Journal of Chromatography B (v.780, #1)

News Section (N1-N2).

Solubilized membrane proteins from HL-60 cells were separated by two-step affinity chromatography. Proteins eluted with MgCl2 in the first heparin-gel were applied to the second heparin-gel and eluted with CaCl2. The eluted proteins were analysed and purified by electrophoresis. N-terminal amino acid sequences of eight proteins on the characteristic bands were determined. Homology search for the sequences indicated that three microsomal proteins, two nuclear proteins and a glycolytic enzyme were eluted with divalent cations, whereas a nuclear ribonucleoprotein and a membrane–cytoskelton linker protein were not dissociated with divalent cations, but with 2 M NaCl. Heparin affinity chromatography combined with differential elution with divalent cations can be a useful method for separation of membrane proteins.
Keywords: Heparin binding proteins; Divalent cations;

Capillary electrophoresis assay of alanine:glyoxylate aminotransferase activity in rat liver by Saori Nishijima; Kimio Sugaya; Makoto Morozumi; Tadashi Hatano; Yoshihide Ogawa (13-19).
We measured hepatic alanine:glyoxylate aminotransferase (AGT) activity using capillary electrophoresis. After rat liver homogenate was incubated in the presence of substrates and pyridoxal 5′-phosphate, the pyruvate and glycine produced by AGT were measured. The AGT activity was 10.02±0.31 μmol pyruvate/h/mg protein and 10.21±0.15 μmol glycine/h/mg protein. This method is relatively simple and shows superior sensitivity, allowing the measurement of enzyme activity in 5 μg of protein. Therefore, it appears to be suitable for laboratory use and may also have advantages for measuring AGT activity in liver biopsy specimens.
Keywords: Alanine:glyoxylate aminotransferase; Enzymes;

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography by D. Teshima; N. Kitagawa; K. Otsubo; K. Makino; Y Itoh; R. Oishi (21-26).
A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1–20 μg/ml, and the limit of quantification was 0.1 μg/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0–98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).
Keywords: Mycophenolic acid;

Nucleosides in human urine and serum have frequently been studied as a possible biomedical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. Fifteen normal and modified nucleosides were determined in 69 urine and 42 serum samples using high-performance liquid chromatography (HPLC). Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached 100%. In the validating set, 95.8 and 92.9% of people were correctly classified into cancer patients and healthy persons when urine and serum were used as the sample for measuring the nucleosides. The results show that the artificial neural network technique is better than principal component analysis for the classification of healthy persons and cancer patients based on nucleoside data.
Keywords: Nucleosides;

Simultaneous determination of phthalate di- and monoesters in poly(vinylchloride) products and human saliva by gas chromatography–mass spectrometry by Tatsuhiro Niino; Tohru Ishibashi; Takeshi Itho; Senzo Sakai; Hajimu Ishiwata; Takashi Yamada; Sukeo Onodera (35-44).
A gas chromatographic–mass spectrometric (GC–MS) method using selected ion monitoring (SIM) is described for the simultaneous determination of phthalate di- and monoesters in poly(vinylchloride) (PVC) products. The method consists of the following four procedures; (1) liquid–liquid extraction with ethyl acetate from the acidified aqueous homogenates of the PVC products, (2) esterification with trimethylsilyldiazomethane (TMSD) and methanol, (3) clean-up using Florisil column chromatography and (4) quantitative determination of methylated phthalate monoesters by GC–MS using SIM. The methylated monoesters show a characteristic mass fragment pattern at m/z 163, 149 and 91. The calibration curves for the monoesters were linear from 0.05 to 10 ng (injection volume 1 μl). Overall recoveries ranged from 86.6 to 94.3%. The limits of detections for these methylated derivatives were in the range of 2.0–5.0 ng/g (S/N=3). This method was applied to phthalate monoesters in PVC toy products. Mono-n-butyl phthalate and mono-2-ethylhexyl phthalate were found at levels of 6.42–11.62 μg/g and 30.50–41.81 μg/g, respectively. No monoethyl phthalate, mono-n-hexyl phthalate and monobenzyl phthalate were found in the toy products. The method was also applied to these compounds in human saliva.
Keywords: Phthalate esters;

N,N-Diethyl-m-toluamide (DEET) is frequently used as an insect repellent by military and civilian populations. Because dermal exposure has resulted in several cases of DEET toxicosis, there is a need to rapidly and reliably determine DEET concentrations in biological matrices. An improved method for the analysis of DEET was developed for determining transdermal diffusion of low levels of DEET following application to an in vitro porcine skin flow-through diffusion cell system. The technical improvement involved the use of disk solid-phase extraction (SPE) instead of packed-bed SPE. The disk SPE method required small volumes of preconditioning, wash, and elution solvent (0.5–1 ml) to extract DEET from perfusate samples containing bovine serum albumin (BSA). The limit of quantitation (LOQ) was estimated as 0.08 μg/ml DEET and recoveries from BSA media samples spiked with DEET ranged from 90.1 to 117% with relative standard deviation (RSD) ranging from 2.0 to 13.1%. This method was used to analyze perfusate samples from skin (n=4) topically exposed to DEET–ethanol formulations. The data from these analyses determined that DEET permeability in porcine skin was 2.55×10−5±0.54×10−5 cm/h.
Keywords: N,N-Diethyl-m-toluamide;

The partitioning of glucose-6-phosphate dehydrogenase (G6PDH) (E.C. and hexokinase (E.C. in polyethylene glycol (PEG)–hydroxypropyl starch (PES) and PEG–phosphate aqueous two-phase systems was investigated with free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, as their affinity ligands. It was found that the free reactive triazine dyes, not bound to phase-forming polymers, preferentially partitioned in the top-PEG phase in the PEG–salt and PEG–PES systems. The effect of various parameters such as type and concentration of affinity ligands, pH of the system, molecular mass of PEG and phase composition on partitioning of the enzymes was estimated. Phosphate is a key factor affecting the enzyme partitioning in the PEG–PES system. Cibacron F3GA changed the partition coefficient of G6PDH from 0.73 to 1.59.
Keywords: Glucose-6-phosphate dehydrogenase; Hexokinase; Enzymes; Triazine dyes;

A GC–MS procedure for the detection of different β-agonists in urine samples based on two consecutive derivatization steps is described. The derivatization procedure is based on the consecutive formation of cyclic methylboronate derivatives followed by a second derivatization step with MSTFA on the same extract, forming TMS derivatives. Injections in the GC–MS system may be carried out after each one of the derivatization steps, obtaining enough information for unambiguous identification. Limits of detection for the two derivatization steps ranged from 0.5 to 5 ng/ml. This procedure was tested with the β-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol, α-hydroxy-salmeterol and terbutaline.
Keywords: β-Agonists;

There is little doubt that mental retardation has been prevented in most babies diagnosed by newborn screening programs for inborn errors and the cost–benefit ratios of these programs have been reported as highly positive. In a previous work we optimised a CE method for quick profiling of organic acidurias, which characterize a large number of inborn errors, so that it permits the separation, detection and even identification in less than 15 min of 22 organic acids in urine samples related to a wide range of metabolic disorders. In the present work we have studied the adequacy of filter paper collection of urine samples to simplify this step, always difficult in babies, when it is not performed by training personnel. The studied parameters were: media and conditions for re-extraction to give the best sensitivity and a more simple procedure when the samples are measured by CE, interferences coming from the diaper, recoveries obtained, possible correction of recoveries with creatinine and stability of the compounds. The whole method we report has the advantages of easy sample collection, easy shipping or delivery, and rapid analysis. Moreover, this method of collection and analysis allows the identification and quantitation of fumaric, methylmalonic, N-acetylaspartic, pyroglutamic and homogentisic acids, as well as glutaric acid for which screening is considered especially advisable.

An efficient liquid chromatographic method for the multiresidue analysis of fluoroquinolone antibiotics in chicken tissue has been developed in which quantitation using fluorescence and confirmation with multiple mass spectrometry (MS n ) was achieved simultaneously. Using this method, eight fluoroquinolones were analyzed in fortified samples of chicken liver and muscle tissue with recoveries at levels of 10–200 ng/g generally in the range of 60–93%, except for desethylene ciprofloxacin, which consistently gave recoveries ≥45%. Relative standard deviations were excellent in all cases, and the limits of detection in ng/g were determined as follows in liver and (muscle): desethylene ciprofloxacin 0.3 (0.1), norfloxacin 1.2 (0.2), ciprofloxacin 2 (1.5), danofloxacin 0.2 (0.1), enrofloxacin 0.3 (0.2), orbifloxacin 1.5 (0.5), sarafloxacin 2 (0.6), difloxacin 0.3 (0.2). Confirmation of the identities of the fluoroquinolones was achieved by monitoring the ratios of two prominent product ions in MS2 (desethylene ciprofloxacin) or MS3 (all others). Levels of confirmation as related to ion ratio variability criteria were established. Enrofloxacin and ciprofloxacin were also determined in enrofloxacin incurred chicken liver and muscle using this method.
Keywords: Fluoroquinolone; Antibiotics;

High-performance liquid chromatographic assay for the determination of the novel C-Seco-taxane derivative (IDN 5390) in mouse plasma by M Zaffaroni; R Frapolli; T Colombo; R Fruscio; E Bombardelli; P Morazzoni; A Riva; M D’Incalci; M Zucchetti (93-98).
A HPLC assay was developed to determine IDN 5390, a new paclitaxel analogue, in mouse plasma. The method involves solid-phase extraction from cyano cartridges (recovery >80%), HPLC separation on Symmetry C18 (4.6×150 mm), on isocratic mobile phase of water–acetonitrile–acetic acid (49:50:1) and detection at 227 nm. Retention times of IDN 5390 and IDN 5517 (internal standard, I.S.) were 9.1 and 10.5 min, respectively. The assay was linear from 0.05 to 5 μg/ml (r 2≥0.995), showed intra- and inter-day precision within 1.0 and 6.2%, and accuracy of 94.7–106.8%. LOQ was 0.050 μg/ml. Using this method IDN 5390 pharmacokinetics was determined in mice.
Keywords: IDN 5390; C-Seco-taxane derivatives;

We have previously described fluorine-18 radiolabeled FCWAY [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) trans-4-fluorocyclohexanecarboxamide] as a high affinity ligand for imaging the 5-HT1A receptor in vivo. In a search for radiopharmaceuticals with unique imaging applications using positron emission tomography (PET), we have also developed three new phenylcarboxamide analogues of FCWAY. Two of these analogues were generated by replacing the fluorocyclohexane carboxylic acid with fluorobenzoic acid (FBWAY) or with 3-methyl-4-fluorobenzoic acid (MeFBWAY). The final analogue was generated by replacing the pyridyl group with a pyrimidyl group and the fluorocyclohexane carboxylate with fluorobenzoic acid (FPWAY). We evaluated the metabolic profile of these compounds using either human or rat hepatocytes to produce metabolites and LC–MS/MS to identify these metabolites. We also compared the metabolic rate of these compounds in human or rat hepatocytes. These in vitro metabolism studies indicate that hydrolysis of the amide linkage was the major metabolic pathway for FPWAY and FBWAY in human hepatocytes, whereas aromatic oxidation is the major metabolic pathway for MeFBWAY. The comparative metabolic rate in human hepatocytes was FPWAY>FBWAY>MeFBWAY. In rat hepatocytes, aromatic oxidation was the major metabolic pathway for all three analogs and the rate of this process was similar for all of the analogues. These in vitro metabolic studies demonstrated species differences prior to the acquisition and interpretation of in vivo results.
Keywords: FCWAY analogues; 5-HT antagonists;

Liquid chromatographic determination of florfenicol in the plasma of multiple species of fish by Chue Vue; Larry J Schmidt; Guy R Stehly; William H Gingerich (111-117).
A simple method was developed for determining florfenicol concentration in a small volume (250 μl) of plasma from five phylogenetically diverse species of freshwater fish. Florfenicol was isolated from the plasma matrix through C18 solid-phase extraction and quantified by reversed-phase high-performance liquid chromatography with UV detection. The accuracy (84–104%), precision (%RSD⩽8), and sensitivity (quantitation limit <30 ng/ml) of the method indicate its usefulness for conducting pharmacokinetic studies on a variety of freshwater fish.
Keywords: Florfenicol;

Validation of a microdialysis–gas chromatographic–mass spectrometric method to assess 8-methoxypsoralen in psoriatic patient dermis by Nathalie Leveque; Patrice Muret; Sophie Mary; Michel Bérard; Safwat Makki; Jean Pierre Kantelip; Philippe Humbert (119-127).
8-Methoxypsoralen (8-MOP) is currently used in PUVA therapy (psoralen+UVA) to treat dermatological diseases such as psoriasis, vitiligo and atopic dermatitis. The aim of this work was to validate a method for collecting 8-MOP from patient dermis by a non invasive technique, microdialysis, and then to assess this molecule by gas chromatography–mass spectrometry (GC–MS). 5-Methoxypsoralen (5-MOP) was used as an internal standard. The calibration curve demonstrated a linear relationship between the peak areas of 8-MOP and 5-MOP over a wide range of 8-MOP concentrations (0.9–100 ng/ml). Within- and between-run precisions were measured, using four different 8-MOP concentrations, which varied from 98.0 to 102.0% and from 98.5 to 101.8%, respectively. The limits of detection and quantification were 0.29 and 0.52 ng/ml, respectively. The method was validated and then applied to determine the pharmacokinetic of 8-MOP in ten psoriatic patient dermis, after oral intake of this drug. The results demonstrated that the association of microdialysis with the GC–MS method was an efficient procedure to collect and assess 8-MOP in human dermis, in vivo.
Keywords: 8-Methoxypsoralen;

Hyperforin is one of the most important active components in St. John’s wort (Hypericum perforatum), a botanical dietary supplement used as an alternative treatment modality for mild to moderate depression. A solid-phase extraction (SPE) and an isocratic high-performance liquid chromatography (HPLC) analysis with ultraviolet (UV) detection were developed to determine hyperforin in human plasma samples. Benzo[k]fluoranthene was used as an internal standard. The absolute recovery for hyperforin was more than 89% for plasma concentrations ranging from 25 to 500 ng/ml. The linearity of calibration curves, inter-day and intra-day relative standard deviations were investigated. The limit of detection (LOD) of hyperforin was 4 ng/ml in plasma and the limit of quantitation (LOQ) was 10 ng/ml. Hyperforin concentrations in human plasma following St. John’s wort administration were analyzed. The result suggests that this method is rapid, sensitive, reproducible and capable of quantitative analysis of hyperforin plasma concentrations.
Keywords: Hyperforin; St. John’s wort;

Fumonisins, mycotoxins produced by Fusarium verticillioides, are potent inhibitors of the de novo sphingolipid biosynthesis via inhibition of the key enzyme ceramide synthase. The cellular response to a fumonisin exposure is obvious as an alteration of the ratio of the sphingoid bases sphingosine (SO) and sphinganine (SA). We developed a new column liquid chromatography/electrospray ionisation-mass spectrometry (LC–ESI-MS) method for the rapid, simultaneous and quantitative determination of these bases in cell cultures of immortalised human kidney epithelial cells (IHKE cells). For sample preparation, cell lysates were only diluted, centrifuged and directly used for LC–MS measurements. Quantification was carried out using phytosphingosine (PSO) as an internal standard. Detecting the protonated molecule [M+H]+ signals of SO (m/z 300) and SA (m/z 302) in the selected ion monitoring (SIM) mode, detection limits of 10 pg for SO (signal-to-noise ratio S/N=3:1) and 25 pg for SA (S/N=3:1) were established. The average recovery for SO and SA was higher than 90% for control IHKE-cells, respectively. The developed LC–ESI-MS method allows the sensitive, selective and rapid monitoring of sphingosine and sphinganine in cell matrices with a drastically reduced time for sample preparation.
Keywords: Mycotoxins; Sphingosine; Sphinganine; Fumonisins;

Human hepatocyte incubations were used to study the metabolism of precursors of testosterone and nortestosterone and to evaluate qualitatively the correlation between in vitro and published in vivo urinary metabolic profiles. Both phase I and phase II biotransformations were observed in vitro: oxidoreduction at C-3 and C-17, reduction at C-4,5, hydroxylation at C-6β and C-16, glucuronidation and sulfation. All major metabolites detected in urine following oral administration of androstenedione and norandrostenedione were present in human hepatocyte incubations. The good correlation between in vitro and in vivo metabolic profiles indicates that hepatocyte incubations can be a useful tool to identify and characterize metabolites that could be potential urinary markers for detection of steroid abuse by athletes.
Keywords: Androstenedione; Norandrostenedione; Steroids;

Simple and rapid high-performance liquid chromatographic method for nelfinavir, M8 nelfinavir metabolite, ritonavir and saquinavir assay in plasma by A Janoly; N Bleyzac; P Favetta; M.C Gagneu; Y Bourhis; S Coudray; I Oger; G Aulagner (155-160).
A simple reversed-phase liquid chromatographic method has been developed to determine protease inhibitors concentrations in plasma. Plasma samples (250 μl) containing protease inhibitors were prepared by a simple deproteinization (recovery: 92, 91, 91 and 90.5% for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite, respectively). Chromatography was accomplished using a Hypersil octadecylsilyl column (100×4.6 mm I.D.) and a mobile phase composed of acetonitrile, tetrahydrofuran and dihydrogenophosphate buffer (pH 4) (32:10:58, v/v). Ultraviolet detection at 210 nm was used. The limit of detection was 200 ng/ml for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite. Calibration curves were linear up to 20 000 ng/ml, with correlation coefficients better than 0.997 for all compounds. Intra- and inter-day coefficients of variation of the assay were ≤6% for all compounds. This method was used to analyse protease inhibitors plasma concentrations after oral administration within the framework of therapeutic drug monitoring and pharmacokinetic studies in AIDS patients.
Keywords: Nelfinavir; M8; Ritonavir; Saquinavir;

A high-performance liquid chromatograph equipped with an evaporative light scattering detector (ELSD) (HPLC–ELSD) was used to assay the ceramides in yeast cells. The HPLC–ELSD method employed a cyanopropyl bonded column (CN column) that effectively separated the main interfering substance ergosterol without any derivatization process; most other interfering substances were also removed. The method can be applied for routine assay of ceramide content in yeast.
Keywords: Ceramides;

A method for the determination of I, a peptide–doxorubicin conjugate that was evaluated for the treatment of prostate cancer, and two of its active metabolites, doxorubicin and leucine–doxorubicin is described. Blood samples were chilled immediately after being drawn in order to prevent ex vivo entry of the metabolites into red blood cells. EDTA (10 mg/ml final concentration) was used to prevent plasma-mediated degradation of the peptide portion of the prodrug. After the addition of internal standard, plasma was prepared for analysis using a C-8 solid-phase extraction column. In order to overcome secondary ionic interactions with the silica-based extraction column, the analytes were eluted with ammonium hydroxide in methanol. The extracts were evaporated to dryness, reconstituted, and assayed by step change, gradient, reverse phase HPLC with fluorescence detection. Two interfering metabolites found in post dose plasma were chromatographically separated by an adjustment of the mobile phase pH. The within-day reproducibility of the doxorubicin and leucine–doxorubicin chromatographic retention times was improved by a brief washing of the analytical column with 90% acetonitrile after each injection. The range of the standard curve was 12.5–1250 ng/ml for doxorubicin and 25–2500 ng/ml for I and leucine–doxorubicin.
Keywords: Peptide–doxorubicin; Doxorubicin; Leucine–doxorubicin;

Simultaneous detection of amphetamine-like drugs with headspace solid-phase microextraction and gas chromatography–mass spectrometry by Stefano Gentili; Alessio Torresi; Remo Marsili; Marcello Chiarotti; Teodora Macchia (183-192).
A headspace solid-phase microextraction and gas chromatography–mass spectrometry (HS-SPME–GC–MS) procedure for the simultaneous detection of methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) in hair has been developed. This method is suitable for the separation of primary and secondary amines, is reproducible, is not time consuming, requires small quantities of sample and does not require any derivatization. It provides sufficient sensitivity and specificity, with limits of detection (LOD) and limits of quantitation (LOQ) for each substance of <0.7 and 1.90 ng/mg, respectively. Intra- and inter-day precision were within 2 and 10%, respectively. This method is suitable for routine clinical, epidemiological and forensic purposes and can be used for the preliminary screening of many other substances (amphetamine, methamphetamine, ketamine, ephedrine, nicotine, phencyclidine, methadone) in hair and other biological matrices such as saliva, urine and blood. We also describe the first application of this HS-SPME–GC–MS procedure to the analysis of hair and saliva samples from young people attending a disco in the Rome area. All positive hair samples were confirmed by the gas chromatography–mass–mass (GC–MS2) technique in positive chemical ionization (PCI) mode. Some examples of the use of the method in detecting different drugs are reported.
Keywords: Amphetamine-like drugs;

Determination of nitrate and nitrite by high-performance liquid chromatography in human plasma by Vera Jedličková; Zoltán Paluch; Štefan Alušı́k (193-197).
A new, accurate, fast and simple method has been implemented by which nitrite and nitrate ions, as stable forms of nitric oxide production were studied. A study of these two ions was carried out by a sensitive and accurate HPLC method with two detectors. The most important advantages of the reported method are: short time of analysis, minimal sample pre-treatment, long life of the analytical column and stable eluent solution. The photodiode array UV–Vis detector detected nitrite and nitrate ions at an absorbance of 212 nm. Much more sensitive electrochemical detection with a WE (glassy carbon) electrode was used for the detection of nitrite ions. An analytical chromatographic column was formed by a sorbent, containing strong base anion-exchange groups bound in Cl form in the hydrophilic hydroxyethyl methacrylate matrix. The anions were analysed in human plasma without deproteinization using 0.02 M sodium perchlorate monohydrate as eluent solution at pH 3.9. At this pH organic substances do not affect the analysis. The retention times for nitrite and nitrate were 3.62 and 3.72 min (by electrochemical detection) and 4.44 min, respectively. The method was linear (r=0.9992, 0.9998, 0.996) within a 1–100 (nitrate), 1–20 μmol/l (nitrite) concentration range.
Keywords: Nitrate; Nitrite;

This paper describes a stable isotope dilution method for quantification of 3-hydroxyglutaric acid (3-HGA) in body fluids. The method comprises a solid-phase extraction procedure, followed by gas chromatographic separation and negative chemical ionization mass spectrometric detection. This method is selective and sensitive, and enables measurement of 3-HGA concentrations in urine-, plasma-, and CSF- samples of controls. The control ranges for 3-HGA were: urine 0.88–4.5 mmol/mol creatinine (n=12); plasma 0.018–0.10 μmol/l (n=10), CSF 0.022–0.067 μmol/l (n=10). We applied this method to measure 3-HGA in body fluids of three patients with glutaric aciduria type I. We also quantified 3-HGA in amniotic fluid of controls (range 0.056–0.11 μmol/l; n=12) and in two samples from fetuses affected with glutaric aciduria type I.
Keywords: 3-Hydroxyglutaric acid;