High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65±2.3 and 8.9±2.4 μmol/l, while post-methionine load gave tHcy levels of 19.9±5.5 and 26.8±5.5 μmol/l, for women and men, respectively (n=40).     

High-performance liquid chromatographic method for the determination of gabapentin in human plasma

A sensitive high-performance liquid chromatography (HPLC) method using UV detection for the determination of gabapentin in human plasma has been developed. In this method, gabapentin was extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge followed by derivatization with phenylisothiocyanate. Analysis was achieved by using a HPLC system that was equipped with a UV detector. The quantitation limit of gabapentin in human plasma was 0.03 microg/ml. The method is sensitive with excellent selectivity and reproducibility and it has been applied to a bioequivalence clinical study with great success.     

High-performance liquid chromatographic method for the determination of dolichols in tissues and plasma

High-performance liquid chromatographic methods for the determination of dolichols in tissues and plasma have been developed. The tissue concentration of dolichols was measured by high-performance liquid chromatography with uv detection and plasma levels of dolichols were determined fluorometrically after derivatization with anthracene-9-carboxylic acid. In both methods, 2,2-didecaprenylethanol was used as an internal standard. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichols in human plasma.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

High-performance liquid chromatographic determination of pentamidine in plasma

This report describes the analysis of pentamidine by isocratic reversed-phase high-performance liquid chromatography (HPLC) using a commercially available compound (melphalan) as the external standard. Previously described assays use ion-pairing HPLC, an internal standard (hexamidine) that is not readily available, and require a relatively large sample size. In the present assay, pentamidine was extracted from plasma using solid-phase extraction and was analyzed using a C₁₈ column and a mobile phase containing 18% acetonitrile, 2% methanol, 0.2 M ammonium acetate and 0.5% triethylamine. The identity of the eluting peaks was verified using a diode array detector. The extraction yield of pentamidine was 82%. The limit of detection was 8.6 ng/ml with a sample size of 100 μl. The inter-day and intra-day coefficients of variation ranged between 0.3% and 10% with an average of 5%. This method was applied to study the pharmacokinetics of pentamidine in rodents.     

High-performance liquid chromatographic determination of fluconazole in plasma

A high-performance liquid chromatographic method with ultraviolet absorbance detection at 260 nm was developed for the analysis of fluconazole in plasma. The method involves sample clean-up by liquid-liquid extraction. The proposed technique is reproducible, selective, reliable and sensitive. Calibration standards were prepared in the range 1.25-20 mg/l. The limit of quantitation was 0.4 mg/l. The coefficients of variation were 5% between measurements of a single extract injected in duplicate, and 7% between two extractions of spiked samples at the same concentrations. The separation between fluconazole and endogenous substances was satisfactory. This method was designed in order to minimise the risk of interference from substances that could be co-administered to critically ill patients undergoing hemodiafiltration. With a run time below 5 min, the present method is rapid and easy to use for later clinical studies, as well as for routine monitoring.     

High-performance liquid chromatographic determination of ganciclovir in plasma

A rapid, selective and sensitive isocratic reversed-phase high-performance liquid chromatographic method for the determination of ganciclovir in plasma samples was developed. This method, which was applied to the analysis of plasma ganciclovir from heart transplant patients under ganciclovir therapy for cytomegalovirus infections, represents a suitable analytical tool for drug monitoring and pharmacokinetic investigations.     

High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues

An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C₈ preparative column with 6% acetonitrile—45% methanol—5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-μm Spin-X cartridges, applied to a reversed-phase C₁₈ column, and eluted in a 0–50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzylamiloride.     

High-performance liquid chromatographic method for determination of 2-difluoromethyl-DL-ornithine in plasma and cerebrospinal fluid

A simple, sensitive, selective and reproducible method based on anion-exchange liquid chromatography with post-column derivatisation was developed for the determination of eflornithine (2-difluoromethyl-DL-ornithine; DFMO) in human plasma and cerebrospinal fluid. The 1-alkylthio-2-alkyl-isoindoles fluorescent derivative of the drug was separated from the internal standard (MDL 77246A) on an anion-exchange column (PRP-X300, 250x2.1 mm, 7-microm particle size: Hamilton, USA), with retention times of 6.9 and 10.7 min, respectively. Fluorescence detection was set at 430/340 nm (emission/excitation wavelength). The elution solvent consisted of a solution of 30 mM potassium dihydrogen phosphate buffer (pH 2.2) and acetonitrile (50:50, v/v), running through the column at a flow-rate of 0.3 ml/min. The chromatographic analysis was operated at 37 degrees C. Sample preparation for either plasma or CSF (100 microl) was done by single-step protein precipitation with 20% trichloroacetic acid after incubation at 4 degrees C for 1 h. Calibration curves for plasma (100, 200, 400, 600, 800 and 1200 nmol/100 microl, and 10, 20, 40, 80, 120 and 160 nmol/100 microl for the high and low concentration range curves, respectively) and CSF (1, 2, 4, 8, 16, 32 nmol/100 microl) were all linear with correlation coefficients better than 0.999. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) at high concentration range was below 15%, whereas at low concentration range was below 20% (% coefficient of variations: %C.V.) Good accuracy was observed for both the intra-day or inter-day assays, as indicated by the minimal deviation of mean values found with measured samples from that of the theoretical values (below +/-15 and +/-20% at high and low concentration range, respectively. The limit of quantification was accepted as 0.1 nmol using 100-microl samples. The mean recovery for DFMO and the internal standard were greater than 95%. The method was free from interference from commonly used drugs including antimalarials and antihelminthics. The method appears to be robust and has been applied to a pharmacokinetic study of DFMO in patients with African trypanosomiasis following oral doses of Ornidyl (Aventis Pharma, Frankfurt, Germany) at 500 mg/kg body weight (125 mg q.i.d.) for 14 days.     

High-performance liquid chromatographic determination of ambroxol in human plasma

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid—liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.     

High-performance liquid chromatographic method for an automated determination of local anaesthetics in human plasma

A method is described that allows the rapid and precise determination of the local anaesthetics bupivacaine and etidocaine from biological fluids. This method uses a fully automated system with solid-phase extraction in combination with a column-switching technique. Both sample extraction on a LiChrocart pre-column and elution onto the analytical LiChrospher column, were performed automatically and concomitantly using conventional HPLC equipment in conjunction with an OSP-2 on-line sample preparator from Merck combined with UV detection. Recoveries were found to be 96.7 and 96.4% for 2 μg/ml bupivacaine and etidocaine, respectively. Lower limits of quantification were found to be 0.05 μg/ml plasma for both of the compounds.     

High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate–methanol (2:1, v/v), these four compounds were successfully separated using a C₁₈ column with a gradient elution of 5 and 25% acetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 μg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16–40, 0.63–160, 0.13–32 and 0.13–30 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.     

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.     

High-performance liquid chromatographic method for sensitive determination of the alkylating agent CB1954 in human plasma

A high-performance liquid chromatography (HPLC) method is described for the measurement of the weak alkylating agent CB1954 in human plasma. CB1954 can be used as an innocuous prodrug designed for activation by bacterial nitroreductases in strategies of gene-directed enzyme–prodrug therapy, and becomes activated to a potent bifunctional alkylating agent. The HPLC method involves precipitation and solvent extraction and uses Mitomycin C (MMC) as an internal standard, with a retention time for MMC of 5.85±0.015 min, and for CB1954 of 10.72±0.063 min. The limit of detection for CB1954 is 2.9 ng/ml, and this compares favourably with systems involving direct analysis of plasma (limit of detection 600 ng/ml, approximately). The method is now being used for pharmacokinetic measurements in plasma samples from cancer patients entering phase I clinical trials of CB1954. Results using serial plasma samples from one patient are presented. The patient was treated intravenously with CB1954 (6 mg/m²), and plasma clearance of the drug showed biphasic kinetics with α half-life 14.6 min, and β half-life 170.5 min.     

High-performance liquid chromatographic determination of thiopentene enantiomers in sheep plasma

An HPLC method was developed to determine the plasma concentrations of R(+)- and S(−)-thiopentone for pharmacokinetic studies in sheep. The method required separation of the thiopentone enantiomers from the corresponding pentobarbitone enantiomers which are usually present as metabolites of thiopentone. Phenylbutazone was used as an internal standard. After acidification, the plasma samples were extracted with a mixture of ether and hexane (2:8). The solvent was evaporated to dryness and the residues were reconstituted with sodium hydroxide solution (pH 10). The samples were chromatographed on a 100 mm × 4 mm I.D.. Chiral AGP-CSP column. The mobile phase was 4.5% 2-propanol in 0.1 M phosphate buffer (pH 6.2) with a flow-rate of 0.9 ml/min. This gave k′ values of 1.92, 2.92, 5.71, 9.30 and 11.98 for R(+)-pentobarbitone, S(−)-pentobarbitone, R(+)-thiopentone, S(−)-thiopentone, and phenylbutazone, respectively. At detection wavelength of 287 nm, the limit of quantitation was 5 ng/ml for R(+)-thiopentone and 6 ng/ml for S(−)-thiopentone. The inter-day coefficients of variation at concentrations of 0.02, 0.1 and 8 μg/ml were, respectively, 4.8, 4.4 and 3.5% for R(+)-thiopentone and, respectively, 5.0, 4.3 and 3.9% for S(−)-thiopentone (n = 6 each enantiomer). At the same concentrations, the intra-day coefficients of variation from six sets of replicates (measured over six days) were, respectively, 8.0, 8.0 and 8.8% for R(+)-thiopentene and 8.8, 7.4 and 9.6% for S(−)-thiopentone. Linearity over the standard range, 0.01–40 μg/ml, was shown by correlation coefficients> 0.998. This method has proven suitable for pharmacokinetic studies of thiopentone enantiomers after administration of rac-thiopentone in human plasma also and would be suitable for pharmacokinetic studies of the pentobarbitone eantiomers.