Simple High-Performance Liquid Chromatographic Assay, with Post-Column Derivatization, for Simultaneous Determination of Mycophenolic Acid and its Glucuronide Metabolite in Human Plasma and Urine

Two simple high-performance liquid chromatographic (HPLC) methods have been established for simultaneous determination of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in human urine, and of their total and unbound forms in human plasma. For total MPA and MPAG analysis sample preparation entailed precipitation of protein with acetonitrile and isolation of the free analytes from the plasma by ultrafiltration. For urine samples, fivefold dilution with water was used. MPAG was determined by UV detection whereas MPA was quantified by fluorescence detection after post-column derivatization with 0.2 M sodium hydroxide solution. For plasma, response was found to be linearly dependent on concentration over the ranges 0.1–40 μg mL^-1 and 0.01–1 μg mL^-1 for total and free MPA, respectively, and 10–200 μg mL^-1 and 2.5–100 μg mL^-1 for total and free MPAG, respectively. For urine, linearity was observed from 0.1 to 50 μg mL^-1 for MPA and 10 to 500 μg mL^-1 MPAG in the urine before dilution. The methods reported were found to be accurate and reproducible for quantifying the level of MPA and MPAG and can thus be used for clinical pharmacokinetic studies and for therapeutic drug monitoring. Contributed equally to this work An erratum to this article is available at .     

High Pressure Liquid Chromatographic Determination of Mecillinam in Human Plasma and Urine

Abstract

A simple, specific, rapid and sensitive method for the analysis of mecillinam in plasma and urine using high pressure liquid chromatography is described. The assay is performed by direct injection of a plasma protein free supernatant or a dilution of urine. A μBondapak phenyl column with an eluting solvent of 16% CH₃CN-0.2% H₃PO₄ was used, with UV detection of the effluent at 220 nm. Desacetyl-cephalothin was used as the internal standard and quantitation was based on peak height ratio of mecillinam to that of the internal standard. The lowest concentration detectable without extraction was 0.25 μg/ml for plasma and 8.9 μg/ml for urine. No interference from plasma and urine was noted.     

Routine Analysis of Catecholamines and Metabolites in Urine by a Liquid Chromatographic Column Switching System

Abstract

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electro-chemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.     

Chromatographic Tandem Mass Spectrometric Detection of Physostigmine and its Major Metabolites in Rat Urine

Abstract

A rapid, sensitive, and specific liquid chromatographic‐electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of physostigmine and its metabolites in rat urine. 300 µg kg^–1 of physostigmine were used as a safe oral gavage dose for studies on its metabolites. 0–24 h urine was purified using a C18 solid‐phase extraction cartridge, and then detected by an on‐line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their MSⁿ spectra with physostigmine. Six metabolites and unchanged physostigmine existed in rat urine. All of the metabolites were reported for the first time.     

Simultaneous Determination of Salicylic Acid and Salicyluric Acid in Plasma and Urine by High Pressure Liquid Chromatography

Abstract

A sensitive, rapid, and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of salicylic (SA) and salicyluric (SU) acids in plasma and urine. The compounds are extracted into ethyl ether at acid pH, evaporated, and reconstituted prior to instrumental separation. Overall recovery of both compounds is 90 ± 5%, and the sensitivity limits are 150 ng of SU and 300 ng SA per ml of biological fluid. The assay was used for the determination of both compounds in plasma and urine of man following oral doses of 40 mg/kg of sodium salicylate.     

A New Assay for Argininosuccinate in Urine Samples by High Performance Liquid Chromatography

Abstract

A rapid, sensitive and simple procedure has been developed for determination of argininosuccinate (ASA). The assay is based upon the enzymatic cleavage of ASA in the presence of excess of argininosuccinate lyase (ASAL), HPLC separation and UV-detection of fumarate formed. The calibration curve, obtained using standards, derives from a linear correlation between the reaction rate and ASA concentration. The range of HPLC detection corresponds with the level of normal excretion of argininosuccinate in urine from the patients with argininosuccinate lyase deficiency (ASLD).     

Liquid Chromatographic Assay for Salicylic Acid in Liquid and Semisolid Formulations

Abstract

A high performance liquid chromatographic assay for quantitating salicylic acid (S) in lotion, collodion, ointment and cream formulations is described. The method involved direct determination of S in the liquid dosage forms after appropriate dilutions with the mobile phase. Prior extraction of S from ointments and creams was carried by chloroform. Metronidazole was used as the internal standard and a μ-Bondapak phenyl column with a mobile phase made of 30%, methanol in 5 mM solution of tetrabutylammonium phosphate (pH 7.5) led to an efficient separation. Retention times of about 3 and 7.5 min were obtained for the internal standard and S respectively. The recovery of S from the dosage forms was tested by adding known amounts of S to each preparation and mixing before determination. The mean percentage recovery was in the range of 98–101.1%. The method was found to be accurate, simple and reproducible.     

高效液相色谱法测定大鼠血浆中的原儿茶酸

建立了大鼠血浆中原儿茶酸含量测定的高效液相色谱方法。采用的色谱柱为DiamondsilTM C18 柱(150 mm×4.6 mm,5 μm);流动相为乙腈-水(体积比为9∶91,用H3PO4 调pH至2.5);流速1.2 mL/min;检测波长260 nm;内标为对羟基苯甲酸。原儿茶酸的线性范围为0.050~3.20 mg/L,线性相关系数为0.9978，最低定量限为0.050 mg/L,日内和日间测定的精密度（以相对标准偏差表示）均低于7.0%,准确度（以相对误差表示）为-1.4%～2.6%；在0.050,0.40,3.20 mg/L低、中、高3个添加浓度水平下，血浆样品的提取回收率分别为83.4%,87.3%,91.1%。该方法简便,灵敏,准确,适用于大鼠体内原儿茶酸的药物动力学研究。     

High Performance Liquid Chromatographic Separation and Determination of Ciprofloxacin and its Metabolites in Urine

An improved HPLC procedure was described for separation of ciprofloxacin and its four metabolites in urine using the reversed phase chromatography. For determination of the optimum condition, the effect of ion pairing reagents was investigated as a view point of its type and concentration.     

High-Performance Liquid Chromatographic Assay for Chlorquine and its Two Major Metabolites,Desethylchloroquine and Bidesethylchloroquine in Biological Fluids

Abstract

A high-performance liquid chromatography (HPLC) method for the determination of chloroquine and its two major metabolites in biological fluids is described. Hydroxychloroquine is used as an internal standard (I.S.). Drug, metabolites and I.S. were extracted as bases with diethyl ether by a single step procedure. After drying and evaporation of the organic phase, the residue was dissolved into the mobile phase and injected into the chromatographic system. Separation was performed using a normal phase column (Inertsil sill with mixture of acetonitrile, methanol and ammonia as mobile phase. The detection was carried out by fluorescence measurement : excitation wavelength was set at 325 nm and emission at 380 nm. The limit of detection was near 3.7 ng ml^?1 for chloroquine and metabolites. No chromatographic interference could be detected by endogenous compounds or other antimalarial drugs. Because of the good accuracy of the method, concentrations were determinated with a relative standard deviation lower than 7% at the 25 ng ml^?l level for all substances.

An excellent precision was obtained over the range of concentrations tested, 25–1000ng ml^?l. This method can be applied to therapeutic, pharmacokinetic and epidemial studies.     

水杨酸分子印迹分离介质的水相制备及色谱行为

以水杨酸为模板分子,2-乙烯基吡啶为功能单体,在甲醇/水体系中制备了水杨酸分子印迹聚合物,并将其作为高效液色谱分离介质研究了其对水杨酸及水杨酸的位置异物体———对羟基苯甲酸的色谱行为。结果表明,该聚合物固定相表现出良好的分离选择性,能够使水杨酸与对羟基苯甲酸快速基线分离。通过对流动相组成与色谱分离效果关系的探讨,证实溶质与印迹固定相的疏水相互作用是分子识别的主要作用力。     

高效液相色谱法测定食品添加剂中水杨酸

提出了高效液相色谱法测定食品添加剂中水杨酸含量的方法。样品采用含0.1%(体积分数)甲酸的甲醇-水(9+1)混合溶剂溶解,超声提取后经0.22μm滤膜过滤,滤液供高效液相色谱荧光仪测定。采用ZORBAX SB-C18色谱柱(4.6mm×250mm,3.5μm)分离,用不同配比的(A)甲酸-乙腈(0.1+99.9)和(B)甲酸-水(0.1+99.9)的混合溶液为流动相梯度洗脱,在激发波长为290nm、发射波长为400nm处检测。水杨酸的质量浓度在41.60～1 664μg·L-1范围内与其对应的峰面积呈线性关系,检出限(3S/N)为1.55μg·L-1,方法的回收率在90.4%～101.7%。     

血清中阿斯匹林和水杨酸浓度的快速高效液相色谱法测定

A fast method for determining concentrations of aspirin and salicylic acid in serum using reversedphase high performance liquid chromatography with Spherisorb C18 column,MeOH i H2O: n-BuOH : H3PO4 (300:200 :10 :0. 5,volume ratio) eluent and UV-237 detector was eatablished. The linear range of the method is 1-20μg/mL (r=0. 9996)for aspirin and 2 30μg/mL (r=0. 9981) for salicylic acid. The averagerecoveries are 96. 0 %-106. 0% and 92. 0%-119.5% ,respectively.     

The Simultaneous Assay of Naproxen and Salicylic Acid in Serum Using High Pressure Liquid Chromatography

Abstract

A rapid, sensitive, reverse-phase high pressure liquid chromatographic assay was developed for the simultaneous determination of naproxen and salicylic acid. These compounds are extracted from serum and then separated on a reverse-phase column using an acidified methanol eluent. Utilization of a flow-through fluorescence detector in series with a variable wavelength micro-UV detector enhances the sensitivity of the assay. Application of the assay to therapeutic levels of the drugs in human serum is demonstrated.     

Method for Determination of Codeine and Morphine in Rat Whole Blood by High Pressure Liquid Chromatography

Abstract

A HPLC method to simultaneously determine codeine and morphine in rat whole blood has been developed and evaluated. This method is based on a selective extraction and reversed-phase chromatography which results in chromatograms with 220 to 3500 theoretical plates for morphine and codeine. Detection is by electrochemical oxidation at +1.2V vs Ag/AgCl. In this method, the procedure of blood centrifugation for plasma preparation is eliminated. Therefore, the blood volume required is decreased and the sensitivity of analysis is considerably increased. Concentrations of codeine and morphine are low as 2ng/ml can be quantitated in as little as 100 mcl of rat whole blood.     

HPLC Assay for Trimethoprim in Plasma and Urine

Abstract

A sensitive HPLC method for the quantitation of trimethoprim in plasma and urine was developed using fluorescence detection. Plasma (or urine) samples were made basic by the addition of 3.8N sodium hydroxide and extracted with chloroform:2-propanol (95:5). After evaporation of the organic layer, a portion of the residue was analyzed by HPLC with fluorescence detection. The minimum detectable quantity is 0.1 μg/ml for this method. This method has been applied to the analysis of plasma and urine obtained from subjects after a single 160 mg dose of trimethoprim.     

Determination of Sparfloxacin in Human Urine by Reversed-Phase High Performance Liquid Chromatography With Nitrous Acid and Hydroiodic Pre-Column Derivatization

Quinolone drugs, due to the aromatic systems with benzoheterocycle in their molecules, can usually emit strong fluorescence. However, contrary to this, sparfloxacin (SPFX) as the fourth generation quinolone products, can only emit weak fluorescence, although it possesses a similar structure to quinolones. At present, the majority of SPFX determinations have been carried out using techniques such as colorimetry1 and spectrophotometry2-3. For the high performance liquid chromatography (HP…     

High Pressure Liquid Chromatographic Microassay for Simultaneous Measurement of Doxapram and its Metabolites in Premature Newborn Infants

Abstract

A simple, specific, rapid and sensitive high pressure liquid chromatographic microassay is described for the simultaneous measurement of doxapram and its metabolites. Doxapram appears rapidly metabolized to ketodoxapram in premature newborn infants with apnea. Therapeutic drug monitoring for doxapram and its metabolites appears warranted during doxapram therapy of neonatal apnea.     

A Simple High-performance Liquid Chromatographic Assay for Loracarbef in Human Plasma

Abstract

A simple, rapid and reproducible high-performance liquid chromatographic (HPLC) method for the determination of loracarbef in human plasma has been developed and evaluated. Plasma protein was precipitated with ammonium sulfate. The drug and the internal standard (Cefetamet) were eluted from a μ-bondapak C-18 column with a mobile phase consisting of acetonitrile:methanol:water:glacial acetic acid (2.5:17.5:79.2:0.8%, v/v). The column eluent was monitored at 265 nm. Quantification was achieved by the measurement of the peak-height ratio of the analyte to the internal standard and the limit of quantification for loracarbef in plasma is 0.5 ug/ml. The within-day coefficient of variation (CV) ranged from 2.28% to 3.67%, and between-day CV from 2.38% to 5.59% at three different concentrations. The absolute recoveries ranged from 91.1% to 93.88%, and the relative recoveries from 93.4% to 108% at three different concentrations. Preliminary stability tests showed that loracarbef is stable for at least 5-weeks in human plasma after freezing. The method is applied for the determination of the pharmacokinetic parameters of loracarbef after oral administration to 2 beagle dogs.     

测定酚醛树脂中水杨酸的高效毛细管区带电泳法

介绍了应用高效毛细管区带电泳技术测定酚醛树脂中水杨酸含量的新方法。缓冲液采用 75％ (φ )乙醇为溶剂,水杨酸不需提取,直接测定。该法的线性范围为 5.8× 10－ 6～ 1.0× 10－ 4 mol/L,检出限为 8.0× 10－ 7 mol/L（ S/N=3）, RSD为 2.7％（ n=5）,实验操作简便、迅速、准确。