Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Chiral gas chromatographic assay with flame ionization detection for amphetamine enantiomers in microsomal incubates

A chiral assay for amphetamine enantiomers in rat liver microsomal incubates is based on derivatization with (S)-(-)-N-(trifluoroacetyl)-prolyl chloride (S-TFPC), capillary chromatographic separation of the diastereomeric amide derivatives, and detection by a flame ionization detector. The method is capable of detecting low levels of S- or R-amphetamine. The assay is linear from 5 to 250 micrograms/mL for each enantiomer, and the limit of detection is 0.5 microgram/mL. The analytical method affords the average recoveries of 77.53 +/- 5.22% for R-amphetamine and 74.47 +/- 3.08% for S-amphetamine. The method allows the study of the metabolic depletion of S- and R-amphetamine in rat liver microsomal incubates. The time-dependent concentration of amphetamine enantiomers in rat liver microsomes was determined, and the stereoselectivity of amphetamine phase I metabolism was observed.     

Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection

Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.     

Triazolines. XXIII. High-performance liquid chromatographic assay in rat blood for a novel triazoline anticonvulsant (ADD17014)

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the analysis of 1-(4-chlorophenyl)-5-(4-pyridyl)-delta 2-1,2,3-triazoline (ADD17014, I), a novel anticonvulsant agent, in rat blood is described. Compound I and the internal standard (dipyridamole) were extracted into diethyl ether (5 ml) from alkalinised blood (0.25 ml of blood plus 0.75 ml of pH 10.7 buffer), with extractability nearing 100% under these conditions. The assay is based on reversed-phase HPLC (25 cm x 0.46 cm I.D. Spherisorb 5-ODS) using a mobile phase of methanol-acetonitrile-McIlvaine's citric acid-phosphate buffer (pH 8.0, 0.005 M) (30:30:40, v/v) and ultraviolet detection at 290 nm. Calibration curves were linear and reproducible (correlation coefficient greater than 0.999). Measurement of I in rat blood (250 microliters sample size) was linear in the range 0-40 microgram/ml and the coefficient of variation was less than 5%. The minimum detectable level was about 0.1 microgram/ml; however, a larger blood sample size (1-2 ml) allowed measurement of levels as low as 10 ng/ml, especially for estimation of drug levels in samples withdrawn at later time points (24 h).     

High-performance liquid chromatographic determination of 1,2-diethyl-3-hydroxypyridin-4-one and its 2-(1-hydroxyethyl) metabolite in rat blood.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the analysis of 1,2-diethyl-3-hydroxypyridin-4-one (CP94, I) and its 2-(1-hydroxyethyl) metabolite (II) in rat blood is described. I, II and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95, III) were extracted into dichloromethane (3 x 5 ml, with the addition of 1 g of sodium chloride) from blood (0.25 ml plus 0.75 ml of pH 7.0 morpholinopropanesulphonic acid buffer). Extractability approached 100% for I and III, and approximately 65% for II under these conditions. Chromatographic analysis was carried out using a Hypercarb porous graphitised carbon HPLC column (10 cm x 0.46 cm). The mobile phase was 14:86 (v/v) acetonitrile-NaH2PO4 buffer (10 mM, containing 2 mM EDTA, pH adjusted to 3 with phosphoric acid) and detection was by ultraviolet at 280 nm. Calibration curves were linear (correlation coefficient greater than 0.99) and reproducible over the concentration range 0-80 micrograms/ml and the coefficient of variation was less than 16% even at low (1 microgram/ml) concentrations. The minimum quantifiable level was 0.5 microgram/ml for both I and II.     

Simultaneous Determination of Diazepam,Oxazepam and Temazepam in Spiked Urine By Hplc

ABSTRACT|Oxazepam and temazepam are two minor metabolites of diazepam. These three benzodiazepines may be found in presence of each other in biological fluids.

Therefore, in this study an HPLC method was developed to separate and analyze them. Benzodiazepines have ability to form inclusion complexes with p-cyclodextrin (β-CyD). According to the degree of binding constant with β-CyD, these compounds can be separated by β-CyD bound to silica (cyclobond column) as the stationary phase using HPLC.

The development and validation of the HPLC procedure for the separation and determination of these compounds in mixtures were studied. The mobile-phase system consisted of phosphate buffer (pH 7): methanol [75:25], with flow rate 0.8 ml min^?1 and UV detection at 240 nm was used.

The calibration graphs were rectilinear from 0.1-2.5 μg/ml and coefficients of variation were <2% for the three compounds in bulk forms.

The method was used to analyse these bezodiazepines in spiked urine containing all three compounds in combination. Recoveries were 97-99.8%

The limit of detection and limit of quantitation were 0.05 μg/ml and 0.1 μg/ml, respectively.

The described method is selective, rapid, simple, reproducible and accurate.     

Simultaneous detection of S-adenosylmethionine and S-adenosylhomocysteine in mouse and rat tissues by capillary electrophoresis.

A capillary electrophoresis method for the determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in rat liver and kidney and mouse liver is described. The method can also be used to determine SAM in whole blood. The method provides rapid (approximately 16 min sample to sample) resolution of both compounds in perchloric extracts of tissues. Separation was performed by using an uncoated 50 microm ID capillary with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV and the separation running buffer was 200 mM glycine pH 1.8 (with HCl). The method compares favorably to HPLC methods (r (2) = 0.994 for SAM, r (2) = 0.998 for SAH) and has a mass detection limit of about 10 fmol for both SAM and SAH at a signal-to-noise ratio of 3. The method is linear over ranges of 1-100 microM SAM and 1-250 microM SAH. This method can be used to determine tissue concentrations of SAM and SAH, two metabolites that can provide insight into many biological processes.     

对甲氧基苯磺酰氯柱前衍生测定尿中游离羟脯氨酸与脯氨酸

以对甲氧基苯磺酰氯为衍生试剂,建立了分离测定人尿中游离羟脯氨酸和脯氨酸的高效液相色谱方法。考察了衍生温度、衍生缓冲液pH值、衍生时间、衍生剂用量对衍生反应的影响以及流动相组成、流动相缓冲液浓度、pH值和柱温对分离的影响。在优化条件下,羟脯氨酸和脯氨酸分别在5～100μmol/L和5～250μmol/L范围内呈良好线性,相关系数分别为0.999 4、0.999 5,检出限(S/N=3)分别为0.50 nmol/L和0.20 nmol/L,回收率(n=5)分别为95%～99%和96%～102%,相对标准偏差分别为2.1%～4.3%和2.0%～4.8%。该方法可用于人尿中游离羟脯氨酸和脯氨酸的定性定量分析。     

High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of mitoxantrone in plasma using electrochemical detection. Bisantrene was chosen as the internal standard. A reversed-phase, 10-microns muBondapak C18 analytical column (30 cm X 3.9 mm) with an isocratic mobile phase of 28% acetonitrile in 80 mM sodium formate buffer (pH 3.0) was used. The eluent was monitored by both electrochemical detection at an applied potential of +0.75 V vs. Ag/AgCl and visible absorbance at 660 nm. Only electrochemical detection was able to quantitate the internal standard and provided ten times higher sensitivity than visible absorbance for mitoxantrone with a detection limit as low as 0.1 ng/ml. Calibration curves in the range 0.1-1000 ng/ml showed good linearity (r = 0.998) and precision (coefficient of variation less than 10%). This HPLC method utilized a reproducible and inexpensive liquid-liquid extraction procedure. Using methylene chloride, the extraction efficacy of mitoxantrone from plasma was 85.3% with a coefficient of variation less than 2.1%. This new assay was then applied to measure mitoxantrone concentrations in plasma obtained from two leukemic patients receiving 12 mg/m2 mitoxantrone as a 1-h infusion.     

High-performance liquid chromatographic method for the routine determination of sulphadimidine, its hydroxy metabolites and N4-acetylsulphadimidine in body fluids and cell culture media.

A simple high-performance liquid chromatographic method is presented for the determination of trace amounts of sulphadimidine (SDD), its hydroxylated metabolites and N4-acetyl-SDD in blood plasma, urine, hepatocyte culture media and microsomal incubations. The synthesis of 5-hydroxy-SDD and an improved method for the isolation of 4-methylhydroxy-SDD from urine are described and their respective specific absorption coefficients at 265 nm are calculated by on-line radiochemical and ultraviolet detection. The limit of detection of the analytical method is 0.05 micrograms/ml for SDD and its hydroxy metabolites and 0.2 micrograms/ml for N4-acetyl-SDD. Linear calibration graphs for SDD and its metabolites were constructed from 0.2 to 50 micrograms/ml. The method has been applied to biotransformation studies in vivo and in vitro.     

High-performance liquid chromatographic analysis of trilostane and ketotrilostane in rat plasma

A simple high-performance liquid chromatographic (HPLC) method for assaying trilostane, a synthetic steroid, and one of its metabolites, ketotrilostane, in small volumes of rat plasma has been developed. A single liquid-liquid extraction was used to isolate the two compounds from acidified plasma prior to the quantitative analysis. The HPLC conditions involved the use of a Spherisorb ODS column (250 mm x 4.6 mm I.D.) and a mobile phase of 1,4-dioxan-Sorenson's buffer at pH 5.0 (52:48, v/v). Ethisterone was used as an internal standard. Trilostane and ketotrilostane were detected by their ultraviolet absorbance at 255 nm. Recoveries greater than 80% and detection limits of 50 ng/ml were obtained for both compounds. Inter-day coefficients of variation were less than 10%.     

Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.     

Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection.

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C18 reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C18 ODS Hypersil analytical column (5 microns particle size; 250 mm x 4.6 mm I.D.) using an acetonitrile-water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N',N'-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75-400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Determination of clobazam, N-desmethylclobazam and their hydroxy metabolites in plasma and urine by high-performance liquid chromatography

Owing to the pharmacological and clinical importance of the determination of plasma and urine levels of the hydroxy metabolites of clobazam and N-desmethylclobazam in healthy volunteers and in epileptic patients, a high-performance liquid chromatographic (HPLC) method was developed that permits the determination of all these compounds in the same plasma or urine sample. The method involved ether extraction at pH 13 with diazepam as internal standard for the measurement of clobazam and N-desmethylcobazam, followed by ether extraction at pH 9 with nitrazepam as internal standard for the measurement of the hydroxy derivatives. The limit of detection was about 10-20 ng/ml for each of these compounds. Applications to patients were limited by chromatographic interferences between the hydroxy metabolites and some medications currently associated with clobazam in the treatment of epilepsy. The only interference in clobazam and N-desmethylclobazam analysis was from N-desmethyldiazepam. Despite these inconveniences, this HPLC procedure appears to be the only available method for the simultaneous quantification of clobazam and its three main metabolites.     

Microdetermination of hyaluronic acid in human urine by high performance liquid chromatography.

A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (delta Di-HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of delta Di-HA and delta Di-0S, which is derived from the reaction of chondroitin with the enzyme, have been studied. The established chromatographic conditions were as follows--column: a stainless steel tube (4 mm i.d. x 250 mm) packed with TSKgel NH2-60; eluent: a mixture of acetonitrile and 0.1 M Tris-HCl buffer containing 0.1 M boric acid and 10 mM sodium sulphate, pH 7.0 (64:36, v/v). The strong fluorescence of unsaturated disaccharide after the reaction with 2-cyanoacetamide in alkaline medium was used for post-column detection. The calibration curve for delta Di-HA was linear in the range 5 pmol-5nmol with a practical detection limit of 2 pmol. The assay coefficients of variation (n = 5) at 200 pmol for delta Di-HA and delta Di-0S were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.     

Determination of sparfloxacin in serum and urine by high-performance liquid chromatography.

A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 x 4.0 mm I.D., 5 microns particle size) protected by a guard column (Perisorb RP-18, 30 x 4.0 mm I.D., 30-40 microns particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5-100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) - 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0-97.8%; method comparison c(bioassay) = 1.092c(HPLC) - 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.     

Improved high-performance liquid chromatographic method for analysis of cyclosporin A using an automated sample processor

Transplant patients receiving the immunosuppressive drug cyclosporin A require regular monitoring to maintain levels within a narrow therapeutic range. A stable, accurate and reproducible high-performance liquid chromatographic method for analysis of cyclosporin A in whole blood has been developed using the Varian Advanced Automated Sample Processor. Starting with 200 microliters of blood, absolute recovery of both cyclosporin A and the internal standard was 81% with a detection limit of 12.5 ng/ml. The assay is perfectly linear over the range 0-1000 ng/ml (r2 = 1.0). At a concentration of 250 ng/ml, the coefficient of variation, both between samples and between assays, is 1.87%. Chromatographic cycle time is 10.2 min per sample. Up to eighty samples can be processed by one person in a working day, with final results within 16 h.     

Pre-column derivatization with fluorescamine and high-performance liquid chromatographic analysis of drugs. Application to tocainide

The quantitative determination of tocainide, a new antiarrhythmic agent, by high-performance liquid chromatography (HPLC) is reported. The drug and a chemically similar internal standard were extracted from blood plasma with acetonitrile under salting-out conditions obtained by saturation of the aqueous medium with sodium chloride-sodium carbonate. The organic extract, without evaporation, was treated with borate buffer (pH 8.2) and fluorescamine. The resulting derivatives were chromatographed on an ODS reversed-phase column using a methanol-phosphate buffer (pH 7.0) mixture as mobile phase and were detected fluorometrically by monitoring the emission at 485 nm, with excitation at 395 nm. The intra-assay coefficients of variation were 3.0 and 4.3% for ten replicate 0.25 and 1.00 microgram/ml samples, respectively, and the inter-assay coefficient of variation was 3.6% for ten replicate 1.00 microgram/ml samples. The procedure is simple, rapid, sensitive, and specific. Several other drugs and drug metabolites also were derivatized with fluorescamine and chromatographed successfully. Pre-column derivatization with fluorescamine followed by HPLC with fluorometric detection may have significant advantages in drug analysis.     

棉花植株中多杀菌素及其代谢物残留量的检测

建立了同时测定棉花植株中杀菌素及其代谢产物残留量的高效液相色谱法。在碱性条件下用甲醇提取棉花植株中的多杀菌素及其代谢产物,改变pH值与液-液萃取结合固相萃取的方法去除杂质,以甲醇和含乙酸铵的乙腈混合溶液作流动相,ODC-C18柱分离,利用紫外检测器检测。在最佳条件下,棉花植株中各组分的平均加标回收率为76.9%~108.0%,相对标准偏差为1.3%~9.9%,最低检出浓度为0.01mg/kg。