Small blood volumes from children for quantitative sotalol determination using high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method using fluorescence detection has been developed for sotalol determination in small plasma samples of children and newborns with limited blood volume. In sample sizes of 100 μl of plasma, sotalol was extracted using an internal standard and solid-phase extraction columns. Chromatographic separation was performed on a Spherisorb C₆ column of 150×4.6 mm I.D. and 5 μm particle size at ambient temperature. The mobile phase consisted of acetonitrile–15 mM potassium phosphate buffer (pH 3.0) (70:30, v/v). The excitation wavelength was set at 235 nm, emission at 300 nm. The flow-rate was 1 ml/min. Sotalol and the internal standard atenolol showed recoveries of 107±8.9 and 97±8.1%, respectively. The linearity range for sotalol was between 0.07 and 5.75 μg/ml, the limit of quantitation 0.09 μg/ml. Precision values expressed as percent relative standard deviation of intra-assay varied between 0.6 and 13.6%, that of inter-assay between 2.4 and 14.4%. Accuracy varied between 86.1 and 109.8% (intra-assay) and 95.4 and 103.3% (inter-assay). Other clinically used antiarrhythmic drugs did not interfere. As an application of the assay, sotalol plasma concentrations in a 6-year-old child with supraventricular tachycardia treated with oral sotalol (3.2 mg/kg per day) are reported.     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm × 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)—acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 μl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Determination of sotalol in human cardiac tissue by high-performance liquid chromatography

A sensitive and quantitative reversed-phase HPLC method for the analysis of -sotalol in human atria, ventricles, blood and plasma was developed. Sotalol was determined in about 100 mg of human right atria, left ventricles, and in 500 μl of blood and plasma samples of patients undergoing coronary bypass surgery or heart transplantation. Patients were taking 80–160 mg of sotalol as an antiarrhythmic agent. Atenolol was used as an internal standard certifying high precision of measurement. Sotalol blood and plasma concentrations correlated linearly to the obtained signals from 26.5 ng/ml to 2.12 μg/ml. Sotalol tissue concentrations showed linearity between 0.27 ng/mg and 10.6 ng/mg wet weight. The limit of quantitation was 0.27 ng/mg at a signal-to-noise ratio of 10. Sotalol was extracted from homogenized tissue with a buffer solution (pH 9) and the remaining pellet was extracted with methanol. The methanol extract was evaporated under nitrogen and reconstituted in buffer (pH 3). The whole extract was cleaned by solid-phase column extraction, eluted with methanol, evaporated again, reconstituted in the mobile phase (acetonitrile-15 mM potassium phosphate buffer pH 3, 17:83, v/v) and injected onto the HPLC column (Spherisorb C₆ column, 5 μm,, 150×4.6 mm I.D). For the detection of sotalol, the UV wavelength was set to 230 nm. Recoveries of sotalol and atenolol in atria and ventricles were 65.6 and 75.0%, respectively. Intra- and inter-assay coefficients of variation for tissue concentrations were 3.38 and 6.14%, respectively. Intra- and inter-assay accuracy for determined tissue sotalol concentrations were 94.9±6.3 and 99.6±4.1%.     

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated β-cyclodextrin as chiral mobile phase additive and solid-phase extraction

A sensitive and stereospecific HPLC method was developed for the analysis of (−)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-β-cyclodextrin (S-β-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)–absolute ethanol (80:20, v/v) containing 10 mM S-β-CD at a flow-rate of 0.7 ml/min. Recoveries of (−)- and (+)-pentazocine were in the range of 91–93%. Linear calibration curves were obtained in the 20–400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9–7.0% and 1.2–6.2%, respectively, for the (−)-enantiomer and 0.8– 7.6% and 1.2–4.6%, respectively, for the (+)-enantiomer (n=3).     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

Liquid chromatographic determination of urinary 2-thiothiazolidine-4-carboxylic acid, a biomarker of carbon disulphide exposure

An effective gradient high-performance liquid chromatographic method for baseline separation of urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), with photodiode array detection at 271 nm was described. o-Methylhippuric acid was used as an internal standard (I.S.). A 1-ml urine sample was saturated with 300 mg of sodium sulphate, acidified with 100 μl of 6 M hydrochloric acid, extracted twice with 2 ml of diethyl ether, and after evaporation, the residue was taken up in 1 ml of 0.1% (v/v) phosphoric acid. The two mobile phases used for gradient elution were: (A) 10 mM ammonium dihydrogenphosphate (pH 3.5) and (B) same concentration of buffer but containing 20% (v/v) of methanol (pH 4.8). The flow-rate was set at 1.0 ml/min. TTCA and I.S. were detected at 2.2 and 9.1 min, respectively. The method was validated with urine samples collected from normal subjects and workers occupationally exposed to carbon disulphide. The present method enables the detection of urinary TTCA at a concentration of 0.025 mg/l. Analytical recovery and reproducibility generally exceeded 90%. The proposed method is considered more sensitive, specific and reliable than other existing methods.     

High-performance liquid chromatographic method for the quantitative determination of butorphanol, hydroxybutorphanol, and norbutorphanol in human urine using fluorescence detection

A sensitive, quantitative reversed-phase high-performance liquid chromatographic method has been established for the simultaneous determination of butorphanol, a synthetic opioid, and its metabolites, hydroxybutorphanol and norbutorphanol, in human urine samples. The method involved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (pH 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the HPLC mobile phase, acetonitrile—methanol—water (20:10:70, v/v/v), containing 10 mM ammonium acetate and 10 mM TMAH (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5-μm column. The analysis was accomplished by detection of the fluorescence of the three analytes, at excitation and emission wavelengths of 200 nm and 325 nm, respectively. The retention times for hydroxybutorphanol, norbutorphanol, the internal standard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1–100 ng/ml for butorphanol and hydroxybutorphanol, and 2–200 ng/ml for norbutorphanol in urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of study samples. The method was applied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.     

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

A high-performance liquid chromatographic method for the determination of the histamine H₁-receptor antagonist cetirizine in human urine was developed. Cetirizine and the internal standard are extracted from acidified (pH 5) urine (0.5 ml) into chloroform and the organic layer is evaporated to dryness. The residue is chromatographed on a Spherisorb 5ODS-2 column using Pic A (5 mM aqueous tetrabutylammonium phosphate)—methanol—tetrahydrofuran (33:65:2, v/v) as the mobile phase with ultraviolet detection (230 nm). The calibration graph is linear from 0.1 to 10 μg/ml and using 0.5 ml of urine the detection limit is 20 ng/ml. The within-run relative standard deviation is <6% and the accuracy is within 10% of the theoretical value at concentrations between 0.1 and 10 μg/ml in urine. There is a good correlation (r = 0.99606) with a previously described capillary gas chromatographic assay.     

Determination of zolpidem in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating zolpidem in rat serum microsamples (50 μl). The separation used a 2.1 mm I.D. reversed-phase OD-5-100 C₁₈ column, 5 μm particle size with an isocratic mobile phase consisting of methanol–acetonitrile–26 mM sodium acetate buffer (adjusted to pH 2.0 with 40% phosphoric acid) containing 0.26 mM tetrabutylammonium phosphate (13:10:77, v/v/v). The detection limit was 3 ng/ml for zolpidem using an ultraviolet detector operated at 240 nm. The recovery was greater than 87% with analysis performed in 12 min. The method is simple, rapid, and applicable to pharmacokinetic studies of zolpidem after administering two intravenous bolus doses (1 and 4 mg/kg) in rats.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Determination of isofezolac in biological fluids by reversed-phase liquid column chromatography

A rapid, sensitive, and specific reversed-phase high-performance liquid chromatography assay was developed for the determination of 1,3,4-triphenylpyrazole-5-acetic acid (isofezolac) in plasma and urine. The assay involves extraction into diethyl ether from plasma buffered at pH 4.4. The organic phase is evaporated and the residue, dissolved in the mobile phase [acetonitrile—water—0.2 M phosphate buffer (pH 3) (65 : 15 : 20)] is chromatographed at a flow-rate of 1.5 ml/min. The drug is detected by its UV absorption (detection limit 100 ng/ml) or its very intense fluorescence (detection limit 10 ng/ml). Absolute analytical recoveries for isofezolac varied from 92.9 to 100.4%. The accuracy is ca. 1%. Each separation requires about 6 min. This method was applied successfully to the determination of isofezolac in humans for pharmacokinetic studies.     

Determination of a new non-narcotic analgesic, DA-5018, in plasma, urine and bile by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new non-narcotic analgesic, DA-5018 (I), in rat plasma, urine and bile samples, using propranolol for plasma samples and protriptyline for urine and bile samples as internal standards. The method involved extraction followed by injection of 100 μl of the aqueous layer onto a C₁₈ reversed-phase column. The mobile phases were 5 mM methanesulfonic acid with 10 mM NaH₂PO₄ (pH 2.5)-acetonitrile, 70:30 (v/v) for plasma samples and 75:25 (v/v) for urine and bile samples. The flow-rates were 1.0 ml/min for plasma samples and 1.2 ml/min for urine and bile samples. The column effluent was monitored by a fluorescence detector with an excitation wavelength of 270 nm and an emission wavelength of 330 nm. The retention time for I was 4.8 min in plasma samples and 10.0 min in urine and bile samples. The detection limits for I in rat plasma, urine and bile were 20, 100 and 100 ng/ml, respectively. There was no interference from endogenous substances.     

Determination of clozapine and its metabolite, N-desmethylclozapine, in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single solvent extraction step high-performance liquid chromatographic method is described for quantitating clozapine and its metabolite, N-desmethylclozapine, in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Symmetry C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–28.6 mM sodium acetate buffer, pH 2.6 (10:20:70, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 230 nm. The method was used to study the pharmacokinetics of clozapine after an intravenous bolus dose (2.5 mg/kg).     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatization with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate at 35°C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)–acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 μm particle size, 10 cm×0.46 cm I.D.) with a mobile phase of phosphate buffer–methanol–acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV–VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 μg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 μg/ml (RSD <10%). In the range 0.145–20 μg/ml, intra-day coefficients of variation were 1.0–7.0% and inter-day coefficients of variation were 0.4–16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 × 2.1 mm I.D., 5 μm Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate—acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 μl sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean ± S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 ± 1.2% and 91.4 ± 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.