Simultaneous determination of isoline and its two major metabolites using high-performance liquid chromatography

A simple and reliable high-performance liquid chromatographic (HPLC) assay was developed for a simultaneous determination of isoline, a potent hepatotoxic pyrrolizidine alkaloid, and its two major metabolites, namely M1 (bisline) and M2 (bisline lactone, a new pyrrolizidine alkaloid). The latter two metabolites were produced during in vitro metabolism of isoline by rat and mouse microsomal enzyme systems. The analysis was conducted by a direct injection of aliquots of supernatant of the microsomal reaction mixture treated with the equal volume of ice-cold methanol onto a conventional reversed-phase analytical column (150 x 4.6 mm). The analytes were separated by a gradient elution with mobile phases A (0.01 M dihydro-potassium phosphate, pH 4.8) and B (acetonitrile). The assay has shown excellent precision and accuracy with less than 10% of overall intra- and interday variations and higher than 94% of overall accuracy. The developed HPLC method was successfully applied for the determination of the intact isoline and its two pH- and thermally labile metabolites produced in rat and mouse liver microsomal incubations.     

Simultaneous determination of serum flecainide and its metabolites by using high performance liquid chromatography

Simultaneous determination of serum flecainide and its oxidative metabolites was carried out by using high performance liquid chromatography (HPLC) equipped with conventional octadecylsilyl silica (ODS) column and fluorescence detector. Flecainide and its metabolites, m-O-dealkylated flecainide (MODF) and m-O-dealkylated lactam of flecainide (MODLF) in serum were extracted with ethyl acetate. The recoveries of flecainide, MODF and MODLF were greater than 92, 93, and 60% with the coefficient of variations (CVs) less than 3.2, 5.8, and 5.3%, respectively. The calibration curves were linear at the concentration range of 50–1500 ng/mL for flecainide and 10–500 ng/mL for MODF and MODLF (r>0.999). The CVs for intra-day assay were 2.7–5.3% for flecainide, 3.0–4.2% for MODF, and 3.7–4.3% for MODLF, respectively. The CVs for inter-day assay were 7.0–8.4% for flecainide, 3.3–6.7% for MODF, and 4.4–7.7% for MODLF, respectively. This assay method can be used for assessing the metabolic ability of flecainide in the patients with tachyarrhythmia.     

An assay for methotrexate and its metabolites in serum and urine by ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic assay for methotrexate and its metabolites, 7-hydroxy-methotrexate, 4-amino-4-deoxy-N10-methyl-pteroic acid and 7-hydroxy-4-amino-4-deoxy-N10-methyl-pteroic acid in the range 10 microg/l to 50 mg/l (2.2 x 10(-8) to 1.1. x 10(-4)M) has been developed using L-tryptophyl-L-glutamic acid as internal standard. Extraction was performed using an anion exchange resin (Dowex 1-X2) with subsequent ion-pair chromatography of the appropriate eluent fraction. The method has been found to be sensitive and precise for the analysis of both serum and urine, and may also be used for the quantitation of polyglutamyl metabolites.     

Determination of amoxapine and its metabolites in human serum by high-performance liquid chromatography

The simultaneous quantitative determination of amoxapine, 7-hydroxyamoxapine and 8-hydroxyamoxapine in human serum was established, with good recoveries, using reversed-phase high-performance liquid chromatography (HPLC). Prior to analysis by high-performance liquid chromatography, the enzymic hydrolysis with β-glucuronidase/arylsulphatase of sera from healthy volunteers receiving the drug snowed that each conjugate of two hydroxyamoxapines was 75–90% of the amount determined by the present method. The concentrations of amoxapine and its hydroxylated metabolites were measured against time in sera from the volunteers who were given the antidepressant orally for 2 weeks. The serum levels of 8-OH-amoxapine were markedly higher than the drug itself and the 7-OH-derivative. Whereas the levels of the drug were little increased during the continuous administration, the levels of 8-OH-amoxapine were linearly increased until the fourth day after the administration was started. In addition, the ratio of each hydroxylated metabolite to the drug and the time-course of their serum levels varied interindividually.     

高效液相色谱法同时测定人血清中茶碱与苯巴比妥、苯妥英、卡马西平的含量

本文报道茶碱与苯巴比妥（ＰＢ）、苯妥英（ＤＰＨ）、卡马西平（ＣＢＺ）两类（４种）不同药物血浓度的ＨＰＬＣ同时测定法．采用国产色谱柱ＹＷＧＣ１８（４．６×２５０ｍｍ），检测波长为２５４ｎｍ，流动相为甲醇－水（５０：５０，ｖ／ｖ），流速：１ｍｌ／ｍｉｎ，以４－氨基安替匹林作内标，各药物的平均回收率分别为茶碱９９．０２％，ＰＢ１００．７３％，ＤＰＨ１０１．０％，ＣＢＺ１０１．７７％。对各药血清标准液的峰高比测量的变异系数（ｎ＝１０），分别为茶碱（１０μｇ／ｍｌ）６．３％，ＰＢ（２０μｇ／ｍｌ）６．１％，ＤＰＨ（２０μｇ／ｍｌ）４．０％，ＣＢＺ（１０μｇ／ｍｌ）５．２％。本法具有快速、准确、适用性广的特点，用于治疗药物的监测，效果满意，大大提高了实验室的工作效率。     

Simultaneous determination of albendazole and its principal metabolites in plasma by normal phase high-performance liquid chromatography

A sensitive, specific and reproducible high-performance liquid chromatography procedure using normal phase is described for the simultaneous determination of albendazole, albendazole sulphoxide and albendazole sulphone in sheep plasma, with mebendazole as an internal standard. Analysis of plasma requires only 100 mul of sample, which is extracted with ethylacetate and injected directly onto a 5-mum normal phase column, using hexane-ethanol (445:55, v/v) as eluent, with detection at 225 nm. The standard curves in plasma were linear for both albendazole and its metabolites at concentrations from 0.1 to 10 mug/ml. The method has been applied to the determination of plasma levels of albendazole and metabolites during preliminary pharmacokinetic studies in sheep.     

Quantitative determination of verapamil and metabolites in human serum by high-performance liquid chromatography and its application to biopharmaceutic investigations

A specific and sensitive high-performance liquid chromatographic method for the quantitative analysis of verapamil and N-desmethylverapamil in human serum is described. The analytes were extracted from serum using diethylether under alkaline conditions, followed by back extraction into dilute hydrochlorid acid for chromatographic analysis on a reversed-phase column with a mobile phase consisting of acetonitrile, water and perchloric acid at a flow rate of 1 l/min. The analytes were detected by fluorescence detection, the influence of temperature on retention is discussed. The method is linear, quantitative and reproducible for two calibration ranges in serum (2.5 ng/ml-100 ng/ml and 12.5 ng/ml-500 ng/ml) using peak area ratios analyte/internal standard for quantification. At ultimate sensitivity, concentrations down to 250 pg/ml could be assayed. The method was selective to 6 other metabolites of verapamil and common exogenous interferences. It was applicated to the serum samples of a comparative 120 mg - verapamil hydrochloride tablet single dose two-way cross-over study comprising 18 volunteers. The pharmacokinetic data for both formulations are presented.     

The determination of D-penicillamine and its disulphide in plasma by reversed-phase ion-pair high-performance liquid chromatography

Reversed-phase ion-pair conditions are used for the determination of D-penicillamine and penicillamine disulphide. Two chromatographic systems were employed, one for penicillamine and the other for penicillamine disulphide. The procedures permit the determination of total penicillamine (protein-bound, free and as disulphides) in whole plasma, and total penicillamine (free and as disulphides) in plasma ultrafiltrate, using an incubation step in the presence of dithiothreitol. Free penicillamine and penicillamine disulphide may be determined independently by direct injection of plasma ultrafiltrate. Both solutes may be measured at an on-column sensitivity of 10 ng, utilizing an electrochemical detector based on a glassy carbon electrode.     

Simultaneous plasma determination of floctafenin and its major metabolites by high-performance liquid chromatography: preliminary observations in children

An isocratic reversed-phase ion-pair liquid chromatography with UV detection at 350 nm for the determination in human plasma of floctafenin (F) and its three main metabolites--floctafenic acid (FA), hydroxyfloctafenin (HOF), and hydroxyfloctafenic acid (HOFA)--is reported. Analytes and internal standard were extracted from acid plasma into ethyl acetate, and this organic phase was evaporated to dryness. This extraction yielded plasma drug recoveries of greater than 72%. Using 1 ml of plasma, the lower quantification limit was 0.05 microgram ml-1 with excellent linearity up to 0.8 microgram ml-1 for HOF and HOFA and up to 4.0 micrograms ml-1 for F and FA. The reproducibility and the selectivity of the method for several drugs thought likely to be administered in conjunction with F, were demonstrated. This method has been successfully applied to a pharmacokinetic study with a single 10 mg kg-1 oral dose in ten children.     

Fluorometric determination of isoniazid and its metabolites in urine by high-performance liquid chromatography using in-line derivatization

A rapid, simple, and accurate method has been developed for the determination of isoniazid and its metabolites (isonicotinic acid, isonicotinylglycine, and acetylisoniazid) in human urine by high-performance liquid chromatography. Isoniazid and its metabolites are separated by reversed-phase ion-exchange chromatography with a mobile phase containing hydrogen peroxide as a fluorogenic reagent and butanesulfonate as a hydrophobic ion exchanger, and are detected by fluorometry (excitation at 317 nm and emission at 415 nm) using in-line derivatization at high temperature (160 degrees C). The detection limits are isonicotinic acid, 0.5 mumol/L; isonicotinylglycine, 1 mumol/L; acetylisoniazid, 1 mumol/L; and isoniazid, 1.5 mumol/L. This method can be applied for acetylator phenotyping.     

Simultaneous determination of cefepime and grepafloxacin in human urine by high-performance liquid chromatography

A liquid chromatographic method with UV detection for simultaneous determination of cefepime and grepafloxacin has been developed. The method uses a C18 column, equipped with a pre-column of the same material, and acetonitrile-0.1 M phosphoric acid/sodium hydroxide buffer (pH 3.0)-0.01 M n-octylamine (pH 3.0) as mobile phase in gradient mode. Mobile flow rate and sample volume injected were 1.3 mL min(-1) and 20 microL, respectively. Detection wavelengths were 259 nm for cefepime and 278 nm for grepafloxacin. The retention times were 4.03 min for cefepime and 8.85 min for grepafloxacin, with detection limits of 1.0 and 1.1 microg mL(-1), respectively. The method was applied to the determination of both antibiotics in spiked samples of human urine.     

An expedient assay for determination of gemcitabine and its metabolite in human plasma using isocratic ion-pair reversed-phase high-performance liquid chromatography

An expedient method is presented for determination in human plasma of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) by ion-pair reversed-phase HPLC. Samples were simply prepared by protein precipitation. Separation was processed on a Thermo Hypersil column (250 x 4.6 mm, 5 microm Hypersil BDS C18) with UV detection at 272 nm. The mobile phase consisted of 17% methanol and 83% phosphate buffer (20 mM, pH 3.1) containing 10 mM sodium 1-heptanesulfonate with a flow rate of 0.8 mL/min. The lower limit of quantification (LLOQ) of gemcitabine was 0.08 microg/mL with linear response over the range 0.08-20.0 microg/mL, and LLOQ of dFdU was 0.1 microg/mL with linear response over the range 0.1-50.0 microg/mL. Assay accuracy for both compounds was within +/- 4%. The coefficient of variation (CV %) for intra- and interday precision for both compounds was <7%. The correlation coefficients (r2) were greater than 0.9996 for all standard curves. The simple method with adequate sensitivity has been successfully used in phase I and II gemcitabine pharmacokinetic and pharmacodynamic studies in an Asian population.     

Simultaneous determination of lamotrigine, phenobarbitone, carbamazepine and phenytoin in human serum by high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous estimation of the antiepileptic drugs (AEDs) lamotrigine (LTG), phenobarbitone (PB), carbamazepine (CBZ) and phenytoin (PHT) in human serum. The procedure involves extraction of the AEDs by mixing 200 microl of serum with 200mul of acetonitrile containing 10 microg/ml of pentobarbitone as internal standard (IS). After centrifugation, 10 microl of the supernatant was injected onto a NOVA PAK C-18 column (250 mm x 4.6mm, 5 microm Hypersil ODS) and eluted with a mobile phase consisting of phosphate buffer (10 mM)-methanol-acetonitrile-acetone in the ratio of 55:22:12:11 (v/v) adjusted to pH 7.0. A UV detector set at 210 nm was employed for detection. The AEDs were well resolved from the human serum constituents and the internal standard. The method can quantify LTG, PB, CBZ, and PHT at concentrations as low as 0.2 microg/ml. The method was quantitatively evaluated in terms of linearity, accuracy, precision, recovery, selectivity, sensitivity, and specificity. The method is simple, convenient, and suitable for the analysis of AEDs from human serum.     

HPLC-MS/ESI+测定人血浆中喹硫平及其代谢产物

目的建立同时测定人血浆中喹硫平及其磺氧化-、7-羟基-和7-羟基-氮-去烷基-代谢产物浓度的高效液相色谱-电喷雾电离质谱联用法.方法采用Kromasil C18 反相柱(250 mm×4.6 mm,5 μm),以水(含甲酸1.70 mmol·L-1, 醋酸铵5.8 mmol·L-1)-乙腈(6535)为流动相,流速0.95 mL·min-1. 质谱采用电喷雾电离源正离子模式(ESI+),选择离子监测(SIR)各物质准分子离子峰,样品用固相萃取法处理.结果喹硫平和磺氧化喹硫平在10～2 000 μg·L-1,7-羟基-喹硫平和7-羟基-氮-去烷基喹硫平在1～200 μg·L-1线性关系良好,萃取回收率均＞85%,方法回收率均＞95%,日内、日间RSD均＜15%.结论该方法专一性强、灵敏度高、简单,可用于研究喹硫平的代谢机制以及药物动力学.     

The determination of metoclopramide in plasma by reversed-phase ion-pair high-performance liquid chromatography

Methodology based on reversed-phase ion-pair high-performance liquid chromatography is described for the determination of metoclopramide in plasma. The chromatography was optimized in terms of the peak shape for the drug and its resolution from endogenous plasma components by investigating the effects of quaternary ammonium (competing) ions and alkylsulphate (pairing) ions in an acidic mobile phase containing acetonitrile (20%) and 20 mM acetic acid. Optimum chromatographic conditions were obtained with an ODS-Hypersil column and a mobile phase containing 20% acetonitrile, 20 mM acetic acid, 0.6 mM sodium octylsulphate and 0.5 mM tetrabutylammonium chloride. A simplified method of sample preparation is described in which only 1 ml of plasma is required. The limit of detection (at 310 nm) was 7 ng/ml and no interference from endogenous plasma components or from any drugs commonly used in the treatment of cancer was observed. Consequently the methodology should be applicable to pharmacokinetic studies on metoclopramide, when used clinically to control the gastro-intestinal side-effects of chemotherapy.     

Simultaneous determination of Aprepitant and two metabolites in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection

A method for the simultaneous determination of Aprepitant, I (5-[[2(R)-[1(R)-(3,5-bistrifluoromethylphenyl)ethoxy]-3(S)-(4-fluorophenyl) morpholin-4-yl]methyl]-2,4-dihydro-[1,2,4]triazol-3-one) and two active metabolites (II and III) in human plasma has been developed. The method was based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in positive ionization mode using a heated nebulizer interface. The analytes and internal standard (IV) (Fig. 1) were isolated from basified plasma using liquid–liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS/MS system.

The analytes were chromatographed on a narrow bore (50 mm×2.0 mm, 3 μm) Keystone Scientific’s Prism R.P. analytical column, with mobile phase consisting of acetonitrile (ACN):water containing trifluoroacetic acid with pH adjusted to 3 (40:60, v/v) pumped at a flow rate of 0.5 ml/min. The MS-MS detection was performed on a Sciex API 3000 tandem mass spectrometer operated in selected reaction monitoring mode. The precursor→product ion combinations of m/z 535→277, 438→180, 452→223 and 503→259 were used to quantify I, II, III, and IV, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–5000 ng/ml for I and II and 25–5000 ng/ml for III when 1 ml of plasma was processed. The precision of the assay (expressed as coefficient of variation, CV) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. Matrix effect experiments were performed to demonstrate the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. This assay was utilized to support a clinical study where multiple oral doses of I were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of Aprepitant. Concentrations of the two most active metabolites, which if present in high concentrations would increase the neurokinin-1 (NK₁) receptor occupancy level and therefore potentially contribute to the antiemetic action of Aprepitant, were determined.