Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 μM and the calibration curve was linear at least up to 5 μM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 μM and from 3.5 to 6.7 at 5 μM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 μM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.     

Determination of clozapine and its major metabolites in human plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance detection

An isocratic high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of clozapine (8-chloro-11-(4′-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two major metabolites in plasma and red blood cells (RBCs). The method involves sample clean-up by liquid-liquid extraction with ethyl acetate. The organic phase was back-extracted with 0.1 M hydrochloric acid. Loxapine served as the internal standard. The analytes were separated by HPLC on a Kromasil Ultrabas C₁₈ analytical column (5 μm particle size; 250×4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm. The limits of quantification were 20 ng/ml for clozapine and N-desmethylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma or RBCs proved to be higher than 62%. Precision, expressed as % C.V., was in the range 0.6–15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine and two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeutic drug monitoring.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Field-amplified sample stacking in capillary electrophoresis for the determination of clozapine, clozapine N-oxide, and desmethylclozapine in schizophrenics' plasma

A method of field-amplified sample stacking in capillary electrophoresis is described for the simultaneous determination of clozapine (CZP) and its metabolites, clozapine N-oxide (CNO), and desmethylclozapine (DMC), in human plasma. Plasma (0.2 mL) was extracted with organic solvents (ethyl acetate/n-hexane/isopropyl alcohol, 8/1/1 by volume) and centrifuged. An aliquot of supernatant was evaporated and suitably reconstituted with water for CE analysis. An untreated fused-silica capillary was used (31.2 cm; effective length, 20 cm; 50 microm i.d.) for the analysis. The background buffer was phosphate buffer (400 mM, pH 3.0) containing 50% ethylene glycol. The separation voltage was 25 kV with a detection wavelength of 214 nm. In the method validation, the calibration curves were linear (r > or = 0.98) over a range of 50-800 ng/mL for CZP, 30-180 ng/mL for CNO, and 25-600 ng/mL for DMC. The relative standard deviation (R.S.D.) and relative error (R.E.) were all less than 11% for the intra- and inter-day assays. The limits of detection (S/N = 3, electric-driven injection, 99.9s) of CZP, DMC, and CNO were 5, 5, and 10 ng/mL, respectively. After continuing treatment with the CZP tablets, a blood sample from one male schizophrenic patient (41-year-old, 62 kg) who had been receiving ongoing treatment with the CZP tablets was prepared and analyzed. The levels of CZP, DMC, and CNO were determined and the feasibility of the method's application in clinical treatment was proven.     

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCl. The maximum plasma concentrations were 235–465 ng/ml (MDE), 67–673 ng/ml (HME) and 7–33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.     

Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A method for the determination of 25-hydroxyvitamin D₃, the major metabolite of vitamin D₃ in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n=25) and from patients with chronic renal failure (n=12). 25-Hydroxyvitamin D₃ 3-sulfate, a major conjugated metabolite of 25-hyroxyvitamin D₃, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.     

Analysis of cysteine and N-acetylcysteine in human plasma by high-performance liquid chromatography at the basal state and after oral administration of N-acetylcysteine

A high-performance liquid chromatographic method for the determination of free reduced cysteine and N-acetylcysteine in human plasma at the basal state and after oral administration of N-acetylcysteine is described. The method is based on acid-catalysed conversion of plasma thiols to the corresponding S-nitroso derivatives by excess of nitrite and their subsequent cation-pairing RP-HPLC with detection at 333 nm. Recovery rates of cysteine and N-acetylcysteine added to human plasma were 94.6 and 99.6%, respectively. Inter- and intra-day precision were below 6%. In healthy humans (n=5), free reduced cysteine was determined to be (mean±S.E.) 10.0±0.96 μM. No N-acetylcysteine was detected in plasma of these subjects above the limit of detection (e.g. 170 nM). The method was successfully applied to a pharmacokinetic study on orally administered N-acetylcysteine to healthy volunteers.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

Determination of O-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic assay for O⁶-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were constituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O⁶-Benzylguanine peak heights were compared to peak heights of O⁶-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O⁶-benzylguanine.     

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 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 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 Z-butylidenephthalide in plasma by high-performance liquid chromatography

An HPLC assay is described for the determination of Z-butylidenephthalide (Z-Bdph) in plasma. Plasma samples were cleaned up by extraction with 2% chloroform in n-hexane. Z-Bdph was separated on a normal-phase silica column with a mobile phase of chloroform-n-hexane (1:1). The limit of quantitation with UV detection at 254 nm for Z-Bdph in plasma was 0.01 μg/ml. The recovery of Z-Bdph by organic solvent extraction of plasma was 99.5%. The intra-day and inter-day coefficients of variation and relative errors for Z-Bdph determination in plasma were both less than 10%. The present method was applied to pharmacokinetic studies of Z-Bdph in plasma after intravenous administration to rabbits.     

Determination of ketorolac in human plasma by reversed-phase high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

An improved high-performance liquid chromatographic method has been developed to measure human plasma concentrations of the analgesic nonsteroidal anti-inflammatory drug ketorolac for use in pharmacokinetic studies. Samples were prepared for analysis by solid-phase extraction using Bond-Elut PH columns, with nearly complete recovery of both ketorolac and the internal standard tolmetin. The two compounds were separated on a Radial-Pak C₁₈ column using a mobile phase consisting of water–acetonitrile–1.0 mol/l dibutylamine phosphate (pH 2.5) (30:20:1) and detected at a UV wavelength of 313 nm. Using only 250 μl of plasma, the standard curve was linear from 0.05 to 10.0 μg/ml.     

Determination of N,N-dimethylarginine in human plasma by high-performance liquid chromatography

N^G,N^G-Dimethylarginine (asymmetric dimethylarginine, ADMA) can be directly separated and measured from deproteinized human plasma using o-phthaldialdehyde-mercaptoethanol (OPA reagent) as a fluorogenic reagent by reversed-phase high-performance liquid chromatography. The mean recovery of ADMA was over 96% and the inter- and intra-assay coefficients of variation of amounts were lower than 3.80% and those of retention time were below 0.37% for five runs. The detection limit of the assay is 1 pmol when the signal-to-noise is 3:1. It was observed that the concentration of ADMA was significantly elevated in plasma of patients with pregnancy induced hypertension (PIH) in contrast to healthy pregnant women.     

Determination of stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of a new antiretroviral agent, stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), in human plasma. Didanosine (2′,3′-dideoxyinosine, ddI) was used as the internal standard. The very selective sample pretreatment involved solid-phase extraction using silica gel columns. Chromatography was carried out on a μBondapak phenyl column, using a mobile phase of 0.005 M phosphate buffer (pH 6.8)—methanol (90:10, v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The detection limit is 10 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used in a single pharmacokinetic experiment in a rat to investigate the applicability of the method in vivo.     

Determination of rifampin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifampin in human plasma. Rifampin and sulindac (internal standard) are extracted from human plasma using a C₂ Bond Elut extraction column. A 100-μl volume of 0.1 M HCl is added to the plasma before extraction to increase the retenction of the compounds on the extraction column. Methanol (1 ml) is used to elute the compounds and 0.5 ml of 3 mg/ml ascorbic acid in water is added to the final eluate to reduce the oxidation of rifampin. Separation is achieved by reversed-phase chromatography on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate-acetonitrile (55:45, v/v). Detection is by ultraviolet absorbance at 340 nm. The retention times of rifampin and internal standard are approximately 4.4 and 7.8 min, respectively. The assay is linear in concentration ranges of 50 to 35 000 ng/ml. The quantitation limit is 50 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Determination of rifabutin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifabutin in human plasma. Rifabutin and sulindac (internal standard) are extracted from human plasma using a C₈ Bond Elut extraction column. Methanol (1 ml) is used to elute the compounds. The methanol is dried down under nitrogen and reconstituted in 250 μl of mobile phase. Separation is achieved by HPLC on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium acetate at pH 4.0-acetonitrile (53:47, v/v). Detection is by ultraviolet absorbance at 275 nm. The retention times of rifabutin and internal standard were approximately 10.8 and 6.9 min, respectively. The assay is linear over the concentration range of 5–600 ng/ml. The quantitation limit was 5 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.