Journal of Chromatography B (v.775, #2)

News Section (N1-N2).

A sensitive and rapid HPLC assay for determining tazobactam and piperacillin in fatty tissue and serum is described. While the common methods need liquid–liquid extraction before the injection in a automated column switching HPLC, the new method works by direct injection of the filtered tissue extract or diluted serum in a automated column switching HPLC without any other pre-treatment. This was performed by the use of a NH2-precolumn and enrichment/transfer at different pH-level. During the analyses, the NH2-precolumn was automatically regenerated with acetonitrile–water. The chromatogram peaks for piperacillin and tazobactam were identified by the retention time and quantified by peak area. The calibration curve was linear between 1 and 16 μg/ml. The quantification limit of tazobactam was about 1 μg/ml in fatty tissue extracts and in diluted serum (calculated for pure serum 2 μg/ml), respectively. For piperacillin it was less. The described procedure allows sample clean-up and determination of the antibiotic within 35 min. The chromatograms with this easy sample treatment had the same quantity of matrix peaks and in contrast to liquid–liquid extraction no loss of piperacillin. Because of the automatically rinsing of the NH2-precolumn during the chromatographic separation, more than 50 different biological samples could be measured with one NH2-precolumn without loss of performance.
Keywords: Tazobactam; Piperacillin;

Solid-phase extraction, utilizing a 96-well plate format, was used to isolate an alpha-1a receptor antagonist and internal standard from human plasma. Following the isolation procedure, the analyte and internal standard were separated and detected using reversed-phase HPLC coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry operated in the positive ion multiple reaction monitoring (MRM) mode. Based upon the peak area ratio (analyte: internal standard) the analyte was quantified over a concentration range of 0.02–2 ng/ml. Assay validation results including parameters such as precision and accuracy are presented. The validated method was subsequently used to support human pharmacokinetic studies.
Keywords: α-Receptor antagonist;

Behaviour of vanadate and vanadium–transferrin complex on different anion-exchange columns. Application to in vivo 48V-labelled rat serum by Koen De Cremer; Rita Cornelis; Karel Strijckmans; Richard Dams; Norbert Lameire; Raymond Vanholder (143-152).
The behaviour of free [48V]vanadate and [48V]vanadium–transferrin complex was investigated on five different anion-exchange columns (Mono Q 5/5 HR, Hitrap Q HP, Sepharose Q FF, Sepharose DEAE FF and Hitrap Q XL). The recovery of both V-compounds was quantitative. The peak shape and retention time of vanadate varied according to the type of column. The vanadium–transferrin complex also showed different elution patterns depending on the type of column. Especially in case of the Sepharose Q FF, Mono Q 5/5 HR and Hitrap Q XL columns the vanadium–transferrin binding was degraded during elution on the column. The results clearly prove that care should be taken as to the choice of column for speciation purposes of vanadium compounds in order to prevent various artefacts showing up in the chromatograms. A Hitrap Q HP column was used to fractionate different vanadium compounds in rat serum.
Keywords: Vanadate; Vanadium–transferrin complex;

Simultaneous quantitation of fatty acids, sterols and bile acids in human stool by capillary gas–liquid chromatography by Ashok K Batta; Gerald Salen; Priti Batta; G Stephen Tint; David S Alberts; David L Earnest (153-161).
A simple method for the simultaneous gas–liquid chromatographic quantitation of fatty acids, sterols and bile acids from human fecal samples is described. The various compounds are directly converted into the n-butyl ester-trimethylsilyl ether derivatives, without prior isolation from the stool. Under these conditions, fecal bile acid derivatives are well resolved from each other and from those of fecal fatty acids and sterols without overlaps. The method was found to be reproducible and recoveries were similar to those obtained after exhaustive solvent extraction of fecal sterols, fatty acids and bile acids. Optimum derivatization conditions that allowed maximum recovery of fecal components with minimal destruction and application of the method for simultaneous bile acid, fatty acid and sterol analysis in human stool are described.
Keywords: Fatty acids; Sterols; Bile acids;

Mirtazapine is a new centrally acting noradrenergic and specific serotonin antidepressant, with an active demethyl metabolite. For toxicological purposes, a specific and accurate RP-HPLC assay was developed for the simultaneous plasma determination of these compounds. A linear response was observed over the concentration range 50–500 ng/ml. A good accuracy (bias <10%) was achieved for all quality controls, with intra-day and inter-day variation coefficients less than 8.3%. The lower limit of quantification was 20 ng/ml, without interferences with endogenous or exogenous components. This rapid method (run time <12 min) was used to manage three intoxications involving mirtazapine.
Keywords: Mirtazapine; Demethylmirtazapine;

This study described the application of liquid chromatography–tandem mass spectrometry for the quantitation of wogonin and its major metabolite in rat plasma. Only one conjugated metabolite with glucuronic acid was identified by chromatographic and electrospray multi-stage mass spectrometric assay. A derivatization reaction with 2-chlorethanol further demonstrated that the metabolite was wogonin-7β-d-glucuronide (W-7-G), not wogonin-5β-d-glucuronide. Other conjugated metabolites, e.g., sulfates and glucosides, were not detected. The plasma concentration of free wogonin was determined using atmospheric pressure chemical ionization source in the selected reaction monitoring mode. The method had a lower limit of quantitation of 0.25 ng/ml for wogonin, which offered increased sensitivity, selectivity and speed of analysis over an existing method. Incubation of the plasma samples with β-glucuronidase allows the quantitation of W-7-G. This quantitation method was successfully applied to a preclinical pharmacokinetic study of wogonin and its major metabolite, W-7-G, after an oral administration of 5 mg/kg wogonin to rats.
Keywords: Wogonin; Wogonin-7β-d-glucuronide;

Application of size-exclusion chromatography–inductively coupled plasma mass spectrometry for fractionation of element species in seeds of legumes by Richard Koplı́k; Markéta Borková; Oto Mestek; Jana Komı́nková; Miloslav Suchánek (179-187).
Fractionation of soluble species of P, Mn, Fe, Co, Ni, Cu, Zn, Se and Mo in pea and lentil seeds was made by on-line hyphenation of size-exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry. Seed samples were extracted with 0.02 mol l−1 Tris–HCl buffer solution, pH 7.5. SEC was performed on Superdex 75 and Superdex Peptide columns (300×10 mm) with the same buffer solution as the mobile phase. Monitoring of oxide ion 47(PO)+ was used for detection of phosphorus compounds. Other elements were detected as ions of 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se and 95Mo nuclides. Elements in individual elution zones were quantified using external calibration. Complete chromatographic recoveries of elements were found in cases of phosphorus, nickel and copper. Substantial parts of manganese and zinc, as well as traces of cobalt, selenium and molybdenum are retained on the column. Injection of EDTA solution removes these elements from the column. Chromatographic profiles of pea and lentil samples are very similar for all elements except Mo. Main element species in the high-molecular-mass region (approx. 190 000 rel. mol. mass unit) were detected in case of Fe. Low-molecular-mass species (<2000 rel. mol. mass unit) as major element forms are typical for Cu and Zn.
Keywords: Phosphorus; Trace elements;

Validated liquid chromatographic method for the determination of bexarotene in human plasma by N.C van de Merbel; J.H van Veen; G Wilkens; G Loewen (189-195).
A new liquid chromatographic method is described for the determination of the anti-tumour agent bexarotene in human plasma over the range 0.500–1500 ng/ml, using 1 ml of sample. Sample preparation consists of liberating the analyte from plasma lipids by adding acetonitrile, followed by acidification of the plasma and liquid extraction using a mixture of isoamyl alcohol and pentane or hexane. Separation and quantitation are performed by reversed-phase column liquid chromatography with fluorescence detection. Parameters affecting the performance of these steps are discussed. Validation results on linearity, selectivity, accuracy, precision, recovery and stability are shown, as well as the application of the method to samples from clinical trials.
Keywords: Bexarotene;

A liquid chromatographic column-switching method for the enantioselective determination of (RS)-sotalol in plasma was developed and validated. The method is based on the on-line coupling of a precolumn filled with the restricted access material LiChrospher ADS to a cellobiohydrolase I-based chiral stationary phase (CSP). The plasma samples were injected onto the precolumn using a mobile phase containing 1% methanol in 10 mM phosphate buffer at pH 7.4 for 10 min for the removal of matrix components. The analytes were transferred to the CSP for their enantiomeric separation by backflushing the precolumn with 15% 2-propanol in 10 mM phosphate buffer (pH 7.0) including 0.05 mM EDTA. The quantitative determination of the sotalol enantiomers was possible upon addition of the internal standard (S)-atenolol. The method was validated showing a good linearity in the concentration range from 25 to 1000 μg l−1 for each enantiomer. The average values of the intra- and inter-day variability were 1.17% and 3.42%, respectively, for (R)-sotalol and 1.24% and 1.99%, respectively, for (S)-sotalol. The applicability of the method to real world samples has been proven by means of two pharmacokinetic studies. They revealed that the pharmacokinetic properties of the sotalol enantiomers do not differ significantly neither for healthy young volunteers after single dose application nor for elder patients in the steady state.
Keywords: Sotalol;

The improvement in the sensitive determination method of estradiol using HPLC with electrochemical detection is described. The improvement was due to the optimization of the potential applied to the electrode of the analytical cell and employment of a guard cell. The detection conditions were optimized from the electrochemical properties of estradiol in acidic and alkaline eluents. The employment of the guard cell drastically decreased the background noise without any reduction in the response of estradiol, and contributed to improvement in the sensitivity. The optimized method combined with pretreatment by liquid–liquid extraction was applied to the determination of estradiol in rat plasma. The detection limit of 8 pg for the standard solution and 24 pg for the plasma sample, which was about 6–8-fold more sensitive compared to the previous reports, was attained.
Keywords: Estradiol;

Background: Despite some clinical applications of 2-methylcitric acid (2-MCA) determination in urine and amniotic fluid, a diagnostic use of 2-MCA levels in serum is not common practice. This could be related to the complexity of the assay, or possibly to unawareness of other feasible clinical applications. Methods: The levels of the diastereomers 2-MCA I and II in human serum were determined by GC–MS based on a method using a pulsed splitless injection technique. A stable isotope dilution principle was modified considering the diastereomer ratio and impurities of the internal standard. Precision parameters as well as recovery rates of the assay were determined. Reference intervals for 2-MCAtotal, 2-MCA I and II levels were obtained in 52 healthy volunteers (31 female, 21 male, mean age 41.7±14.4 years). Results: 2-MCA was readily detected in each sample of serum, as well as in urine, cerebrospinal fluid and amniotic fluid. The limit of detection was 10 nmol/l for 2-MCAtotal. The internal standard showed a diastereomer ratio of 2-MCA II-d3 to 2-MCA I-d3 of 0.83±0.05, its chemical purity had to be corrected to 90.5±0.5%. In concentrations of 446, 750 and 1256 nmol/l 2-MCAtotal, recovery rates of 98.5, 93.7 and 88% with a mean intra-assay RSD of 1.5% were determined. The day-to-day precision was 10% RSD (SD 40 nmol/l) for 2-MCAtotal obtained with a pooled serum sample at a concentration of 401 nmol/l 2-MCAtotal over a period of 5 months (n=17). The normal range for 2-MCAtotal in human serum was calculated as 81–266 nmol/l confirming previous findings. Conclusions: The GC–MS assay using a pulsed splitless injection procedure ensures a good response to differing concentrations of 2-MCA in various specimens. Considering exact determination of the diastereomer ratio as well as the purity of the internal standard, the assay offers good precision and recovery for 2-MCA I and II levels in serum.
Keywords: 2-Methylcitric acid;

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 205 nm has been validated for the determination of indinavir, ritonavir and lopinavir (ABT 378) in human plasma. The ritonavir analogue A-86093.0 was used as internal standard. Good chromatographic separation was achieved using a stainless steel column packed with 5 μm Phenomenex phenyl hexyl material operated at 40 °C, and a mobile phase consisting of acetonitrile–10 mM potassium phosphate buffer (50:50, v/v). The calibration curve for indinavir was linear over the range of 50 to1000 μg/l while the ritonavir and lopinavir calibration curves were linear over the range of 100 to 15000 μg/l. The lower limit of quantitations for indinavir, ritonavir and lopinavir were 50, 100 and 100 μg/l, respectively, using 500 μl of human plasma. The validation data showed that the assay is sensitive, specific and reproducible for determination of indinavir, ritonavir and lopinavir. This method is being used in a therapeutic drug monitoring service to quantitate these therapeutic agents in patients infected with human immunodeficiency virus.
Keywords: Indinavir; Ritonavir; Lopinavir;

Determination of 9-nitrocamptothecin and its metabolite 9-aminocamptothecin in human plasma using high-performance liquid chromatography with ultraviolet and fluorescence detection by Nadja E Schoemaker; Hilde Rosing; Sindy Jansen; Patrick Schöffski; Jinee Rizzo; Jan H.M Schellens; Jos H Beijnen (231-237).
A high-performance liquid chromatography assay is described for the determination of the investigational anti-cancer drug 9-nitrocamptothecin (9-NC) and its metabolite 9-aminocamptothecin (9-AC) as the total of their lactone and carboxylate forms. The sample pre-treatment consisted of a deproteinisation step and a quantitative acid-catalyzed conversion of all 9-NC and 9-AC into their lactone forms and a subsequent solid-phase extraction. Redissolved extracts were analyzed on a Prodigy analytical column, using a mixture of methanol–phosphate buffer (pH 2.5). Detection was concomitantly performed with UV for 9-NC and fluorimetrically for 9-AC. The lower limit of quantifications were 10 ng/ml and 2.5 ng/ml for the determination of 9-NC and 9-AC, respectively, using 500 μl of plasma. The presented method was successfully applied to a clinical pharmacokinetic study.
Keywords: 9-Nitrocamptothecin; 9-Aminocamptothecin;

The applicability of capillary zone electrophoresis (CZE) for the determination of cephalosporin antibiotics has been studied. In the case of the separation conditions optimised for fourteen cephalosporins, the precision of migration times was smaller than 1.3% RSD, and the values of the limit of detection ranged between 0.42 and 1.62 μg/ml. The proposed CZE method was applied to study the stability of cephalosporins in water at different temperatures (+25, +4 and −18 °C). It was established that the degradation of most cephalosporins was not higher than 20% at room temperature within 4 h of dissolution of these antibiotics.
Keywords: Cephalosporins;

Simple liquid chromatography–electrospray ionization mass spectrometry method for the routine determination of salmon calcitonin in serum by Keon-Hyoung Song; Heung-Man An; Hack-Joo Kim; Sung-Hoon Ahn; Suk-Jae Chung; Chang-Koo Shim (247-255).
A simple liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) method was developed for the quantification of salmon calcitonin (sCT) in serum. Serum samples from rats and dogs were deproteinized and freeze-dried. The residue was then reconstituted with 57% acetonitrile in water containing 0.1% trifluoroacetic acid and 0.005% benzalkonium chloride. A 20-μl aliquot of the reconstituted solution was injected onto a polymer based RP-C18 column, The outlet was connected to an ion-trap mass spectrometer equipped with an ESI source, and spectra were recorded in a positive-ion, selected-ion monitoring mode. The limit of quantification of the method was 10 ng/ml. Biexponential curves were observed for the temporal serum concentration of sCT following intravenous administration of sCT to rats (100 μg/kg) and dogs (250 μg/kg), resulting in reasonable pharmacokinetic parameters. The present method appears applicable to routine analysis of serum sCT in pharmacokinetic studies with good selectivity, accuracy and precision.
Keywords: Calcitonin;