Quantitative determination of antipyrine and its metabolites in urine by the method of microcolumn HPLC

Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 23, No. 3, pp. 351–354, March, 1989.     

HPLC analysis of diuron and metabolites in blood and urine

A high-pressure liquid chromatographic method for the determination of diuron and its metabolites in human urine and blood is presented. The synthesis of different metabolites and of a suitable internal standard is described and the structure of the compounds is determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The method is applied to an overdose case.     

液相色谱串联质谱法测定人血浆中奥马曲拉及其代谢物(英文)

目的:建立液相色谱串联质谱法测定人血浆中奥马曲拉(BMS-186716)及其四种代谢物(BMS-196087,BMS-225308,BMS-198433和BMS-253653)。方法:以甲基丙烯酸酯对奥马曲拉、BMS-196087和BMS-253653结构中的游离巯基进行衍生化处理后,采用液相进行测定。结果:奥马曲拉及其代谢物在下列范围内具有良好的线性关系:BMS-186716,0.2-250μg/L;BMS-196087,0.5-250μg/L;BMS-225308,1-250μg/L;BMS-198433,2-250μg/L;BMS-253653,10-2500μg/L。最低检测浓度分别为0.2μg/L、0.5μg/L、1.0μg/L、2.0μg/L和10μg/L;平均萃取回收率分别为60.5％、88.％、76.3％、71.2％、26.6％;日内精密度和日间精密度(RSD％)均在±15％之内,相对回收率在85％-115％之间;血浆样品可耐受3次冻融,室温下可 稳定放置6小时,萃取后的样品室温下放置24小时没有任何异常变化,长期稳定性实验显示血浆样品在-30℃条件下可冻存3个月。结论:本方法准确、灵敏、快速、选择性好,能用于奥马曲拉及其代谢物的临床药代动力学研究。     

HPLC法测定人血浆中伏立康唑及其代谢物的浓度

目的 建立一种快速、灵敏、准确、稳定的测定人血浆中伏立康唑及其代谢物浓度的方法,用于伏立康唑临床治疗药物监测。方法 采用高效液相色谱（HPLC）-紫外（UV）检测法,以罂粟碱为内标,乙腈蛋白沉淀法处理血浆样品。色谱柱为ACE5C₁₈-AR （150 mm×4.6 mm）,流动相为0.025 mol/L磷酸二氢钠（含三乙胺400 μl/L,用0.25 mol/L的氢氧化钠调至pH=7.0）-乙腈（67：33）,流速为l ml/min,柱温为40℃,检测波长为255、276 nm （双波长检测）,进样量为20 μl。结果 伏立康唑氮氧化物、伏立康唑和罂粟碱的保留时间分别为4.5、11.3、13.7 min;血浆中伏立康唑、伏立康唑氮氧化物线性范围均为0.5~20.0 μg/ml （r=0.999 5）,定量下限均为0.5 μg/ml,批内、批间RSD均<11%,定量下限、低、中、高梯度浓度提取回收率在90.3%~109.9%之间。结论 该方法操作简便,结果准确,适用于伏立康唑的临床治疗药物监测和对其主要代谢物的测定。     

On-line immunoaffinity extraction and HPLC analysis of flunitrazepam and its main metabolites in serum

A sensitive, simple, and rapid method for the determination of flunitrazepam and its major metabolites (7-aminoflunitrazepam, 7-acetamidoflunitrazepam, and norflunitrazepam) in serum and plasma is presented. The on-line procedure uses an immobilized, highly reusable antibody against benzodiazepines for selective extraction from serum followed by analysis by high-performance liquid chromatography with ultraviolet detection. This reliable method provides a limit of detection of 1 ng/mL serum, and results are obtained in less than 40 min.     

Quantitative determination of tolbutamide and its metabolites in human plasma and urine by high-performance liquid chromatography and UV detection

An isocratic, high-performance liquid chromatography method has been developed for simultaneous determination of the oral antidiabetic tolbutamide and two of its metabolites, 4-hydroxytolbutamide and carboxytolbutamide, in human plasma and urine. The method was based on simple one-step liquid-liquid extraction with tertiary-butyl methyl ether as extraction solvent. The chromatographic eluent was 23:77 (v/v) methanol: 0.01 M aqueous sodium acetate buffer pH 3.0, and the UV detection was performed at a wavelength of 230 nm. The limit of detection was 0.1 microM for tolbutamide in plasma and 1.5 microM, 0.5 microM, and 0.75 microM for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The limit of quantitation was 0.5 micro for tolbutamide in plasma and 2 microM, 0.75 microM, and 1.25 microM for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The overall mean recoveries ranged from 91% to 109% for tolbutamide in plasma and from 80% to 98% in urine for all three compounds.     

Stability studies of vorinostat and its two metabolites in human plasma, serum and urine

Effects of storage time and freeze-thaw procedure on the stability of vorinostat (suberoylanilide hydroxamic acid) and its two metabolites, vorinostat O-glucuronide (M1) and 4-anilino-4-oxobutanoic acid (M2), in human plasma, serum and urine have been examined using high turbulence liquid chromatography (HTLC) online extraction and tandem mass spectrometry (MS/MS) [L. Du, D.G. Musson, A.Q. Wang, Rapid Commun. Mass Spectrom. 19 (2005) 1779–1787]. Vorinostat was demonstrated not to be stable in human plasma during the process of sample processing and storage. Acidifying the plasma sample to prevent possible enzymatic hydrolysis and using plasma with different anticoagulants were evaluated to increase the stability of vorinostat, but neither of these approaches improved stability. Human serum was then used as an alternative to plasma to monitor drug concentration, and vorinostat and its two metabolites maintained consistent concentrations in human serum after 3 freeze-thaw cycles and more than 1 year storage at −70 °C. By comparing the stability results of serum, EDTA plasma and heparin plasma, it was deduced that clotting proteins of plasma might be a major cause of vorinostat degradation. The stability of the three analytes during the process of serum sample collection was verified indicating that prolonged sample collection (up to 180 min) has no effect on the integrity of these analytes.     

High-pressure liquid chromatographic analysis of ticrynafen and one of its metabolites in urine and serum

A method is described for the extraction of ticrynafen, a new hypotensive agent, and its reduced metabolite from serum and urine. Drug-related material is extracted from biological fluids with ether under strongly acidic conditions and then back-extracted into an alkaline aqueous phase, which is subjected to high-pressure liquid chromatographic analysis. Separations are performed on a reversed-phase column with a mobile phase consisting of phosphate buffer-acetonitrile. This accurate and reproducible method measures serum concentrations of ticrynafen and its reduced metabolite as low as 1.0 and 0.4 microgram/ml, respectively.     

用液相色谱-串联质谱法测定人血清和尿液中非那西丁及其代谢物浓度

目的:建立测定人体血清和尿液中非那西丁及其代谢产物的液相色谱-串联质谱法。方法:用乙酸乙酯萃取血清和尿液中非那西丁及其代谢产物。色谱柱为Restek Allure PFPP柱(100 mm×2.1 mm,5μm),流动相为乙腈-0.1%甲酸水溶液(30∶70,V/V),流速0.3 mL/min。以磺胺甲氧嘧啶为内标,非那西丁、对乙酰氨基酚和内标的多反应监测(MRM)离子对分别为m/z180→138、152→110和281→156。测定13名健康受试者口服非那西丁1.25 g后血清和尿液中非那西丁及其代谢产物的浓度。结果:非那西丁、对乙酰氨基酚和内标的保留时间分别为4.1、1.4和2.8 min。在0.2~50μg/mL范围内,非那西丁和对乙酰氨基酚的浓度与峰面积相关性良好(r>0.999 0)。非那西丁和对乙酰氨基酚的准确度分别为87.1%~101.5%和89.5%~107.3%,精密度分别为0.85%~7.83%和2.53%~8.74%,提取回收率为53.0%~76.0%,基质效应为92.0%~106.3%,均符合生物样本分析方法的有关要求。除万古霉素和甲泼尼龙外,其他临床常用药物对测定无干扰。13名健康受试者血清中非那西丁和总对乙酰氨基酚浓度为(5.94±4.79)和(8.81±2.91)μg/mL;尿液中非那西丁和总对乙酰氨基酚浓度为(3.25±1.10)和(570.45±187.13)μg/mL。结论:该方法简便、灵敏、特异性强,具有临床实用价值。     

Quantitation of phenobarbital and its main metabolites in human urine

Summary A method for the quantitative determination of phenobarbital and free and conjugatedp-hydroxyphenobarbital in urine samples is described. The method includes initial extraction, purification on a small chromatographic column and finally determination by gas chromatography. The barbituric acids are methylated by trimethylanilinium hydroxide which serves as a flash heater methylating agent. The conjugate ofp-hydroxyphenobarbital, which appears to be a glucuronide, is hydrolysed with hydrochloric acid.     

Determination of theophylline and its metabolites in human urine by high-performance liquid chromatography

High-performance liquid chromatographic method with UV detection was developed for the determination of theophylline and its metabolites, in human urine using β-hydroxyethyl theophylline (β-HET) as an internal standard. For extraction of urine sample, quality control sample and xanthine-free blank urine were mixed with decylamine (ion-paring reagent) and β-HET. After saturation with ammonium sulfate, the mixture was then extracted with organic solvent at pH values of 4.0∼4.5. All separations were performed with ion-pair chromatography using decylamine as an ion-pairing reagent and 3 mM sodium acetate buffered mobile phase (pH 4.0) containing 1% (v/v) acetonitrile and 0.75 mM decylamine. The detection limits of theophylline, 1,3-DMU, 1-MU, 3-MX and 1-MX in human urine were 0.17, 0.17, 0.39, 0.19 and 0.19 μg/ml, based on a signal-to-noise ratios of 3.0. The mean intraday coefficients of variation (C.V.s) of each compound on nine replicates were lower than 2.0%, while mean interday C.V.s on three days were lower than 1.6%. All separations were finished within 40 minutes.     

高效液相色谱法测定尿中咖啡因主要代谢物浓度

本文建立了测定尿中咖啡因5种主要代谢物（AFMU、1X、1U、17X、17U）浓度的高效波相色谱法。尿样用硫酸铵饱和后加氯仿及异丙醇混合液（85：15）提取2次，空气流吹干，蒸馏水溶解进样。以ShimPackC18为固定相，甲醇-动腈-0．05％醋酸（10：1：89）为流动相，扑热息痛为内标，流速为1．2ml／min，在280um处定量检测。本法精确稳定可靠。用此方法测定了120名健康志愿者口服咖啡后的尿样，对人体内N-乙酰化转移酶和CYP1A2酶活性作了初步分析。     

Determination of salicylazosulphapyridine, sulphapyridine and its metabolites in serum and urine

Methods for the determination of salicylazosulphapyridine (Salazopyrin(R)), sulphapyridine, N-acetylsulphapyridine, sulphapyridine-O-glucuronide and N-acetylsulphapyridine-O-glucuronide in serum and urine have been developed. In one method samples were diluted with methanol to precipitate proteins present and thereafter injected directly onto the liquid chromatographic column. The sulphapyridine-O-glucuronide could not be determined by this method. For the determination of all the main sulphapyridine metabolites the sulphapyridine aglycones were formed after treatment with beta-glucuronidase. These sulphapyridine compounds were then extracted with isobutylmethyl ketone at pH 5, and re-extracted to a phosphate buffer at pH 13 prior to injection. Separation and retention of all compounds was affected both by pH and concentration of methanol in the mobile phase. The proposed method made determinations down to 1 mug ml(-1) possible, which was found to be sufficient. Comparisons were made with spectrophotometric methods.     

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.     

高效液相色谱法测定人血清中西替利嗪及其药物动力学

的:建立高效液相色谱法测定人血清中西替利嗪。方法:用HPLC法检测8名健康自愿者血中西替利嗪浓度,经3P87程序拟合,计算药动学参数。结果:西替利嗪药动学参数T1/2α,T1/2β,Tmax,Cmax,V/F和AUC分别为(1.6±0.8)h,(7.2±1.1)h,(1.3±0.4)h,(374.7±61.8)ng·ml-1,(39.1±8.8)L和(3188.4±246.1)ng·h·ml-1。结论:该方法简单、快速、准确,适用于药动学及药效学研究。     

Determination of trazodone and its metabolite, m-CPP, in serum and urine by HPLC

A sensitive and rapid method for the analysis of trazodone (TZD) and its metabolite, 1-(m-chlorophenyl)piperazine (m-CPP), in the serum and urine of rabbits treated with the drug was developed. The assay requires extraction from the biological fluids with adequately buffered organic solvents followed by HPLC. The assay allows good reproducibility, fair recovery, and excellent linearity in the range of 0.6 to 10 micrograms/mL for TZD and 1.2 to 20 micrograms/mL for m-CPP.     

HPLC determination of D and L moxalactam in human serum and urine

A high-pressure liquid chromatographic procedure was developed to determine the D and L isomers of moxalactam in human plasma and urine. After protein precipitation with hydrochloric acid the sample was extracted with ethyl acetate. It was then back extracted into tromethamine buffer (pH 8.0) and washed with octanol. Extraction recovery from plasma ranged from 73-81%. An aliquot of the tromethamine buffer was then injected onto a C18-muBondapak column. The mobile phase was 3% acetonitrile in 0.05 M ammonium acetate pH 6.5 buffer. Samples were quantitated by UV detection at 275 nm and 0.01 aufs. The lower limit of detection was 0.5 microgram/ml for each isomer. Preliminary stability studies were performed to assess proper sample handling and storage conditions. The procedure was evaluated in a clinical setting to demonstrate its applicability to the study of moxalactam pharmacokinetics in critically ill patients.     

Quantitative liquid chromatographic determination of bromadoline and its N-demethylated metabolites in blood, plasma, serum, and urine samples

Bromadoline and its two N-demethylated metabolites were extracted into ether:butyl chloride after the addition of internal standard and basification of the various biological fluids (blood, plasma, serum, and urine). These compounds were then extracted into dilute phosphoric acid from the organic phase and separated on a reversed-phase chromatographic system using a mobile phase containing acetonitrile and a buffer of 1,4-dimethylpiperazine and perchloric acid. The overall absolute extraction recoveries of these compounds were approximately 50-80%. The background interferences from the biological fluids were negligible and allowed quantitative determination of bromadoline and the metabolites at levels as low as 2-5 ng/mL. At mobile phase flow rate of 1 mL/min, the sample components and the internal standard were eluted at the retention times within approximately 7-12 min. The drug- and metabolite-to-internal standard peak height ratios showed excellent linear relationships with their corresponding concentrations. The analytical method showed satisfactory within- and between-run assay precision and accuracy, and has been utilized in the simultaneous determination of bromadoline and its two N-demethylated metabolites in biological fluids collected from humans and from dogs after administration of bromadoline maleate.