Solid-phase extraction of vinblastine and vincristine from plasma and urine: variable drug recoveries due to non-reproducible column packings

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the determination of vinblastine and vincristine in plasma and urine is described. The drugs are isolated from 1.0 ml of the biological fluid with a solid-phase extraction column (Bond-Elut Diol). The HPLC method was combined with electrochemical detection at +850 mV versus an Ag/AgCl reference electrode. The detection limit is 100 pg for vinblastine and 250 pg for vincristine with a signal-to-noise ratio of 3, which permits the determination of these compounds in biological fluids at the nanogram level. Evaluation of the isolation method revealed that the drug recoveries and the reproducibility of the extraction procedure depend on the batch number of the solid-phase extraction column used.     

High-performance liquid chromatographic determination of vinblastine, 4-O-deacetylvinblastine and the potential metabolite 4-O-deacetylvinblastine-3-oic acid in biological fluids.

Procedures for the determination of vinblastine (VBL), 4-O-deacetylvinblastine (DVBL) and 4-O-deacetylvinblastine-3-oic acid (DVBLA) in biological samples using high-performance liquid chromatography (HPLC) combined with selective sample clean-up are presented. VBL and DVBL were determined in plasma and urine using ion-exchange normal-phase HPLC with fluorescence detection. The limit of detection was 1 microgram/l for both compounds using a 500-microliter sample. Successful chromatographic analyses of DVBLA were achieved by using a glass column packed with 5-microns Hypersil ODS and acetonitrile-0.05 M phosphate buffer (pH 2.7) (23:77, v/v). Positive identification was supported by the use of diode-array detection. The limit of detection (at 270 nm) was 10 micrograms/l using 1-ml samples.     

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

Determination of vinca alkaloids in plasma and urine using ion-exchange chromatography on silica gel and fluorescence detection

The development of a method for the determination of the antineoplastic vinca alkaloids vinblastine and vindesine in biological samples is described. The selectivity of the assay is high owing to the use of solid-phase extraction on a cyanopropyl extraction column prior to isocratic chromatography on unmodified silica gel with fluorescence detection. The influence of acetonitrile concentration and mobile phase pH on the capacity factors of the drugs was studied in order to optimize the separation between the drugs and endogenous components. The effect of varying the type and concentration of competing cations in the mobile phase was also examined. The limit of determination (signal-to-noise ratio = 3) for vinblastine is 0.5 ng/ml in plasma and urine and for vindesine 2.5 ng/ml. The assay is suitable for determining the concentrations of both compounds in plasma and urine samples from patients.     

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.     

Detection of oxilofrine in plasma and urine by high-performance liquid chromatography with electrochemical detection and comparison with gas chromatography-mass spectrometry

A high-performance liquid chromatographic (HPLC) method with electrochemical detection for the determination of oxilofrine [1-(4-hydroxyphenyl)-2-methylaminopropanol] in human plasma and urine (before and after cleavage of the metabolic conjugates) is described. Isolation from biological fluids is performed batchwise by weak acid cation exchange. Separation of plasma and urine components is achieved on a reversed-phase C18 column as an ion pair with heptanesulphonic acid. For amperometric detection the potential of the electrode was set at 0.95 V versus an Ag/AgCl reference electrode. The detection limit for oxilofrine in plasma is 1 ng/ml and in urine 12.5 ng/ml at a signal-to-noise ratio of 2.0 using 1.0 ml of plasma and 0.02 ml of urine. The method was compared with a gas chromatographic-mass spectrometric method and showed a good concordance for plasma (r = 0.996) and urine (r = 0.994). With the HPLC method it is also possible to determine related sympathomimetic drugs, e.g., etilefrine, norefenefrine or octopamine, after a slight modification of the mobile phase.     

High-performance liquid chromatographic determination of vinca-alkaloids in plasma and urine

A liquid chromatographic method is described for separating and determining vinblastine, vincristine and vindesine in plasma and urine. The drugs are extracted from the biological material using an ion-pair extraction, with sodium octylsulphate as counter-ion at pH 3. The extracts are injected on a reversed-phase system with a cyano column as stationary phase and a mobile phase composed of acetonitrile-phosphate buffer, pH 3 (65:35, vol. %). Stability studies are carried out for stock solutions of the drugs in water at different temperatures and pH values. The stability of these compounds in plasma is also investigated in the presence of an antioxidant. The method is applied to determine drug levels of vindesine and vinblastine in preliminary pharmacokinetic studies, using vincristine as the internal standard.     

High-performance liquid chromatographic determination of pipotiazine in human plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of pipotiazine in human plasma and urine. After selective extraction, pipotiazine and the internal standard (7-methoxypipotiazine) and chromatographed on a column packed with Spherosil XOA 600 (5 micrometers) using a 7:3 (v/v) mixture of diisopropyl either--isooctane (1:1, v/v + 0.2% triethylamine and diisopropyl ether--methanol (1:1, v/v) + 0.2% triethylamine + 2.6% water. The eluted compounds are measured by fluorescence detection. The sensitivity of the method was established at 0.25 ng/ml pipotiazine in plasma and 2 ng/ml pipotiazine in urine (C.V. less than 5%). The method has been successfully applied to a pharmacokinetic study following a single oral administration of 10 mg of pipotiazine.     

Determination of clomipramine and its hydroxylated and demethylated metabolites in plasma and urine by liquid chromatography with electrochemical detection

A procedure for the determination of clomipramine and its 8-hydroxy, demethyl, 8-hydroxydemethyl and didemethyl metabolites in plasma and urine by high-performance liquid chromatography with electrochemical detection is described. A 1-ml plasma or urine sample is made alkaline with a carbonate buffer (pH 9.8) and extracted with 20% ethyl acetate in n-heptane. After back-extraction into an acid phosphate buffer (pH 2.4), an aliquot is injected into a 5-microns ion-paired reversed-phase column and eluted with a mobile phase containing a phosphate buffer with tetramethylammonium chloride-acetonitrile (57:43). The detection is coulometric with a first cell at +0.40 V, a second at +0.73 V and a guard cell set at 0.75 V for oxidation of the mobile phase. The method provides recoveries in the general range of 80-110% and a day-to-day precision of 3.7-8.8%, depending on the compound. The minimum quantifiable level for all compounds was 0.2 ng/ml with a 20-microliters injection. Steady-state plasma concentration data and urinary levels are reported for 24 depressed patients receiving daily either 75-150 mg orally or 50-75 mg by infusion.     

HPLC for stress-free screening of potential prostate cancer marker catechol estrogens in urine using a diamond-electrode electrochemical and a fluorescence detector

Improvement of the sensitivity and specificity of a simultaneous stress-free screening method for catechol estrogens as a potential prostate cancer marker in urine has been accomplished by HPLC with a diamond-electrode electrochemical detector and a fluorescence detector. Since taking urine samples generates less stress (or pain) than the drawing of blood, the method can readily be applied to almost any patient, and will also assist in improving the sensitivity and specificity of the prostatic specific antigen test. Catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestriol) and estrogens (estrone, estradiol, estriol) were separated on an Inertsil ODS-II column with acetonitrile-potassium dihydrogen phosphate (pH 3.0). The diamond-electrode electrochemical detector used had the great advantage of being a maintenance-free system, and could sequentially analyze hundreds of samples. Fluorescence detection improved the sensitivity 10-500 times (e. g., the LOD of 2-hydroxyestriol was improved 250 times) compared to previous electrochemical detection reports, and dual detection improved peak identification in the urine samples. The proposed method was applied to the simultaneous determination of catechol estrogens in spiked urine in a preliminary study on estrogens and PSA values in biopsy and prostate cancer patients.     

Simultaneous determination of inulin and p-aminohippuric acid (PAH) in human plasma and urine by high-performance liquid chromatography

Inulin and p-aminohippuric acid (PAH) clearances are used for the estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid high-performance liquid chromatography (HPLC) method with UV detection is described for the simultaneous determination of inulin and PAH in the same chromatogram in the plasma and urine of humans. Plasma and urine samples were hydrolyzed with perchloric acid (0.7%) in boiling water. The mobile phase consisted of 0.01 M potassium dihydrogenphosphate with 0.02 M tetramethylammonium chloride and o-phosphoric acid (pH 3)-acetonitrile (94:6, v/v), pumped at a rate of 1.2 ml min-1 on a C8 reversed-phase column. Tannic acid was used as the internal standard and UV detection at 285 nm was employed. The calibration curves were linear over the concentration range of 12.5-100 mg l-1 for inulin and 6.25-50 mg l-1 for PAH with determination coefficients greater than 0.997. The method is accurate (bias < 13%) and reproducible (intra- and inter-day relative standard deviation less than 11%), with a limit of quantitation of 12.5 mg l-1 and 6.25 mg l-1 for inulin and PAH, respectively. Analytical recoveries from urine and plasma were ranged from 81 to 108% for both compounds. This fully validated method, which allows the simultaneous determination of inulin and PAH clearances, is simple, rapid (total run time < 10 min) and requires only a 200 microliters plasma or urine sample.     

Simultaneous determination of clarithromycin and 14(R)-hydroxyclarithromycin in plasma and urine using high-performance liquid chromatography with electrochemical detection.

A sensitive method for the simultaneous high-performance liquid chromatographic determination of clarithromycin and its active metabolite in plasma and urine is described. Alkalinized samples were coextracted with an internal standard and analyzed on a C8 column using electrochemical detection. Recoveries were greater than or equal to 85% and consistent. Standard curves for plasma were linear in the range 0-2 micrograms/ml for both compounds (r greater than 0.99), with limits of quantification of approximately 10.03 micrograms/ml (0.5-ml sample). Within-day and day-to-day precision were good, with coefficients of variation mostly within +/- 5%; accuracy for both compounds were routinely within 90-110% of theoretical values. Standard curves for urine were linear in the range 0-100 micrograms/ml with limits of quantification of 0.5 micrograms/ml (0.2-ml sample). Urine assays also had similar within-day and day-to-day precisions and accuracy.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.     

Direct analysis of the dopamine agonist (-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin hydrochloride in plasma by high-performance liquid chromatography using two-dimensional column switching.

A reversed-phase, two-dimensional, liquid chromatographic method incorporating column switching and electrochemical detection was used for the direct analysis of the dopamine (D2) agonist (-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin hydrochloride in plasma. Sample work-up consisted of addition of internal standard, filtration, then direct injection of the plasma sample onto an internal surface reversed-phase (ISRP) guard column where the dopamine agonist and internal standard were separated from plasma proteins. An automated pneumatic valve was then used to switch to a stronger eluent which stripped the retained substances from the ISRP support onto a C18 analytical column where the analytes were separated from endogenous biological interferences. A dual-electrode electrochemical detector was used to minimize interferences and provide the desired sensitivity. The method has a detection limit of 1.5 ng/ml and requires a total assay time of 20 min per plasma sample. The method is linear from 1.5 to 1000 ng/ml and yielded greater than 80% drug recovery for plasma concentrations greater than 10 ng/ml. Precision for the method at 100 ng/ml yielded a relative standard deviation of 4.4%. Reproducibility was within 6.5% on a 20 ng/ml spiked plasma sample assayed on different days by different people. The method has successfully been applied to human plasma samples and for pharmacokinetic studies in rats and monkeys.     

The determination of cysteamine in physiological fluids by HPLC with electrochemical detection

Cysteamine, an amino thiol, was separated by rapid isocratic cation exchange chromatography and detected by electrochemical oxidation at a platinum electrode maintained at +0.45 V relative to an Ag/AgCl reference electrode. Eluent pH and electrode working potentials were optimized and the effects of alternative buffers and organic modifiers have been examined. On column sensitivity for cysteamine was 1.5 pmol at a signal-to-noise ratio of 5. Although the specificity was good, plasma samples required maximal sensitivity whereas urine samples required greater selectivity, which was achieved by use of lower working potentials. Cysteamine concentrations were determined in serial samples of plasma and urine from volunteers who had received a single oral dose of 200 mg of the drug. Cysteamine was rapidly oxidized in vivo, and detection required prior reduction with dithiothreitol before analysis.     

Selective clean-up method using an immunoaffinity column following radioimmunoassay of prostaglandin F2 alpha in biological fluids.

A selective clean-up method using an immunoaffinity column followed by radioimmunoassay (RIA) was developed for determining prostaglandin F2 alpha (PGF2 alpha) in human urine and plasma. Polyclonal antibody raised against PGF2 alpha, obtained from rabbits, was coupled to a tresyl-activated support based on a synthetic hydrophilic resin, TSKgel Tresyl-Toyopearl 650M, and used as the stationary phase for the immunoaffinity column. A human urine or plasma sample was introduced to this column, and PGF2 alpha was eluted with methanol-water (50:50, v/v) after the column had been washed. The eluate was subjected to competitive RIA for PGF2 alpha. The cross-reactivities of the RIA to a number of endogenous prostanoids, except PGD2, were negligible and the sensitivity was 4 pg/tube (p less than 0.05), giving a detection limit of 40 pg/ml when 1 ml of plasma or urine was available. The recoveries of plasma and urine samples were 98-108% and 96-106%, respectively, and their assay variances were 7-23%. The concentrations of endogenous PGF2 alpha in plasma and urine used here were estimated to be 72 and 98 pg/ml, respectively. This method should be very useful for various biological samples because of its good specificity, sensitivity, reliability and reproducibility.     

Determination of exifone in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of exifone in human plasma and urine. Exifone was extracted from acidified plasma or neutralized urine with diethyl ether and the evaporated extracts were analysed on a C18 reversed-phase column. The compound was eluted in about 8 min with acetonitrile-0.3 M orthophosphoric acid (15:85, v/v) at a flow-rate of 0.9 ml/min. This method gave accurate and reproducible results; the calibration graphs were linear (r greater than 0.99) over the range of 2.8-360 nmol/l for plasma and 0.18-36 mumol/l for urine, and concentrations as low as 1 nmol/l in plasma could be quantified. These results allowed this assay to be used for determinations in single-dose pharmacokinetic studies.     

The stable o‐phthalaldehyde‐ferrocene/6‐ferrocenyl‐1‐hexanethiol pre‐column derivatization high‐performance liquid chromatography of dimethylarginine by novel dual fluorescence and electrochemical detector

Monomethylarginine, asymmetric dimethylarginine and symmetric dimethylarginine were separated on a Wakopak Combi ODS with an acetonitrile–100 mm potassium phosphate buffer (pH 7.0; 1:1, v/v). Dimethylarginines were derived from o‐phthalaldehyde for the fluorescence detector and from 6‐ferrocenyl‐1‐hexanethiol for the electrochemical detector. The detection limits of the dimethylarginines in spiked plasma were 0.3–0.5 pmol by electrochemical detection and 1–2 pmol by fluorescence detection. The detection limits were improved over 30 times by electrochemical detection and 10 times by fluorescence detection compared with previous reports. In previous derivatization liquid chromatography, the reaction solutions, o‐phthalaldehyde, 2‐mercaptethanol and dimethylarginines were unstable and required quick derivatization at 4°C. By our proposed pre‐column methods, the dimethylarginines were derivatized at room temperature and the fluorescent products were stable for 6 h. The manipulation performance was greatly advanced compared with previous LC reports. This is the first report on stable and sensitive dimethylarginines by dual detection. The selectivity was also improved by dual detection. The proposed method was applied to preliminary monitoring of dimethylargines in plasma and urine. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of cabergoline in plasma and urine by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of cabergoline in plasma and urine has been developed. After buffering plasma and urine samples, cabergoline was extracted with a methylene chloride-isooctane mixture, back-extracted into 0.1 M phosphoric acid, then analysed by reversed-phase high-performance liquid chromatography. Quantitation was achieved by electrochemical detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the biological matrices (human plasma and urine) was observed. The assay was still inadequate in terms of sensitivity for the quantitation of cabergoline plasma concentrations after a single oral dose of 1 mg of the drug to humans, but was successfully used in the determination of the urinary excretion of the drug.     

Determination of Propylene Glycol in Low Volume Plasma and Urine Samples of Neonates by LC with Photodiode Array Detection

In order to enhance the sensitivity and to develop a method suitable for quantification of propylene glycol (PG) in low volume neonate plasma and urine samples, several steps in earlier described high performance liquid chromatographic methods were optimised. Chromatographic separation on a reversed-phase column and ultraviolet detection resulted in cleaner chromatograms without interfering compounds. Linearity of the standard curves was validated in the concentration range 0.25?C50 mg L^?1. The lower limit of quantification was 20 times lower than in earlier described methods. Presented method was suitable for quantification of PG concentrations in low volume neonate plasma (15?C46 mg L^?1) and urine samples (20?C175 mg L^?1) enabling us to document very low renal versus non-renal contribution of PG clearance in neonates.