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

An improved, rapid and specific high-performance liquid chromatographic assay was developed for the determination of famotidine in human plasma and urine. Plasma samples were alkalinized and the analyte and internal standard (cimetidine) extracted with water-saturated ethyl acetate. The extracts were reconstituted in mobile phase, and injected onto a C18 reversed-phase column; UV detection was set at 267 nm. Urine samples were diluted with nine volumes of a mobile phase-internal standard mixture prior to injection. The lower limits of quantification in plasma and urine were 75 ng/ml and 1.0 microg/ml, respectively; intra- and inter-day coefficients of variation were < or =10.5%. This method is currently being used to support renal function studies assessing the use of intravenously administered famotidine to characterize cationic tubular secretion in man.     

Determination of moniliformin by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) was investigated as a technique for determination of moniliformin, a toxic secondary metabolite of various Fusarium species. Two HPLC procedures gave satisfactory results. In the first procedure, separation was achieved on a strong anion exchange column (10 microns, 4 mm id x 25 cm) with an eluting solvent consisting of 0.01 M sodium dihydrogen phosphate (pH 5.0) and a flow rate of 1.0 mL/min. The second procedure made use of paired ion chromatography on a reverse phase column (10 microns, 4 mm id x 25 cm). Elution was done at 2 mL/min with 0.005 M tetrabutylammonium hydrogen sulphate in a mixture containing 8% methanol and 92% 0.1 M sodium phosphate buffer (pH 7.0). Moniliformin was detected in both procedures by ultraviolet absorbance at 229 nm. The lower detection limit of pure moniliformin was 1 ng per injection. Water extraction of moniliformin from dried Fusarium cultures grown on maize was found to be efficient, giving a 95% recovery from a spiked sample. Peak height and retention time reproducibility was good for both procedures. In the analysis of maize containing on the order of 1 mg/kg moniliformin, background interference made interpretation of chromatograms difficult. The cleanup procedure giving the most promising results is described. HPLC methods have been applied successfully in screening fungal cultures for moniliformin production and for monitoring the steps in isolating moniliformin from mold material.     

Determination of individual bile acids in biological fluids by thin-layer chromatography and fluorimetry

A method is described for the separation and determination of individual bile acids in serum and in intestinal contents, employing thin-layer chromatography and fluorimetry. The mean recovery from intestinal juice was 94% and from serum 82%.Normal values for the individual bile acids of serum and examples of clinical applications of the method are given.     

Assay of 2-naphthol in human urine by high-performance liquid chromatography

This paper describes a novel liquid chromatographic method for the quantitation of 2-naphthol in human urine. Urine samples were extracted after enzymatic hydrolysis of glucuronides and sulfates; 2-naphthol was then separated using reversed-phase high-performance liquid chromatography. The corresponding detection limits were 0.04 ng/ml for the standard sample in acetonitrile and 0.13 ng/ml for urine samples. The level of urinary 2-naphthol in 100 Korean shipyard workers was analyzed using this new method. The level ranged from 0.21 ng/ml (0.26 micromol/mol creatinine) to 34.19 ng/ml (59.11 micromol/mol creatinine), and the mean+/-standard deviation was 5.08 ng/ml (6.60 micromol/mol creatinine)+/-5.75 ng/ml (9.22 micromol/mol creatinine). The mean+/-standard deviation of urinary 2-naphthol level of smokers, 7.03 ng/ml (8.49 micromol/mol creatinine)+/-6.16 ng/ml (10.23 micromol/mol creatinine), was significantly higher than that of non-smokers, 2.49 ng/ml (4.10 micromol/mol creatinine)+/-3.92 ng/ml (7.03 micromol/mol creatinine).     

Determination of hypericin in plasma by high-performance liquid chromatography

Hypericin, a polycyclic dianthroquinone, is one of the characteristic ingredients of Hypericum perforatum extracts (St. John's wort, HP), which has antidepressant effects. Hypericin and the internal standard (I.S.), dansylamide, were extracted from plasma utilizing solid-phase extraction (SPE). Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with fluorescence end-point detection. The calibration curve was linear over the range 5-100 ng per ml of plasma. The sensitivity for hypericin was 75 pg on column. Mean inter- and intra-assay coefficients of variation (C.V.s) over the range of the standard curve were less than 10%. The absolute recovery for hypericin averaged 72.6%.     

Routine analysis of amphetamine class drugs as their naphthaquinone derivatives in human urine by high-performance liquid chromatography

We describe a simple HPLC method which is suitable for the routine confirmation of immunoassay positive amphetamine urine samples. The precolumn derivisation method employing sodium naphthaquinone-4-sulphonate was found to have adequate sensitivity, selectivity and precision for the measurement of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxyethylamphetamine (MDEA) at 500 microg/l cutoff level for confirmatory analysis of amphetamines in urine. The specificity of the method is enhanced by detecting the peaks at two different wavelengths. The ratios of the peak heights measured at the two wavelengths were different for each of the 5 amphetamines analysed. There was no interference from other phenylethylamine analogues that are commonly found in "over the counter" preparations. The HPLC method is compared to a commercial TLC system for detecting amphetamines in urine of drug abusers attending drug rehabilitation programmes. The HPLC confirmatory method described is a viable alternative to GC or to the more complex and costly GC-MS techniques for confirming amphetamine, methamphetamine, MDMA, MDA and MDEA in urine of drug abusers especially when used in a clinical care setting.     

Simultaneous analysis of anthranilic acid derivatives in pharmaceuticals and human urine by high-performance liquid chromatography with isocratic elution

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of mefenamic acid (MFA), flufenamic acid (FFA) and tolfenamic acid (TFA) is presented for application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-pentylammonium bromide (TPAB) as an ion-pair reagent. Urine samples were purified by solid-phase extraction using a silica-based strong anion-exchanger, Bond-Elut SAX cartridge. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of 1.9 g of TPAB dissolved in 1:1 of a mixture of acetic acid-sodium acetate buffer solution, pH 5.0, and acetonitrile (11:9, v/v). The calibration curves of MFA, FFA and TFA showed good linearity in the concentration range of 33-167 microg/ml with a wavelength of 280 nm for pharmaceuticals, and in the low concentration range (1.7-30.1 microg/ml) with a wavelength of 230 nm for biological fluids. The correlation coefficients were better than 0.9999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2 ng for MFA, 3.5 ng for FFA and 2.5 ng for TFA. The procedure described here is rapid, simple, selective and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.     

Determination of terbutaline enantiomers in human urine by coupled achiral-chiral high-performance liquid chromatography with fluorescence detection

A coupled achiral-chiral high-performance liquid chromatographic system with fluorescence detection at excitation/emission wavelengths of 276/306 nm has been developed for the determination of the enantiomers of terbutaline, (S)-(+)-terbutaline and (R)-(-)-terbutaline in urine. Urine samples were prepared by solid-phase extraction with Sep-pak silica, followed by HPLC. The terbutaline was preseparated from the interfering components in urine on Phenomenex silica column and the terbutaline enantiomers and betaxolol were resolved and determined on a Sumichiral OA-4900 chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The precolumn was used to concentrate the terbutaline in the eluent from the achiral column before back flushing onto the chiral phase. For each enantiomer the assay was linear between 1 and 250 ng/ml (R2=0.9999) and the detection limit was 0.3 ng/ml. The intra-day variation was between 4.6 and 11.6% in relation to the measured concentration and the inter-day variation was 4.3-11.0%. It has been applied to the determination of (S)-(+)-terbutaline and (R)-(-)-terbutaline in urine from a healthy volunteer dosed with racemic terbutaline sulfate.     

Determination of a chemopreventive agent,Oltipraz, in rat plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemopreventive agent, Oltipraz, in rat plasma and urine. The sample preparation was simple; 2 volumes of acetonitrile were added to deproteinize the biological sample. A 50-microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase, acetonitrile : 0.5 mM ammonium acetate (55: 45, v/v for rat plasma and 45 : 55, v/v for rat urine), was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 305 nm. The retention times for Oltipraz in rat plasma and urine were approximately 5.8 and 8.6 min, respectively. The detection limits of Oltipraz in rat plasma and urine were 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 4.65%) in concentration ranges from 0.02 (0.05) to 10 microg/ml for rat plasma and urine. No interference from endogenous substances was found.     

Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection.

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C18 reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2'-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O6-benzylguanine in a phase I clinical trial. Previously, O6-benzyl-8-oxoguanine was identified as the primary metabolite of O6-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O6-benzylguanine.     

Determination of stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume

Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2',3'-didehydro-3'-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025-25 and 2-150 microg/ml in plasma and urine, respectively. An aliquot of 200 microl of plasma was extracted with solid-phase extraction using Oasis cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 microl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.     

Precise analysis of primary amino acids in urine by an automated high-performance liquid chromatography method: comparison with ion-exchange chromatography

A precise, simple and rapid method for the quantitative determination of primary amino acids in urine based on high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. All primary urinary amino acids could be determined within 49 min (injection to injection). Amino acid concentrations in 40 urinary samples were measured by this method and the results were compared with those measured by ion-exchange chromatography. The correlation coefficient for the common amino acids was greater than 0.90. This is the first study in which such a detailed comparison has been made on urine samples. It appeared that the method described is an excellent alternative to the classical ion-exchange method for the quantitation of urinary amino acids.     

Determination of sialic acids in biological fluids using reversed-phase ion-pair high-performance liquid chromatography

A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C18 column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva.     

Determination of cetirizine in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of cetirizine in human plasma is presented. The method is based on liquid-liquid extraction with dichloromethane and reversed-phase chromatography with spectrophotometric detection at 232 nm. Gradient elution was used to remove late eluting peaks. Diazepam was used as the internal standard. The limit of quantitation was 10 ng/ml using 0.5 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 10% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of ciprofloxacin in human serum by high-performance liquid chromatography

A high-performance liquid chromatography method was developed for the rapid determination of ciprofloxacin in human serum. After protein precipitation, chromatographic separation of ciprofloxacin in plasma was achieved with an octyl column and 20 mmol/L phosphate buffer, methanol and acetonitrile 76/12/12 (v/v/v), pH 4, as mobile phase. Quantitative determination was monitored on a fluorescence detector using an excitation and emission wavelength of 278 and 450 nm, respectively. Enrofloxacin was used as internal standard. Calibration curve is linear over the range 0,12 to 20 microg/mL (r=0,9992). Limit of detection and limit of quantification are 0,04 and 0,12 microg/mL, respectively. Within-day coefficient of variation (2,0 to 3,1 %) and day-to-day coefficient of variation (4,3 to 5,5 %) at three different concentrations are sufficient. Relative recovery ranged from 91,1 to 94,0 % at three different concentrations. Each analysis requires no longer than 7 min.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid--liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0-5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0-4000.0 ng/ml.     

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography.

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 microg/ml for both MPA and MPAG in plasma and from 1 to 50 microg/ml of MPA and 5 to 2000 microg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 microg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Determination of urinary methylated purine pattern by high-performance liquid chromatography

We describe the group selective separation and quantification of unmodified and modified purines in human urine by high-performance reverse phase liquid chromatography. The pattern of oxypurines and methylated purines: hypoxanthine (Hx), xanthine (X), 1-methyl hypoxanthine (1-MHx), 1-methyl guanine (1-MG), 3-methyl guanine (3-MG), 7-methyl guanine (7-MG), 1-methyl xanthine (1-MX), 3-methyl xanthine (3-MX), 7-methyl xanthine (7-MX), 1,7-dimethyl guanine (1,7-dMG), 1,3-dimethyl xanthine (1,3-dMX), 1,7-dimethyl xanthine (3,7-dMX) and 1,3,7-trimethyl xanthine (1,3,7-tMX) were determined in a single run in urine of a healthy subject and a gout patient before and after treatment with allopurinol. This method may be useful to investigate the urinary pattern of methylated bases in diseases involving purine metabolism.     

Determination of cefmenoxime in human body fluids by high-performance liquid chromatography

A rapid and reliable method for the quantitative determination of cefmenoxime in serum and urine by reversed phase high-performance liquid chromatography is described. Serum was deproteinized with acetonitrile. Urine was diluted with dilute acetic acid (17.5 mmol/l). Separations were performed in isocratic mode using a C18 type column and a precolumn packed with Perisorb RP/8. The eluant consisted of a mixture of acetonitrile and 25.0 mmol/l acetic acid in a ratio of 32/69 (vol/vol). In normal subjects cefmenoxime was well separated from endogenous compounds and various added drugs. Its complete separation was confirmed by selective degradation with beta-lactamase fromBacillus cereus and UV spectrophotometry. The detection limit was 0.3 mg/l at a detection wave-length of 254 nm. Peak areas gave linear results up to concentrations of 500 mg/l. Within-batch precision (coefficient of variation) ranged from 1.1 to 6.2 %. Recovery rates varied from 99.0 to 103.3%. Results of a standard microbiological assay correlated well with those obtained by the present HPLC method. Eight healthy volunteers who were given a single intravenous dose of 1 g cefmenoxime excreted 86.3 ± 5.8 % of the unchanged drug within 24 h in urine.     

Determination of lamotrigine in human plasma by high-performance liquid chromatography.

The method involves precipitation of plasma proteins with acetonitrile and analysis of the supernatant by high-performance liquid chromatography using a 5 microm Zorbax C8 column. Quantitation was performed by measurement of the UV absorbance at a wavelength of 306 nm. The method was linear in the range of 1-20 microg/ml, with a mean coefficient of determination (r2=0.998). The limit of detection was 0.6 microg/ml and the lower limit of quantitation was 1 microg/ml using 200 microl of plasma. Within- and between-day accuracy and precision were below 6% at all analysed concentrations except at the limit of quantitation. No interfering peaks were found by commonly monitored antiepileptic drugs. Recovery was found to be > or =99%. Satisfactory performance was obtained in the evaluation of epileptic patient samples, whose results of plasma concentration measurements are briefly discussed. We conclude that this is a reliable method for the routine monitoring of lamotrigine concentration in plasma in the clinical setting.