Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

Simple High-Performance Liquid Chromatographic Method for the Analysis of Quinine in Human Plasma without Extraction

Abstract

A simple, rapid and sensitive assay, capable of quantitating quinine (Q) in human plasma samples is reported. The assay uses a reversed-phase C18 HPLC column packed with 5 μ ODS Hypersil. The chromatographic separation was accomplished with an isocratic mobile phase comprising acetonitrile-aqueous phosphate buffer pH 2 (50:50, v/v) containing 25 mM sodium dodecyl sulfate and 3 mM tetrabutylammonium bromide at a flow rate of 0.5 ml/min. The eluant was monitored by a fluorescence detector (excitation wavelength at 350 nm and emission wavelength at 450 nm). The assay was based on a simple plasma protein precipitation technique. To 200 μ of plasma sample, 400 μ of internal standard (cinchocaine 30 μ/ml in methanol) was added. After brief vortexing and centrifugation, the clear supernatant was 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 Q in plasma was 18 ng/ml.     

Determination of camptothecin in biological fluids using reversed-phase high-performance liquid chromatography with fluorescence detection

Camptothecin, a plant alkaloid with antitumor activity, is a potent and rapidly acting inhibitor of DNA synthesis. The objective of this study was to develop a sensitive high-performance liquid chromatographic (HPLC) method for the detection and estimation of the camptothecin concentration in biological fluids. Using HPLC coupled with fluorescence detection, at an excitation wavelength of 370 nm and an emission wavelength of 434 nm, we found that the lower limits of detection for camptothecin in aqueous, plasma and urine samples were 0.5, 1 and 10 ng/ml, respectively. The ideal mobile phase used was methanol-10 mM potassium phosphate (75:25, v/v, pH 4.0). To determine the utilization of the method in a biological system, we studied the pharmacokinetics of camptothecin in mice. Elimination of camptothecin from mice blood was triphasic and followed first-order kinetics. The half-life of camptothecin in mouse blood was 25.7 min. Our studies indicate that HPLC with fluorescence detection for the determination of camptothecin in different media is a simple, rapid, sensitive and reproducible method.     

Development and validation of rapid and sensitive LC methods with PDA and fluorescence detection for determination of piribedil in rat plasma and brain tissues and their pharmacokinetic application

Simple, selective and sensitive high‐performance liquid chromatographic (HPLC) bioanalytical methods using fluorescence (FL) and photodiode array (PDA) detectors were developed and validated for determination of piribedil (PBD), an anti‐Parkinson's drug, in rat plasma and brain samples, with telmisartan as internal standard (IS). Protein precipitation technique was used to extract PBD from both biological matrices. Chromatographic separation was achieved on a Phenomenex Kinetex C₁₈ end‐capped column (250 × 4.6 mm, 5 μm), with 38:62 v/v acetonitrile and ammonium acetate buffer (pH 5.0) as mobile phase at 1.0 mL/min flow rate. Linear response in the concentration ranges 5–300 and 150–3000 ng/mL in plasma, and 15–900 and 450–9000 ng/g in brain tissue were achieved in FL and PDA detectors, respectively. The chromatograms were extracted at 239 nm in case of PDA detection and at excitation wavelength of 239 nm and emission wavelength of 385 nm in case of FL detection. FL detection was found to be more sensitive compared with PDA detection. The developed methods were successfully employed in determining the plasma time course, brain distribution and the pharmacokinetic parameters of PBD following intravenous bolus administration of the drug in male Wistar rats.     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

Determination of 1-hydroxypyrene in human urine by acid hydrolysis coupled to solid-phase microextraction and semi-microcolumn liquid chromatography.

This study developed an acid hydrolysis coupled to a solid-phase microextraction method employing a semi-microcolumn liquid chromatography system, instead of enzyme hydrolysis with solid-phase extraction for the pretreatment of human urine samples, to detect urinary 1-hydroxypyrene (1-OHP). The complete separation and detection of urinary 1-hydroxyprene was performed using a high-performance liquid-chromatography fluorescence detection system with an analytical C(18) semi-microcolumn, 60% (v/v) aqueous acetonitrile elution, and a lambda(ex/em) = 348/388 nm pair detection wavelength. Calibration graphs were linear with very good correlation coefficients (r = 0.9997), and the detection limit was 1.0 ng/L. These important parameters for acid hydrolysis and solid-phase microextraction were investigated. The total recovery was above 83% in acid hydrolysis with solid-phase microextraction. The proposed method provided a relatively simple, convenient, and practical procedure to determine the level of urinary 1-hydroxypyrene in biological samples, and was successfully applied to detect the urine of students.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.     

Ion-paired high-performance liquid chromatographic determination of mitoxantrone in physiological fluids

Mitoxantrone is one of the newer anthracenedione derivatives which has already been studied in phase I and II trials, where it has shown significant antitumor activity against a variety of human tumours. To determine the prolonged terminal half-life of mitoxantrone, we developed a sensitive high-performance liquid chromatographic method, providing a detection limit of 1 ng/ml of extracted serum. This system uses a C18 reversed-phase column. The mobile phase consists of a mixture of acetonitrile (30%, v/v) and an ammonium formate buffer (70%, v/v) with a pH of 2.7. Hexane sulphonic acid is added as an ion-pair former. Detection at a wavelength of 658 nm provides a highly selective system, showing no interfering peaks. Ametantrone, another anthracenedione derivative, is used as an internal standard. The extraction procedure for serum also uses hexane sulphonic acid in an ion-paired system. Because of the highly selective detection wavelength, urine samples can be injected without a sample clean-up procedure. This very sensitive method, combined with high selectivity and a fast and inexpensive serum clean-up procedure, has allowed us to document the prolonged terminal plasma half-life of mitoxantrone (levels of 2-5 ng/ml of plasma can still be detected six days after an intravenous infusion of 15 mg/m2 over 30 min). In urine an as yet unidentified metabolite was discovered.     

Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection

A selective, accurate and precise high-performance liquid chromatographic assay coupled to fluorescence detection was developed for the detection of some angiotensin II receptor antagonists (ARA II): Losartan, Irbesartan, Valsartan, Candesartan cilexetil and its metabolite Candesartan MI. The analytes and the internal standard (bumetanide, a high-ceiling diuretic) were extracted from plasma under acidic conditions by means of solid-phase extraction using C8 cartridges. This procedure allowed recoveries close to 80% for all these drugs excluding Candesartan cilexetil (70%) which presented adsorption processes on glass and plastic walls. The analytes and potential interferences were separated on a reversed-phase column, muBondapak C18, at room temperature. A gradient elution mode was used to carry out the separation, the optimal mobile phase being composed of acetonitrile-5 mM acetate buffer, pH 4, at variable flow-rates (from 1.0 to 1.2 ml/min). Fluorescence detector was set at an excitation wavelength of 250 nm and an emission wavelength of 375 nm. Intra- and inter-day relative standard deviations for all the compounds were lower than 8% except for Losartan (12%) and the method assesses a quite good accuracy (percentage of relative error approximately 6% in most of the cases). The limit of quantitation for these compounds was 3 ng/ml for Candesartan cilexetil and M1, 16 ng/ml for Losartan and 50 ng/ml for Irbesartan and Valsartan, which allows their determination at expected plasma concentration levels. This assay method has been successfully applied to plasma samples obtained from hypertensive patients under clinical studies after oral administration of a therapeutic dose of some of these ARA II compounds.     

Analysis of 5-fluorouracil in plasma and urine by high-performance liquid chromatography.

A relatively simple, sensitive and rapid high-performance liquid chromatographic method is described for measuring the anticancer drug 5-fluorouracil (5-FU) in human plasma and urine. The procedure includes liquid-liquid extraction using ethyl acetate-methanol (95:5) and preparative column chromatography to separate 5-FU from constituents normally occurring in these biological samples. The columns contained a specially modified form of diatomaceous earth, which requires no pre-conditioning washes. Reversed-phase high-performance liquid chromatography was performed on a C18 column (70 mm x 4.6 mm I.D.) with a mobile phase of water-methanol (95:5) and ultraviolet detection (268 nm). The overall recovery from plasma and urine was 91 and 94%, respectively, at the concentration of 50 ng/ml. The determination limit of the assay for 5-FU was 10 ng/ml of plasma and urine. Concentrations of 5-FU between 10 and 500 ng/ml were measured in plasma and urine with a relative standard deviation of 6.8%. In order to evaluate the procedure, plasma and urine samples from three patients treated with 5-FU by continuous intravenous perfusion, were investigated.     

Determination of streptomycin residue in cucumber and Chinese cabbage by high-performance liquid chromatography with postcolumn derivatization and fluorometric detection

A sensitive and accurate method was developed for the determination of streptomycin using HPLC followed by postcolumn derivatization and fluorometric detection. The analyte was extracted, using aqueous solution from cucumber and Chinese cabbage, by a two-step SPE procedure. The extraction, cleanup, and chromatography conditions were optimized, and the performance of the analysis method was evaluated. The conditions of chromatography were as follows: the separation was performed on a C18 column; the isocratic mobile phase consisted of acetonitrile and a mixed solution containing 10 mM sodium 1,2-naphthoquinone-4-sulfonate and 0.4 mM sodium 1-heptanesulfonate (25+75, v/v); and the flow rate was 1 mL/min. The fluorescence detector was set at an excitation wavelength of 263 nm and an emission wavelength of 435 nm. The calibration curve was linear over the range of 50-2000 ng/mL, with a correlation coefficient of 0.9995. The LOD and LOQ were 10 and 30 ng/g, respectively, in both cucumber and Chinese cabbage. The method was validated for selectivity, linearity, precision, and accuracy. The intraday and interday precision and accuracy were within 10%. The mean recoveries from spiked samples were more than 75%, with RSD lower than 10%.     

Determination of the new morpholino anthracycline MX2.HCl and its metabolites in biological samples by high-performance liquid chromatography.

Methods for determining concentrations of a new morpholino anthracycline MX2.HCl and its metabolites in biological samples using reversed-phase high-performance liquid chromatography and fluorescence detection are described. The limits of detection were less than 1 ng/ml for all compounds after extraction from 0.5 ml of plasma using C18 Sep-Pak cartridges and consecutive solvent extraction. The recoveries from rat plasma ranged from 72.0 to 89.3%. The peak-height ratio of the fluorescence intensities of these compounds versus internal standard showed a linear correlation for concentrations up to at least 500 ng/ml in the plasma (correlation coefficient r greater than 0.999). The within-day and between-day precisions of this assay were in the range 0.8-8.7% (n = 5) and 2.0-3.5% (n = 5), respectively. The concentrations of these compounds in the blood and urine can be also determined by a slight modification of the extraction procedure.     

Monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine by high-performance liquid chromatography after pre-column derivatization with glyoxal reagents

A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.     

Separation of fifteen quinolones by high performance liquid chromatography: application to pharmaceuticals and ofloxacin determination in urine

A simple chromatographic method is described for assaying 15 quinolones and fluoroquinolones (pipemidic acid, marbofloxacin, enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, nalidixic acid, flumequine and piromidic acid), in urine and pharmaceutical samples. The determination was achieved by LC using an RP C18 analytical column. A mobile phase composed of mixtures of methanol-ACN-10 mM citrate buffer at pH 3.5 and 10 mM citrate buffer at pH 4.5, delivered under an optimum gradient program, at a flow rate of 1.5 mL/min, allows to accomplish the chromatographic separation in 26 min. For detection, diode-array UV-Vis at 280 nm and fluorescence detection set at excitation wavelength/emission wavelength: 280/450, 280/ 495, 280/405 and 320/360 nm were used. Detection and quantification limits were between 0.3-18 and 0.8-61 ng/mL, respectively. The method was validated in terms of interday (n = 6) and intraday (n = 6) precision and accuracy. The procedure was successfully applied to the analysis of human and veterinary pharmaceuticals. Also, ofloxacin was determined in human urine samples belonging to a patient undergoing treatment with this active principle, among others.     

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.     

Rapid assay for hard tissue collagen cross-links using isocratic ion-pair reversed-phase liquid chromatography

Following a detailed study, a rapid and sensitive assay for the naturally fluorescent collagen cross-links pyridinoline and deoxypyridinoline has been developed using ion-pair reversed-phase high-performance liquid chromatography in the presence of 1-octanesulphonic acid (OSA). Pyridinoline and deoxypyridinoline were separated on an Exsil 100 ODS, 5-microns column (100 mm X 4.6 mm I.D.) using 25 mM sodium formate, 5 mM OSA and 1 mM ethylenediaminetetraacetic acid adjusted to pH 3.25, containing 20% (v/v) methanol. The mobile phase flow-rate was 1.5 ml/min. Compounds were detected by their natural fluorescence (xenon lamp; excitation wavelength 290 nm, emission wavelength 400 nm). Peak areas were linear to 25 pmol injected for pyridinoline and 20 pmol injected for deoxypyridinoline (r = 0.99). Intra-assay coefficients of variation for urinary extracts were 7.65 and 9.07% (n = 10), respectively. Limit of detection (signal-to-noise ratio = 5) was 200 fmol injected. Quantification of the cross-links in acid hydrolysates and human urine samples was possible in under 15 min.     

Improved quantitation of 13-cis- and all-trans-acitretin in human plasma by normal-phase high-performance liquid chromatography.

A reliable analytical method has been developed for measurement of 13-cis- and all-trans-acitretin (Neotigason) in human plasma by normal-phase high-performance liquid chromatography, with ultraviolet detection. Human plasma was obtained after centrifugation of whole blood samples and deproteinized by ethanolic denaturation. After liquid-liquid extraction with water-n-hexane, and aliquot ws chromatographed on a silica column using isocratic elution with n-hexane-methylsalicylate-acetic acid (200:18:0.6, v/v). The wavelength was set at 360 nm, and for plasma samples a limit of quantification of 3-4 ng/ml was obtained. All manipulations were carried out under dim light conditions to prevent photoisomerization.     

Determination of AJ-3941, a possible agent for the treatment of cerebrovascular disorders, in plasma and brain by means of high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method with fluorescence detection is described for the determination of AJ-3941 (I), a possible agent for the treatment of cerebrovascular disorders, in plasma and brain tissue. A simple hexane extraction was used for plasma, and for brain homogenate the hexane extract was further purified by solid-phase extraction. The determination limit was ca. 3 ng/ml for both plasma (0.5 ml) and 10% (w/v) brain homogenate (1 ml). The method was applied to the determination of I in plasma and brain samples of experimental animals.     

Determination of fluoxetine and its N-desmethyl metabolite in human plasma by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of the antidepressant fluoxetine and its active metabolite norfluoxetine in human plasma using paroxetine as internal standard. After liquid-liquid extraction, the compounds were separated on a C18 column using as mobile phase acetonitrile and 40 mM potassium dihydrogen phosphate buffer (pH 2.3) in the ratio 31:69 (v/v). The quantification of fluoxetine and norfluoxetine was made by fluorescence detection at Ex/Em 230/312 nm. The assay for each analyte was linear over the ranges 1-39 and 0.9-36 ng/ml, respectively. For both compounds intra- and inter-day accuracy and precision ranged between −7.9-12.4 and 0.7-14.7%, respectively. The method was applied to the analysis of plasma samples obtained from healthy subjects treated with one single oral dose of 40 mg fluoxetine.