High-performance liquid chromatography of histamine and 1-methylhistamine with on-column fluorescence derivatization.

An on-column fluorometric derivatization method was developed for the determination of histamine and 1-methylhistamine (HMs) by high-performance liquid chromatography. The system for the derivatization consisted only of a commercially available single-plunger pump and a reversed-phase C18 column supported on synthetic polymer with a mobile phase of acetonitrile and alkaline borate buffer solution containing o-phthalaldehyde as a derivatization reagent. It required no additional reaction system as for a post-column derivatization method. Injected HMs might be derivatized to a fluorophore on the inlet site of the high-performance liquid chromatographic column, followed by chromatography on the same column. Optimization of the on-column reaction conditions resulted in a simple and sensitive analytical method for the determination of HMs with excellent reproducibility and linearity of 0.05-5 micrograms/ml of both HMs. Application of this method to the determination of HMs in food samples resulted in a limit of quantification of 0.05 mg/100 g and in a greater than 95% overall mean recovery at a fortification of 0.1 mg/g of both HMs. This method was furthermore applicable to the determination of histamine released from rat peritoneal mast cells.     

Novel assay method for mitoxantrone in plasma, and its application in cancer patients

Mitoxantrone is an anthracene derivative that acts as a cytostatic in a variety of cancers. A quantitative analytical method has been established for the determination of mitoxantrone in plasma. The method employed C18 reversed-phase ion-pair chromatography with an isocratic mobile phase of 50.0% methanol in 10 mM phosphate buffer (pH 3.0) plus 0.09% 1-pentanesulphonic acid and ultraviolet detection. Sample preparation consisted of two extraction steps using same organic solvent system at different pH to remove plasma impurities efficiently. Potential adsorption of mitoxantrone onto glassware was considered. Silanization of all glassware with 5% dichlorodimethylsilane in chloroform increased the extraction recovery in plasma from 50 to 85% with high reproducibility. Mitoxantrone was unstable in human plasma. To maintain plasma sample integrity, each millilitre of sample should be fortified with 0.1 ml of 5% vitamin C (in citrate buffer) and kept frozen until analysis. Using this new method, the calibration curve of mitoxantrone in plasma in the range of interest (1-500 ng/ml) showed good linearity (r = 0.996) and precision (both between-day and within-day coefficients of variation less than 10%). The lower detection limit of this assay method was 1 ng. The application of this method allowed us to study the stability of mitoxantrone in plasma, and the pharmacokinetics of mitoxantrone in nasopharyngeal carcinoma patients receiving 12 mg/m2. The study revealed a prolonged terminal phase half-life for mitoxantrone.     

Validation of a high-performance chromatographic method for determination of cefotaxime in biological samples

An analytical method for detecting and quantifying cefotaxime in plasma and several tissues is described. The method was developed and validated using plasma and tissues of rats. The samples were analyzed by reversed phase liquid chromatography (HPLC) with UV detection (254 nm). Calibration graphs showed a linear correlation (r > 0.999) over the concentration ranges of 0.5–200 μg/mL and 1.25–25 μg/g for plasma and tissues, respectively. The recovery of cefotaxime from plasma standards prepared at the concentrations of 25 μg/mL and 100 μg/mL was 98.5 ± 3.5% and 101.8 ± 2.2%, respectively. The recovery of cefotaxime from tissue standards of liver, fat and muscle, prepared at the concentration of 10 μg/g was: 89.8 ± 1.2% (liver), 103.9 ± 6.5% (fat) and 97.8 ± 2.1% (muscle). The detection (LOD) and quantitation (LOQ) limits for plasma samples were established at 0.11 μg/mL and 0.49 μg/mL, respectively. The values of these limits for tissues samples were approximately 2.5 times higher: 0.3 μg/g (LOD) and 1.25 μg/g (LOQ). For plasma samples, the deviation of the observed concentration from the nominal concentration was less than 5% and the coefficient of variation for within-day and between-day assays was less than 6% and 12%, respectively. The method was used in a pharmacokinetic study of cefotaxime in the rat and the mean values of the pharmacokinetic parameters are given.     

Development of a selective sample clean-up method based on immuno-ultrafiltration for the determination of deoxynivalenol in maize

This paper describes the development of a highly selective analytical method for the determination of deoxynivalenol (DON) in maize. The developed method is based on immuno-ultrafiltration (IUF) and is the first application of IUF as a clean-up strategy in food analysis. Quantification of DON was carried out by high-performance liquid chromatography with ultraviolet detection. In contrast to immunoaffinity chromatography, in IUF the antibodies are not bound to a solid support material but used in free form, thus making it possible to avoid the critical immobilisation step. Sample clean-up by IUF proved to be as selective as clean-up using commercially available immunoaffinity columns. The limit of detection (S/N=3) of the analytical method was found to be 74 ng DON/g maize. Repeated analysis of a certified maize reference material on four different days resulted in a mean recovery of 93% with a standard deviation of 10%.     

High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of mitoxantrone in plasma using electrochemical detection. Bisantrene was chosen as the internal standard. A reversed-phase, 10-microns muBondapak C18 analytical column (30 cm X 3.9 mm) with an isocratic mobile phase of 28% acetonitrile in 80 mM sodium formate buffer (pH 3.0) was used. The eluent was monitored by both electrochemical detection at an applied potential of +0.75 V vs. Ag/AgCl and visible absorbance at 660 nm. Only electrochemical detection was able to quantitate the internal standard and provided ten times higher sensitivity than visible absorbance for mitoxantrone with a detection limit as low as 0.1 ng/ml. Calibration curves in the range 0.1-1000 ng/ml showed good linearity (r = 0.998) and precision (coefficient of variation less than 10%). This HPLC method utilized a reproducible and inexpensive liquid-liquid extraction procedure. Using methylene chloride, the extraction efficacy of mitoxantrone from plasma was 85.3% with a coefficient of variation less than 2.1%. This new assay was then applied to measure mitoxantrone concentrations in plasma obtained from two leukemic patients receiving 12 mg/m2 mitoxantrone as a 1-h infusion.     

Chiral separation and determination of propranolol enantiomers in rat or mouse blood and tissue by column switching high performance liquid chromatography with ovomucoid bonded stationary phase

Resolution of propranolol (PL) enantiomers in biological samples was accomplished by column switching high performance liquid chromatography using a short precolumn and an analytical column of ovomucoid chiral phase. Plasma, whole blood or tissue homogenate sample was directly injected into the precolumn, and PL was adsorbed on Butyl Toyopearl 650-M. After column switching, the PL was backflushed and transferred to the analytical column (Ultron ES-OVM) by the eluant. Fluorometric detection was carried out at lambda ex = 297 nm and lambda em = 340 nm with a detection limit of 0.5 pmol (signal to noise ratio = 2). The recovery (98.8-103%), reproducibility (coefficient of variance less than 3%) and enantiomer resolution (separation factor 1.15) were satisfactory using as eluant 50 mM sodium dihydrogenphosphate (pH 4.6) containing 12% ethanol. The time course of elimination of PL enantiomers in rat or mouse blood and tissues was also studied.     

A high-performance liquid chromatographic method for determination of the niguldipine analogue DHP-014

A simple and reliable reversed-phase high-performance liquid chromatography method was developed and validated for the determination of DHP-014, a niguldipine analogue with potent P-glycoprotein inhibitory and negligible calcium channel blocking properties, in rat plasma. DHP-014 and niguldipine hydrochloride (the internal standard) were extracted from rat plasma by liquid extraction using hexane. DHP-014 was then separated by HPLC on a C18 column and quantified by ultraviolet detection at 238 nm. The mobile phase consisted of acetonitrile-aqueous 5 mM phosphate buffer (65:35, v/v) containing 0.4% (v/v) triethylamine adjusted to pH 7.0. The mean extraction efficiency of DHP-014 was 109.0 +/- 12.9, 97.7 +/- 8.0 and 102.9 +/- 7.5% for DHP-014 concentrations of 10, 50 and 100 nM, respectively (n = 5). The method was linear over the concentration range 2.5-200 nM with a regression coefficient of 0.998. The limit of detection of DHP-014 in rat plasma was 1.0 nM. The intra- and inter-day coefficients of variation for DHP-014 in rat plasma were 4.7-7.9 and 6.9-9.9%, respectively. The intra- and inter-day accuracy was 98.2-99.5 and 97.9-103%, respectively. The bioanalytical technique was used to determine DHP-014 in plasma samples in a pharmacokinetic study of DHP-014 administered to female Sprague-Dawley rats.     

A simple high-performance liquid chromatography assay for the major cisapride metabolite, norcisapride, in human urine

A simple high-performance liquid chromatography assay using fluorescence detection for the major metabolite of the gastric prokinetic drug cisapride, norcisapride, is presented. Analysis is performed using an Alltech Platinum EPS C8 column with a mobile phase made up of methanol and 0.02M sodium dihygrogen phosphate (45:55, v/v) containing triethylamine (1 g/L). Complete resolution is achieved among norcisapride, the internal standard (metoclopramide), and endogenous urinary components. The assay is linear over the range 50-2000 ng/mL with a mean recovery of 71.2% across the analytical range following solvent extraction with toluene-isoamyl alcohol (95:5, v/v). Intraday coefficients of variation (precision) determined at 200 and 1000 ng/mL are 6.0 and 9.8%, respectively, and interday coefficients of variation are 8.8 and 6.6%, respectively. Intra- and interassay accuracy (as mean relative error) determined at the same concentrations is within 10% in all cases. An analysis of urine samples from a healthy volunteer following the administration of a single 10-mg oral dose of cisapride is shown.     

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.     

High Performance Liquid Chromatographic Assay of Phenacemide in Tablets

Abstract

A rapid, specific and reliable high-performance liquid chromatographic assay of phenacemide in tablets has been developed. Reversed-phase chromatography was conducted using a mobile phase of acetonitrile and acetate buffer, pH 4.2 (50% V/V) and detection at 254 nm. The recovery and coefficient of variation from six placebo samples of 100 mg were 100.2% and 0.57%, respectively. The percent recovery of 10 replicate commercial tablets was 101.1% of the label amount and its coefficient of variation was 0.95%. Regression analyses of three standard plots in the concentration range of 15-150 μ g/ml obtained on three different days gave a correlation coefficient > 0.999 and the coefficient of variation for their slopes <2%. The HPLC method is rapid as it takes - 1 hour to analyze six commercial tablets compared with 6 hours consumed by the USP method. The assay was precise within day and between days as indicated by ANOVA test.     

Determination of 2′,3′-Dideoxyinosine in Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatography analysis method has been developed for the quantitation of 2′,3′-dideoxyinosine (DDI) in plasma. Proteins were precipitated from plasma samples with acetonitrile containing the internal standard, 6-methylaminopurine riboside. The treated samples were evaporated to dryness and reconstituted in mobile phase for the analysis. Separation of the components was achieved on a 5 μm octadecylsilane column with ultraviolet detection at 254 nm. The method was validated at nine concentrations between 0.015 and 150 μg/mL. Using 500 μL of human plasma, the limit of quantitation was 120 ng/mL and the limit of detection was 60 ng/mL. The mean intra-day precision of the method was 1.6%. The mean accuracy of the method was within 2% of the actual values. This method is currently being used for pharmacokinetic studies in the rat.     

Validation of a high-performance chromatographic method for determination of cefotaxime in biological samples

An analytical method for detecting and quantifying cefotaxime in plasma and several tissues is described. The method was developed and validated using plasma and tissues of rats. The samples were analyzed by reversed phase liquid chromatography (HPLC) with UV detection (254 nm). Calibration graphs showed a linear correlation (r > 0.999) over the concentration ranges of 0.5–200 μg/mL and 1.25–25 μg/g for plasma and tissues, respectively. The recovery of cefotaxime from plasma standards prepared at the concentrations of 25 μg/mL and 100 μg/mL was 98.5 ± 3.5% and 101.8 ± 2.2%, respectively. The recovery of cefotaxime from tissue standards of liver, fat and muscle, prepared at the concentration of 10 μg/g was: 89.8 ± 1.2% (liver), 103.9 ± 6.5% (fat) and 97.8 ± 2.1% (muscle). The detection (LOD) and quantitation (LOQ) limits for plasma samples were established at 0.11 μg/mL and 0.49 μg/mL, respectively. The values of these limits for tissues samples were approximately 2.5 times higher: 0.3 μg/g (LOD) and 1.25 μg/g (LOQ). For plasma samples, the deviation of the observed concentration from the nominal concentration was less than 5% and the coefficient of variation for within-day and between-day assays was less than 6% and 12%, respectively. The method was used in a pharmacokinetic study of cefotaxime in the rat and the mean values of the pharmacokinetic parameters are given. Received: 25 May 1998 / Revised: 27 July 1998 / Accepted: 1 August 1998     

Pre-Column Derivatization HPLC Procedure for the Quantitation of Aluminium Chlorohydrate in Antiperspirant Creams Using Quercetin as Chromogenic Reagent

This article describes the development and validation of a selective high-performance liquid chromatography method that allows, after liquid–liquid extraction and pre-column derivatization reaction with quercetin, the quantification of aluminium chlorohydrate in antiperspirant creams. Chromatographic separation was achieved on an XTerra MS C18 analytical column (150 × 3.0 mm i.d., particle size 5 μm) using a mobile phase of acetonitrile:water (15:85, v/v) containing 0.08 % trifluoroacetic acid at a flow rate of 0.30 mL min^?1. Ultraviolet spectrophotometric detection at 415 nm was used. The assay was linear over a concentration range of 3.7–30.6 μg mL^?1 for aluminium with a limit of quantitation of 3.74 μg mL^?1. Quality control samples (4.4, 17.1 and 30.6 μg mL^?1) in five replicates from five different runs of analysis demonstrated intra-assay precision (% coefficient of variation <3.8 %), inter-assay precision (% coefficient of variation <5.4 %) and an overall accuracy (% recovery) between 96 and 101 %. The method was used to quantify aluminium in antiperspirant creams containing 11.0, 13.0 and 16.0 % (w/w) aluminium chlorohydrate, respectively.     

Combination of a sub‐3 μm superficially porous particle packed column with charged aerosol detection for the simple and sensitive measurement of nine macrolides in human urine

Due to the lack of chromophores in many macrolides, analytical methods based on mass spectrometry and electrochemical detection coupled to liquid chromatography have been suggested to be suitable for the quantification of macrolides in complex matrices. In this study, a simple and sensitive analytical method was established for the simultaneous measurement of nine macrolides in human urine by combining a sub‐3 μm superficially porous particle packed column with charged aerosol detection. After thorough investigation of various sample preparation methods, including two liquid–liquid extraction methods and four solid‐phase extraction methods, HLB solid‐phase extraction was selected and further optimized. Absolute recovery of the optimized sample preparation method ranged from 99.5–110.2%, indicating its very high extraction/clean‐up efficiency. For chromatography, parameters influencing macrolide separation were systematically optimized, and the resulting conditions allowed baseline separation of nine macrolides within 24 min using a very simple mobile phase. The established method was validated for linearity, limit of detection, limit of quantification, absolute recovery, and precision. Based on its limit of detection (0.025–0.100 μg/mL), the method had similar or greater sensitivity than most methods based on electrochemical detection. It was found that the current method was appropriate for application to real human urine samples after drug administration.     

Determination of Ascorbic Acid in Blood Plasma or Serum and in Seminal Plasma Using a Simplified Sample Preparation and High-Performance Liquid Chromatography Coupled with Uv Detection

Abstract

A simplified method of sample preparation and high-performance liquid chromatography procedure using UV detection is described for the determination of ascorbic acid (AA) in blood plasma or serum and seminal plasma. Within two hours from collection samples are treated with dithioerythritol (DTE) and then stored under Argon at -80°C. Prior to analysis, protein precipitation is initiated with the addition of cold methanol. AA elution is carried out on a C₁₈ reverse phase column using dodecyltrimethylammonium bromide as an ion-pairing agent. the detection is accomplished by measuring ultraviolet absorption at 265 nm. the analysis time for sample is 10.5 min, the retention time of AA being 9.7 min. Within- and between-day coefficients of variation are 2.9% and 4.9% for blood serum, 1.0 % and 2.3 % for seminal plasma. Mean analytical recovery of 102.5 ± 3.7% was found analyzing a serum pool after addition of a standard amount of AA. AA levels are stable for at least 43 days under the described storage conditions.     

Development of an analytical system for the simultaneous determination of anabolic macrocyclic lactones in aquatic environmental samples

An analytical procedure that enables routine analysis for trace determination of six anabolic macrocyclic lactones (zearalenone, alpha- and beta-zearalenol, zearalanone, zeranol, and taleranol) in sewage treatment plant (STP) samples has been developed. The method uses solid-phase extraction, followed by high-performance liquid chromatography with on-line tandem mass spectrometry using atmospheric pressure chemical ionization (LC/APCI-MS/MS). The extraction of these compounds from filtered water samples was performed off-line with C(18) solid-phase cartridges. The detection was achieved by isocratic reversed-phase high-performance liquid chromatography coupled with an heated nebulizer (HN) APCI interface operating in negative ion mode. Mean recovery of the analytes in STP effluent samples generally exceeded 81%. This method was used to determine the occurrence of target analytes in the aquatic environment. In the selected STP effluent samples, zearalenone and alpha-zearalenol were detected in the ng/L range.     

HPLC analysis of in vivo intestinal absorption and oxidative metabolism of salicylic acid in the rat

In vivo absorption and oxidative metabolism of salicylic acid in rat small intestine was studied by luminal perfusion experiment. Perfusion through the lumen of proximal jejunum with isotonic medium containing 250 μm sodium salicylate was carried out. Absorption of salicylate was measured by a validated HPLC‐DAD method which was evaluated for a number of validation characteristics (specificity, repeatability and intermediate precision, limit of detection, limit of quantification, linearity and accuracy). The method was linear over the concentration range 0.5–50 μg/mL. After liquid–liquid extraction of the perfusion samples oxidative biotransformation of salicylate was also investigated by HPLC‐MS. The method was linear over the concentration range 0.25–5.0 μg/mL. Two hydroxylated metabolites of salicylic acid (2,5‐dihydroxybenzoic acid and 2,3‐dihydroxybenzoic acid) were detected and identified. The mean recovery of extraction was 72.4% for 2,3‐DHB, 72.5% for 2,5‐DHB and 50.1% for salicylic acid, respectively. The methods were successfully applied to investigate jejunal absorption and oxidative metabolism of sodium salicylate in experimental animals. The methods provide analytical background for further metabolic studies of salycilates under modified physiological conditions.     

Microassay for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine by high-performance liquid chromatography

An improved method for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine using reversed-phase high-performance liquid chromatography with ultraviolet detection is described. Following solid-phase extraction, chromatography was performed using a column containing an octadecylsilica-coated packing, eluted with 6% acetonitrile in phosphate buffer, pH 2.1, and detected at 233 nm. Using 80-microliters samples, the detection limit is 18 ng/ml for benzoylecgonine and benzoylenorecgonine and 35 ng/ml for cocaine and norcocaine. The coefficients of variation range from 3.5% (benzoylecgonine) to 7.0% (norcocaine). The procedure has been applied to samples of guinea pig plasma, urine and amniotic fluid and human urine.     

Analysis of ochratoxin A in milk after direct immunoaffinity column clean-up by high-performance liquid chromatography with fluorescence detection

The present work describes a new analytical method for direct immunoaffinity column clean-up of ochratoxin A (OTA) in milk samples followed by determination of the toxin using high-performance liquid chromatography with fluorescence detection (HPLC-FD). Two different immunoaffinity cartridges (IAC) were investigated, and Ochraprep columns were chosen because they showed the best results. An average recovery of 89.8% and a mean RSD of 5.8% for artificially contaminated cow's milk in the range of 5-100 ng/L were attained. The calculated limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.5 and 5 ng/L, respectively. This new easy and fast method avoids a previous liquid-liquid extraction step and therefore the use of toxic chlorinated solvents. Chromatograms of the final extracts were clean and OTA could be easily detected at a retention time of 8.4 min without interferences. To assess the presence of the toxin in cow's milk eight samples of skimmed and four samples of whole milk were analysed and OTA was not detected over the established detection limit.     

Determination of coenzyme Q10 in over-the-counter dietary supplements by high-performance liquid chromatography with coulometric detection

A rapid and sensitive method is described for the determination of coenzyme Q10 (Q10) in over-the-counter dietary supplements by automated high-performance liquid chromatography (HPLC) with coulometric detection. Sample solutions of powder-filled capsules, oil-based softgels, and tablets were prepared by serial dilution with 1-propanol. After dilution, a known volume of sample solution containing Q10 and the internal standard, coenzyme Q9 (Q9), was directly injected into the HPLC system. Most of electrochemically active compounds in the injection were oxidized at the precolumn conditioning cell and postcolumn guard cell. Q9 and Q10 were monitored at an analytical cell that contained 2 coulometric electrodes, where Q9 and Q10 were reduced to the corresponding ubiquinol-9 and -10 and then oxidized to produce currents. This method produced a linear detector response for peak height measurements over the concentration range of 0.05-8 microg/mL (r > 0.999). The lower limit of detection was 5 ng/mL (signal-to-noise ratio, > or =3). The mean recovery was 98.9 +/- 0.6%; coefficients of variation for intra- and interday precisions were 1.8-4.0%. The proposed method was successfully applied to the determination of Q10 in marketed products.