Journal of Chromatography B (v.773, #2)

News Section (N1-N2).

A rapid flow system for automatic sample conditioning for the determination of phenol compounds in human urine has been developed and optimised. Free phenols are detected directly in urine samples while total phenols require acid hydrolysis to convert their conjugate fraction into free phenols, all compounds then being cleaned up and preconcentrated by solid-phase extraction. Separation and determination are done by gas chromatography, using mass spectrometry operating in the selective ion monitoring mode for quantitation. The linear range was 1–160 ng/ml of urine for most of the phenols. Limits of detection for phenol compounds (phenol, alkylphenols and chlorophenols) in the nanogram-per-millilitre range (0.3–0.6 ng/ml) are thus achieved by using 1 ml of urine; also, the repeatability, as RSD, is less than 6.5%. Based on the results for urine samples from unexposed individuals, 2-methylphenol, 2-chlorophenol and 2,4-dichlorophenol are largely detected in hydrolysed urine samples, whereas phenol and 4-methylphenol are detected in hydrolysed and unhydrolysed urine. Other chlorophenols such as trichlorophenols and pentachlorophenol are not detected. The results obtained in the analysis of urine from an individual before and after dietary intake reveal that the levels of phenol compounds in urine look related to food intake.
Keywords: Phenols; Chlorophenols; Alkylphenols;

Application of liquid chromatography–mass spectrometry to the quantification of bisphenol A in human semen by Koichi Inoue; Megumi Wada; Tae Higuchi; Shigeru Oshio; Takashi Umeda; Yoshihiro Yoshimura; Hiroyuki Nakazawa (97-102).
The potential risks to human health and reproduction from the xenoestrogen bisphenol A (BPA) have not been well established. This is due in part to the absence of accurate analytical methods to quantify BPA in biological samples. In this study we establish an accurate, sensitive and selective analytical method for the quantification of BPA in human semen. To quantify BPA we compared the techniques of liquid chromatography–mass spectrometry (LC–MS) and enzyme-linked immunosorbent assay (ELISA). In addition we have taken steps to eliminate BPA contamination during sample extraction and preparation. Results show that the ELISA method gives an over-estimate of BPA concentration, which may be due, at least in part, to non-specific interactions with the BPA-antibodies. LC–MS gave much more accurate results and proved to be more sensitive with a detection limit of 0.5 ng ml−1 compared to 2.0 ng ml−1 by ELISA.
Keywords: Bisphenol A;

The validation of a method for the determination of mevalonic acid (MVA; after conversion to the lactone, MVAL) in human plasma, using high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS–MS), is reported. MVAL and deuterated internal standard were extracted from human plasma samples using automated solid-phase extraction. Analysis was conducted by column-switching, reversed-phase LC–MS–MS, using two hyper-cross-linked styrene–divinylbenzene copolymer sorbent reversed-phase columns. An assay range of 0.2–35 ng/ml and a lower limit of quantitation (LLOQ) of 0.2 ng/ml were achieved with acceptable accuracy and precision. MVA was stable in plasma under a variety of storage conditions.
Keywords: Mevalonic acid;

Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.
Keywords: Cetirizine;

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine drugs. A genetic polymorphism is responsible for large inter-individual differences observed in TPMT activity. We report a new HPLC technique, which avoids an extraction step and the use of radioactive reagents, based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-l-methionine (SAM) as methyl donor in red blood cell lysates (RBC). Intra- and inter-assay variation, within-day, within-run, between-day, and between-run variations showed high precision. The formation of 6-MMP was linear with respect to the lysate concentration and time. In a blinded assay of 61 samples, the results of HPLC method correlated with those of the radiochemical method (r 2=0.82, P<0.0001). Using a cut-off point of 8.5 nmol/h/ml packed RBC, positive predictive value of HPLC was 100% for heterozygous patients. Because of the absence of extraction step, this new HPLC technique of TPMT activity determination reduces analysis variation and is time-saving. This rapid, sensitive, and reproducible method is suitable for routine monitoring of TPMT activity and for fundamental studies.
Keywords: Thiopurine methyltransferase; Enzymes;

Sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of stavudine in human plasma by J.L. Wiesner; F.C.W. Sutherland; M.J. Smit; G.H. van Essen; H.K.L. Hundt; K.J. Swart; A.F. Hundt (129-134).
A sensitive method for the determination of stavudine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak®Vac, 100 mg, tC18 ® solid-phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery® C18, 5 μm, 150×2 mm column with a mobile phase consisting of ammonium acetate (0.01 M)–acetonitrile–methanol (800:100:100, v/v/v) at a flow-rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC–MS–MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for stavudine was 94% with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS–MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been described.
Keywords: Stavudine;

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a β-diketonate europium chelate by Kazuko Matsumoto; Yoshie Tsukahara; Tomonari Uemura; Kinichi Tsunoda; Hidehiro Kume; Seiji Kawasaki; Jutaro Tadano; Takeshi Matsuya (135-142).
A new fluorescent europium chelate labeling reagent, 5-(4″-chlorosulfo-1′,1″-diphenyl-4′-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl3, TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17β-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0–105.1% with the RSD of 1.9–5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC–MS method and a high degree of correlation (r≧0.98) was observed.
Keywords: Estrogens; β-Diketonate europium;

Rapid and sensitive determination of nalmefene in human plasma by gas chromatography–mass spectrometry by Shan Xie; Raymond F. Suckow; Barbara J. Mason; David Allen; Thomas B. Cooper (143-149).
A rapid gas chromatography–mass spectrometric method for the determination of nalmefene in human plasma is described. The procedure involves protein precipitation, extraction with ethanol–chloroform mixture and derivatization with pentafluropropionic anhydride. The deuterated analog of nalmefene, 6β-naltrexol-d 7, was used as the internal standard. Quantitation was achieved on a HP-1 column (12 m×0.2 mm I.D.) with negative chemical ionization (NCI) using methane:ammonia (95:5) as the reagent gas. The standard curves were fitted using a quadratic equation with the curve encompassing a range of 0.5 to 200 ng/ml, and the intra- and inter-assay variations for three different nalmefene levels were less than 10% throughout. The limit of quantitation was found to be 0.5 ng/ml. The method described is highly specific and reproducible, and could also be applied for the determination of naltrexone and 6β-naltrexol. Application of the method to actual human plasma samples is demonstrated.
Keywords: Nalmefene;

The use of micellar electrokinetic chromatography for the determination of lormetazepam and its metabolite, lorazepam, in serum samples at a concentration range of therapeutic interest was investigated. The separation was carried out at 30 °C and 25 kV, using a 15 mM borate–phosphate buffer (pH 8) with 30 mM sodium dodecyl sulfate as the separation electrolyte and 15% methanol as organic modifier. The analyses were carried out in 20 min under these conditions. Detection limits of 0.5 mg l−1 were achieved for both benzodiazepines in serum. This method was employed for the quantitative resolution of both drugs (at different concentration ratios) in serum with very good recoveries.
Keywords: Lormetazepam; Lorazepam;

We attempted to devise a preparation method for clinical samples that could be used for all antibiotics and antivirals. We studied thirteen antibiotics, including five penicillins, four cephalosporins, metronidazole, ofloxacin, and sulfamethoxazole and four protease inhibitors including indinavir, retonavir, nelfinavir, and sequinavir. We compared four sample preparation techniques including solvent precipitation, filtration and resin column. We employ HPLC methods based on a minimal number of columns and mobile phases. We were unable to find one sample preparation method that could be used for all antibiotics and antivirals. But, we did develop an algorithm for determining optimal processing procedures for all drugs.
Keywords: Antibiotics; Antivirals;

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides by Marina Krstanović; Ruža Frkanec; Branka Vranešić; Đurdica Ljevaković; Vesna Šporec; Jelka Tomašić (167-174).
The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-l-Ala-d-isoGln-meso-DAP(ωNH2)-d-Ala-d-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH2-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides l- and d-(adamant-2-yl)-Gly-l-Ala-d-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-l-alanine amidase and an l,d-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.
Keywords: Peptides; Peptidoglycan monomers; Adamantyltripeptides;

Gas chromatographic determination of d-/l-arabinitol ratio in healthy Polish children by Teresa J Stradomska; Zbigniew Mielniczuk (175-181).
The d-/l-arabinitol enantiomers ratio (a marker of disseminated candidiasis of Candida species) in urine was determined by gas chromatography (GC) in 198 healthy Polish children ranging in age from 0 to 18 years. The urine samples were dry and trifluoroacetic anhydride (TFAA)-treated. Enantiomers derivatives were separated on a chiral column (β-Dex 120, 60 m×0.25 mm I.D.). A glass “solid-phase” injector and electron capture detector (ECD) were used. The ECD response was linear with correlation coefficients 0.999. The limit of detection was 0.02 μmol/l. Good results in terms of reproducibility, accuracy (RSD<10%, bias<6%), and linearity were obtained from real urine samples containing up to 400 μmol/l d-arabinitol. TFA–arabinitol derivatives in biological samples were stable from 1 to 5 days (depending on the arabinitol contents), while TFA–arabinitol standard derivatives were stable for 2 weeks. The identity of d- and l-arabinitol were confirmed by GC–MS analysis. The mean d-/l-arabinitol ratios ranged from 2.48 to 1.65 in the examined groups. The d-/l-arabinitol ratio was found to be exponentially regressive with age. A few cases of diagnosis of disseminated candidiasis by the GC method and confirmed by blood culture are described. The described GC method was also used for monitoring antifungal treatment of patients with disseminated candidiasis.
Keywords: Arabinitol;

Determination of the docetaxel vehicle, polysorbate 80, in patient samples by liquid chromatography–tandem mass spectrometry by Alex Sparreboom; Ming Zhao; Julie R. Brahmer; Jaap Verweij; Sharyn D. Baker (183-190).
A new simple method was developed for the quantitative determination of the docetaxel (Taxotere) vehicle, polysorbate 80 (Tween 80), in human plasma. Calibration curves were constructed in the range of 1–100 μg/ml, using paclitaxel (0.01 mM) as internal standard, and were analyzed using a power fit with equal weighting. Sample pretreatment involved a one-step extraction with acetonitrile–n-butyl chloride (1:4, v/v). The analytes were separated on a Waters X-Terra MS column (50×2.1 mm I.D.) packed with 3.5-μm ODS material, and eluted with methanol–water (9:1, v/v) containing 0.1% formic acid. The column effluent was monitored by tandem mass spectrometry with electrospray ionization. The overall extraction efficiency was 50–60%, with values for precision and accuracy of ≤16% and <15% relative error, respectively. Our current method is ∼60–100-fold more sensitive than previous assays, and will be used to define Tween 80 disposition in patients receiving Taxotere.
Keywords: Docetaxel; Polysorbate 80;

Determination of olanzapine in plasma by high-performance liquid chromatography using ultraviolet absorbance detection by Leon J. Dusci; L. Peter Hackett; Linda M. Fellows; Kenneth F. Ilett (191-197).
A rapid method for the determination of olanzapine in plasma using high-performance liquid chromatography with ultra violet detection is described. Olanzapine was extracted from plasma with a mixture of hexane/dichloromethane (85:15), and then back extracted into phosphate buffer pH 2.8. Separation was achieved on a RP Select B C18 column and commonly administered drugs did not interfere with the assay. The limit of quantitation was 1.5 μg/l and the inter-day and intra-day relative standard deviations were less than 10%. Olanzapine was shown to be stable in plasma for up to 7 days when stored at 4 °C. Moreover, the addition of ascorbic acid was not necessary for the achievement of chemical stability during storage, or during the assay procedure. The method has been used to measure olanzapine concentrations in patients treated with various doses of the drug varying from 5 to 40 mg/day.
Keywords: Olanzapine;