HPLC Assay for S-2-(3-Aminopropylamino)ethyl Phosphorothioate (WR 2721) in Plasma

Abstract

A specific HPLC assay has been developed for determination of the radioprotective drug WR 2721. The method is based on precolumn derivatization of plasma with fluorescamine, separation with a C-18 cartridge and detection by fluorescence. An external standard was used for calibration, and values were adjusted based upon recovery of added ¹⁴C-labeled WR 2721. WR 2721 had a retention time of about 13 minutes using a mobile phase of acetonitrile/water (22:78), 0.01 M in dibutylammonium phosphate, at a flow rate of 2 mL/min. Sensitivity of the assay was characterized to 2 μg/mL, and detector response was linear over the range of 2 to 1100 μg/mL. The assay requires 90 μL of plasma and has a total chromatography time of about 45 minutes. 2-(3-Aminopropylamino)ethanethiol (WR 1065) and bis- [2- (3-aminopropylamino)ethyl]disulfide (WR 33278), metabolites of the drug, and a variety of primary amines were shown not to interfere with the assay. Suitability of this assay for pharmacokinetic studies was demonstrated in preliminary experiments with a beagle dog.     

Measurement of S-2-(3-Aminopropylamino)ethanethiol (WR1065) in Blood and Tissues

Abstract

A procedure for the analysis of blood and tissue specimens for WR1065, the dephosphorylated metabolite of the radioprotective drug WR2721, has been developed. The method includes the use of a perchloric acid/EDTA extraction step at 0°C followed by chromatographic analysis using a mercury/gold thin film electrochemical detection liquid chromatography system. The extraction technique was designed to assure the stability of both WR1065 and any WR2721 present in blood or tissues. Using the described chromatography conditions and an analog of WR1065, 3-(4-aminopropylamino)propanethiol (WR251833), as an internal standard the respective retention times of these two compounds are 6.2 and 8.3 minutes. Experiments showing the applicability of this method to pharmacokinetic studies of WR2721 and WR1065 and to investigation of the kinetics of WR2721 hydrolysis in biological fluids such as stomach juice are described.     

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.     

High-Performance Liquid Chromatographic Assay of Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in Human Plasma using a Solid-Phase Extraction Technique

A selective and sensitive liquid chromatographic method was developed for the determination of zonisamide in small volumes of plasma. Zonisamide and the internal standard methyl 4-hydroxybenzoate were extracted from 0.2 mL of plasma with solid-phase extraction columns and eluted with methanol. Analysis of the extracts was performed on a Symmetry C₁₈ column with ultra-violet spectrophotometric detection. The calibration curve was linear over the concentration range of 0.05–5 g mL^–1 in plasma. Recoveries were reasonable for routine analyses; the limit of quantification was 0.05 g mL^–1 with a signal-to-noise ratio of 5. This method could be useful for the pharmacokinetic study of zonisamide in a limited volume of human plasma and for therapeutic drug monitoring.     

A Rapid High Pressure Liquid Chromatographic Assay of Benoxaprofen in Plasma

Abstract

A rapid high-performance liquid chromatographic assay for the determination of the anti-inflammatory drug benoxaprofen in human plasma, is described. Plasma samples of 1.0 ml, to which benoxaprofen, and warfarin as an internal standard, had been added, were extracted with ether under acidic conditions. The samples were analyzed on a MicroPak CN-10 column using 25% acetonitrile in water (pH 2.5 with phosphoric acid). Detection was made on a variable wavelength UV absorbance detector at 309 nm.

Samples containing 0.5–10 μg benoxaprofen gave a mean extraction recovery from control plasma of 90.6 ± 6.8% (n=18). Stability tests have shown that benoxaprofen in plasma is stable for at least two weeks after freezing.     

High Performance Liquid Chromatographic Determination of (-)-N-Formylnorephedrine in Plasma

Abstract

An HPLC procedure for the detection and quantitative estimation of (-)-N-formylnorephedrine in rabbit plasma had been developed. The procedure involved the extraction of (-)-N-formylnorephedrine from plasma spiked with the internal standard (phenacetin), using ethyl acetate. The ethyl acetate extract is evaporated under nitrogen and the residue is reconstituted in water and injected onto the column. A u-Bondapak-C18 column 30 cm × 3.9 mm ID was used. The mobile phase is 20% acetonitrile in water; at a flow rate of 1.5 ml/min and uv detection at 256 nm. A linear relationship between concentration and peak height ratio (I/internal standard) was obtained (r = 1.00). The reported procedure allows the measurement of (-)-formylnorephedrine in concentrations as low as 150 ng/ml of plasma with total procedure time of about 10 min. The applicability of the procedure to pharmacokinetic studies is illustrated and metabolites are shown not to interfere with the assay procedure.     

A Liquid Chromatographic Method for the Determination of Atenolol in Human Plasma

Abstract

A reliable, highly reproducible, accurate and time-efficient high performance liquid chromatographic (HPLC) method to measure atenolol concentration in human plasma was developed and validated. Sample clean-up consists of simple and efficient liquid-liquid extraction (mean recovery 103%) which allows a high sample throughput. Chromatography on a CN-propyl column yields symmetrical and well resolved peaks for atenolol and for the internal standard (metoprolol) without any interference from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection of 12.6 ng/ml (calculated at a 99.9% confidence level) with %CV (precision) ≥ 8.8% and bias (accuracy) ≥ 3.8% for concentrations in the range of 10 – 1000 ng/ml. We now routinely use this method in human pharmacokinetic studies of atenolol dosage forms.     

Assay of Yohimbine in Human Plasma Using High Performance Liquid Chromatography with Electrochemical Detection

Abstract

Yohimbine is a selective α₂ adrenoreceptor antagonist used in the study of α₂ adrenoreceptors in man. In order to better improve administration regimens for the study of yohimbine in man, we have developed an assay for the determination of yohimbine in plasma utilizing reverse phase high performance liquid chromatography with electrochemical detection. Using a C₁₈ column and a methanol:acetate (60:40) mobile phase, we detected yohimbine in plasma following a simple chloroform extraction. Reserpiline was used as an internal standard. The assay was linear over a concentration range of 50–250 ng/ml in spiked plasma and had a lower limit of sensitivity of 10 ng/ml. It was used to detect yohimbine in plasma sampled from 4 volunteers during an infusion of the alkaloid.     

Improved High-Performance Liquid Chromatographic (HPLC) Assay Method for Ceftizoxime

Abstract

We improved a high-performance liquid chromatographic method for the quantitative determination of ceftizoxime in human serum and urine using cefotaxime as internal standard. It employs a μ Bondapak Alkyl Phenyl column, elution with acetonitrile-phosphate buffer and measurement of UV absorption at 254 nm. Results obtained using the HPLC assay were compared to those obtained using a microbiological assay. The correlation coefficient was 0.987 (n:25). The method is rapid, accurate and reproducible with a sensitivity of 2.5 μg/ml of ceftizoxime. Cefotaxime and its major metabolite, the desacetylcefotaxime, can also be quantitated by this procedure.     

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

Improved High Pressure Liquid Chromatographic Method for Phenylpropanolamine in Human Plasma

Abstract

A high-pressure liquid chromatographic analysis of phenylpropanolamine in plasma following extraction, back extraction and pre-column derivitization with O-phthalaldehyde is presented. The method is improved by the use of phenylethanolamine as internal standard. Using fluorescence detection, the method is sufficiently sensitive to quantitate 5 ng/ml in 0.5 ml plasma with a standard error of estimate of 2.7 ng/ml when calibrated over the O to 240 ng/ml range. Analysis of over 2000 clinical samples have shown the method to be highly specific and reliable.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.     

高效液相色谱电化学法检测人体血浆中芹菜素和相关黄酮类化合物

建立了同时检测人体血浆中芹菜素等5种黄酮类化合物含量的反相高效液相色谱法。血浆样品经固相萃取柱提取后,用Hypersil C18色谱柱分离样品,流动相为甲醇/磷酸水溶液(V/V,2/3,pH2.25),等度洗脱,流量为1 mL/min,柱温为40℃,用电化学检测器在直接模式下,1100 mV分析检测待测物,内标法定量。对于全部待测组分r均大于0.99;槲皮素、毛地黄黄酮、山奈酚、芹菜素和异鼠李黄素线性范围分别为3~7000、3~5900、3~7000、5~7000和3~7400 nmol/L;检出限为1.4~4.8 nmol/L。方法的实际样品加标回收率为86.8%~102.9%;相对标准偏差(RSD)低于7.4%(n=3)。方法简便、快速、准确,可用于芹菜素等黄酮类化合物在人体内的生物利用度及膳食干预研究。     

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.     

High Performance Liquid Chromatography Assay of Itraconazole in Human Plasma

Abstract

A high performance chromatographic method, using internal standard quantification, for the analysis of Itraconazole in human plasma is described. The standard curve was linear over the concentration range tested. The detection limit of the method was 250 ng. An authentic sample of both Itraconazole and the internal standard (ketoconazole) were used to establish the calibration curve.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

A Rapid Liquid Chromatographic Method for Determination of Flecainide in Human Blood Plasma Using Ultraviolet Detection

Abstract

A liquid chromatographic method for the assay of the antiarrhythmic drug flecainide in plasma has been developed. The method is rapid, simple and with sufficient detection sensitivity to render it suitable for therapeutic drug monitoring. Flecainide and added internal standard, a non-fluorinated analogue, were extracted by a single ether extraction from alkalinized plasma followed by a back-extraction of the ether with dilute phosphoric acid. A portion of the acid extract was then applied directly to a 30 cm ODS column eluting isocratically with 30% acetonitrile in water containing 0.01M dibutylamine phosphate. Monitoring was by ultraviolet detection at 214 nm and the total run time was 8 min. This method is specific and can quantitate plasma levels to less than 30 ng/ml (free base) from 0.5 ml of plasma without interference from antiarrhythmic drugs commonly used in therapy.     

A New High Performance Liquid Chromatographic Assay for Fluspirilene in Dosage Forms

Abstract

A new method for the assay of fluspirilene (R 6218) in dosage forms using HPLC has been developed. The instrument used had a low volume positive displacement pump, a universal injector, a single wavelength (254 nm) detector, and a data module. The column was stainless steel 30 cm × 4 mm i.d. packed with microparticulate silica. The mobile phase consisted of equal volumes of chloroform and methanol at a flow rate of 1 ml per min. A linear relationship (r = 0.999) was obtained between peak area and concentration of fluspirilene in the range 10–200 μg per ml; no internal standard was used. Fluspirilene was extracted from injectable aqueous suspensions by membrane filtration, drying and dissolution in chloroform-methanol (1:1). Results of assaying fluspirilene in two commercial injectable suspensions by this method were 99.73 and 99.78% of labelled amount.