained in the analysis of 56 blood samples from non-diabetics. This average value is lower than that reported by most of the methods currently used in routine analyses.
Keywords: Haemoglobin A1c;
Determination by liquid chromatography with fluorescence detection of total 7-ethyl-10-hydroxy-camptothecin (SN-38) in beagle dog plasma after intravenous administration of liposome-based SN-38 (LE–SN38) by W. Guo; A. Ahmad; S. Khan; F. Dahhani; Y.F. Wang; I. Ahmad (85-92).
An HPLC– fluorescence method to quantitate total 7-ethyl-10-hydroxy-camptothecin (SN-38) in beagle dog plasma spiked with liposome based formulation of SN-38 (LE–SN38) and using camptothecin (CPT) as the internal standard (I.S.) was developed and validated to support pharmacokinetics/toxicokinetics studies. Sample preparation was done by protein precipitation using acetonitrile with 0.5% acetic acid. The supernatant was evaporated, and reconstituted in acetonitrile–20 mM ammonium acetate, pH 3.5 (20:80, v/v). When injected onto a Zorbax SB-C18 HPLC column SN-38 as well as I.S. were detected by fluorescence using an excitation at 368 nm and emission at 515 nm. The SN-38 concentrations in samples were calculated from a standard curve of peak area ratios of SN-38 to the I.S. using weighted linear regression. The sensitivity limit for SN-38 was 1.00 ng/ml in beagle dog plasma with a precision (expressed as relative standard deviation) of 12.4% and an accuracy (expressed as analytical recovery) of 104%. The assay was linear within the standard curve range of 1–750 ng/ml. Acceptable precision and accuracy were also obtained for concentrations over the balance of the standard curve range from between-run and within-run calculations.
Keywords: SN-38; 7-Ethyl-10-hydroxy-camptothecin; Liposome-based SN-38;
Measurement of piperaquine in plasma by liquid chromatography with ultraviolet absorbance detection by Te-Yu Hung; Timothy M.E Davis; Kenneth F Ilett (93-101).
Piperaquine (PQ) is an antimalarial drug enjoying a resurgence of use in combination with an artemisinin derivative because of parasite resistance to standard treatments. Its pharmacokinetic properties have not been characterised. An assay for PQ in plasma was developed using solvent extraction and liquid chromatographic separation on a Waters XTerra™ RP18 column, with a mobile phase of 7% acetonitrile in water (containing 0.025% trifluoroacetic acid, 0.1% NaCl and 0.008% triethylamine) and UV detection at 340 nm. The assay was linear up to 1000 μg/l. Intra- and inter-day relative standard deviations were <10% (5–500 μg/l) and <21% (5–500 μg/l), respectively. Inter-day limits of quantitation and detection were 5 μg/l and 3 μg/l, respectively. A preliminary pharmacokinetic study in a patient who received 2.56 g of PQ phosphate orally with dihydroartemisinin as four doses over 32 h found an apparent steady-state volume of distribution of 447 l/kg, an apparent oral clearance 0.93 l/h/kg and a terminal half-life of 17.3 days.
Keywords: Piperaquine; Antimalarial drugs;
Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography by Bilal Yılmaz; Yücel (Yaşar) Kadıoğlu; Yılmaz Aksoy (103-109).
Gemcitabine (dFdC) is a pyrimidine antimetabolite with broad spectrum activity against tumors. In this paper, a normal-phase high-performance liquid chromatographic method was developed for the determination of the parent drug (dFdC) and its metabolite (dFdU) in human plasma. The described sample preparation procedure for determination of dFdC and dFdU is rapid, sensitive, reproducible and simple. The linear regression equations obtained by least square regression method, were area under the curve=0.0371 concentration (ng ml−1)+192.53 and 1.05·10−4 concentration (ng ml−1)−1.2693 for dFdC and dFdU, respectively. The assay for dFdC and dFdU described in the present report has been applied to plasma samples from a bladder cancer patient.
Keywords: Gemcitabine; 2′-Difluoro-2′,2′-deoxyuridine;
Development and validation of a liquid chromatographic method for Casiopeina IIgly® in rat plasma by Leobardo Reyes; Inés Fuentes-Noriega; Lena Ruiz-Ramı̀rez; Lucı́a Macı́as (111-116).
A sensitive and specific liquid chromatographic method using solid-phase extraction with Sep-pak cartridges has been developed for the determination of Casiopeina IIgly and validated over the linear range 2.5–50 μg/ml in rat plasma. The analysis was performed on a Symetry C18 (5 μm) column with a Phenomenex C18 precolumn. The mobile phase was methanol–water (58:42, v/v). The column effluent was monitored at 273 nm. The results showed that the assay is sensitive at 2.5 μg/ml. Maximum intra-day coefficient of variation was 11.47%. The recovery based upon addition of internal standard to rat plasma was 80.98%. The method was used to perform preclinical pharmacokinetic studies in rat plasma and was found to be satisfactory.
Keywords: Casiopeina IIgly;
Determination of polychlorinated biphenyls in serum using gas chromatography–mass spectrometry with negative chemical ionization for exposure estimation by Helena Kontsas; Kaija Pekari (117-125).
A sensitive and selective method for the determination of 24 polychlorinated biphenyls (PCBs) by gas chromatography–mass spectrometry with negative chemical ionization (GC–MS-NCI) was applied for the recent needs of occupational exposure in waste incineration. The three most abundant ions were used in determining compounds with at least five chlorine atoms in the PCB molecule. Selecting ions Cl35 and Cl37 for di-, tri-, and tetrachlorinated PCBs resulted in reliable quantification of these compounds. The detection limits for the 24 individual compounds varied from 0.01 to 0.08 μg/l. The recovery of the method was 113±16%. Stability tests showed no degradation of the compounds studied during 6 weeks. The sum of 24 PCB compounds measured from the sera of workers in a disposal plant was 1.9–10.9 μg/l, and 0.3–3.0 μg/l for controls, respectively. The mean proportion of the low chlorinated PCB compounds (with four or less chlorine atoms) was 20% for workers in the disposal plant and 14% for the controls.
Keywords: Polychlorinated biphenyls;
Determination of 3-keto-4-ene steroids and their hydroxylated metabolites catalyzed by recombinant human cytochrome P450 1B1 enzyme using gas chromatography–mass spectrometry with trimethylsilyl derivatization by Junghan Song; Lalita Wadhwa; Bassem A Bejjani; William E O’Brien (127-135).
A protocol utilizing gas chromatography with selected ion monitoring mass spectrometric detection (GC–SIM-MS) using a simplified trimethylsilyl (TMS) derivatization protocol was developed and validated for the determination of hydroxylated metabolites of 3-keto-4-ene steroids such as testosterone, progesterone and androstenedione. Hydroxylated metabolites catalyzed by human CYP1B1 were extracted with methylene chloride and derivatized with BSTFA–10% TMCS. To get an optimal derivatizing condition, the effect of various incubation times and temperatures was evaluated. When the incubation temperature and time in the presence of the TMS derivatizing agent were increased, the 3-keto group became derivatized with TMS to form a 3-TMS derivative. To minimize the formation of the TMS ether on the 3-keto group, a reaction condition of 56 °C for 10 min was used for the routine measurement of the steroids and their hydroxylated metabolite. Performance studies including linearity of calibration curves, extraction efficiency and precision were performed. Linearity of the calibration curves was satisfactory from 0.125 to 5 μM for most compounds except 21-hydroxyprogesterone and 16α-hydroxyandrostenedione which deviated from linearity at the lower concentrations. Mean percentage extraction recoveries were greater than 80% for all compounds. Most compounds showed good precisions with C.V.s of within-day precision of less than 5% and C.V.s of between-day precision of less than 10%. The selected ion chromatograms from the recombinant human CYP1B1 incubations with testosterone, progesterone and androstenedione showed evidence of 6β-, 16α-, 2α-, and 15α-hydroxytestosterone, 6α- and 16α-hydroxyprogesterone and 6α- and 16α-hydroxyandrostenedione, respectively. There was no significant interference associated with Escherichia coli membrane extracts in detecting hydroxylated metabolites. This procedure provides a rapid and sensitive method for the evaluation of steroid hydroxylation by CYP isoenzymes.
Keywords: 3-Keto-4-ene steroids; Cytochrome P450 1B1; Enzymes;
Simultaneous determination of six HIV nucleoside analogue reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance detection by Naser L. Rezk; Richard R. Tidwell; Angela D.M. Kashuba (137-147).
An accurate, sensitive and specific reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors zalcitabine, lamivudine, didanosine, stavudine, zidovudine, and abacavir with the non-nucleoside reverse transcriptase inhibitor nevirapine in human blood plasma is described. The new Polarity dC C18 silica column used in this method provides better resolution and peak shape than all other columns tested. Also, four different ultraviolet wavelengths were used for accurate and specific quantitation of the analytes. The method was validated over the range of 10–10 000 ng/ml for all analytes except zalcitabine (10–5000 ng/ml). This method is accurate (average accuracies of three different concentrations ranged from 97.2 to 105%), and precise (within- and between-day precision measures ranged from 0.5 to 5.1% and 0.5 to 5.6%, respectively), and is currently being used for determination of plasma drug concentrations in our laboratory.
Keywords: Zalcitabine; Lamivudine; Didanosine; Stavudine; Zidovudine; Abacavir; Nevirapine;
Sedimentation field flow fractionation purification of immature neural cells from a human tumor neuroblastoma cell line by C. Lautrette; P.J.P. Cardot; C. Vermot-Desroches; J. Wijdenes; M.O. Jauberteau; S. Battu (149-160).
The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, useable and purified immature neural cell fraction without inducting cell differentiation.
Sensitive method for the quantitative determination of gemfibrozil in dog plasma by liquid–liquid cartridge extraction and liquid chromatography–tandem mass spectrometry by Brad A. Roadcap; Don G. Musson; J.Douglas Rogers; Jamie J. Zhao (161-170).
A sensitive LC–MS/MS assay for the quantitative determination of gemfibrozil in dog plasma has been developed and validated and is described in this work. The assay involved the extraction of the analyte from 0.5-ml aliquots of dog plasma using Chem Elut cartridges and methyl tert.-butyl ether (MTBE). Chromatography was performed on a Metasil Basic column (50×2 mm I.D., 3 μm) using a mobile phase that consisted of 70:30 acetonitrile–ammonium acetate (1 mM, pH 5.0) with a flow-rate of 0.2 ml min−1. The method showed excellent reproducibility with an inter- and intra-assay precision of <8.9% (%RSD), as well as excellent accuracy with an inter- and intra-assay accuracy between 99 and 101%. This method has a lower limit of quantitation (LLOQ) of 1.0 ng ml−1 with a linear calibration range from 1.0 to 250 ng ml−1. This new assay offers higher sensitivity and a much shorter run time over earlier methods.
Simultaneous determination of bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography–mass spectrometry by Keiko Kudo; Akiko Kiyoshima; Yukio Ohtsuka; Noriaki Ikeda (171-177).
We devised a sensitive and simple method to simultaneously determine bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography–mass spectrometry. Bromvalerylurea and allylisopropylacetylurea were extracted using an Extrelut® column with an internal standard, 2-bromohexanoylurea, followed by derivatization with heptafluorobutyric anhydride. The derivatized extract was submitted to GC–MS analysis of EI-SIM mode. The calibration curves of both compounds were linear in the concentration range from 0.01 to 10 μg/ml in both blood and urine samples. The lower limits of detection of bromvalerylurea and allylisopropylacetylurea were 0.005 and 0.005 μg/ml, respectively. This method proved most useful in accurately identifying these drugs in blood and urine from an autopsied individual.
Keywords: Bromvalerylurea; Allylisopropylacetylurea;
Simultaneous enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4′-hydroxymephenytoin in human plasma by liquid chromatography by Britt Jansson; Ulrika S.H. Simonsson; Michael Ashton (179-191).
A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4′-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C2 column in tandem with a chiral α1-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation <13%, accuracy <±20%). The method was validated for human plasma in the concentration range 10–2000 ng/ml for each of the six compounds. The method allows for the simultaneous characterisation of the metabolic capacity of two human drug-metabolising enzymes, CYP2C19 and CYP2B6, and may be used when investigating polymorphisms or changes in activity of these two enzymes.
Keywords: Mephenytoin; Nirvanol; 4′-Hydroxymephenytoin;
Determination of glycosaminoglycan monosaccharides by capillary electrophoresis using laser-induced fluorescence detection by Victoria Ruiz-Calero; Lluı́s Puignou; Maria Teresa Galceran (193-202).
A newly developed capillary electrophoretic method using laser-induced fluorescence detection (CE–LIF) for the analysis of monosaccharides released from acid hydrolysis of glycosaminoglycans was studied. The method was compared with a previously published method using indirect LIF detection (CE–ILIF). For the CE–LIF method, electrophoretic conditions for the separation of the monosaccharides derivatised with 8-aminopyrene-1,3,6-trisulfonate (APTS) were optimised. The best separations were obtained using 100 mM acetate at pH 4.5 as running buffer. The influence of the injection vial volume on the precision and stability of the sample in different conditions was studied. The detection limits of the CE–LIF method were found to be 0.4–0.6 nM, while those obtained by CE–ILIF ranged from 11.4 to 14.3 μM. Other quality parameters of the method, such as run-to-run precision, day-to-day precision, and linearity were also determined. Finally, the new method was applied to the analysis of the acid hydrolysis products from a glucosaminoglycan (heparin) and a galactosaminoglycan (dermatan sulfate) and cross-contamination between the two solutions was determined. The high sensitivity of the new method allows the determination of dermatan sulfate contaminations in a heparin raw sample down to 0.04% (w/w) and broadens the practical applicability of CE–LIF for the quantitation of the endogenous levels of glycosaminoglycans in animal samples and for pharmacokinetic control after therapeutical heparin administration.
Keywords: Glycosaminoglycan monosaccharides;
Development and validation of a capillary zone electrophoresis method for the determination of ephedrine and related compounds in urine without extraction by Lidia Mateus-Avois; Patrice Mangin; Martial Saugy (203-216).
A capillary zone electrophoresis (CZE) method, with UV detection and in the presence of dimethyl-β-CD, was optimized by means of an experimental design for the separation and the simultaneous quantitation of ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine) and norpseudoephedrine (cathine) in urine without any extraction. In this application, the optimization of the analytical conditions with an experimental design was preferred to a univariate study. Therefore, a central composite design was used and the following factors were investigated and varied simultaneously: buffer concentration, buffer pH and dimethyl-β-CD concentration. In order to evaluate the influence of each experimental parameter on the analytical separation, the resolutions between the four compounds, as well as the separation time and generated current were observed and established as responses of the experimental design. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. After acceptance of the mathematical models, the most favorable conditions were determined by maximizing the resolutions between the four compounds and by setting the other responses at threshold values. Successful results were obtained with a 260 mM Tris–phosphate buffer at pH 3.5 in the presence of 13.3 mM dimethyl-β-CD at 25 °C and with an applied voltage of 30 kV. Under these optimized conditions, a baseline separation of the four compounds was achieved in less than 6 min. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of these compounds in urine samples without any extraction as well as in nutritional supplements.
Keywords: Ephedrine; Pseudoephedrine; Norephedrine; Norpseudoephedrine;
Liquid chromatography with amperometric detection using functionalized multi-wall carbon nanotube modified electrode for the determination of monoamine neurotransmitters and their metabolites by Wen Zhang; Yunfeng Xie; Shiyun Ai; Fangli Wan; Jian Wang; Litong Jin; Jiye Jin (217-225).
The fabrication and application of a novel electrochemical detection (ED) method with the functionalized multi-wall carbon nanotubes (MWNTs) chemically modified electrode (CME) for liquid chromatography (LC) were described. The electrochemical behaviors of dopamine (DA) and other monoamine neurotransmitters at the CME were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results indicated that the CME exhibited efficient electrocatalytic effects on the current responses of monoamine neurotransmitters and their metabolites with high sensitivity, high stability and long-life activity. In LC–ED, DA, norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) had good and stable current responses at the CME. The linear ranges of seven analytes were over four orders of magnitude and the detection limits were 2.5·10−10 mol/l for DA, 2.5×10−10 mol/l for NE, 5.0·10−10 mol/l for MHPG, 3.0·10−10 mol/l for DOPAC, 3.5·10−10 mol/l for 5-HT, 6.0·10−10 mol/l for 5-HIAA, 1.25·10−9 mol/l for HVA. The application of this method coupled with microdialysis sampling for the determination of monoamine neurotransmitters and their metabolites in Parkinsonian patients’ cerebrospinal fluid was satisfactory.
Keywords: Monoamine neurotransmitters;
Liquid chromatographic assay for the antiviral nucleotide analogue tenofovir in plasma using derivatization with chloroacetaldehyde by Rolf W. Sparidans; Kristel M.L. Crommentuyn; Jan H.M. Schellens; Jos H. Beijnen (227-233).
A sensitive and selective reversed-phase liquid chromatographic assay for tenofovir in human plasma has been developed and validated. Tenofovir was isolated from a 200 μl plasma sample using protein precipitation with trichloroacetic acid. The fluorescent 1,N 6-etheno derivative is formed at 98 °C in the buffered extract with chloroacetaldehyde. This derivative was analysed using gradient ion-pair liquid chromatography and fluorescence detection at 254 nm for excitation and 425 nm for emission. In the evaluated concentration range (20–1000 ng/ml), the intra-day precision was 4% and the inter-day precision was 5–6%. An accuracy of between 97 and 110% was determined. The lower limit of quantification was 20 ng/ml with an inter-day precision of 11%, an intra-day precision of 12% and an accuracy of 103%. The assay is subject to interference from co-administered abacavir. The usefullness of the assay was demonstrated for samples obtained from an HIV-infected patient treated with tenofovir.
Keywords: Tenofovir; Chloroacetaldehyde;
Multidimensional gas chromatography–mass spectrometry for tracer studies of fatty acid metabolism via stable isotopes in cultured human trophoblast cells by Alexandra Muth; Armin Mosandl; Adelheid Bursen; Rolf Marschalek; Adrian C Sewell; Hans Böhles (235-244).
The determination of placental fatty acid metabolism using stable isotope-labeled tracers was investigated in the human placental choriocarcinoma (JAR) cell line. Stable isotope incorporation was measured by MDGC–MS. The cultured trophoblast cells incorporated and metabolized the essential fatty acids to long-chain polyunsaturated fatty acids. The described method enables the detection of a low Δ6-desaturase activity in this human placental cell line. The developed MDGC–MS method allows the assessment of long-chain polyunsaturated fatty acid biosynthesis in cultured cells with high sensitivity and selectivity. In this respect, tracer studies with MDGC–MS will be a powerful tool to clarify the significance of placental fatty acid metabolism.
Keywords: Fatty acids;
Quantification of tamoxifen and three metabolites in plasma by high-performance liquid chromatography with fluorescence detection: application to a clinical trial by Kyung-Hoon Lee; Bryan A. Ward; Zeruesenay Desta; David A. Flockhart; David R. Jones (245-253).
A sensitive and reproducible assay employing liquid–liquid extraction and high-performance liquid chromatography with fluorescence detection for the quantification of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, and Z-4-hydroxy-N-desmethyltamoxifen in human plasma is described. The compounds and internal standard, propranolol, were separated with a cyano column and a mobile phase of acetonitrile–20 mM potassium phosphate buffer (pH 3; 35:65, v/v) then detected with fluorescence using a modified version of a method originally described by Fried and Wainer [J. Chromatogr. B 655 (1994) 261]. The coefficients of variation for the midpoint of the standard curve for each compound were less than 10%. This method was applied to a pharmacokinetic study of tamoxifen disposition in breast cancer patients.
Keywords: Tamoxifen; N-Desmethyltamoxifen; 4-Hydroxytamoxifen; Z-4-Hydroxy-N-desmethyltamoxifen;
Development of a liquid chromatographic method for bioanalytical applications with sildenafil by Ming-Thau Sheu; An-Bang Wu; Geng-Cheng Yeh; Angel Hsia; Hsiu-O Ho (255-262).
An improved HPLC method was developed for the determination of sildenafil concentrations in plasma. Analysis of sildenafil in plasma samples was simplified by utilizing a one-step liquid–liquid extraction after alkaline treatment of only 1 ml of plasma. The lower limit of quantitation was 10 ng/ml with a coefficient of variation of less than 20%. A linear range was found to exist from 10 to 1000 ng/ml. This HPLC method was validated with precisions (coefficient of variation, C.V.) for inter- and intra-day runs of 0.41–11.15% and 0.36–8.05%, respectively, and accuracies (the relative error of the mean, REM) for inter- and intra-day runs of −8.72–6.81% and 0.41–11.15%, respectively. A bioavailability study of sildenafil was performed on one normal healthy male volunteer by analyzing sildenafil plasma concentrations with this validated HPLC method. Results demonstrated that this HPLC method is appropriate for applications to bioavailability studies of sildenafil. In addition, an example of the influence of the co-administration of grapefruit juice on sildenafil pharmacokinetics in a healthy volunteer is presented.
Enhanced purification of plasmid DNA using Q-Sepharose by modulation of alcohol concentrations by Wen-Chi Tseng; Fu-Lyn Ho (263-272).
Ion-exchange chromatography is one of the most commonly used methods for plasmid preparation. In this study a modified method was used to purify plasmid from bacterial lysate using Q-Sepharose. Incorporation of alcohols into the washing buffers enhanced the separation of plasmid from RNA and proteins. The use of isopropanol and ethanol achieved a high yield and purity whereas the use of methanol failed to improve the plasmid purification using Q-Sepharose by batch adsorption–desorption. Stepwise elution containing various concentrations of isopropanol and NaCl was used in preparative chromatography to enhance the plasmid purification. The same stepwise elution was applied to the chromatography columns packed with 0.5, 20, and 200 ml of Q-Sepharose for plasmid purification from 7.5, 300, and 3000 ml bacterial broth, respectively. Complete separation of DNA from RNA and proteins was achieved under gravity flow by modulation of the alcohol concentrations in the stepwise elution. These three scales of chromatography maintained an approximate plasmid yield and the purified plasmid contained undetectable levels of RNA and protein.
Keywords: DNA; Alcohols;
Egg yolk phosvitin: preparation of metal-free purified protein by fast protein liquid chromatography using aqueous solvents by O Castellani; V Martinet; E David-Briand; C Guérin-Dubiard; M Anton (273-284).
Two chromatographic methods for hen egg yolk phosvitin purification avoiding organic solvents were evaluated. Hydrophobic interaction and ion-exchange chromatographies were applied to isolated phosvitin. Hydrophobic interaction chromatography has better capacity than ion-exchange chromatography to fractionate phosvitin in their different polypeptides, but its protein yield was lower (0.7 vs. 1.7% of egg yolk dry matter). Finally, ion-exchange chromatography was selected and allowed to fractionate phosvitin polypeptides, including the recovering of phosphoproteins with high electrophoretic mobility: phosvettes. Highly purified (>98%) and free metal protein was obtained in reduced time. Phosvitin polypeptide heterogeneity was evidenced.
Keywords: Egg yolk proteins; Phosphoprotein; Phosvitin;
Simultaneous determination of N-butyramide-tacrine and tacrine in mouse plasma and brain homogenate by high-performance liquid chromatography with a simple gradient solvent system by Y. Jiang; Y. Zhang; Z.R. Zhang (285-290).
A novel reversed-phase HPLC method was developed for the simultaneous determination of tacrine (THA) and the newly synthesized prodrug (N-butyramide-THA, BTHA) in mouse plasma and brain homogenate. The assay involves deproteinisation and subsequent detection at 240 nm with a gradient solvent system. Retention times were 18.5 and 9.3 min for BTHA and THA, respectively. Average recoveries for the analytes were 80.7% (BTHA) and 76.6% (THA) from plasma, and 75.0% (BTHA) and 68.4% (THA) from brain homogenate. Linear responses were observed over a wide range (0.25–20 μg/ml for BTHA in plasma and in brain homogenate, 0.025–20 μg/ml for THA in both matrices). Both BTHA and THA degraded from the prodrug can be detected even 12 h after intravenous administration of BTHA, indicating that BTHA is a promising prodrug for brain targeting.
Keywords: N-Butyramide; Tacrine;
Liquid chromatography studies on the pharmacokinetics of phentermine and fenfluramine in brain and blood microdialysates after intraperitoneal administration to rats by Amal Kaddoumi; Mihoko N Nakashima; Toshihide Maki; Yoshihiro Matsumura; Junzo Nakamura; Kenichiro Nakashima (291-303).
A highly sensitive and simple HPLC method with fluorescence detection for the determination of phentermine (Phen), fenfluramine (Fen) and norfenfluramine (Norf, the active metabolite of Fen) in rat brain and blood microdialysates has been developed. The brain and blood microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) in the presence of carbonate buffer (0.1 M, pH 9.0) at room temperature. The chromatographic conditions consisted of an ODS column and mobile phase composition of acetonitrile and water (65:35, v/v) with flow rate set at 1.0 ml/min. The detection was performed at excitation and emission wavelengths of 325 and 430 nm, respectively. Under these conditions, the DIB-derivatives of Phen, Fen and Norf were well separated and showed good linearities in the studied ranges (5–2000 nM for Phen and 10–2000 nM for Norf and Fen) with correlation coefficients greater than 0.999. The obtained detection limits were less than 23 fmol on column (for the three compounds) in both brain and blood microdialysates at a signal-to-noise ratio of 3 (S/N=3). The intra- and the inter-assay precisions were lower than 10%. The method coupled with microdialysis was applied for a pharmacokinetic drug–drug interaction study of Phen and Fen following individual and combined intraperitoneal administration to rats. In addition, since the role of protein binding in drug interactions can be quite involved, the method was applied for the determination of total and free Phen and Fen in rat plasma and ultrafiltrate, respectively. The results showed that Fen and/or Norf significantly altered the pharmacokinetic parameters of Phen in both blood and brain but did not alter its protein binding. On the other hand, there was no significant difference in the pharmacokinetics of Fen when administered with Phen.
Keywords: Phentermine; Fenfluramine;
Improved simultaneous determination method of β-carotene and retinol with saponification in human serum and rat liver by Keisuke Hosotani; Masahiro Kitagawa (305-313).
Among the many simultaneous determination methods for carotenoid and retinoid, there are only a few reports including the saponification process. However, the yields of β-carotene and retinol were higher when using this process. In this study, the analytical conditions, including saponification, were investigated. The extraction solvent was n-hexane and the sample solvent was HPLC mobile phase in the β-carotene and retinol analysis. BHT as an antioxidant was added at concentrations of 0.125 and 0.025%, respectively, to ethanol and n-hexane phase in the extraction process for serum. The recovery rates were 99.7, 93.7 and 98.3% for β-carotene, retinol and retinyl palmitate in serum, respectively, and 107.1, 92.8 and 98.8% for β-carotene, retinol and retinyl palmitate in liver, respectively. The within-day coefficients of variation (C.V.) were 6.0% for serum and 4.7% for liver in the case of β-carotene, 7.1% for serum, and 5.1% for liver in the case of retinol. The between-day coefficients of variation were 2.7% for serum and 2.7% for liver in the case of β-carotene, and for retinol, 6.4% for serum and 2.7% for liver.
Keywords: β-Carotene; Retinol;
Analysis of secretory immunoglobulin A in human saliva by laser-induced fluorescence capillary electrophoresis by Cheng-Ming Liu; Kuo-Hua Tung; Tsui-Hua Chang; Chen-Chin Chien; Mao-Hsiung Yen (315-321).
The utility of capillary electrophoresis (CE) has been demonstrated for the analysis of secretory immunoglobulin A (sIgA) in human saliva. The amount of sIgA in saliva correlates with immune status. For detecting salivary sIgA, laser-induced fluorescence was conducted in this report for signal amplification. sIgA and anti-sIgA antibody were labeled with cyanine fluorescence (Cy5) for competitive immunoassay and non-competitive analysis, respectively. Cy5 was excited by He–Ne laser with a wavelength of 635 nm, with maximum emission at 670 nm. Migration time during electrophoresis depended on whether sIgA–Cy5 was mixed with antibody or anti-sIgA–Cy5 mixed with sIgA to form Ag–Ab complex. The results indicated that CE competitive immunoassay was effective for analyzing serum sIgA, but not for salivary sIgA. However, salivary sIgA can be analyzed by complex formation assay. The peak area of the complex was proportional to the amount of sIgA added. A standard linear regression curve was generated using purified sIgA. From this standard curve, the amount of sIgA from saliva of either normal or immunocompromised patients can be calculated from the Ag–Ab complex peak area.
Keywords: Immunoglobulin A;
Guidelines for analytical method development and validation of biotechnological synthesis of drugs by Johan Lindholm; Monika Johansson; Torgny Fornstedt (323-336).
In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients. In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field. Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9α-hydroxyprogesterone in E. coli using progesterone as substrate. The process liquid was extracted and analyzed using an HPLC–DAD system. The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107%. The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions. The tolerances (C.V. values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process). In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.
Liquid chromatographic determination of minocycline in brain-to-plasma distribution studies in the rat by Milena Colovic; Silvio Caccia (337-343).
An isocratic reversed-phase high-performance liquid chromatographic procedure was developed for the determination of minocycline in rat plasma and brain and applied to brain-to-blood (plasma) distribution studies. The procedure is based on isolation of the compound and the internal standard (either demeclocycline or tetracycline may be used) from plasma and brain constituents using the Oasis HLB cartridge, with satisfactory recovery and specificity, and separation on a Symmetry Shield RP8 (15 cm×4.6 mm, 3.5 μm) column coupled with a UV detector set at 350 nm. The assay was linear over a wide range, with a lower limit of quantification of 50 ng ml−1 or g−1, using 0.2 ml of plasma and about 200 mg of brain tissue. Precision and accuracy were acceptable. In the rat minocycline crossed the blood–brain barrier slowly, achieving mean brain concentrations between 30 and 40% of the equivalent systemic exposure, regardless of the dose and route of administration.
Human hemoglobin-derived peptides exhibit antimicrobial activity: a class of host defense peptides by Cornelia Liepke; Susann Baxmann; Cornelia Heine; Nicole Breithaupt; Ludger Ständker; Wolf-Georg Forssmann (345-356).
Hemoglobin is a known source of biologically active peptides with various functions. In the present study, we report for the first time the existence of natural processed hemoglobin fragments exhibiting antimicrobial activity in humans. Two antimicrobial hemoglobin-derived peptides were purified from a human placental peptide library by consecutive chromatographic steps tracking the maximum growth inhibitory activity against Escherichia coli BL21. These peptides, consisting of 17 and 36 amino acid residues, were identified as being C-terminal fragments of γ-hemoglobin and β-hemoglobin, respectively. The antimicrobial β-hemoglobin fragment was also purified from lysed erythrocytes, demonstrating that proteolytic degradation of hemoglobin into small bioactive peptides already starts inside erythrocytes. The identified peptides inhibit the growth of Gram-positive and Gram-negative bacteria and yeasts in micromolar concentrations. Moreover, by LPS-binding, the β-hemoglobin fragment reduces biological activity of endotoxins. In contrast, even at high concentrations, the identified antimicrobial hemoglobin peptides do not exhibit toxic activity on human primary blood cells. We conclude that antimicrobial hemoglobin-derived peptides could be important effectors of the innate immune response killing microbial invaders.
Technique validation by liquid chromatography for the determination of acyclovir in plasma by Marcos Fernández; Jacqueline Sepúlveda; Teobaldo Aránguiz; Carlos von Plessing (357-363).
In this research project, a high-performance liquid chromatography (HPLC) method was developed for the determination of acyclovir (ACV) in plasma. The plasma samples, recharged with acyclovir and in presence of 5′-N-methylcarboxyamidoadenosine (MECA) as an internal standard, were purified using a solid-phase extraction technique with Waters Oasis HLB columns. The separation of the components from the extract was carried out in a LiChrospher 100 RP-18 column for further ultraviolet detection at a wavelength range of 250–260 nm. The mobile phase composition was 18% acetonitrile, sodium dodecylsulphate 5 mM and phosphate buffer at pH 2.6 with an analysis time of 13 min per sample. The average retention time for acyclovir was of 5.0 min and for the internal standard 11.2 min. The calibration curve was linear ranging between 0.05 and 1.80 μg/ml. The detection limit was 0.006 μg/ml with a quantification limit of 0.020 μg/ml. The ACV recuperation percentage for 250 μl of plasma was between 94.7 and 109.7% with a coefficient of variation not higher than 5.2%. This method was developed and validated for use in bioavailability and bioequivalence studies.
Inactivation of acrolein by sodium 2-mercaptoethanesulfonate using headspace-solid-phase microextraction gas chromatography and mass spectrometry by Satoshi Takamoto; Nobuo Sakura; Mikio Yashiki; Tohru Kojima (365-369).
Acrolein, the metabolite of cyclophosphamide and ifosphamide, is an irritant of mucous membranes and seems to play an important role in hemorrhagic cystitis. Several methods are available to reduce the risk of hemorrhagic cystitis. Mesna is a regional detoxificant which inactivates acrolein. However, the interaction of mesna and acrolein has never been reported because no available method can detect acrolein. In this study, we measured acrolein to evaluate the effect of mesna in urine or phosphate-buffered saline using a headspace-solid-phase microextraction gas chromatography and mass spectrometry method which we had previously established. We also investigated the effect of mesna at different conditions of pH. Mesna was effective in a dose-dependent (10 μM to 20 mM) fashion in both urine and phosphate-buffered saline and completely inactivated acrolein at concentrations over 10 mM. Furthermore, mesna was more effective in alkaline conditions than in acid.
Keywords: Sodium 2-mercaptoethanesulfonate (Mesna); Acrolein;
Sensitive method for the quantification of urinary pyrimidine metabolites in healthy adults by gas chromatography–tandem mass spectrometry by Ute Hofmann; Matthias Schwab; Sonja Seefried; Claudia Marx; Ulrich M. Zanger; Michel Eichelbaum; Thomas E. Mürdter (371-380).
Enzyme deficiencies in pyrimidine metabolism are associated with a risk for severe toxicity against the antineoplastic agent 5-fluorouracil. To assess whether urinary levels of pyrimidines and their metabolites can be used for predicting patients’ individual phenotype, a new gas chromatographic–tandem mass spectrometric method was developed which allows the simultaneous determination of uracil and thymine and their metabolites dihydrouracil, dihydrothymine, β-ureidopropionic acid, β-ureidoisobutyric acid, and the amino acids β-alanine and β-aminoisobutyric acid in human urine. Small aliquots (2–20 μl) of the urine samples were evaporated and derivatized to the tert.-butyldimethylsilyl derivatives before quantification, using the respective stable isotope-labelled analogues as internal standards. Analytical variation was acceptable with an intra-day imprecision (RSD) below 10%, for β-ureidoisobutyric acid below 15%. The method was used for investigating the stability of urine samples and the influence of urine collection at different times.
Pharmaceutical profiling method for lipophilicity and integrity using liquid chromatography–mass spectrometry by Edward H. Kerns; Li Di; Susan Petusky; Teresa Kleintop; Donna Huryn; Oliver McConnell; Guy Carter (381-388).
A method is described for the simultaneous profiling of sample lipophilicity, integrity, and purity. The method is rapid and is applicable to high throughput profiling of pharmaceutical properties in drug discovery. A short Polaris C18 column is used with a rapid, wide-polarity mobile phase gradient, UV detection, and MS analysis. The lipophilicity of each component is estimated from a calibration curve using six drug or organic compounds and plotting their respective measured retention time versus Log D 7.4 (literature). The correlation of Log D 7.4 (literature) to Log D 7.4 (HPLC) for 60 structurally diverse drugs has a correlation coefficient r 2 of 0.89. The method is applicable to compounds with MW>200 and retention time>1.5 min for rapid, initial pharmaceutical profiling in drug discovery.
Simultaneous determination of levomepromazine, midazolam and their major metabolites in human plasma by reversed-phase liquid chromatography by P.G.J. ter Horst; N.A. Foudraine; G. Cuypers; E.A. van Dijk; N.J.J. Oldenhof (389-398).
A sensitive and reliable high-performance liquid chromatographic (HPLC) assay is a prerequisite for pharmacokinetic analysis of continuous infusion of levomepromazine adjuvant to midazolam. We developed such a method to determine the levels of levomepromazine, midazolam and their major metabolites (levomepromazinesulfoxide, desmethyl-, didesmethyllevomepromazine, O-desmethyllevomepromazine and α-hydroxy-midazolam) simultaneously. Desmethylclomipramine was used as an internal standard (I.S.). The lower limit of quantification of this assay was set for levomepromazine 4.1 μg/l, levomepromazinesulfoxide 4.9 μg/l, O-desmethyllevomepromazine 18.4 μg/l, α-hydroxymidazolam 26.6 μg/l, midazolam 23.4 μg/l, didesmethyllevomepromazine 15.8 μg/l, and desmethyllevomepromazine 6.6 μg/l. The between- and within day assay variations were commonly below 5%. The recovery in human plasma for the different analytes varied between 85 and 11%. The accuracy of this assay varied between 95 and 105% for the different concentrations. The linearity of this assay was set between 25 and 800 μg/l (r 2>0.999 of the regression line). The first results of pharmacokinetic analysis of midazolam indicated that half-life varied between 1.1 and 1.9 h. Pharmacokinetic analysis using a one-compartment model of levomepromazine revealed that the apparent volume of distribution was 4.1±2.4 l per kg lean body mass and the metabolic clearance was 309±225 l per hour per 70 kg. This assay proved to be robust and reproducible. It can reliably be used for further study of the pharmacokinetics of continuous infusion of levomepromazine.
Keywords: Levomepromazine; Midazolam;
Determination of 13C and 15N enrichments of urea in plasma by gas chromatography–combustion isotope ratio mass spectrometry and gas chromatography–mass spectrometry using the 2-methoxypyrimidine derivative by W. Kulik; M.J.S. Oosterveld; R.M. Kok; K. de Meer (399-405).
We describe a GC–MS and GC-c-IRMS method for the determination of labeled urea tracer enrichments in plasma as a result of combined 13C- and 15N2-urea infusion experiments in piglets. Urea was converted into 2-methoxypyrimidine, a stable derivative, suited for analyses by both GC–MS and GC-c-IRMS. Using calibration curves for the respective working ranges (13C-urea: 0–1% APE; 15N2-urea: 0–7% MPE) enrichments were established in single point measurements; for 15N2-urea as values±0.15% MPE (95% confidence interval); for 13C-urea as values±0.02% APE (95% confidence interval). 15N1-urea enrichments were determined by measurement of the same sample with GC-c-IRMS and GC–MS. Subtraction of the 13C specific GC-c-IRMS data from the nondiscriminating GC–MS data for the sum of 13C- and 15N1-urea resulted in 15N1-urea enrichments±0.15% MPE (95% confidence interval). Application of the method in a combined 13C-urea bolus and 15N2-urea primed constant infusion experiment in piglet was demonstrated.
Pullulan–cyclodextrin microspheres by Gheorghe Fundueanu; Marieta Constantin; Doina Mihai; Fabrizio Bortolotti; Rita Cortesi; Paolo Ascenzi; Enea Menegatti (407-419).
Pullulan microspheres containing cyclodextrin (CyD) were obtained by chemical crosslinking with epichlorohydrin of an alkaline solution of pullulan (Pul) and α-, β- or γ-CyD. The amount of α-, β- and γ-CyD in microspheres was 120, 156, and 138 μmol/g, respectively, as determined from the percentage of iodine incorporated in the hydrophobic cavity of CyD’s. Microspheres were packed in a glass column and the liquid chromatographic behaviour by isocratic elution of different drugs or typical organic compounds (TOC), taken as model drugs, was investigated. The increase of the retention volume (V R) of each compound, depending on the interaction(s) between CyD’s cavity and the considered molecule, is characterized by a broadening of the peaks. The interaction coefficient K, corresponding to the ratio between the V R value of each tested molecule on Pul–α-, Pul–β- and Pul–γ-CyD active stationary phase and the V R value of benzoic acid on St/maltodextrin neutral stationary phase, was determined. According to K values, the accurate prediction can be done on the potential drugs to be conditioned in suitable CyD cavity. Values of K allow to anticipate the release profiles of drugs considered.
Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection by Frank Niedorf; Hans-Herbert Bohr; Manfred Kietzmann (421-426).
An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 μl) are cleaned up via liquid–liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile–0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.
Keywords: Ketamine; Xylazine;
Liquid chromatographic determination of hydroxyproline in tissue samples by Paul R. Hutson; Mark E. Crawford; Ronald L. Sorkness (427-430).
We describe a reversed-phase assay of hydroxyproline in rat lung tissue using sarcosine for the internal standard and pre-injection reaction with both o-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC). Intra-assay variability in the concentration range of 25–500 μM hydroxyproline was less than 1%. Normal rat (left) lung was found to have a hydroxyproline content of 1.08±0.18 mg/lung. This ability to measure minute amounts of hydroxyproline is being applied to the measure of collagen and pathological fibrosis.
Keywords: Hydroxyproline; Amino acids;
Author Index to Vol. 791 (431-434).
Compound Index to Vol. 791 (435-438).