Journal of Chromatography B (v.783, #1)

News Section (N1-N2).

Assay methodology for the quantitation of unbound ertapenem, a new carbapenem antibiotic, in human plasma by Donald G Musson; Kimberly L Birk; Chester J Kitchen; Jin Zhang; John Y.K Hsieh; Wei Fang; Anup K Majumdar; John D Rogers (1-9).
Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree® centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree® device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 μg/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 μg/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 μg/ml and stored at −70 °C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. (n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).
Keywords: Ertapenem; Carbapenem;

This paper describes the development of a HPLC–MS–MS (ESI) method with baseline separation of oxytetracycline, 4-epi-oxytetracycline, α-apo-oxytetracycline and β-apo-oxytetracycline using an XTerra column and an MeOH–MilliQ-water (containing 8 mM formic acid) mobile phase. Limits of quantification for aqueous standards were in the range of 0.004 to 0.008 μM. The linear range tested was 0.003 to 0.5 μM and in one case up to 17 μM. An experiment simulating the degradation of oxytetracycline in manure was set up and free concentrations of the four antibiotics were determined during 6 months. Oxytetracycline (>0.02 μM) was observed up till 6 months after spiking. No important increase in free concentrations of the degradation products was observed.
Keywords: Oxytetracycline; 4-Epioxytetracycline; Apooxytetracycline;

Determination of fluoxetine and norfluoxetine in human plasma by high-performance liquid chromatography with ultraviolet detection in psychiatric patients by Adrián LLerena; Pedro Dorado; Roland Berecz; Antonio González; Marı́a Jesús Norberto; Alfredo de la Rubia; Macarena Cáceres (25-31).
A rapid high-performance liquid chromatographic method is described for the simultaneous determination of the widely used antidepressant drug, fluoxetine and its principal metabolite norfluoxetine in plasma. After liquid–liquid extraction the compounds were separated in a reversed-phase column and assayed by ultraviolet absorption at 226 nm. The analytical interference from psychoactive drugs and their metabolites was also studied. The extraction recoveries were 93 and 87% for norfluoxetine and fluoxetine, respectively. The limit of quantitation under the described conditions was 14 nmol/l for both compounds. The method was found to be reproducible with coefficients of variation less than 10%. A great variability in plasma concentrations of fluoxetine and norfluoxetine as well as in fluoxetine/norfluoxetine ratios was found among the 29 patients studied. This result suggests the implication of genetically polymorphic enzymes, presumably CYP2D6, CYP2C9 and CYP2C19 in the metabolism of fluoxetine to norfluoxetine. Therapeutic drug monitoring should thus be useful in patients treated with regular doses.
Keywords: Fluoxetine; Norfluoxetine;

Quantification of mesna and total mesna in kidney tissue by high-performance liquid chromatography with electrochemical detection by Miranda Verschraagen; Marjolein Bosma; T.H.Ursula Zwiers; Emine Torun; Wim J.F van der Vijgh (33-42).
A sensitive and selective assay for the determination of mesna and total mesna in tissue was developed and validated. After a simple homogenization, extraction and deproteinization step, mesna could be measured immediately by HPLC with an electrochemical detector provided with a sensitive wall-jet gold electrode. Total mesna (i.e., free mesna and mesna present in mesna disulfides and mixed mesna disulfides) could be measured after pre-column reduction with sodium borohydride to free mesna. The lower limit of quantification of mesna and total mesna was for both compounds 10 nmol/g. The assays for mesna and total mesna in tissue were linear over the ranges of 10–3000 and 10–10 000 nmol/g, respectively. The within-day and between-day precisions of both methods were better than 9%. The within-day and between-day accuracy of the mesna assay ranged from 103.7 to 113.6%, whereas the accuracies of the total mesna assay ranged from 97.8 to 106.7%. Mesna in an EDTA containing tissue homogenate or in deproteinized tissue homogenate stored at −80 °C was stable for at least 12 weeks. Total mesna was stable under all conditions measured. The developed assays will be applied for the determination of the distribution of mesna and total mesna in tissues of the rat after administration of mesna or BNP7787.
Keywords: Mesna; BNP7787;

Carboplatin is a platinum analogue that is used in a number of chemotherapeutic regimens for solid tumors, such as lung and ovarian carcinomas. Most often characterization of carboplatin’s pharmacokinetic properties is based on measurement of platinum, rather than intact carboplatin. We have developed a sensitive LC–MS method for the determination of intact carboplatin in plasma ultrafiltrate and in tumor tissue. Carboplatin was extracted from rat plasma ultrafiltrate and tumor samples using solid-phase extraction cartridges and analyzed using reversed-phase chromatography with positive electrospray ionization followed by mass spectrometric detection. Using 50 μl of plasma ultrafiltrate or 140 μl of tumor homogenate supernatant, the extraction afforded a recovery of 58.7 and 45.8% for plasma and tumor, respectively. The mobile phase was 5% acetonitrile in 0.5% acetic acid at 0.2 ml/min that yielded a retention time of carboplatin of 2.2 min. The method has been validated at carboplatin plasma ultrafiltrate concentrations from 0.07 to 2.5 μg/ml, and from 0.03 to 1.3 μg/ml in tumor homogenates. The main advantages of this method compared with earlier methods are the ability to measure intact carboplatin in a sensitive and specific manner.
Keywords: Carboplatin;

A method for the determination of the synthetic narcotic analgesic piritramide in human serum utilizing high-performance liquid chromatography with atmospheric pressure chemical ionization two-stage mass spectrometry (HPLC–APCI-MS–MS) is presented. Pipamperone is used as the internal standard. Serum samples are prepared by liquid–liquid extraction under basic conditions with 1-chlorobutane. The chromatographic separation is achieved on an RP-18 stationary phase applying gradient elution with methanol–0.02% trifluoroacetic acid in water. Detection is carried out in the MS–MS single reaction monitoring mode of the ion-trap mass spectrometer. The limit of detection is 0.3 ng/ml and the calibration covers the range of 1–80 ng/ml. The intra-day RSDs are 6.1 to 7.3% over the calibration range, whereas the inter-day RSDs are 9.6 to 12.8%.
Keywords: Piritramide;

Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography by Kenneth J. Fountain; Martin Gilar; Yeva Budman; John C. Gebler (61-72).
Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50×4.6-mm column packed with porous, 2.5 μm C18 sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of purification. By-products of oligonucleotide synthesis were characterized by liquid chromatography with mass spectrometry detection (LC–MS). We purified oligonucleotides labeled with 6-carboxyfluorescein (6FAM), hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxytetramethylrhodamine (TAMRA) and indodicarboxycyanine (Cy™3) dyes, as well as dually-labeled TaqMan™ probes. Purification of a 0.1-μmole oligonucleotide synthesis in a single injection was demonstrated.
Keywords: Oligonucleotides;

This work presents a high-throughput selected reaction monitoring (SRM) LC–MS method for the determination of methylphenidate (MPH), a central nervous stimulant, and its de-esterified metabolite, ritalinic acid (RA) in rat plasma samples. A separation of these two compounds was achieved in 15 s by employing a 3.5-ml/min flow-rate, a porous monolithic column and a TurboIonSpray source compatible with relatively high flow-rates. In addition, a relatively fast autosampler and a new data acquisition system resulted in a time lag of less than 17 s between consecutive injections. Overall, 768 protein-precipitated rat plasma samples (eight 96-well plates) containing both MPH and RA were analyzed within 3 h and 45 min. The partial method validation described in this report included an assessment of linearity, intra and inter-assay precision and accuracy, and method robustness. Deuterated internal standards for the target compounds, d3-MPH and d5-RA, were employed. The calibration curves ranged from 0.1 to 50 ng/ml for MPH and from 0.5 to 50 ng/ml for RA. The limit of quantification (LOQ) for MPH and RA was 0.1 and 0.5 ng/ml, respectively. For both analytes, the intra- and inter-assay precision (relative standard deviation, % C.V.) and accuracy (relative error) did not exceed 15% for the quality control samples (QCs) QC1, QC2 or QC3 (0.3, 1.5 and 40 ng/ml for MPH and 0.15, 15 and 40 ng/ml for RA) for either analyte and did not exceed 20% at the lower limit of quantitation (LOQ) level. No carry-over from the autosampler was detected. The retention times remained constant throughout the experiment. Baseline resolution of MPH and RA was consistently observed throughout the plates analyzed. The described method demonstrates the feasibility for employing monolithic HPLC columns to effect rapid bioanalytical SRM LC–MS analysis of representative biological samples.
Keywords: Methylphenidate; Ritalinic acid;

Peroxisomicine A1 is a potential antineoplastic substance extracted from plants of the genus Karwinskia. An RP-HPLC–DAD method was developed and validated for the separation and quantification of four isomers of this compound. These isomers coelute in the preparative procedure and are present at a proportion ranging between 3 and 5% in the peroxisomicine A1 purified in the laboratory. The desirability coefficient of the method described here was enhanced 140% with respect to the previously reported method.
Keywords: Peroxisomicines; Isoperoxisomicines;

A simple, highly sensitive and specific method based on gas-chromatography-selected ion monitoring (SIM) mass spectrometry has been developed for the quantitation of corticosterone in rat and mouse plasma. After extraction of the plasma with ethyl acetate, the residue was trimethy-silylated with pentafluorobenzyl hydroxylamine-trimethylsilyl (PFBO-TMS). Detection of the derivatives was accomplished by a quadruple mass spectrometer in the selected ion monitoring mode (m/z of 316, 648, 663 and 678). The detection limit of the assay was 0.1 pg on column. The results show that in the plasma of non-stressed animals, only minor amounts of corticosterone were found; whereas in the plasma of stressed animals, it was dramatically increased. The method developed here can be used to examine corticosterone levels as a marker of stress in rats and mice and may also be used for estimation of the effect of stress-release medications.
Keywords: Corticosterone;

To establish pharmacokinetic/pharmacodynamic relationships, a selective and specific high-performance liquid chromatographic method was developed for the quantitation of remifentanil and its metabolite in dog plasma. The assay involves a solid-phase extraction and a reversed-phase chromatographic separation with ultraviolet detection (λ=210 nm). The calibration curves are linear in the range of 7.89–1500 ng ml−1. Intra-day assay variability is less than 7% for all standards evaluated. Good recovery, linearity, accuracy, and precision were achieved with the assay that proved readily applicable to pharmacokinetic studies in dogs.
Keywords: Remifentanil;

This study presents the optimization of a microemulsion electrokinetic chromatographic (MEEKC) electrolyte solution by using UV detection and with the method, simultaneous separations of chemically, biochemically and pharmaceutically related anionic and cationic compounds. Representatives of the compound groups were from isoflavonoids, benzodiazepines, metanephrines, diuretics and peptide hormones. The MEEKC separations under basic conditions were first optimized using a two-component isoflavonoid mixture as the sample and an electrolyte containing 10 mM tetraborate as the main buffer (pH 9.5). The stable microemulsion phase was adjusted with various amounts of octane, 1-butanol and sodium dodecyl sulfate (SDS). An only acidified electrolyte solution used in the study was made of phosphoric acid (pH 1.8) containing octane, SDS and ethyl acetate. The analyses with isoflavonoids showed that electrophoretic mobilities of the investigated compounds were highly related to the concentrations of SDS and 1-butanol with linear and parabolic correlation, respectively. However, addition of octane gave linear correlation only at low concentrations. In most cases four to six structurally related compounds and even 13 diuretics with various polar properties were separated from each other in basic microemulsion medium. The acidified MEEKC electrolyte gave good resolution for anionic metanephrines.
Keywords: Polar compounds;

A gas chromatographic–mass spectrometric assay was developed for the determination of benzidine (BZ)–hemoglobin adducts. Adducts were released from hemoglobin by alkaline hydrolysis and extraction at pH 8 with ethyl ether. The dried extract was completely derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)–NH4I (1000:3) under catalysis of dithioerythritol. The recovery of BZ, acetylbenzidine (ABZ) and diacetylbenzidine (DABZ) in the extraction procedure was 76–98%. The detection limits of the assay were 0.1 ng/g for both BZ and ABZ, and 0.5 ng/g for DABZ based upon assayed hemoglobin of 0.1 g. The method was applied to the determination of BZ–hemoglobin adducts formed in young female Sprague–Dawley rats after treatment for 1, 2 and 3 weeks with 0.008% BZ via the drinking water. Two adducts were detected by proposed procedure. The structure of these adducts could be assigned to BZ and ABZ. After 1 week, the total mean amount of adducts determined was 2.8 ng/g hemoglobin. The adduct levels increased up to about 7.5 ng/g after a week and, thereafter, remained essentially constant. The relative contribution of BZ and ABZ to the total hemoglobin adduct level was strongly treatment time-dependent. After 1 week, the BZ and ABZ adducts were formed at similar levels, whereas after 3 weeks the ABZ adducts was predominant. Treatment of rats for 3 weeks in the dose range 12.2–36.8 mg of BZ in drinking water resulted in a dose-proportional increase in the total amount of hemoglobin adducts formed.
Keywords: Benzidine–hemoglobin adducts;

A new method, using high-performance liquid chromatography/ion electrospray (negative ion) mass spectrometry, has been developed for the determination of a hydrophilic liver-specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin in human plasma. In this method, plasma samples were prepared by a solid-phase extraction on C18 Bond Elut cartridge. Chromatography was carried out with a Zorbax C8 column. Simple isocratic chromatography conditions were used. The method has been validated in a linear range of 0.25–300 ng/ml with a coefficient of variation of 0.6–3.4%. The overall recovery was 90.5% for pravastatin and 90.8% for the internal standard β-hydroxy-lovastatin. The method is simple and reliable with a total run time of less than 2 min.
Keywords: Pravastatin;

Zilpaterol is an adrenergic drug currently licensed in Mexico and South Africa as a feed additive for cattle close to consignment. In this study an analytical method to detect zilpaterol in commercial feeds was set up. The influence of extraction solvent and matrix was evaluated. The drug as a trimethylsilyl derivative was characterized by GC–MS, on a quadrupole detector, in the electron impact mode. Acidic extraction, solid-phase extraction C18 non-endcapped clean-up and mass characterization on ions m/z 308, 291, 405, 390 provided zilpaterol recoveries >75.3% and repeatability <3.3% in feeds spiked in the range 30.0–120.0 ng/g. The limits of detection and quantification were 7.5 and 25.0 ng/g, respectively. Such limits are well below the dose of 5.0–20.0 μg/g proposed as effective.
Keywords: Zilpaterol;

A simple methodology based on fluorescence and gel exclusion chromatography (GEC) has been developed to assay recombinant Interleukin-2 (rIL-2) associated with vesicles. A Sephadex G75 column was used to separate the liposomes from non-entrapped rIL-2. The elution of the rIL-2 liposomes was monitored by coupling fluorescent and light scattering detection. The solubilisation of the vesicles with octylglucoside (OG) before the assay was necessary to avoid interference from light scattering. This methodology can be automated to yield an on-line system that can separate, solubilise and quantify rIL-2 in liposome samples. It can be extended to any protein associated with vesicles provided that the former can be detected by fluorescence.
Keywords: Interleukin-2;

A sensitive and selective LC–MS–MS method for the determination of DPC 423 (I), an antithrombotic agent, is described. This method used a solid-phase extraction from 0.1 ml plasma with an Isolute C2 cartridge. HPLC separation was carried out on a YMC ODS-AQ C18 column (50×2 mm) at a flow-rate of 300 μl/min with an analysis time of 5 min. Compounds were eluted using a mobile phase of H2O/CH3CN/HCOOH: 66:34:0.1 (v/v/v), pH 4.0. A structural analogue of I was used as the internal standard to account for variations in recovery and instrument response. Mass spectrometric detection was carried out with a PE Sciex API III+ triple quadrupole mass spectrometer equipped with a Turbo IonSpray™ source as the LC–MS interface. Good intra-day and inter-day assay precision (<10% CV) and accuracy (<10% difference) were observed over a concentration range of 0.005–2.5 μM in plasma. The extraction recoveries were approximately 90% and the method was found to be linear for the assay (r 2>0.999). The method has been successfully applied to discovery and preclinical pharmacokinetic studies, including a dose range-finding study and toxicokinetic exposure studies in rat and dog.
Keywords: Antithrombotic agents; DPC 423;

Histone acetylation and methylation are processes that are generally considered to play crucial roles in the chromatin-based regulatory mechanism. Characterization of the histones as well as their modification sites has become increasingly important. In this paper, the use of LC–MS and peptide mapping methods to analyze chicken core histones and identify the modification sites is reported. Microbore C4 HPLC separated the core histones into H2A, H2B, H3 and H4 using HFBA as the ion-pairing agent. The four subclasses of histones and their putative acetylated or methylated isoforms were identified by LC–MS simultaneously. MALDI-TOF and tandem mass spectrometry provided peptide mapping of the modification sites of the histones through trypsin digestion of the HPLC eluents. This approach is straightforward and prospective for further application in the field of chromatin research.
Keywords: Histones;

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC–thermospray mass spectrometry. The HPLC–thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa+ and MH+–H2O ions. As the other minor ions, MK+, MH+ and m/z 282 ions were detected. Although the preponderance of MNa+ ions compared with the MH+–H2O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH+–H2O ions based on the elimination of the hydroxyl group introduced at the α-position of acyl-portion compared with the MNa+ ions was detected in α-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-d-sphingosine (f=0.45), N-palmitoyl-d-sphingosine (f=0.40), N-stearoyl-d-sphingosine (f=0.39), N-nervonoyl-d-sphingosine (f=0.39) and N-lignoceroyl-d-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100 000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa+/MH+–H2O ions corresponding to several ω-hydroxyacyl-ceramides were detected.
Keywords: Ceramide;

Atypical mitochondrial creatine kinase (creatine N-phosphotransferase, CK, EC was detected in the serum of a patient with carcinoma of germ cell origin, probably hepatoid yolk sac tumor. The pI of the oligomeric atypical mitochondrial CK (Mi-CK) was found at the acidic side compared to that of the typical ubiquitous Mi-CK (uMi-CK), while the molecular size of the atypical Mi-CK was similar to that of the typical uMi-CK. The pIs of the oligomeric and the dimeric atypical Mi-CKs became the same as those of the typical uMi-CK upon treatment with 2-mercaptoethanol. Therefore, the atypical Mi-CK was suggested to be an oxidized form of uMi-CK, and the oxidation might have occurred in the mitochondria because the oligomeric atypical Mi-CK had atypical pIs. The physicochemical characteristics of the oxidized uMi-CK were similar to those of the typical uMi-CK.
Keywords: Enzymes; Creatine kinase;

An analytical method for the determination of benazepril and its active metabolite, benazeprilat, in human plasma by capillary gas chromatography–mass-selective detection, with their respective labelled internal standard, was developed and validated according to international regulatory requirements. After addition of the internal standards, the compounds were extracted from plasma by solid-phase extraction using automated 96-well plate technology. After elution, the compounds were converted into their methyl ester derivatives by means of a safe and stable diazomethane derivative. The methyl ester derivatives were determined by gas chromatography using a mass-selective detector at m/z 365 for benazepril and benazeprilat and m/z 370 for the internal standards. Intra- and inter-day accuracy and precision were found to be suitable over the range of concentrations between 2.50 and 1000 ng/mL.
Keywords: Benazepril; Benazeprilat;

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C18) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10–500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.
Keywords: Celecoxib; Hydroxycelecoxib; Carboxycelecoxib;

Determination of risperidone and 9-hydroxyrisperidone in human plasma by liquid chromatography: application to the evaluation of CYP2D6 drug interactions by Adrián LLerena; Roland Berecz; Pedro Dorado; César Sanz de la Garza; Marı́a Jesús Norberto; Macarena Cáceres; José Ramón Gutiérrez (213-219).
A high-pressure liquid chromatography with ultra-violet detection method for the simultaneous determination of risperidone and 9-hydroxyrisperidone in plasma after liquid–liquid extraction has been developed. The limit of quantitation was 5 nmol/L, and the inter-day coefficient of variation was less than 8% for both compounds. The mean recoveries of risperidone and 9-hydroxyrisperidone added to plasma were 96.8 and 99.4%, with an intra-day coefficient of variation of under 5 and 6%, respectively. Studies of analytical interference showed that the most commonly co-administered antidepressants and benzodiazepines did not interfere. The method was used for the determination of the plasma concentrations of a schizophrenic patient treated daily with an oral dose of 4.5 mg risperidone. The patient suffered severe extrapyramidal side-effects after adding risperidone to his previous medication of haloperidol and levomepromazine. The risperidone plasma concentration was well above the average (182 nmol/L), which suggests that a pharmacokinetic interaction occurred, presumably due to inhibition of the enzyme CYP2D6.
Keywords: Risperidone; 9-Hydroxyrisperidone; CYP2D6;

In this paper, a new high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICP-MS) methodology for the analysis of organotin compounds in complex matrices is described. Earlier studies had failed to show baseline resolution between dibutyltin (DBT) and triphenyltin (TPhT). The data presented in this paper show that, by using a different C-18 stationary phase material (Ace C-18) with decreased particle size, baseline resolution of DBT and TPhT can be achieved, with the resultant separation of a third interfering component. In addition, the Ace C-18 stationary phase yields a significant increase in the number of theoretical plates, and, combined with changes in the mobile phase composition, a reduction in run-time by ∼25%. It is shown that the minor compounds detected are present in the sample and not artefacts of the analytical procedure. The accuracy and precision of the proposed HPLC–ICP-MS method was demonstrated for the determination of TBT in oyster tissue during the BCR “MULSPOT” international interlaboratory certification project.
Keywords: Organotin compounds;

Sensitive liquid chromatography–mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma by Robert A Parise; Ramesh K Ramanathan; William C Zamboni; Merrill J Egorin (231-236).
We have developed a high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS) method for quantifying docetaxel and paclitaxel in human plasma. The assay fulfills the need for defining the lower plasma concentrations of these antineoplastic agents that result from a number of changes in how these agents are used clinically. The assay uses paclitaxel as the internal standard for docetaxel, and vice versa; solid-phase extraction; a Phenomenex Hypersil ODS (5 μm, 100×2 mm) reversed-phase analytical column; an isocratic mobile phase of 0.1% formic acid in methanol–water (70:30, v/v); and mass spectrometric detection using electrospray positive mode electron ionization. The assay has a lower limit of quantitation (LLOQ) of 0.3 nM and is linear between 0.3 nM and 1 μM for docetaxel. For paclitaxel, the LLOQ was 1 nM, and the assay is linear between 1 nM and 1 μM. We demonstrated the suitability of this assay for docetaxel by using it to quantify the docetaxel concentrations in plasma of a patient given 40 mg/m2 of docetaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. In a similar manner, the suitability of the assay for paclitaxel was demonstrated by using it to quantify the concentrations of paclitaxel in the plasma of a patient given 15 mg/m2 of paclitaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. The LC–MS assay, which proved superior because of its greater sensitivity and relatively short (7 min) run time, should be an important tool for future pharmacokinetic analyses of docetaxel and paclitaxel.
Keywords: Docetaxel; Paclitaxel;

This paper describes a simple, fast, sensitive and reliable method for the simultaneous determination of albendazole sulfoxide (ASOX) and albendazole sulfone (ASON), the two most important metabolites of the drug albendazole (ABZ), in plasma samples using liquid chromatography and tandem mass spectrometry. After liquid–liquid extraction with dichloromethane, the two albendazole metabolites and the internal standard phenacetin were resolved in a CN column using the mobile phase methanol–water (4:6, v/v) acidified with 1% acetic acid. Detection by electrospray mass spectrometry was carried out in the positive ion mode. The method was linear up to 2500 and 250 ng/ml for ASOX and ASON, respectively, with mean recoveries of more than 85%. The precision and accuracy data, based on within- and between-day variations over 5 days, were lower than 15%. The quantitation limits of 0.5 and 5.0 ng/ml for ASON and ASOX are low enough for the method to be suitable for pharmacokinetic studies. Pharmacokinetic data obtained with the proposed method following oral administration of ABZ to a patient with neurocysticercosis are also reported.
Keywords: Albendazole sulfoxide; Albendazole sulfone;

Angiotensin I converting enzyme (ACE) plays a major role in blood pressure regulation, catalyzing the conversion of angiotensin I to the vasoconstrictor angiotensin II. In this report we describe a two-step affinity chromatography method for preparative purification of ACE from pig lung using Concanavalin-A Sepharose 4B and affinity chromatography on Lisinopril Sepharose 6B. The same purification scheme was used to obtain Cobalt-ACE, where zinc ion located at the active site is replaced by cobalt. Cobalt-ACE visible spectrum shows a characteristic broad peak from 500 to 600 nm. The shape and maximum absorptivity of this peak changes in presence of ACE inhibitors that bind at the catalytic site.
Keywords: Enzymes; Angiotensin converting enzyme;

A method based on high-performance liquid chromatography with UV detection in combination with solid-phase extraction for sample pretreatment has been developed for the simultaneous analysis of the antiepileptic drug oxcarbazepine and its main metabolites in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective solid-phase extraction procedure using hydrophilic–lipophilic balance cartridges. The separation was obtained on a reversed-phase column (C18, 150×4.6 mm I.D., 5 μm) using a phosphate buffer–acetonitrile–methanol–triethylamine mixture (final apparent pH* 3.5) as the mobile phase. Under these chromatographic conditions, oxcarbazepine and its metabolites 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine and the internal standard are baseline separated in less than 9 min. The extraction yield values were >94% for all analytes and the precision, expressed by the RSD%, was in the low percentage range. For the entire method, including sample pre-treatment and HPLC determination, the linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.995; the limit of quantitation was 15 ng ml−1. The method was applied to plasma samples from patients undergoing chronic treatment with oxcarbazepine, both in monotherapy and in polytherapy. Based on the analytical parameters precision, accuracy, limit of quantitation and analysis time the method is suitable for routine application in therapeutic drug monitoring.
Keywords: Oxcarbazepine;

A sensitive, specific and reproducible gas chromatographic assay utilizing mass-selective detection has been developed for the stereoselective determination of mephenytoin (MP) in human urine. Following extraction of urine samples using methyl tert.-butyl ether, separation of R- and S-MP was achieved with a chiral capillary column; detection and quantitation were accomplished by mass spectrometry in the single ion monitoring mode (m/z 104 and 189). Excellent linearity was observed for both enantiomers over the concentration range of 5–1000 ng/ml with corresponding correlation coefficients (r)>0.99. The intra- and inter-day precision and accuracy were within ±5%. This method employs a simplified processing procedure, demonstrates improved extraction recovery, and provides at least 5-fold greater sensitivity than previously reported assays. This method is well suited for the phenotypic evaluation of CYP2C19 activity using mephenytoin.
Keywords: CYP2C19; Mephenytoin;

A reversed-phase high-performance liquid chromatography method with electrospray ionization and mass spectral detection is described for the determination of capecitabine, 5′-deoxy-5-fluorocytidine and 5′-deoxy-5-fluorouridine in human plasma with 5-chloro-2′-deoxyuridine as the internal standard. An on-line sample clean-up procedure allows dilution of the plasma sample with the initial mobile phase. The linear dynamic range is 0.0500–10.0 μg/ml for capecitabine, and 0.0500–25.0 μg/ml for the metabolites, 5′-deoxy-5-fluorocytidine and 5′-deoxy-5-fluorouridine, respectively. This method has been used to analyze plasma samples from patients receiving capecitabine in combination with oxaliplatin.
Keywords: Capecitabine; 5-Deoxy-5-fluorocytidine; 5-Deoxy-5-fluorouridine;

Curcumin in plasma and urine: quantitation by high-performance liquid chromatography by Dennis D Heath; Milagros A Pruitt; Dean E Brenner; Cheryl L Rock (287-295).
Curcumin, a derivative of the plant Curcuma longa, is used extensively in the food industry. It is a major component of curry powder, and research has shown that curcumin may prevent cancer and other chronic diseases. We have developed a robust automated analytical method for the determination of curcumin in plasma and urine. The method involves extracting the curcumin from 0.2 ml sample volume with ethyl acetate/methanol organic solvents, and use of an internal standard, β-17-estradiol acetate. Analysis utilizes a reversed-phase C18 column and UV detection at 262 nm. Performance characteristics have been assessed. The assay is linear from 0.2 to 7.0 μg/ml. The coefficient of variation for intra- and inter-day assays is <7.5%. The average recovery of curcumin from plasma and urine is greater than 96%. The data presented in this report demonstrate that the method provides rapid, sensitive, precise and accurate measurements of curcumin concentrations in plasma and urine.
Keywords: Curcumin;

Simple liquid chromatographic method for the determination of cefotaxime in human and rat plasma by Sharon Sheue Nee Ling; Kah Hay Yuen; Susan A Barker (297-301).
A high-performance liquid chromatographic method with ultraviolet (UV) detection was developed for measuring cefotaxime in rat and human plasma. The method used direct injection of the plasma supernatant after deproteinization with 70% perchloric acid. Degradation of cefotaxime in acidic medium was retarded by adding phosphate buffer before centrifuging the sample. The mobile phase was 0.05 M aqueous ammonium acetate–acetonitrile–tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5. Analysis was run at a flow-rate of 1.0 ml/min, and a detection wavelength of 254 nm was used. The method has a quantification limit of 0.20 μg/ml. The within- and between-day coefficients of variation and accuracy values were less than 8% and ±3%, respectively, while the recovery values were greater than 87% over the concentration range tested (0.20–50 μg/ml). The speed, sensitivity, specificity and reproducibility of this method make it particularly suitable for the routine determination of cefotaxime in human plasma. Moreover, only a relatively small sample plasma volume (100 μl) is required, allowing this method to be applied to samples taken from neonates.
Keywords: Cefotaxime;

A rapid, sensitive and selective LC–MS method was developed for the simultaneous determination of midazolam (MDZ) and 1′-hydroxymidazolam (1′-OHMDZ) in plasma taken from 54 patients undergoing methadone maintenance therapy, most of whom were multidrug users. Samples spiked with prazepam, the internal standard, and were extracted into diethyl ether. Compounds were separated on a Phenomenex Luna C18 column and a mobile phase of acetonitrile–ammonium acetate buffer (10 mM, pH 4.7) (52:48, v/v) at a flow-rate of 1 ml/min. The limit of detection was 0.65 and 0.68 (ng/ml) for MDZ and 1′-OHMDZ, respectively. Within-day relative standard deviations were less than 8%.
Keywords: Midazolam; 1′-Hydroxymidazolam;