High-performance liquid chromatographic determination of cocaine and its metabolites in serum microsamples with fluorimetric detection and its application to pharmacokinetics in rats

A sensitive, selective and simple HPLC method with fluorimetric detection is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). Chromatographic separation is achieved on a Hypersil BDS C₁₈ column (100×2.1 mm, 5 μm) with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.6, containing 1.0·10⁻⁴ M tetrabutylammonium phosphate (14:10:76, v/v/v). The detection limit (0.5 ng/ml) for all the compounds, using direct fluorometric detection operated at excitation and emission wavelengths of 230 and 315 nm, respectively, was approximately five-times lower than that of using a UV detector operated at 235 nm. The effects of ratio of 2-propanol to chloroform in extraction solvents on the recovery and precision for cocaine and its metabolites were systematically examined. The method was used to study the pharmacokinetics of cocaine after administration of intravenous 2 mg/kg and oral 20 mg/kg doses.     

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.     

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).     

Study of the solid-phase extraction of diclofenac sodium, indomethacin and phenylbutazone for their analysis in human urine by liquid chromatography

A selective semi-automated solid-phase extraction (SPE) of the non-steroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone from urine prior to high-performance liquid chromatography was investigated. The drugs were recovered from urine buffered at pH 5.0 using C₁₈ Bond-Elut cartridges as solid sorbent material and mixtures of methanol–aqueous buffer or acetonitrile–aqueous buffer as washing and elution solvents. The extracts were chromatographed on a reversed-phase ODS column using 10 mM acetate buffer (pH 4.0)–acetonitrile (58:42, v/v) as the mobile phase, and the effluent from the column was monitored at 210 nm with ultraviolet detection. Absolute recoveries of the anti-inflammatory drugs within the range 0.02–1.0 μg/ml were about 85% for diclofenac and indomethacin, and 50% for phenylbutazone without any interference from endogenous compounds of the urine. The within-day and between-day repeatabilities were in all cases less than 5% and 10%, respectively. Limits of detection were 0.007 μg/ml for diclofenac sodium and indomethacin and 0.035 μg/ml for phenylbutazone, whereas limits of quantitation were 0.02 μg/ml for diclofenac and indomethacin and 0.1 μg/ml for phenylbutazone.     

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 method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C₈ cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C₁₈ stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% (n = 10) for plasma (0.2 μg/ml) and lower than 12.4% (n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Sensitive and specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of cocaine and its metabolites in rat plasma

A sensitive, specific and precise HPLC–UV assay was developed to quantitate cocaine (COC) and its metabolites benzoylecgonine (BE), norcocaine (NC) and cocaethylene (CE) in rat plasma. After adding 50 μl of the internal standard solution (bupivacaine, 8 μg/ml) and 500 μl of Sørensen's buffer (pH 6) to 100 μl of rat plasma sample, the mixture was extracted with 10 ml of chloroform. The organic layer was transferred to a clean test tube and was evaporated under nitrogen. The residue was reconstituted in 100 μl of mobile phase and 35 μl was injected onto the HPLC column. The mobile phase consisted of methanol–acetonitrile–50 mM monobasic ammonium phosphate (5:7:63, v/v/v) and was maintained at a flow-rate of 0.4 ml/min. Separation of COC and its metabolites was achieved using a Supelcosil ABZ+plus deactivated reversed-phase column (250×2.1 mm I.D., 5 μm). Calibration curves were linear over the range of 25–5000 ng/ml for COC and its three metabolites. The absolute extraction efficiencies for BE, COC, NC, CE and bupivacaine were 56.6%, 78.6%, 61.1%, 76.4% and 67.0%, respectively. COC and its metabolites were stable in mobile phase for 24 h at room temperature and in rat plasma for 2 weeks at −20°C. The limits of detection for BE, COC, NC and CE were 20, 24, 15 and 12.9 ng/ml, respectively. These values correspond to 0.70, 0.84, 0.525 and 0.452 ng of the according compound being injected on column. The within-day coefficient of variation for the four compounds ranged from 3.0% to 9.9% while the between-day precision varied from 3.6% to 14%. This method was used to analyze rat plasma samples after administration of COC alone and in combination with alcohol. The pharmacokinetic profiles of COC and its metabolites in these rats are also described.     

High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C₁₈ cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol–0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2–50 μg/g (r²=0.9998) and for flumequine in the range of 0.3–50 μg/g (r²=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 μg/g for oxolinic acid and 0.3 μg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.     

High-performance liquid chromatographic assay of formycin A in plasma after solid-phase extraction

A new, simple and accurate high-performance liquid chromatography (HPLC) method for the determination of formycin A in plasma is presented. The samples were chromatographed on a LiChrosorb RP-18 column after purification using a Bakerbond SPE column. The mobile phase was methanol–0.067 M phosphate buffer, pH 4.20 (1:4, v/v) containing 0.005 M sodium hexanesulfonate. Azathioprine was applied as an internal standard. UV detection was carried out at 293 nm. The method was tested for linearity (over the range 0.1–9.0 μg/ml). The recovery was 91.89% (mean). The described method has been successfully applied to the quantitative determination of formycin A in plasma and should be useful for clinical and bioavailability investigations.     

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 the anticonvulsant felbamate and its three metabolites in brain and heart tissue of rats

An automated, internal standard high-performance liquid chromatographic method for the simultaneous quantitation of felbamate and its three metabolites in adult and neonatal rat brain and heart tissue homogenates was developed and validated. The homogenates prepared from one part of the tissue and four parts of water were extracted with ethyl acetate, and the extract was evaporated to dryness and redissolved in mobile phase. Separation was accomplished on a Waters Resolve C₁₈, 5 μm, 300 mm × 3.9 mm I.D. column with a mobile phase consisting of 0.01 M phosphate buffer, pH 6.8—acetonitrile—methanol (800:150:50, v/v/v). Eluting peaks were monitored with an ultraviolet detector at 210 nm. The linear range of the assay for felbamate and the metabolites was 0.20–50.00 μg/ml of homogenate or 1–250 μg/g of brain or heart tissue. The lower limit of quantitation for all four analytes was 0.20 μg/ml of homogenate or 1.00 μg/g of tissue.     

Determination of cocaine and its metabolites 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 cocaine and its three metabolites in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Brownlee C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.2, containing 1.29·10⁻⁴M tetrabutylammonium phosphate (12.5:10:77.5, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 235 nm. The method was used to study the pharmacokinetics of cocaine after an intravenous (i.v.) bolus dose (4 mg/kg).     

Rapid and simple high-performance liquid chromatographic determination of nimesulide in human plasma

A high-performance liquid chromatographic method for the quantitation of nimesulide in human plasma is presented. The method is based on protein precipitation with methanol and reversed-phase chromatography with spectrophotometric detection at 404 nm. The separation was performed on a Nucleosil 120-5 C₁₈, 50×4-mm I.D. column and the mobile phase consisted of acetonitrile–methanol–15 mM potassium dihydrogenphosphate buffer, pH 7.3 (30:5:65, v/v). Only 250 μl of plasma are used for sample preparation and no internal standard is necessary. The limit of quantitation is 80 ng/ml and the calibration curve is linear up to 10 000 ng/ml. More than 20 samples can be analysed within 1 h. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C₁₈ Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 mM ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C₁₈, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 mM ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/z 291 for idarubicin and idarubicinol, m/z 321 for daunorubicin and daunorubicinol, m/z 361 for epirubicin and doxorubicin, m/z 363 for doxorubicinol and m/z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs (r≥0.992) and 200 ng/ml for the active metabolites (r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.     

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.     

Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in 100 μl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C₁₈ J’Sphere ODS-M80 (150×4.0 mm) column with mobile phase acetonitrile–phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 μM and 13.5% at 1.00 μM. The inter-assay precision was 3.3% at 12.00 μM and 15.6% at 1.00 μM. The lower limit of quantification was 1.00 μM. The limit of detection was 0.50 μM.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

Quantitative determination of cefepime in plasma and vitreous fluid by high-performance liquid chromatography

An isocratic reversed-phase HPLC method was developed to determine cefepime levels in plasma and vitreous fluid. Cefepime and the internal standard cefadroxil were separated on a Shandon Hypersil BDS C18 column by using a mobile phase of 25 mM sodium dihydrogen phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet detection was carried out at 270 nm. The retention times were 4.80 min for cefepime and 7.70 min for cefadroxil. This fast procedure which involves an efficient protein precipitation step (addition of HClO₄), allows a quantification limit of 2.52 μg ml⁻¹ and a detection limit of 0.83 μg ml⁻¹. Recoveries and absolute recoveries of cefepime from plasma were 96.13–99.44% and 94–102.5% respectively. The intra-day and inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50 μg ml⁻¹ (n=10).The method was proved to be suitable for determining cefepime levels in human plasma and was modified to measure vitreous fluid samples.     

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.