High-performance liquid chromatographic determination of ibuprofen and its major metabolites in biological fluids

A sensitive and selective high-performance liquid-chromatographic assay for ibuprofen and its major metabolites in biological fluids is described. To ensure good chromatographic separation the drug and metabolites were run on a gradient elution system and detected with a variable wavelength detector set at 220 nm. A second, more rapid, isocratic system is also described for the detection of only ibuprofen.     

High-performance liquid chromatographic determination of thiopurine metabolites of azathioprine in biological fluids

A selective and sensitive reversed-phase liquid chromatographic method for the analysis of thiopurine bases, nucleosides and nucleotides in biological samples was developed. A simple and rapid sample treatment procedure using perchloric acid deproteinization with dithiothreitol for the analysis of thiopurine bases and nucleosides is presented. The addition of dithiothreitol during sample collection and treatment improves recoveries. This procedure also allows the determination of thiopurine nucleotides by hydrolysis to their free bases after heating of the perchloric acid extract. The method was applied to the analysis of thiopurine metabolites in plasma and erythrocytes from lung-transplant patients under azathioprine therapy.     

High-performance liquid chromatographic analysis of sulfinpyrazone and its metabolites in biological fluids

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of sulfinpyrazone and its sulfone and p-hydroxy metabolites in plasma and urine. The method uses two different procedures for sample preparation: (1) a rapid and convenient procedure using a single extraction with 1-chlorobutane and subsequent back-extraction into sodium hydroxide solution for the analysis of sulfinpyrazone and its sulfone metabolite, and (2) a more time consuming procedure using triple extraction with ethylene dichloride, a buffer wash, and back extraction into the base for the additional analysis of the p-hydroxy metabolite. The lower limit of sensitivity for sulfinpyrazone is 50 ng/ml. Concentrations of sulfinpyrazone between 0.05 to 0.1 and 50 μg/ml were measured with an average coefficient of variation of 3.9%, ranging from 1.5 to 6.1%.     

High-performance liquid chromatographic determination of sotalol in biological fluids

A sensitive, selective and reproducible reversed-phase high-performance liquid chromatographic method is described for the quantification of sotalol in human serum and urine. Sotalol and the internal standard, atenolol, were extracted from alkalinized serum and urine (pH 9.0) into 1-butanol—chloroform (20:60, v/v). The organic phase was evaporated, and to the residue was added 0.1 M sulphuric acid (serum analysis) or mobile phase (urie analysis). The mobile phase consisted of 0.01 M phosphate buffer (pH 3.2) and acetonitrile (20:80, v/v) containing 3 mM n-octylsodium sulphate. The flow-rate was 1.5 ml/min. The retention times of atenolol and sotalol were 7 and 10 min, respectively. Ultraviolet detection at 226 nm made it possible to achieve a detection limit of 0.03 μmol/l.     

High-performance liquid chromatographic determination of quinine in rat biological fluids

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of quinine in rat biological fluids is described. Due to its selectivity and sensitivity, the proposed method can be used in the case of such rat biological fluids as cerebrospinal fluid (CSF) and perilymph for which the accessible volumes are limited to 100 μl and 10 μl, respectively. Consequently, the assay method has been applied to the measurements of quinine concentration in rat plasma, CSF and perilymph samples.     

High-performance liquid chromatographic determination of hypoxanthine and xanthine in biological fluids

A rapid and selective reversed-phase high-performance liquid chromatographic method for the simultaneous determination of hypoxanthine and xanthine in biological fluids was developed. The identification of hypoxanthine and xanthine was confirmed by xanthine oxidase reaction. This method was applied to the investigation of purine metabolism in subjects with xanthine oxidase deficiency or gout. Hypoxanthine concentrations three to ten times higher than those determined in plasma were found in erythrocyte samples from normal subjects and from patients with xanthine oxidase deficiency or hyperuricemia under allopurinol therapy.     

High-performance liquid chromatographic method for determination of gentamicin in biological fluids

A selective and sensitive method for the determination of gentamicin in plasma and urine by high-performance liquid chromatography has been developed. Following deproteinization, the gentamicin is reacted with fluorescamine to produce a fluorescent derivative. This reaction mixture is directly chromatographed on a cation-exchange column using as mobile phase acetonitrile—phosphoric acid (7:3). The gentamicin components elute as a single peak. Using 0.1 ml of plasma, quantitation of gentamicin concentrations as low as 1 mg/l are possible. Possible interference from other aminoglycosides and antibiotics is discussed.     

High-performance liquid chromatographic determination of isoniazid, acetylisoniazid and hydrazine in biological fluids

The basic principle of derivatization of a hydrazide moiety with an aldehyde as applied in the method developed by Lacroix et al. [J. Chromatogr., 307 (1984) 137–144] for the quantitation of isoniazid and acetylisoniazid was imppoved by modification, standardization and extension to allow quantitation of hydrazine in patient samples. It could be shown that 40 μl of 1% methanonic cinnamaldehyde per 200 μl of deproteinized analysate gave maximal chromophoric isoniazid-cinnamaldehyde conjugate, read at 340 nm. The hydrolytic loss of isoniazid, crucial to the quantitation of acetylisoniazid, could be compensated for by introduction of an appropriate set of calibration curves. Although the method described here allows quantitation of monoacetylhydrazie and diacetylhydrazine, in addition to hydrazine, in mono-spiked samples, the method cannot be used for the quantitation of the acetylated metabolites of hydrazine in patient samples because of a lack of specificity. Linear calibration curves in the range 1–25 μg/ml for isoniazid and acetylisoniazid, 10–400 ng/ml for hydrazine and 50–1000 ng/ml for mono-acetylhydrazine and diacetylhydrazine, could be constructed; analyte recoveries approaching 100% could be achieved in all instances.     

High-performance liquid chromatographic analysis of methocarbamol enantiomers in biological fluids

Methocarbamol enantiomers in rat and human plasma were quantified using a stereospecific high-performance liquid chromatographic method. Racemic methocarbamol and internal standard, (R)-(−)-flecainide, were isolated from plasma by a single-step extraction with ethyl acetate. After derivatization with the enantiomerically pure reagent (S)-(+)-1-(1-naphthyl)ethyl isocyanate, methocarbamol diastereomers and the (R)-flecainide derivative were separated on a normal-phase silica column with a mobile phase consisting of hexane—isopropanol (95:5, v/v) at a flow-rate of 1.6 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm. The resolution factor between the diastereomers was 2.1 (α = 1.24). An excellent linearity was observed between the methocarbamol diastereomers/internal standard derivative peak-area ratios and plasma concentrations, and the intra- and inter-day coefficients of variation were always <9.8%. The lowest quantifiable concentration was 0.5 μg/ml for each enantiomer (coefficients of variation of 9.8 and 8.8% for (S)- and (R)-methocarbamol, respectively), while the limit of detection (signal-to-noise ratio 3:1) was approximately 10 ng/ml. The assay was used to study the pharmacokinetics of methocarbamol enantiomers in a rat following intravenous administration of a 120 mg/kg dose of racemic methocarbamol and to evaluate plasma and urine concentrations in a human volunteer after oral administration of a 1000-mg dose of the racemate. The method is suitable for stereoselective pharmacokinetic studies in humans as well as in animal models.     

High-performance liquid chromatographic determination of carbamazepine and metabolites in human hair

To study the use of hair analysis in monitoring drug compliance and historical changes in pharmacokinetics we developed a method for the quantitative determination of the anti-epileptic drug carbamazepine (CBZ) and trans-10,11-dihydro-10,11-dihydroxy-carbamazepine (CBZ-diol) in hair from carbamazepine users. Digestion by 1 M NaOH was found to be the best method for isolating CBZ and CBZ-diol from hair, followed by solid-phase extraction and reversed-phase HPLC with UV detection. Recoveries from spiked hair samples were 76–86%. Within-day precision (C.V.; n=10) for CBZ and CBZ-diol in hair of a CBZ user containing 10.9 μg/g CBZ and 3.2 μg/g CBZ-diol were 1.7 and 5.0%, respectively. Sectional hair analysis of a patient on a constant dosage of CBZ demonstrates an exponential decrease in hair concentrations of CBZ and CBZ-diol with increasing distance from the root, probably caused by shampooing. No CBZ-10,11-epoxide (CBZ-epox) could be detected. However, one component in the chromatogram is probably CBZ-β-hydroxythioether, an adduct of CBZ-epox with cysteine, or acridinethioacetal, its rearrangement product. The concentration of this component does not decrease with increasing distance from the root.     

High-performance liquid chromatographic determination of 2-hydroxypropyl-gamma-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence

A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-gamma-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5-15 microg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.     

High-performance liquid chromatographic analysis of cytosine arabinoside and metabolites in biological samples

A rapid, non-radioactive method to quantitate therapeutically realistic levels of 1-β- -arabinofuranosylcytosine (Ara-C) and its metabolites would be useful both in the clinic, for monitoring drug levels, and in the laboratory for correlating drug levels with cellular and molecular perturbations. Liquid chromatographic analysis of arabinose-nucleoside analogs in biological samples is complicated by the presence of interfering nucleosides and nucleotides. We report the development of two analytic procedures to measure Ara-C and metabolite levels in biological samples. One method uses a quaternary ammonium type anion-exchange resin to achieve isocratic separation in less than one hour. The second method utilizes a boronate-derivatized polyacrylamide column which binds cis-diols to selectively retain cytosine and uridine, while arabinose compounds are eluted with recovery approaching 100%. The eluted compounds are then easily quantitated on a reversed-phase C₁₅ column. The sensitivity of both procedures was sufficient to obtain pharmacokinetic data on Ara-C and uracil-arabinose levels in serum and urine and on Ara-C triphosphate levels in tumor cells.     

High-performance liquid chromatographic method for determination of amodiaquine,chloroquine and their monodesethyl metabolites in biological samples

A high-performance liquid chromatographic method for determination of amodiaquine (AQ), desethylamodiaquine (DAQ), chloroquine (CQ) and desethylchloroquine (DCQ) in human whole blood, plasma and urine is reported. 4-(4-Dimethylamino-1-methylbutylamino)-7-chloroquinoline was used as internal standard. The drugs and the internal standard were extracted into di-isopropyl ether as bases and then re-extracted into an acidic aqueous phase with 0.1 M phosphate buffer at pH 4.0 for AQ samples and at pH 2.5 for CQ filter paper samples. A C(18) column was used and the mobile phase consisted of methanol-phosphate buffer (0.1 M, pH 3)-perchloric acid (250: 747.5:2.5, v/v). The absorbance of the drugs was monitored at 333 nm and no endogenous compound interfered at this wavelength. The limit of quantification in whole blood, plasma and urine was 100 nM for AQ and DAQ (sample size 100 microliter) as well as for CQ and DCQ in blood samples dried on filter paper. For 1000 microliter AQ and DAQ samples, the limit of quantification was 10 nM in all three biological fluids. The within-assay and between-assay coefficients of variations were always <10% at the limits of quantification. Plasma should be preferred for the determination of AQ and DAQ since use of whole blood may be associated with stability problems.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

High-performance liquid chromatographic analysis of estradiol-17β and metabolites in biological media

A method was developed to resolve radiolabeled estradiol-17β and its various metabolites in biological fluids and tissues. After a rapid initial clean-up step, samples were analyzed with the sequential use of reversed-phase and normal-phase high-performance liquid chromatographic systems. Approximately 25 conjugated and non-conjugated standards could be resolved by the combined use of six systems. Radiolabeled parent compound and metabolites from biological samples were separated and tentatively identified by comparing their retention times to those of known standards. The method was found to be reproducible and quantitative for the majority of the estrogens and their conjugates, and semiquantitative for some of the more polar and di-conjugated estrogens.