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 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 method for the determination of histamine and 1-methylhistamine in biological buffers

A method for the determination of histamine and its catabolite 1-methylhistamine (1-MH) was developed, using HPLC with fluorescence detection. Derivatization of both compounds occurred on-column with o-phthaldialdehyde dissolved in an alkaline borate buffer, followed by separation on a reversed phase C18 column. Histamine and 1-MH could be detected with comparable sensitivity (limit of quantification, 50 nM). The method was proven suitable to investigate catabolism of histamine by epithelia of pig colon. The method should be useful in research on histamine metabolism.     

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 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 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 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 method for the simultaneous determination of monoethylglycinexylidide and lignocaine

An accurate and sensitive high-performance liquid chromatographic method with UV detection was developed for the simultaneous measurement of monoethylglycinexylidide (MEGX) and lignocaine in human plasma and serum, using organic solvent extraction and trimethoprim (TMP) as an internal standard. The mean recoveries for MEGX, TMP and lignocaine were 86.1 ± 3.7, 98.3 ± 1.8 and 77.0 ± 4.7%, respectively (n = 6). The relative standard deviations for MEGX concentrations of 10 and 200 ng/ml were < 4% and for lignocaine concentrations of 200 and 1200 ng/ml they were < 8%.     

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 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 method for guanylhydrazone compounds

A high-performance liquid chromatographic method has been developed for a series of aromatic guanylhydrazones that have demonstrated therapeutic potential as anti-inflammatory agents. The compounds were separated using octadecyl or diisopropyloctyl reversed-phase columns, with an acetonitrile gradient in water containing heptane sulfonate, tetramethylammonium chloride, and phosphoric acid. The method was used to reliably quantify levels of analyte as low as 785 ng/ml, and the detector response was linear to at least 50 μg/ml, using a 100 μl injection volume. The assay system was used to determine the basic pharmacokinetics of a lead compound, CNI-1493, from serum concentrations following a single intravenous injection in rats.     

High-performance liquid chromatographic determination of histamine in biological samples after derivation with o-phthalaldehyde

A high-performance liquid chromatographic (HPLC) method for the determination of histamine in tissues, based on precolumn derivation with o-phthalaldehyde, is described. Trichloroacetic acid extracts of rat brain, but not of rat stomach or of rat peritoneal mast cells, had to be cleaned-up by a chromatographic step before HPLC. The extracts were allowed to react with o-phthalaldehyde at pH 12.5 and -20 degrees C for 12 h, followed by acidification to pH 2.0. HPLC was performed on a reverse-phase column with isocratic elution using sulfuric acid in methanol as solvent system. A fluorescence detection system was used; excitation was set at 353 nm and emission was read at 451 nm. One chromatographic run was completed in 20 min. The detection limit with the conventional procedure was 1.5 ng histamine per sample, with a scaled-down procedure it was 250 pg per sample. With extracts of rat gastric mucosa the within-run variation was 2.7% and the day-to-day variation 4.5%.     

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 determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.