Journal of Chromatography B (v.794, #2)

News Section (N1-N3).

A stable isotope dilution gas chromatography–mass spectrometry (GC–MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17β]-2H3-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C18 with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l (r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 μg/l (r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 μg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.
Keywords: Estriol;

A systematic optimization of the HPLC separation of a complex mixture containing urinary steroids (anabolics and corticoids), boldenone and bolasterone (synthetic anabolics) by micellar liquid chromatography has been carried out. The isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic strength and flow-rate on the separation has been studied. A micellar mobile phase made of 5% propanol and 40 mM surfactant allowed the separation of 13 steroids in about 23 min. A bivariant optimization method for the micellar mobile phase surfactant-propanol corroborated the above results. The separations obtained show good perspectives for future developments.
Keywords: Corticosteroids; Anabolic steroids;

Simultaneous determination of ticarcillin and clavulanate in rabbit serum and tissue cage fluid by liquid chromatography by Chonghua Li; Qiuming Geng; David P. Nicolau; Charles H. Nightingale (227-236).
A gradient elution HPLC method with a wavelength switch technique was developed to simultaneously analyze the β-lactam ticarcillin and the β-lactamase inhibitor clavulanate in rabbit serum and tissue cage fluid (TCF). A C18 reversed-phase column with a programmable UV detector changing the wavelength from 218 to 254 nm at 9 min was used for chromatographic separation. The mobile phase consisted of acetonitrile, phosphate buffer and tetrabutylammonium hydrogen sulfate by following a gradient elution program at a flow-rate of 1 ml/min. Sample processing was carried out with liquid–liquid extraction. Good linearity, recoveries, precision and accuracy were obtained. The ranges of the standard curves were 1–100 μg/ml for ticarcillin, and 0.2–20 μg/ml for clavulanate. This assay has been successfully applied to analyze ticarcillin and clavulanate in rabbit serum and tissue cage fluid samples from a pharmacokinetic study.
Keywords: Ticarcillin; Clavulanate;

Divergence in urinary 8-iso-PGF (iPF-III, 15-F2t-IsoP) levels from gas chromatography–tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF by Dimitrios Tsikas; Edzard Schwedhelm; Maria-Theresia Suchy; Jonas Niemann; Frank-Mathias Gutzki; Veit J Erpenbeck; Jens M Hohlfeld; Andrzej Surdacki; Jürgen C Frölich (237-255).
Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F2-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC–tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8-iso-PGF (iPF-III, 15-F2t-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF (i.e. 15(S)-8-iso-PGF) has been shown by others to be highly selective and specific for this 8-iso-PGF isomer when quantified by GC–MS. In the present study we established IAC for urinary 8-iso-PGF for subsequent quantification by GC–tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15(S)-8-iso-PGF. 8-iso-PGF was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF was determined to be 291±102 pg/mg creatinine by method A and 141±41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8-iso-PGF at 128±55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF is heterogenous, with 15(S)-8-iso-PGF contributing by ∼50%. PGF and other 8-iso-PGF isomers including 15(R)-8-iso-PGF are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF. We assume that ent-15(S)-8-iso-PGF is also contributing by ∼50% to urinary 8-iso-PGF. This finding may have methodological, mechanistic and clinical implications.
Keywords: Prostaglandins;

Liquid chromatographic determination of the glutathione conjugate and ring-opened metabolites formed from coumarin epoxidation by Jeffrey D. Vassallo; Stephen W. Morrall; Kristi L. Fliter; Sandra M. Curry; George P. Daston; Lois D. Lehman-McKeeman (257-271).
Species differences in the biotransformation of coumarin are thought to play an important role in its toxicity. Since the putative toxic metabolite is coumarin 3,4-epoxide (CE), methods to measure the metabolites of CE were developed. The glutathione (GSH) conjugate of CE (CE-SG) at the 3-position was purified by reversed-phase (RP)-high performance liquid chromatography (HPLC), and characterized by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). An RP-HPLC method was developed to quantify CE-SG in hepatic microsomal mixtures and a separate RP-HPLC method was also developed to quantify the three ring-opened coumarin metabolites; o-hydroxyphenylacetic acid (o-HPAA), o-hydroxyphenylethanol (o-HPE) and o-hydroxyphenylacetaldehyde (o-HPA) in hepatic microsomal mixtures. Detection limits for all four products of coumarin epoxidation exceeded 3.5 ng/ml and recovery from hepatic microsomal mixtures was essentially quantitative with RSD values less than 8%. Species differences in o-HPA detoxification were consistent with sensitivity to coumarin, thereby demonstrating that these methods have utility in addressing the fate of CE and its contribution to toxicity.
Keywords: Glutathione; Coumarin;

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects by Ulrich Friess; Michaela Waldner; Hans-Guenther Wahl; Rainer Lehmann; Hans-Ulrich Haring; Wolfgang Voelter; Erwin Schleicher (273-280).
We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57±14 years, HbA1c 8.0±1.8%) to 42 non-diabetic controls (C) (age 45±17 years). The urinary excretion of total CML in diabetic patients was increased by ∼30% (DM: 0.58±0.21; C: 0.45±0.14 μmol/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36±0.17; C: 0.27±0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).
Keywords: N(ϵ)-(Carboxymethyl)lysine;

Development and validation of a liquid chromatography–mass spectrometry assay for the determination of opiates and cocaine in meconium by S Pichini; R Pacifici; M Pellegrini; E Marchei; E Pérez-Alarcón; C Puig; O Vall; O Garcı́a-Algar (281-292).
A procedure based on liquid chromatography–mass spectrometry (LC–MS) is described for determination of 6-monoacetylmorphine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, cocaine, benzoylecgonine and cocaethylene in meconium using nalorfine as the internal standard. The analytes are initially extracted from the matrix by methanol (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or 0.01 M ammonium hydrogen carbonate buffer (morphine-3-glucuronide, morphine-6-glucuronide). Subsequently a solid-phase extraction with Bondelut Certify columns (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or ethyl solid-phase extraction columns (morphine-3-glucuronide, morphine-6-glucuronide) was applied. Chromatography was performed on a C8 reversed-phase column using a gradient of acetic acid 1%–acetonitrile as a mobile phase. Analytes were determined in LC–MS single ion monitoring mode with atmospheric pressure ionisation-electrospray (ESI) interface. The method was validated in the range 0.005–1.00 μg/g using 1 g of meconium per assay and applied to analysis of meconium in newborns to assess fetal exposure to opiates and cocaine.
Keywords: Opiates; Cocaine;

Analysis of erythromycin and benzoylperoxide in topical gels by liquid chromatography by P Dehouck; E Van Looy; E Haghedooren; K Deckers; Y Vander Heyden; E Adams; E Roets; J Hoogmartens (293-302).
Gels containing a combination of erythromycin and benzoylperoxide are frequently used in the treatment of acne vulgaris. A method was developed to determine the content of both erythromycin and benzoylperoxide in these gels. Erythromycin was extracted from the gel in conditions where the oxidative power of benzoylperoxide was neutralised by addition of ascorbic acid and this extract was analysed on an Xterra RP18 column, with a mobile phase containing acetonitrile–0.2 M K2HPO4–water (35:5:60, v/v/v). The detection wavelength was 215 nm. A second extraction procedure was developed for the analysis of benzoylperoxide. The extraction solution was analysed on a Hypersil C18 BDS column and a mobile phase containing acetonitrile–water (58:42, v/v). Detection was performed at 254 nm. The flow rate was 1.0 ml/min in both methods. The selectivity, repeatability, linearity and recovery of both methods were examined. Special attention was given to determination of the recovery and the uncertainty on the recovery. This allowed evaluation of the bias of the extraction method. The method developed was used to examine the stability of a gel for topical use.
Keywords: Erythromycin; Benzoylperoxide;

Silibinin is the main isomer of a group of flavanoids extracted from the seeds of the milk thistle weed, a common herb that is widely used to maintain liver health and for the treatment of liver disorders. Silibinin when incubated with human liver microsomes produced one major metabolite and at least two minor metabolites. Tandem mass spectrometry (MS) was used to identify the metabolite structures partially. MS studies confirmed that the major metabolite is demethylated silibinin and the two minor metabolites are mono-hydroxy and di-hydroxy silibinin. The K m value for the demethylation shows that silibinin has a strong affinity for the cytochrome P450 enzymes.
Keywords: Silibinin;

Solid-phase enzyme modification via affinity chromatography by Erkan Türker Baran; Nazmi Özer; Vasif Hasirci (311-322).
In the present study antileukemic enzyme l-asparaginase (ASNase) and catalase (as a model enzyme) were modified in solid-phase with activated polyethylene glycol (PEG2) by using ligand-immobilized affinity column systems l-asparagine-Sepharose CL-4B and Procion red-Sepharose CL-4B, respectively. Studies on change of specific activity with modification time showed negligible differences between batches of modified catalase. Modification of ASNase for 1 h resulted in 50.2% recovery of the specific activity and the attachment of 69 molecules of PEG2 per molecule of ASNase forming ‘PEGylated ASNase’. Sequential modification of ASNase by activated PEG and heparin resulted in coupling of about nine molecules of heparin per molecule of PEGylated ASNase. Intravenous (i.v.) administration of PEG2-modified ASNase showed prolonged presence in the blood circulation and no adverse effects or symptoms of anaphylaxis were observed in presensitized mice.
Keywords: Enzymes; L-asparaginase;

Determination of mirtazapine in human plasma by liquid chromatography by P Ptáček; J Klı́ma; J Macek (323-328).
A rapid high-performance liquid chromatographic method for the quantitation of mirtazapine in human plasma is presented. The method is based on a liquid–liquid extraction and reversed-phase chromatography with fluorimetric detection. The separation was performed on a Luna 3-μm C18(2) 50×4.6 mm I.D. column using an isocratic elution. Zolpidem hemitartrate was used as the internal standard. The between-day precision expressed by relative standard deviation was less than 5% and inaccuracy does not exceed 6%. A low limit of quantitation (1.5 ng/ml) and a short time of analysis (4 min) makes this assay suitable for pharmacokinetic studies.
Keywords: Mirtazapine;

We have developed a tandem mass spectrometry (LC–MS–MS) method for measuring tobramycin concentrations in serum samples and have compared it with a fluorescence polarisation immunoassay. After protein precipitation with acetonitrile supernatant was injected into the LC–MS–MS system. A C18 cartridge (4×2 mm) was eluted with a step gradient of 20–100% methanol containing HFBA. The retention times were, tobramycin 1.05 min and sisomycin 1.05 min. The MRM transitions were: m/z 467.8>163 (tobramycin) and m/z 447.8>160 (sisomycin). The limit of quantification was 0.15 mg/l and the assay was linear up to 50 mg/l. Assay precision was <6% within and between batch.
Keywords: Tobramycin;

Determination of amitraz in canine plasma by solid-phase microextraction–gas chromatography with thermionic specific detection by M.E.C. Queiroz; C.A.A. Valadão; A. Farias; D. Carvalho; F.M. Lanças (337-342).
A simple and rapid analytical method is presented for the determination of amitraz in canine plasma samples using solid-phase microextraction (SPME) and gas chromatography with thermionic specific detection (GC-TSD). The best conditions for the SPME procedure were: direct extraction on a polydimethlysiloxane (PDMS) fiber with 100-μm film thickness; 400 μl of sample plasma matrix modified with 4 ml sodium borate solution (0.01 mol l−1, pH 6.5); extraction temperature 70 °C, with stirring at 2500 rpm for 45 min. The method was linear between 20 and 400 ng ml−1 with regression coefficients corresponding to 0.998 and coefficient of the variation of the points of the calibration curve lower than 15%. The lowest limit of quantification (LOQ) for amitraz in plasma was 20 ng ml−1. This LOQ was determined as the lowest concentration on the calibration curve in which the coefficient of variation was lower than 15%. The proposed method was applied to determine amitraz concentrations in canine plasma to look for toxicity after treatment with amitraz in a dipping bath.
Keywords: Amitraz;

A reversed-phase high-performance liquid chromatographic (HPLC) using ultraviolet (UV) absorbance detection method for simultaneous determination of clofibrate (I) and its major metabolite clofibric acid (II) in human plasma has been developed to support a clinical study. I, II and internal standard (I.S., III) are isolated from human plasma by 96-well solid-phase extraction (SPE) C18⋅AR plate and quantified by direct injection of the SPE eluent onto the HPLC with UV detection wavelength at 230 nm. Two chromatographic methods, isocratic and step gradient, have been validated from 1.0 to 100.0 μg/ml and successfully applied to plasma sample analysis for a clinical study. The lower limit of quantitation (LLOQ) is 1.0 μg/ml for both I and II when 500 μl plasma sample is processed. Sample collection and preparation is conducted at 5 °C to minimize the hydrolysis of I to II in human plasma.
Keywords: Clofibrate; Clofibric acid;

A selective and sensitive method based on derivatisation with 2,4-dinitrophenylhydrazine (DNPH) and consecutive HPLC gradient separation is described for the determination of malondialdehyde (MDA) in urine. Preparation of urine samples involved a one-step derivatisation/extraction procedure. Separation was achieved using a Waters Symmetry™C18 column (3.9×150 mm) and linear gradient of acetonitrile in water (from 30% to 70% in 30 min). The overall detection limit of the method was 56 nM of MDA in urine. The recovery of MDA was 94.3±8.6%. MDA in urine of healthy volunteers, measured using the method of standard additions, was 0.019±0.012 μmol/mmol creatinine. MDA in the same samples measured using the 2-thiobarbituric acid (TBA) assay was 0.181±0.063 μmol/mmol creatinine. We demonstrate that the commonly used TBA assay in conjunction with HPLC may overestimate the MDA concentration in human urine by almost 10-fold.
Keywords: Malondialdehyde; 2,4-Dinitrophenylhydrazine; 2-Thiobarbituric acid;

20-HETE is a potent, vasoconstrictive arachidonic acid metabolite with a limited number of published methods for quantitative assessment of microsomal formation rate. The purpose of this study was to evaluate the utility of HPLC–MS (negative ESI) for quantitation of rat microsomal 20-HETE enzyme kinetics. Calibration curves were linear over 0.75–16 ng on-column (r 2>0.996). The intra- and inter-assay precision and accuracy were <15%. Microsomal 20-HETE revealed saturable (100 μM) kinetics (brain K m and V max: 39.9±6.0 μM and 8.7±0.6 pmol/min per mg; liver K m and V max: 23.5±3.2 μM and 775.5±39.8 pmol/min per mg; kidney K m and V max: 47.6±8.5 μM and 1933±151 pmol/min per mg). This paper demonstrates HPLC–MS as an efficient method for quantitating 20-HETE enzyme kinetics in microsomes from rat tissues.
Keywords: 20-Hydroxyeicosatetraenoic acid; Arachidonic acid; CYP4A;

This study describes a capillary GC–MS method for the simultaneous determination of endogenous 6β-hydroxycortisol (6β-OHF) and its stable isotope-labelled analogue, 6β-hydroxy-[1,1,19,19,19-2H5]cortisol (6β-OHF-2H5), in human urine. 6β-Hydroxy-[1,2,4,19-13C4,1,1,19,19,19-2H5]cortisol (cortisol-13C4,2H5) was used as an analytical internal standard. The methoxime trimethylsilyl ether (MO-TMS) derivatization was employed for the GC–MS analysis of 6β-OHF. Quantitation was carried out by selected-ion monitoring (SIM) of the characteristic fragment ion ([M−31]) of the MO-TMS derivative of 6β-OHF. The sensitivity limit of the present GC–MS-SIM method was found to be 25 pg per injection for 6β-OHF (S/N ratio=5.6). The within-day reproducibility in the amounts of unlabelled and labelled 6β-OHFs determined were in good agreement with the actual amounts added, the relative errors being less than 5.30%. The inter-assay RSDs were less than 4.95% for unlabelled and labelled 6β-OHFs.
Keywords: 6β-Hydroxycortisols;

A capillary zone electrophoretic (CZE) method was investigated for the determination of Gleevec and its main metabolite (N-demethylated piperazine derivative) in human urine using a fused-silica capillary (75 μm I.D.×60 cm total length, 10 cm effective length). The separation was performed with an hydrodynamic injection time of 10 s (0.5 p.s.i.) a voltage of −25 kV, a capillary temperature of 25 °C and a 100 mM phosphoric acid adjusted to pH 2 with the addition of triethanolamine. Under these conditions, the analysis takes about 5 min. A linear response over the 0.4–30.0 mg l−1 concentration range was investigated for two compounds. A dilution of the sample was the only step necessary before the electrophoresis analysis. Detection limits of 0.1 mg l−1 for Gleevec and its metabolite (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine Gleevec and its main metabolite in clinical urine samples.
Keywords: Gleevec; Imatinib mesylate;

Triazinic herbicide determination by gas chromatography–mass spectrometry in breast milk by L. Balduini; M. Matoga; E. Cavalli; E. Seilles; D. Riethmuller; M. Thomassin; Y.C. Guillaume (389-395).
A solid-phase extraction procedure using a graphitized carbon black cartridge for extraction and cleaning of a series of five triazines (atrazine, deethylatrazine, deisopropylatrazine, ametryne and prometryne) from breast milk samples was developed. Using a chemometric methodology, the optimisation of both the analysis time and the triazinic herbicide separation by gas chromatography–mass spectrometry (GC–MS) was then carried out with only 18 experiments. Detection and quantification limits for 1 ml breast milk sample were, respectively, 0.3 and 1 ppb for each studied compound. The variation coefficients were less than 5% over the concentration range from 1 to 100 ppb. The accuracy was between 98.63 and 104.62% for each triazinic herbicide. The recovery was between 58.64 and 63.22% for the concentration range from 1 to 100 ppb for each triazinic herbicide. The assay was successfully applied to the analysis of several breast milk samples.
Keywords: Triazines;

An HPLC method was developed for the determination of a new oxazolidinone, DA-7867 (I), in human plasma and urine and in rat tissue homogenates. To 100 μl of biological sample, 300 μl acetonitrile and 50 μl methanol containing 10 μg/ml DA-7858 (the internal standard) were added. After vortex-mixing and centrifugation, the supernatant was evaporated under a gentle stream of nitrogen. The residue was reconstituted in 100 μl of the mobile phase and a 50-μl aliquot was injected directly onto the reversed-phase (C18) column. The mobile phase, 20 mM KH2PO4:acetonitrile (75:25, v/v) was run at a flow rate of 1.5 ml/min and the column effluent was monitored by a UV detector set at 300 nm. The retention times of I and DA-7858 were approximately 6.5 and 8.7 min, respectively. The detection limits of I in human plasma and urine and in rat tissue homogenates were 20, 20, and 50 ng/ml, respectively.
Keywords: DA-7867; Oxazolidinones;