Determination of Reserpine in Plasma Using High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

A high-performance liquid chromatographic method for determining reserpine in plasma has been developed. The procedure involves extraction of reserpine from buffered plasma into benzene, oxidation of reserpine to a fluorophor by treatment with vanadium pentoxide in phosphoric acid, and chromatographic separation of the reserpine fluorophor on an octadecylsilane column by ion-pairing with heptanesulfonate ions. Fluorescence monitoring of the column effluent provides high sensitivity of detection and increases the specificity of the procedure. A detection limit of approximately 100 pg of reserpine per ml of plasma was obtained following analysis of 2 ml samples. Analysis of a number of samples demonstrated the applicability of this method in confirming the presence of reserpine in equine plasma specimens collected at various horse shows and in evaluating the pharmacokinetic behavior of reserpine following intramuscular administration to horses.     

高效液相色谱－荧光检测法测定人血浆中匹伐他汀的含量

 Pitavastatin belongs to the class of coenzyme A reductase inhibitors. Very few methods of assaying pitavastatin from human plasma are available in literature. An analytical method is presented for the determination of the drug from human plasma making use of the fluorescent property of the drug. The drug is extracted from plasma using ethyl acetate under neutral condition and then analyzed by reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection (λEx 245 nm; λEm 420 nm). Analysis of pitavastatin was carried out on a C18 HPLC column using a gradient flow of mobile phase (0.01 mol/L monobasic potassium phosphate (pH 3.20)-acetonitrile, 63∶37, v/v). Fluorescein isothiocyanate was used as internal standard. The dynamic range of assay was 3 to 50 ng/mL. The intraday precision was less than 10% and accuracy ranged from 95.2% to 112.6%. The same for interday check was less than 12% and 92.8% to 105.1%, respectively. The drug was found to be stable under the assay conditions. The developed method is simple, precise, accurate, and stable. This indicates that it can be applied to routine analysis of this drug in human subjects where there are large numbers of samples without the need of specialized instruments like column switching.     

Rapid and Sensitive Method for Citrinin Determination Using High-Performance Liquid Chromatography with Fluorescence Detection

Citrinin is a toxic product of secondary metabolism of fungi, such as certain Aspergillus, Penicillium, and Monascus species that are usually contaminating cereals. A new sensitive liquid chromatographic method with fluorescence detection was developed, validated, and applied for citrinin determination. The method is based on reversed-phase separation at pH 2.5, where citrinin exhibits the highest fluorescence quantum yield. In this setup, no derivatization step is needed. The method shows linearity in the range between 0.2 μg/mL and 0.1 mg/mL. The detection limit reached is 90 ng/mL (3.6 × 10^?7 M). Validated method was successfully applied on analysis of spiked and real cereal samples.     

固相萃取/反相高效液相色谱法对血清中胰岛素的测定

采用固相萃取纯化血清样品,荧光胺柱前衍生,建立了反相高效液相色谱/荧光检测血清中胰岛素的方法.色谱条件:LunaC5柱(100 mm×4.6 mm,5 μm)分离,流动相为乙腈-Tris缓冲溶液(10 mmol/L,pH 8.5)梯度洗脱,流速:1 mL/min.激发波长(λex)273 nm,发射波长(λem)476 nm,进样量20 μL.结果表明:胰岛素在10 ～800 μg/L范围内峰面积和质量浓度呈良好的线性关系,相关系数r为0.997 1;检出限为5 μg/L.日内、日间精密度分别为1.5%、1.0%,回收率为92%.     

高效液相色谱-荧光检测法测定血浆中总同型半胱氨酸

 建立了测定血浆中总同型半胱氨酸的柱前衍生、高效液相色谱-荧光检测的分析方法。以Br omobimane作荧光剂,对巯基进行衍生。同型半胱氨酸的最低检测浓度为0.5 μmol/L,线性 浓度范围是2.5～80.0 μmol/L,回收率为94.0%～112.0%,批内、批间相对标准偏差都小于5. 6%。 关键词：     

Determination of Flecainide in Human Plasma by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, selective, and sensitive high-performance liquid chromatographic (HPLC) method for the monitoring of plasma flecainide levels in a therapeutic or research environment is described. The drug is first separated from plasma by a single-step extraction with hexane and then quantitated by HPLC with fluorescence detection. Two linear ranges have been established; 100–2000 ng/ml for drug monitoring in clinical management of patients and 3–300 ng/ml for pharmacokinetic studies. The intra-day variation is less than 6%.     

Determination of Plasma Pseudo-Ephedrine by Fluorescence Detection and High Performance Liquid Chromatography

Abstract

A sensitive and specific high-performance liquid chromatographic method has been developed for the determination of pseudoephedrine in plasma. The assay was based on the production of a highly fluorescent derivative of pseudoephedrine using 4-chloro-7-nitrobenzo-2, 1, 3-oxadiazole (NBD-Cl) as the derivatization agent. The fluorescent derivatives were separated using normal phase liquid chromatography after an automated, column-switching, sample clean-up procedure. Pseudoephedrine determination was accurate and precise at concentrations as low as 10 nanograms per milliliter of plasma. The chromatographic step separated derivatives of pseudoephedrine from those of norpseudoephedrine and several other amines. Measurement of pseudoephedrine concentrations in plasma following a single 120 milligram oral dose is illustrated.     

Determination of Bendroflumethiazide in Plasma by High Performance Liquid Chromatography and Fluorescence Detection

Abstract

An accurate, precise, and specific assay is described for the determination of bendroflumethiazide (BFTZ) in plasma. The procedure employs a C18 column, a mobile phase consisting of 35% acetonitrile in 0.015M phosphoric acid, and a fluorescence detector with a 254nm excitation filter and a 400nm emission filter. Furosemide is used as the internal standard. Using lml of plasma, this method can detect 10ng/ml of BFTZ.     

高效液相色谱法（荧光检测）分析人血浆中哌啶酸

本工作用丹磺酰氯柱前衍生比反相色谱（荧光检测）法分析了正常人和肝病患者血浆中哌啶酸－２的水平。样品用荧光胺提取法,除去血浆中的其他氨基酸和生物胺对检测的干扰。以哌啶酸－３为内标物,测得平均回收率为８４％。至少在４０ｐｍｏｌ至２ｎｍｏｌ范围内,浓度与响应的线性关系良好。两个哌啶酸的检测极限分别为２．０和２．５ｐｍｏｌ。对操作中应予注意的几个步骤作了讨论。     

Determination of Bumetanide in Human Plasma and Urine by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

This paper describes a new high-pressure liquid chromatographic method used for quantitation of bumetanide in urine and plasma. Compared to previously reported methods, this assay offers the advantages of increased sensitivity, shortened sample preparation time and decreased instrumentation requirements. After addition of the 4-benzyl derivative of bumetanide as the internal standard, both urine and plasma underwent a single extraction with ethyl acetate at an acidic pH. The organic extract was separated, evaporated to dryness, reconstituted with methanol, and chromatographed using a reversed-phase C-18 radial compression cartridge with fluorescence detection. Sensitivity limits are approximately 1 ng of bumetanide per mL of plasma, with a coefficient of variation for identical samples never exceeding 6%. The method lends itself to pharmacokinetic and pharmacodynamic studies of bumetanide in humans following single therapeutic doses.     

高效液相色谱法测定人血浆中总高半胱氨酸含量

建立了快速检测人血浆中总高半胱氨酸含量的高效液相色谱（HPLC）方法。采用反相HPLC分析前,以巯基特异性荧光试剂ABDF对血浆中巯基进行衍生．方法简便、灵敏、准确,无干扰峰影响。平均回收率为97．75％,日内和日间精密度分别为4．49％和9．79％。     

柱切换-荧光检测反相高效液相色谱法测定血浆中特布他林浓度

采用柱切换技术 荧光检测反相高效液相色谱法测定血浆中特布他林 (TB)浓度。使用LunaC8( 2 )和KromasilC18为分析柱 ( 1 5 0mm× 4.6mm ,5 μm)和预处理柱 ( 2 5mm× 4.6mm ,5 μm) ,流动相分别为pH 3 0 ,0 .0 3 3mol/L磷酸盐缓冲液∶甲醇∶乙腈 ( 92∶7∶1 )和水∶甲醇∶乙腈 ( 97∶2∶1 ) ,流速均为 1 .0ml/L。血浆样品经乙腈沉淀蛋白后进样 ,切换时间为 3 .2～ 4.2min。荧光检测 ,λex为 2 80nm ,λem为 3 0 9nm。以沙丁胺醇作内标 ,按内标法定量。标准曲线线性范围为 0 .8～ 3 2 μg/L ;最低定量限为 0 .8μg/L;TB和内标的保留时间分别为 8.7和 9.3min;日内RSD小于 4% ,日间RSD小于 9% ,方法回收率在 93 %～ 1 1 2 %。     

Sensitive High-Performance Liquid Chromatographic Determination of Mebeverine in Plasma Using Fluorescence Detection

Abstract

A sensitive high-performance liquid chromatographic (HPLC) method for mebeverine (MB) determination in plasma is described. Sample preparation involves extraction of MB and Ibuprofen (internal standard) from 0.5 ml plasma. The analysis is carried out on reversed-phase chromatographic system using U-Bondapack C₁₈ column with a mobile phase consisting of water: acetonitrile:acetic acid (59:40:1) mixture. The effluent was monitored using a fluoremetric detection at excitation and emission wave lengths 270 and 362 nm, respectively. The method gave accurate, precise and reproducible results with high sensitivity. The within-day coefficients of variation ranged from 2.5 to 6.1% and between-days from 7.5 to 13.5% at four different concentrations. Injection-volumes containing as small amount of MB as 0.5 ng in plasma was detected. This method was applied to a bioavailability study with a single 10 mg/kg oral dose in two rabbits.     

Determination of Exogenously Administered Dopamine in Infant Plasma Using Liquid Chromatography with Electrochemical Detection

Abstract

A simple, rapid liquid chromatography method is described for the measurement of exogenously administered dopamine in infant plasma, using electrochemical detection and 3,4 dihydroxybenzylamine hydrobromide as the internal standard (IS).

The mobile phase consisted of a monochloroacetate buffer (pH=3.0) with sodium octyl sulfate as the ion-pairing agent, and 2.5% acetonitrile. A biophase II^R reverse phase column was used, and the electrochemical detector was set at +650 mV versus Ag/AgCl. The retention times of IS and dopamine were 3.3 and 6.5 minutes, respectively. The recovery of dopamine following an alumina extraction procedure ranged from 75 to 80%. The inter- and intra-day coefficients of variation using this method were less than 10% and less than 5%, respectively. The major advantages of this method include use of a very small volume of plasma sample (20 μl) and ability to determine very wide ranges of plasma concentrations of dopamine following its administration intravenously. The method was used to measure the plasma concentrations of dopamine in two newborn infants to demonstrate its clinical application.     

A Direct and Sensitive Method for the Determination of Salicylamide in Microplasma Samples by High-Performance Liquid Chromatography Using Fluorescence Detection

Abstract

A quantitative analysis of salicylamide in microplasma volumes by high-performance 1iquid chromatography using fluorescence detection is reported. The procedure is extremely simple and very rapid, involving the direct introduction of the plasma sample on the HPLC column. The assay procedure is linear over the concentration range studied, 0–100 ng/ml with correlation coefficient for the linear regression, r = 0.998. This assay procedure enables the detection of salicylamide as low as 5.0 ng/ml in plasma, using sample volume of 100 μl.     

固相萃取-高效液相色谱荧光检测法测定人血浆3-硝基酪氨酸含量

建立了人血浆中痕量3-硝基酪氨酸的高效液相色谱-荧光检测法. 血浆经乙腈沉淀蛋白后,上清液用氮气吹干,残渣复溶后过C18固相萃取小柱净化浓缩,洗脱液再用氮气吹干,残渣进行荧光衍生化反应.采用高效液相色谱分离,荧光检测器检测,外标法定量.3-硝基酪氨酸在0.50～50.0 nmol/L范围内线性关系良好,相关系数为0.9999;样品加标绝对回收率为89.3%～91.9%,相对回收率为99.2%～107.9%;日内相对标准偏差为1.46%～4.79%,日间相对标准偏差为2.75%～9.20%;在血浆中检出限为0.25 nmol/L;测定正常人血浆中3-硝基酪氨酸平均浓度为118 nmol/L(n=21),Ⅱ型糖尿病病人血浆中3-NT平均浓度为3.63 nmol/L(n=23).本方法灵敏度高,重现性好,适用于临床研究中大样本量的测定.     

Determination of Vitamin B6 Compounds in Foods Using Liquid Chromatography with Post-Column Derivatization Fluorescence Detection

The liquid chromatographic determination of six vitamin B₆-related compounds, the three B₆ vitamers, their corresponding phosphorylated esters, and an excretion product, is optimized using the reversed-phase technique with a stationary phase based on a ligand with amide groups and the endcapping of trimethylsilyl. The isocratic mobile phase consisted of a phosphate buffer, and fluorescence detection involved a post-column derivatization reaction using sodium hydrogensulphite to enhance the fluorescence of the phosphate ester. Peaks were identified by the retention characteristics and fluorescence spectra. Detection limits ranged from 1–25 ng mL^–1. Two extraction procedures using acid hydrolysis and enzymatic hydrolysis were compared. The method was applied to the determination of B₆ vitamin derivatives in different types of food including beef liver, egg yolk, baby food cereals and honey. The natural free vitamers appeared in honey and baby food cereals, while the phosphorylated esters were found in the foods of animal origin. An assay using two certified reference materials gave results within the certified range.     

Improved Liquid Chromatographic Method for Acyclovir Determination in Plasma

Abstract

A simple and reproducible method for determining acyclovir (ACV) in plasma is presented. The method involved the use of acetaminophen as internal standard. A single extraction step was performed using trichloroacetic acid for protein separation. After pH adjustment, samples from the supernatent layer were directly injected into a high pressure liquid chromatograph. Components separation was perfected through manipulation of solvent combinations and pH. The acyclovir and the internal standard retention times were 8.5 and 11 min. respectively. High correlation was obtained between AUC and the drug concentration (r>0.99). Statistical analysis showed that the method is highly reproducible for ACV determination in aqueous solutions or in plasma. The mean drug recovery was better than 88%. The sensitivity obtained should enable the use of the method in future bioavailability and/or pharmacokinetic studies.     

High Performance Liquid Chromatography Determination of Pcmx In Blood Plasma Using Electrochemical Detection

Abstract

A rapid and specific high performance liquid chromatographic (HPLC) procedure has been developed for the determination of p-chloro-mxylenol (PCMX) in blood plasma. the method is based on the extraction of PCMX from plasma with benzene in the presence of a known amount of dichloro-m-xylenol (internal standard). the benzene extract is evaporated to dryness and the residue dissolved in 200 μl of mobil phase. the HPLC system consisted of a reversed-phase column and a 65% methanol:35% ammonium carbonate 0.05% solution as a mobile phase. an electrochemical (EC) detector/glassy carbon electrode set a potential of +0.9V versus Ag/AgCl/3M NaCl is used to monitor the drug. the recovery of PCMX is approximately 98%, the limit of quantitation is 2 ng/ml of plasma for the HPLC-EC procedure. the coefficient of variation is 5.1% over the range of 10-1000 ng/ml of plasma. Data are presented to illustrate the practicality of this method for evaluation of PCMX plasma levels after a single intravenous administration of 500 mg dose of PCMX to five mongrel dogs.     

Determination of Ciprofloxacin in Human Plasma and Urine by Precolumn Derivatization High Performance Liquid Chromatography with Fluorescence Detection

A sensitive, simple and selective high‐performance liquid chromatographic (HPLC) method was developed for the determination of ciprofloxacin in biological fluids. The method is based on the reaction between the drug and 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) in borate buffer of pH 9.0 to yield a highly fluorescent derivative that is measured at 535 nm after excitation at 464 nm. The calibration curves were linear over the concentration ranges of 25–3000 and 50–3000 ng·mL^?1 for plasma and urine, respectively. The mean recovery of ciprofloxacin from plasma and urine was 98.37% and 98.40%, respectively. The method was found to be sensitive, precise, accurate, and reproducible. All of the validation parameters were within the acceptance range.