Journal of Chromatography B (v.819, #1)

Zdeněk Deyl (1934–2005) by Karel Macek (1-2).

This review summarizes all the research efforts in the last decade (1994–2003) that have been spent to the various application of immobilized enzyme reactor (IMER) in on-line high performance liquid chromatography (HPLC). All immobilization procedures including supports, kind of assembly into chromatographic system and methods are described. The effect of immobilization on enzymatic properties and stability of biocatalysts is considered. A brief survey of the main applications of IMER both as pre-column, post-column or column in the chemical, pharmaceutical, clinical and commodities fields is also reported.
Keywords: Immobilized enzyme reactor; HPLC; Chromatographic supports;

Photodegradation products of hexahydroquinoline derivatives (HHQ) have been analysed with gas chromatography–mass spectrometry (GC–MS). The photodegradation was carried out under the conditions recommended in the first version of the document issued by the International Conference on Harmonization (ICH), currently in force in the studies of photochemical stability of drugs and therapeutic substances. The study was performed on the compounds having two chlorine atoms at different positions of the phenyl ring. Photodegradation of dichlorophenyl derivatives of HHQ resulted in formation of one or three photoproducts. The main product of their decomposition was aromatic compound formed as a result of dehydrogenation of the dihydropyridine ring. The most often observed fragmentation pathway of the photoproducts formed was elimination of methyl and methoxy radicals from the ester groups. The fragmentation of the photoproducts containing one chlorine atom at the ortho-position of the phenyl ring occurred through elimination of chlorine radical.
Keywords: Hexahydroquinoline derivatives; Dihydropyridine derivatives; Calcium channel blockers; Mass spectrometry; Photodegradation;

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha lactoalbumin and beta lactoglobulin) and alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000; 1500 and 3350)–potassium phosphate was analysed. Bovine serum albumin and alpha lactoalbumin concentrated in the polyethyleneglycol rich phase with a partition coefficient of 10.0 and 27.0, respectively, while beta lactoglubulin and alpha-1 antitrypsin showed affinity for the phosphate-rich phase with a partition coefficient of 0.07 and 0.01, respectively. An increase of medium pH induced an increase of the partition coefficient of these proteins while the increase in polyethyleneglycol molecular mass induced the opposite behaviour. The system polyethyleneglycol 1500-pH 6.3 showed the best capacity for recovering the alpha-1 antitrypsin with a yield of 80% and a purification factor between 1.5 and 1.8 from an artificial mixture of the milk whey proteins and alpha-1 antitrypsin. The method appears to be suitable as a starting point to isolate proteins expressed in transgenic milk.
Keywords: Whey milk proteins; Protein isolation; Transgenic milk;

Protein expression profiling of CLL B cells using replicate off-line strong cation exchange chromatography and LC–MS/MS by David R. Barnidge; Renee C. Tschumper; Diane F. Jelinek; David C. Muddiman; Neil E. Kay (33-39).
In this study we use replicate 2D-LC–MS/MS analyses of crude membranes from B cells derived from a patient with chronic lymphocytic leukemia (CLL) to examine the protein expression profile of CLL B cells. Protein identifications made by replicate 2D-LC–MS/MS analysis of tryptic peptides from detergent solubilized B cell membrane proteins, as well as replicate LC–MS/MS analysis of single off-line strong cation exchange chromatography (SCX) fractions, were analyzed. We show that despite the variance in SCX, capillary LC, and the data-dependent selection of precursor ions, an overlap of 64% between proteins identified in replicate runs was achieved for this system.
Keywords: CLL; 2D-LC–MS/MS; B cell; Expression profiling; Off-line SCX; Reproducibility;

A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52–1.6%, and the inter-day assay precisions were 3.6–5.8% for rat liver and cerebral cortex (n  = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.
Keywords: Catecholamines; Catechol-O-methyltransferase; Norepinephrine; Normetanephrine;

We present a simple chromatographic method to detect and quantify protease inhibitors (PI), metabolites and non-nucleoside reverse transcriptase inhibitors (NNRTIs) in human plasma of HIV-1 infected patients and in peripheral blood mononuclear cells (PBMCs) using either liquid chromatography coupled with ultraviolet (LC–UV) or liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). A solid–liquid extraction was carried out on 500 μl of plasma as pre-treatment. Calibration curve ranges were from 50 (100) to 5000 ng/ml (indinavir). PBMC pellets from 7 ml of blood were lysed with methanol/tris with a calibration curve ranging from 0.25 to 250 ng/pellet. Simple modifications in the mobile phase composition (slight increase of ammonium acetate concentration and addition of methanol for LC–UV) easily linked the two analytical systems.
Keywords: PI; NNRTI; LC–UV; LC–MS/MS; Plasma; Intracellular;

Peptide profiling in epithelial tumor plasma by the emerging proteomic techniques by Emilia Caputo; Maria Luisa Lombardi; Vincenza Luongo; Ramy Moharram; Pete Tornatore; Giuseppe Pirozzi; John Guardiola; Brian M. Martin (59-66).
The plasma peptide component (PPC) from ten melanoma (Mel), breast cancer (BC) and healthy individuals was examined by a combination of RP-HPLC, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and tandem mass spectrometry. A three peak pattern (2023, 2039, 2053.5 m/z) was primarily observed in melanoma. Two peaks (2236.1 and of 2356.3 m/z) were found only in BC samples. Fibrinogen alpha and inter-α-trypsin inhibitor heavy chain H4 fragments were absent in both tumor samples.
Keywords: Melanoma; Breast cancer; Plasma peptide component; SELDI-TOF MS;

We developed a non-radioactive and sensitive assay method for measurement of the HTL hydrolase (HTLase) activity in biological samples, using OPA as a fluorescent post-labeling agent, l-homocysteine thiolactone (L-HTL) as the substrate, and HPLC to achieve rapid and selective separation of the substrate and product. The method was applied to measure the activity of HTLase in human, rabbit, rat and mouse serum samples. In addition, the correlation between the serum HTLase activity and PON1 polymorphisms in Japanese subjects was also investigated. The serum HTLase activity in humans, as determined by measurement of the enzyme activity in 22 subjects, was found to be in the range of 0.89–2.06 nmol/min mg protein, with a mean activity of 1.44 nmol/min mg protein.
Keywords: Homocysteine thiolactone hydrolase; HPLC; Polymorphism;

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry by Ming Zhao; Carol Hartke; Antonio Jimeno; Jing Li; Ping He; Yelena Zabelina; Manuel Hidalgo; Sharyn D. Baker (73-80).
A rapid, sensitive and specific method was developed and validated using liquid chromatography–tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra™ C18 (50 mm × 2.1 mm i.d., 3.5 μm) analytical column using a mobile phase consisting of acetonitrile–water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1–1000 ng/mL for the human plasma samples and 5–1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of >0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.
Keywords: Gefitinib; ZD1839; LC/MS/MS; Pharmacokinetics;

A novel assay method for theanine synthetase activity by capillary electrophoresis by Ping Li; Xiao-Chun Wan; Zheng-Zhu Zhang; Jian Li; Zuo-Jun Shen (81-84).
The determination of theanine has been performed by micellar electrokinetic capillary chromatography (MECC) using 2,4-dinitrofluorobenzene (DNFB) as a derivative reagent. To achieve the separation, a fused-silica capillary column was used with a borax buffer at 0.03 mol/L pH 9.8 (containing Brij35 and isopropanol) at 17 °C with detection wave length at 360 nm. The factors affecting the efficiency of the sample separation were examined simultaneously. A 40-min reaction at 35 °C between l-glutamate and ethylamine (with Tris–HCl buffer, pH 7.5) was investigated using the theanine synthetase from budding tea seeds. A novel method for the analysis of theanine synthetase activity based on MECC was established. The method shows mean recovery ranged from 87.1 to 105.3% and linearity ranged from 0.2 to 5.0 mmol/L.
Keywords: Micellar electrokinetic capillary chromatography; Theanine; Theanine synthetase; Activity assay;

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography by Yufang Zheng; Guowang Xu; Jun Yang; Xinjie Zhao; Tao Pang; Hongwei Kong (85-90).
A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm × 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm × 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol–water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r  > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.
Keywords: Coupled-column; Urinary nucleosides; High-performance liquid chromatography;

High performance liquid chromatographic determination of N-butyryl glucosamine in rat plasma by Ali Aghazadeh-Habashi; John Carran; Tassos Anastassiades; Fakhreddin Jamali (91-96).
A high performance liquid chromatography (HPLC) method for determination in plasma of N-butyryl glucosamine (GLBU), a highly water-soluble compound with no chromophore was developed. Method: To 100 μL of plasma containing GLBU was added fucose as internal standard. GLBU and fucose were derivatized using 1-phenyl-3-methyl-5-pyrazolone in the presence of sodium hydroxide at 70 °C for 30 min. The solution was neutralized with hydrochloric acid and the excess derivatizing reagent was extracted with chloroform. The aqueous layer was injected into an isocratic HPLC system consisting of an autoinjector, a single pump and a UV detector set at 245 nm. Two different 25 cm reversed phase columns were used, a 4 and a 10 μm C18 columns. The mobile phase was a mixture of phosphate buffer (pH 7) and acetonitrile (80:20), which was run through a pump at a flow rate of 1.0 mL/min at ambient temperature. Results: Derivatized fucose and GLBU appeared 24 and 28 min, and at 34 and 37 min using 4 and 10 μm columns, respectively. The assay was linear over the range of 0.2–200 μg/mL with a limit of quantification of 0.2 and 1 μg/mL for the 4 and 10 μm columns, respectively. The method was applied to the determination of GLBU in rat plasma after oral administration of 233 mg/kg of GLBU. Conclusion: The present assay is precise, and accurate with sufficient sensitivity for pharmacokinetic studies following therapeutically relevant doses.
Keywords: N-Butyryl glucosamine; 1-Phenyl-3-methyl-5-pyrazolone; Rat plasma; High performance liquid chromatography; Glucosamine; Fucose; Post-column derivatization; C18 columns;

Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatography–fluorescence detection by Giampiero Pagliuca; Elisa Zironi; Alberto Ceccolini; Riccardo Matera; Gian Paolo Serrazanetti; Andrea Piva (97-103).
An analytical method based on high-performance liquid chromatography (HPLC) combined with fluorescence detection (FL) has been developed for the simultaneous determination of fumonisin B1 (FB1) and its totally hydrolized metabolite aminopentol-1 (AP1) in pig liver. The sample preparation is based on a single solid phase extraction (SPE). o-Phthalaldehyde (OPA) was used for pre-column derivatization before the programmed reversed-phase analysis on phenylhexyl column. The developed method shows good repeatibility for inter- and intra-day precision as well as adequate linearity of calibration curves (r 2 was 0.9855 for FB1 and 0.9831 for AP1). Average recoveries from the matrix were 93.6% for FB1 and 95.3% for AP1. The limit of quantification (LOQ) in swine liver was 75 μg/kg for FB1 and 42 μg/kg for AP1.
Keywords: Fumonisin B1; Aminopentol-1; High-performance liquid chromatography (HPLC); Fluorescence detection (FL); Solid phase extraction (SPE); Swine liver;

Validation of a liquid chromatography–mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs by C. Arellano; C. Philibert; E.N. Dane à Yakan; C. Vachoux; O. Lacombe; J. Woodley; G. Houin (105-113).
A rapid, sensitive and selective liquid chromatography–mass spectrometry (LC–MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1–2.5 μM (27.1 ng/mL–0.677 μg/mL) for dextromethorphan and 0.005–0.5 μM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL–0.128 μg/mL) and 3-hydroxymorphinan (1.22 ng/mL–0.122 μg/mL). The limits of quantification (LOQ) were 0.0025 μM (12.5 fmoles, 3.4 pg, 5 μL injected) for dextromethorphan; 0.0025 μM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 μL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.
Keywords: Dextromethorphan; LC–MS; Intestine; Metabolism; Cytochrome P450;

The rat brain hippocampus proteome by Michael Fountoulakis; George T. Tsangaris; Antony Maris; Gert Lubec (115-129).
The hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In humans, the CA1 area of hippocampus is one of the first brain areas to display pathology in Alzheimer's disease. A comprehensive analysis of the hippocampus proteome has not been accomplished yet. We applied proteomics technologies to construct a two-dimensional database for rat brain hippocampus proteins. Hippocampus samples from eight months old animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The database comprises 148 different gene products, which are in the majority enzymes, structural proteins and heat shock proteins. It also includes 39 neuron specific gene products. The database may be useful in animal model studies of neurological disorders.
Keywords: Rat brain; Hippocampus; Proteome; Proteomics; Two-dimensional protein database; Mass spectrometry; Memory;

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns by V. Jankowski; R. Vanholder; L. Henning; S. Karadogan; W. Zidek; H. Schlüter; J. Jankowski (131-139).
In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the μmolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.
Keywords: Dinucleoside monophosphates; Isolation; Quantification; Monolithic columns; Vasoregulatory hormones;

Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine by J. Rodríguez Flores; G. Castañeda Peñalvo; A. Espinosa Mansilla; M.J. Rodríguez Gómez (141-147).
A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm × 75 μm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 °C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C18 cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0–6.0 mg L−1 for MTX and 0.5–6.0 mg L−1 for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mg L−1. CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.
Keywords: Capillary zone electrophoresis; Chemotherapeutic agents; Human urine; Methotrexate; Folinic acid; Folic acid;

An isocratic high-performance liquid chromatographic method with detection at 472 nm was developed, optimized and validated for the determination of lycopene in canine plasma. Ethyl-β-apo-8′-carotenoate was used as internal standard. A Hypersil BDS RP-C18 column (150 mm × 4.6 mm), 5 μm particle size, was equilibrated with a mobile phase composed of acetonitrile and methanol (50:50, v/v). Its flow rate was 1.5 ml/min. The elution time for lycopene and ethyl-β-apo-8′-carotenoate was approximately 11 and 5 min, respectively. Calibration curves of lycopene were linear in the concentration range of 3–200 ng/ml in plasma. Limits of detection and quantification in plasma were 1 and 4 ng/ml, respectively. Recovery was greater than 97%. Intra- and inter-day relative standard deviation for lycopene in plasma was less than 1.8 and 3.1%, respectively. This method was applied to the determination of lycopene plasma levels after single dose administration to dogs.
Keywords: Lycopene; RP-HPLC; Canine plasma; Liquid–liquid extraction (LLE);

Analysis of environmental biomarkers in urine using an electrochemical detector by Zhisong Liu; Mary S. Wolff; Jacqueline Moline (155-159).
Phenols are present in the environment and are prevalent in human populations, as environmental contaminants, dietary components, or their metabolites. Many are potential endocrine-altering agents. Currently available methods analyze single components or single families of chemicals as biomarkers of exposure. In order to assess multiple biologically relevant exposures to such substances, we evaluated the feasibility of determining several phenols simultaneously in urine, using an electrochemical detector (ECD) in combination with high performance liquid chromatography (LC). Based on reported analyses in the literature and the ECD response, we selected four xenobiotic residues, including three phytoestrogens (enterolactone, daidzein, and genistein) and bisphenolA [BPA]. These compounds had detection limits below 1 μg/L in urine using the cleanup procedure (glucuronidase hydrolysis and C18 column) and the urine volume (2 mL) we employed. As a pilot study to demonstrate the method's utility, we determined urinary enterolactone, daidzein, genistein and BPA in samples from nine children and 24 adults.
Keywords: Phytoestrogens; BisphenolA; Electrochemical detector; Urine; LC;

In order to improve the sensitivity and stability of human blood samples containing WR-1065 (i.e., active metabolite of the cytoprotective agent amifostine), a high-performance liquid chromatographic method was developed and validated using fluorescent derivatization with ThioGlo™3. Using a sample volume of only 100 μl, the method was specific, sensitive (limit of quantitation = 10 nM in deproteinized blood or 20 nM in whole blood), accurate (error ≤ 3.2%) and reproducible (CV ≤ 8.7%). In addition, the stability of WR-1065 in deproteinized and derivatized blood samples was assured for at least four weeks at −20 °C. This method should be particularly valuable in translating the kinetic–dynamic relationship of WR-1065 in preclinical models to that in cancer patients.
Keywords: Blood samples; Fluorescent derivatization; HPLC; ThioGlo™3; WR-1065;

Qualitative assessment of IC50 values of inhibitors of the neuronal nicotinic acetylcholine receptor using a single chromatographic experiment and multivariate cluster analysis by Krzysztof Jozwiak; Ruin Moaddel; Rika Yamaguchi; Sarangan Ravichandran; Jack R. Collins; Irving W. Wainer (169-174).
It has been widely demonstrated that affinity chromatography can be used to derive binding affinities, and that these affinities can be correlated to data obtained using standard techniques such as membrane binding, ultrafiltration and equilibrium dialysis. The purpose of this study is to evaluate the use of immobilized nicotinic acetylcholine receptor stationary phase in chromatographic experiments to assess the functional activity of series of noncompetitive inhibitors (NCIs) as reflected in their IC50 values. Chromatographically determined retention values and computer generated molecular descriptors were obtained for 29 compounds and the data were analyzed by cluster analysis. The approach qualitatively ranked the test compounds as efficient NCIs (low IC50 values) or poor NCIs (high IC50 values). The data obtained with the 29 compounds used in this study demonstrate that the experimental approach had been able to place 25 of these compounds in the correct IC50 clusters. To our knowledge, this is the first relationship established between chromatographic retention and IC50 for membrane-bound receptors. These results suggest that the chromatographic approach may be useful in development of lead drug candidates including the determination of off-target binding.
Keywords: Affinity chromatography; Immobilized receptors; Nicotinic acetylcholine receptor stationary phase; Drug discovery;

A robust, rapid, selective and sensitive liquid chromatography-negative atmospheric pressure chemical ionization (LC–(APCI)–MS–MS) method has been developed for the quantification of mometasone furoate (MF) in human plasma utilizing a solid-phase extraction clean-up step and 13C-fluticasone propionate as internal standard. The intra- and inter-day coefficients of variation were ≤15% and the lower limit of quantification (LLOQ) was 15 pg/ml. This method is ideally suited for pharmacokinetic investigations of low MF levels following inhalation of MF.
Keywords: HPLC–MS–MS; Glucocorticoid; Drug analysis; Mometasone furoate; Liquid chromatography–tandem mass spectrometry;

Determination of deracoxib in feline plasma samples using high performance liquid chromatography by Sherry K. Cox; Jacob Roark; Adam Gassel; Karen Tobias (181-184).
A new HPLC procedure for the determination of deracoxib, a selective cyclooxygenase-2 inhibitor, has been developed and validated. Following a liquid–liquid extraction using isopropyl alcohol and chloroform, samples were separated by isocratic reversed-phase HPLC on an Atlantis C18 column and quantified using UV detection at 252 nm. The mobile phase was a mixture of 10 mM potassium phosphate (pH 4.5) and acetonitrile, with a flow-rate of 1.0 ml/min. The procedure produced a linear curve over the concentration range 10–1500 ng/ml. The development of the assay allowed the determination of pharmacokinetic parameters after oral administration of deracoxib in cats and would be suitable for other pharmacokinetic studies.
Keywords: COX-2 inhibitors; Deracoxib; HPLC;

A rapid and sensitive LC–MS–MS method for quantifying levodropropizine in human plasma after oral administration of a single-dose (60 mg/day) was developed and validated. The sample preparation used liquid–liquid extraction with a mixture of dichloromethane–diethyl ether (2:3, v/v) in a basic environment. The retention time of levodropropizne and zolmitriptan (used as internal standard) was 1.6 and 1.4 min, respectively. The assay was linear over the range 0.25–500 ng/mL with a LOQ of 0.25 ng/mL. The intra- and inter-day precision were <8.1% and <11.5%, respectively, and the accuracy was in the range 87.6–112%. The levodropropizine concentration profile in human plasma was determined.
Keywords: Levodropropizine; Zolmitriptan;

A method based on a liquid–liquid extraction procedure followed by high-performance liquid chromatography (HPLC) coupled with UV–visible detection is described and validated for the determination of lauroyl-indapamide in rat whole blood. The blood sample was extracted with diethyl ether after the addition of 10% trifluoroacetic acid (aq.). The chromatographic separation was performed on a Chromasil ODS column, using methanol–acetonitrile–tetrahydrofuran–0.2% trifluoroacetic acid (170:20:15:38, v/v/v/v) as the mobile phase. The UV detection wavelength was set at 240 nm. The extraction recovery of lauroyl-indapamide was ranged from 76.5 to 82.6%, and the calibration curve had a good linearity in the range of 0.048–200 μg/ml (r  = 0.9976). The method presents appropriate intra-day and inter-days repeatabilities, showing values below 7.4% in terms of the percentage of relative standard deviation (R.S.D.). The method proposed is simple, rapid and sensitive, being useful for pharmacokinetic studies in rats.
Keywords: Lauroyl-indapamide; HPLC; Rat whole blood; Pharmacokinetic study;

Determination of levetiracetam in human plasma with minimal sample pretreatment by Jens Martens-Lobenhoffer; Stefanie M. Bode-Böger (197-200).
We here present a method for the routine quantification of the novel antiepileptic drug levetiracetam in human serum by HPLC–UV. The procedure is very easy, quick, inexpensive and rugged. The sample preparation consists only in the precipitation of serum proteins by perchloric acid and extraction of unpolar components by cyclohexane. The aqueous phase containing the analyte levetiracetam is injected onto a porous graphitic carbon analytical HPLC-column and separated by gradient elution with diluted phosphoric acid/acetonitrile. Detection is carried out at a wavelength of 205 nm. The calibration function is linear in the range of 1–75 μg/ml. The detection limit is 0.1 μg/ml. Using four quality control sample concentrations, the inter-day relative standard deviations (R.S.D.) are lower than 3% and the accuracies are better than 6%. The respective inter-day values are: R.S.D. < 4% and accuracies better than 2%. Frequently co-administered antiepileptic drugs do not interfere with the assay. The method has been successfully applied to patient samples.
Keywords: Levetiracetam; Antiepileptic drugs; Porous graphitic carbon; HPLC;

Erratum to “Quantitation of tigecycline, a novel glycylcycline, by liquid chromatography” by Chonghua Li; Christina A. Sutherland; Charles H. Nightingale; David P. Nicolau (201).