Simultaneous determination of vinburnine and 6-hydroxyvinburnine in human plasma by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method has been developed to allow the simple and rapid determination of both vinburnine (I) and its main metabolite, 6-hydroxyvinburnine (II), in heparinized human plasma (0.5 ml). Compounds I and II and p-chlorodisopyramide (internal standard) were first extracted with alkalinized ethyl acetate and then with sulphuric acid. Separation was achieved on a reversed-phase muBondapak C18 column with a mobile phase of acetonitrile-water-0.1 M heptanesulphonate in acetic acid and with detection at 254 nm. Each run required 20 min. The within-day coefficients of variation for identical samples (20 ng/ml) were 7 and 6% and between-day coefficients of variation 8 and 26% for I and II, respectively. The detection limit was 5 ng/ml (normal therapeutic concentration, 10-300 ng/ml). The application of the method to drug monitoring was compared to that of a thin-layer chromatographic procedure.     

Rapid, high-sensitivity reversed-phase liquid chromatographic assay for 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-imine and its oxo metabolite in plasma using fluorescence detection

A rapid, sensitive and specific assay for 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-imine (I) and its oxo metabolite (II) in plasma was developed and validated employing reversed-phase high-performance liquid chromatography with fluorescence detection. Sample preparation was achieved by a simple ethyl acetate extraction from plasma buffered at pH 10 (0.1 M boric acid-0.1 M potassium chloride). Chromatographic analyses were performed isocratically on a C18 column, with a mobile phase consisting of methanol-0.2 M sodium acetate buffer, pH 5.0 (67:33, v/v). Chromatographic run time was less than 8 min. The assay was linear (r greater than 0.9998) over the concentration range 1.50-10,000 ng/ml for both I and II; for individual studies, curves covering a range of two orders of magnitude were generally employed. Limits of detection for I and II were 0.5 and 1.0 ng/ml, respectively. A preliminary investigation of the plasma concentrations of I and II in the rat following a single 30 mg/kg oral dose demonstrated the applicability of the method for pharmacokinetic studies.     

Spectrofluorimetric determination of guanethidine sulphate, guanoxan sulphate and amiloride hydrochloride in tablets and in biological fluids using 9,10-phenanthraquinone

A highly sensitive spectrofluorimetric method for the determination of some drugs of the monosubstituted guanidine derivatives in laboratory made tablets, in spiked human serum and in urine samples is presented. The method is based on the reaction of guanethidine sulphate (I), guanoxan sulphate (II) and amiloride hydrochloride (III) with 9,10-phenanthraquinone (IV) to give highly fluorescent derivatives. The linearity ranges were found to be 0.06-0.96 mug/ml for (I) and (II) and 0.04-0.28 mug/ml for (III), with relative standard deviation less than 2%. Mean percentage recoveries for tablets were found to be 99.9 +/- 1.3, 100.5 +/- 1.1 and 100.0 +/- 1.6 for I, II and III, respectively. For I and III the results are highly correlated with the B.P. methods. Using the synchronous fluorimetry, differentiation between I and II was possible. Chloroform, dichloromethane and ethyl acetate have been used to extract I, II and III, respectively from serum and urine at basic pH, followed by applying the proposed fluorimetric method. Percentage recoveries were found to be 95.7-102.2%. The limit of detection is 0.04 mug/ml for I and II and 0.02 mug/ml for III.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.     

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography.

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane-ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile-ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5-500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63-74 and 63-68%, respectively, over a concentration range of 0.5-500 ng/ml.     

Sensitive high-performance liquid chromatographic determination of famotidine in plasma. Application to pharmacokinetic study.

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of famotidine in human plasma is described. Clopamide was used as the internal standard. Plasma samples were extracted with diethyl ether to eliminate endogenous interferences. Plasma samples were then extracted at alkaline pH with ethyl acetate. Famotidine and the internal standard were readily extracted into the organic solvent. After evaporation of ethyl acetate, the residue was analysed by HPLC. The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-water (12:88, v/v) containing 20 mM disodium hydrogenphosphate and 50 mM sodium dodecyl sulphate, adjusted to pH 3. The HPLC microbore column was packed with 5 microns ODS Hypersil. Using ultraviolet detection at 267 nm, the detection limit for plasma famotidine was 5 ng/ml. The calibration curve was linear over the concentration range 5-500 ng/ml. The inter- and intra-assay coefficients of variation were found to be less than 10%. Applicability of the method was demonstrated by a bioavailability/pharmacokinetic study in normal volunteers who received 80 mg famotidine orally.     

Analysis of antiepileptic drugs by capillary gas chromatography with on-column injection

A simple and quick gas chromatographic method has been developed for the determination of up to six commonly used antiepileptic drugs in human serum. The antiepileptics are isolated from serum by solid phase extraction on to a reversed phase sorbent and recovered with ethyl acetate as eluent. The ethyl acetate eluate is suitable for direct on-column injection on to a phenyl methyl siloxane capillary column; hydrogen is used as carrier gas and the compounds are separated with a two-ramp temperature program. Detection is by FID. The reproducibility of the method varies between 2 and 6% RSD, depending on the drug and the level analyzed; limits of detection were found to be 14–51 pg and minimum assayed concentrations in serum were between 14 and 51 ng/ml.     

Sensitive high-performance liquid chromatographic determination of ionic drugs in biological fluids with short-wavelength ultraviolet detection using column switching combined with ion-pair chromatography: application to basic compounds

A highly sensitive and selective high-performance liquid chromatographic method with short-wavelength UV detection is described for the determination of ionic compounds in biological fluids, which was applied to two basic compounds, 2-(3-[4-(4-fluorophenyl)-1-piperazinyl]propyl)-6,7,8,9-tetrahydro-2H-nap htho [2,3-b][1,4]oxazin-3(4H)-one (I) and methyl 2-(4-diphenylmethyl-1-piperazinyl)ethyl (+/-)-1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxyla te (II), in human serum. The method is based on the combination of the column-switching technique and ion-pair chromatography. In the first ODS column, the analyte is pre-separated from endogenous substances in serum by ion-pair chromatography. After column switching, in the second ODS column the heart-cut fraction containing the analyte is further separated by non-ion-pair chromatography from coeluted endogenous substances from the first ODS column. The proposed method offered high sensitivity and selectivity with UV detection at 215 nm for I and 230 nm for II. The detection limits were 0.2 ng/ml for both I and II using 1 ml of serum. The principle of the proposed method would be applicable to both acidic and basic compounds in biological fluids with a suitable ion-pair reagent.     

Determination of megestrol acetate and cyproterone acetate in serum of patients with advanced breast cancer by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with ultraviolet detection of megestrol acetate and cyproterone acetate in human sera is described. The proposed assay is linear up to 1400 ng/ml (r = 0.999) and has a detection limit of 5 ng/ml. Recoveries of both compounds in spiked sera were ca. 95%; inter-assay coefficients of variation were 4.0 and 3.1% and intra-assay values were 1.3 and 1.4%, respectively. For validation of the method we also developed a gas chromatographic-mass spectrometric method for both steroids. The results obtained by the two methods showed good correlation: for megestrol acetate r = 0.98, n = 31, p less than 0.0001, and for cyproterone acetate r = 0.94, n = 0, p less than 0.0001. Large inter-individual differences in the serum concentrations of both substances were found in groups of patients with metastatic breast cancer receiving the same oral load of either steroid.     

Ultratrace molybdenum determination in biological samples by graphite-furnace atomic-absorption spectrometry

A method is described for molybdenum determination in human serum at sub-ng/ml levels by graphite-furnace atomic-absorption spectrometry. Sample preparation involves a nitric acid digestion, chelation with benzohydroxamic acid and extraction into hexanol. A detection limit of 0.1 ng/ml and a characteristic concentration of 0.18 ng/ml for 1% absorption can be achieved. The effectiveness of the method has been demonstrated by analysis of unspiked and spiked human serum, standard reference materials, and comparison with the results obtained by inductively-coupled plasma atomic-emission spectroscopy.     

Measurement of plasma vanillylmandelic acid by liquid chromatography with electrochemical detection

A liquid chromatographic method with electrochemical detection is described for measuring plasma 3-methoxy-4-hydroxymandelic acid (VMA). Plasma is deproteinized by gel filtration and VMA is extracted into ethyl acetate, which is evaporated. VMA is oxidized to vanillin, which is purified by toluene extraction and quantified by high-performance liquid chromatography. The recovery of VMA through the entire procedure is 52 +/- 10% (mean +/- S.D., n = 19). The plasma VMA concentration in healthy young volunteers varies between 4.39 and 14.6 ng/ml, a range that is in excellent agreement with data obtained with mass fragmentography.     

High-performance liquid chromatographic determination of 3,5-dimethylhippuric acid in the occupational exposure to trimethylbenzenes.

A high-performance liquid chromatographic method for the measurement of urinary 3,5-dimethylhippuric acid (3,5-DMHA) in the human biological monitoring of the occupational exposure to trimethylbenzenes has been developed. 3,5-DMHA was extracted from urine with ethyl acetate. The organic phase was dried under vacuum and the resultant product, dissolved in the mobile phase, was analysed by an isocratic system and a programmable photodiode-array detector adjusted to 205 nm. The mobile phase was water-acetonitrile (80:20, v/v) containing 0.1% acetic acid. 3,5-DMHA was chromatographed on a reversed-phase Supelco C18 column (3 microns; 15 cm x 0.46 cm I.D.), and identified by its retention time and ultraviolet spectrum. Quantitation was performed by peak area. The detection limit of the method is 30 ng/ml and the recovery and the accuracy are 96%.     

Alternative Method for Forensic Determination of Lysergic Acid Diethylamide and Related Compounds in Body Fluids by Liquid–Liquid Extraction and HPLC with Fluorescence Detection

An alternative and practical method is described for simultaneous detection and quantification of the potent hallucinogen lysergic acid diethylamide (LSD) and related compounds in urine and serum samples. The procedure is based on liquid–liquid re-extraction with ethyl acetate and reversed-phase liquid chromatography coupled with fluorescence detection (HPLC–FLD). With detection limits in urine and serum samples of ca 0.07 ng mL^–1 for LSD, nor-LSD, and iso-LSD, respectively, the method is well suited to forensic investigations. Application of the method to clinical samples and autopsy material enable selective identification and accurate quantification of LSD and related compounds. Comparison of results with those obtained from an LSD immunoassay (EMIT II) emphasize the need for chromatographic confirmation.Revised: 1 December 2003 and 9 February 2004     

Catalytic Kinetic Determination of Trace Amounts of Copper with Polarographic Detection

Abstract

This method is based on the catalytic effect of copper(II) on the redox reaction between acid chrome dark blue and ascorbic acid in acetic acid-sodium acetate buffer solution and 1,10-phenanthroline as an activator. Acid chrome dark blue exhibits a sensitive polarographic wave at -0.375V Vs. SCE and change of acid chrome dark blue concentration is traced by a polarographic detection technique. The detection limit and the linear range of copper(II) are 2.34ng/ml and 5 to 50ng/ml respectively. This new catalytic method has been applied to the determination of copper in human hair, with satisfactory results.     

Rapid determination of sulphonamides in milk using liquid chromatographic separation and fluorescamine derivatization.

A simple and selective method is presented for the multiple residue determination of eight sulphonamides in consumers' milk. The drugs are sulphisomidine (ID), sulphadiazine (DZ), sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine and sulphaquinoxaline (SQ). The milk sample was deproteinized with the same volume of 2 M hydrochloric acid and filtered. A 1-ml volume of the filtrate was mixed with 1 ml each of 1.25 M sodium acetate solution and a buffer (pH 3.0) for derivatization with 0.6 ml of 0.02% fluorescamine solution in acetone. A high-performance liquid chromatographic analysis was carried out on a C18 column with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C using a fluorescence detector at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. Average recoveries at fortification levels of 2, 5 and 10 ng/ml were 114%, 109% and 106%, respectively. Relative standard deviations were 1-4% at 10 ng/ml for ID, 5 ng/ml for DZ and SQ and 2.5 ng/ml for the other five sulphonamides. The method was applied to 25 milk samples and all appeared to be free from the drugs.     

Determination of risperidone and 9-hydroxyrisperidone in plasma, urine and animal tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the simultaneous determination of the new antipsychotic risperidone and its major metabolite 9-hydroxyrisperidone in plasma, urine and animal tissues. The alkalinized plasma samples were extracted with ethyl acetate and further purified prior to reversed-phase chromatography with ultraviolet detection at 280 nm. The method could also be applied to urine samples and animal tissue homogenates. Quantification limits were 2 ng/ml for plasma and urine and 10 ng/g for animal tissue. The method was applied to pharmacokinetic studies in experimental animals, human volunteers and patients.     

Determination of a potential anxiolytic drug (CGS 20625) in human plasma by high-performance liquid chromatography.

An analytical method employing reversed-phase high-performance liquid chromatography is described for the determination of a potential anxiolytic agent in human plasma. This experimental drug candidate has potent and selective affinity for the central benzodiazepine receptor complex. The compound and internal standard are extracted from buffered plasma (pH 9.0) into ethyl acetate. The solvent is evaporated and the residue is reconstituted in chromatographic mobile phase. Separation is achieved on a 5-microns phenyl column with ultraviolet absorbance detection of the drug and internal standard at 270 nm. Recovery and reproducibility assessments indicate good accuracy (overall relative recovery of 101%) and precision (coefficients of variation from 2.0 to 11%) over the concentration range 10-1000 ng/ml. The limit of quantification for the method is 10 ng/ml. The method is suitable for pharmacokinetic analysis following the administration of 80 mg of drug to normal volunteers.     

High-performance liquid chromatography followed by radioimmunoassay for the determination of a luteinizing hormone-releasing hormone analogue, leuprorelin, and its metabolite

A sensitive method for the determination of leuprorelin (TAP-144), a luteinizing hormone-releasing hormone analogue, and its C-terminal metabolite, M-I, in serum and urine has been developed. Leuprorelin and M-I were extracted from serum or urine samples with Sep-Pak C18 cartridges, and separated completely by high-performance liquid chromatography and determined by radioimmunoassay using [125I]leuprorelin as the labelled antigen. The detection limit of the method was 0.05 ng/ml for leuprorelin and M-I, and the recovery of the compounds added to serum and urine was over 88% with a coefficient of variation (within-assay) of less than 5%. The method was applied to the determination of leuprorelin and M-I-like immunoreactivity in serum or urine after administration of once-a-month injectable microspheres of leuprorelin acetate (TAP-144-SR) to patients with prostate cancer.     

Serum and plasma beta-carotene levels measured with an improved method of high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method specifically developed to allow simple and rapid determination of beta-carotene concentrations in serum and plasma is reported. Using a method modified from a previously published technique, serum and plasma proteins are denatured by exposure to perchloric acid, and beta-carotene is subsequently extracted into an organic matrix consisting of ethyl acetate-tetrahydrofuran (1:1); no evaporation step is required. Separation is achieved using isocratic elution from a reversed-phase C18 column with UV detection at 436 nm. Recovery of beta-carotene from water and plasma was greater than 98.1%; beta-carotene was stable in the extraction matrix for at least 4 h. Three anticoagulants (oxalate, citrate, and EDTA) caused losses of beta-carotene; perchloric acid and tetrahydrofuran could also destroy beta-carotene under certain conditions. Each run required less than 15 min; within-day coefficient of variation for identical samples averaged 2.3%, between-day coefficient of variation was 4.4% and sensitivity was better than 10 ng/ml. Stability of beta-carotene in plasma was also examined. This method permits a simple, rapid, sensitive, precise, and accurate determination of beta-carotene using 0.5 ml of serum or heparinized plasma.     

Quantitation of urapidil and its metabolites in human serum by high performance liquid chromatography

The antihypertensive agent urapidil (Ebrantil Byk-Gulden, Konstanz) can be reliably quantitated with three metabolites in human serum using high performance liquid chromatography. Serum is alkalinized and extracted with ethyl acetate. The organic phase is back-extracted with diluted acid. An aliquot is sampled automatically and chromatographed in an optimized combination of mobile and stationary phase. UV-detection at 273 nm allows a quantitation limit of 5 ng/ml for all analytes. Precise handling of exact volumes facilitates external calibration. The coefficient of variation for spiked samples is less than 5% within and less than 7% between studies. Application of the method to experimental and clinical pharmacokinetic studies of urapidil is illustrated.