High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

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.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.     

Stereospecific high-performance liquid chromatographic assay of ibuprofen: improved sensitivity and sample processing efficiency

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of the non-steroidal anti-inflammatory drug ibuprofen (IB) in plasma was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), samples are acidified and extracted with a mixture of isooctane—isopropanol (95:5, v/v). After evaporation of the organic layer, the drug and I.S. are derivatized with S-(−)-1(1-naphthyl)ethylamine (S-NEA) after addition of ethyl chloroformate as the coupling reagent. Ethanolamine is added 3 min after the addition of S-NEA to react with the excessive ethyl chloroformate. The resultant diastereomers corresponding to IB and I.S. were chromatographed at ambient temperature on a 100 mm × 4.6 mm I.D. C₁₈ reversed-phase column using acetonitrile—water—acetic acid—triethylamine (60:40:0.1:0.02) as the mobile phase pumped at a flow-rate of 1.2 ml/min. Detection of the fluorescent chromophore was at 280 and 320 nm for excitation and emission, respectively. The suitability of the assay for clinical pharmacokinetic studies of IB was determined by the analysis of plasma samples obtained from a healthy volunteer, following administration of a single 400-mg oral dose of racemic IB.     

Determination of ceftazidime in plasma using high-performance liquid chromatography and electrochemical detection: Application for individualizing dosage regimens in elderly patients

This study describes a sensitive HPLC–electrochemical detection method for the analysis of ceftazidime, a third-generation cephalosporin, in human plasma. The extraction procedure involved protein precipitation with 30% trichloroacetic acid. The separation was achieved on a reversed-phase column (250×4.6 mm I.D., 5 μm) packed with C₁₈ Kromasil with isocratic elution and a mobile phase consisting of acetonitrile–25 mM KH₂PO₄–Na₂HPO₄ buffer, pH 7.4 (10:90, v/v). The proposed analytical method is selective, reproducible and reliable. The assay has a precision of 0.2–15.1% (C.V.) in the range of 5–200 μg ml⁻¹. (corresponding to 0.5 to 20 ng of ceftazidime injected onto the column), and is optimised for assaying 50 μl of plasma. The extraction recovery from plasma was approximately 100%. The method was highly specific for ceftazidime and there was no interference from either commonly administered drugs or endogenous compounds. This assay was used to measure ceftazidime in elderly patients for therapeutic drug monitoring.     

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 assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C₁₈ columns, plasma analysis; 15 cm×4.6 mm I.D., 5 μm particle size, urine analysis; 7.5 cm×4.6 mm I.D., 5 μm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)–methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.     

Determination of nadolol diastereomers in dog plasma using chiral derivatization and reversed-phase high-performance liquid chromatography with fluorescence detection

A stereospecific high-performance liquid chromatographic method has been developed for the determination of four diastereomers of nadolol in plasma. After the nadolol diastereomers were extracted from plasma using an Extrelut-1 solid-phase extraction cartridge, they were derivatized with (R)-(−)-1-(1-naphthyl)ethylisocyanate to form urea derivatives. These derivatives were then separated on a YMC-AM-303 ODS column using water—acetonitrile (60:40, v/v). The calibration curves of (SR)-, (RS)-, (SS)- and (RR)-nadolol were linear over the range 2.5–200 ng/ml, and the correlation coefficient (r) of the curves were higher than 0.9991 for each diastereomer. The limit of quantification was 2.5 ng/ml for each diastereomer in plasma. This method was used for a pharmacokinetic study in four dogs after oral administration of nadolol (1 mg/kg). The plasma concentrations of nadolol diastereomers showed no significant differences in C_max, T_max or AUC values. The assay appears to be readily applicable to the study of diastereoselective nadolol pharmacokinetics in animals and humans.     

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid—liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm × 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol—Sörensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol—water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.     

Determination of a new oral iron chelator, ICL670, and its iron complex in plasma by high-performance liquid chromatography and ultraviolet detection

ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe–[ICL670]₂ with one ferric iron. A simple and rapid HPLC–UV method for the separate determination of ICL670 and Fe–[ICL670]₂ in the plasma of iron-overloaded patients is described. Plasma samples were prepared as rapidly as possible, the tubes being kept at 4°C. Plasma proteins were precipitated with methanol. The supernatant was diluted with water and placed on the refrigerated sample rack of an autosampler before injection. The chromatographic separations were achieved on an Alltima C₁₈ column using 0.05 M Na₂HPO₄ and 0.01 M tetrabutylammonium hydrogen sulfate–acetonitrile–methanol (41:9:50, v/v/v) as mobile phase. The analytes were detected at 295 nm. Calibration and quality control samples were prepared in normal human plasma. The mean accuracy (n=6) over the entire investigated concentration range 0.25–20 μg/ml ranged from 91 to 109% with a coefficient of variation (C.V.) from 4 to 8% for ICL670, and from 95 to 105% with a C.V. from 2 to 20% for the iron complex. The dissociation of the complex during analysis was shown to be marginal. The iron removal from plasma of iron-overloaded patients by free ICL670 during analysis was low. The in vitro iron transfer from the iron pools of iron-overloaded plasma onto ICL670 was shown to be a slow process.     

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl)etoperidone and 1-(3-chlorophenyl)piperazine, in plasma

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid—liquid extraction procedure. The resulting sample is chromatographed on a C₁₈ column (10 cm × 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2–1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are ≤ 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

Determination of a novel sigma receptor antagonist, DuP 734, in rat plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A selective high-performance liquid chromatographic (HPLC) assay for a sigma receptor antagonist, DuP 734 (I), in rat plasma has been developed. Compound I and internal standard, XC031 (I.S.), were first extracted from plasma into an ethyl acetate—toluene mixture (3:7, v/v) and then back-extracted into freshly prepared phosphoric acid (0.03 M). Separation of I and I.S. with no interference from endogenous substances was achieved on a reversed-phase octyl column and detection was by UV at 229 nm. The mobile phase consisted of acetonitrile—glacial acetic acid—triethylamine—0.05 M ammonium acetate (670:4:2:2000, v/v). Using 0.5 ml of rat plasma for extraction, the limit of quantitation was 43 ng/ml and the assay was linear from 43 to 8536 ng/ml. The intra- and inter-day coefficients of variation ranged from 0.7 to 3.0%, and from 1.4 to 14.5%, respectively, over the entire concentration range. The accuracy was within 16.1% of the spiked concentrations. I was stable in frozen plasma at −20°C for at least 68 days.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C₁₈ column (250 × 4.6 mm I.D.) using 10 mM phosphoric acid—acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 μg/ml and the detection limits were 0.06 μg/ml for indomethacin and 0.08 μg/ml for mefenamic acid, for 50μl plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

Fluorodensitometric evaluation of gentamicin from plasma and urine by high-performance thin-layer chromatography

High-performance thin-layer chromatographic (HPTLC) analysis of gentamicin by in situ fluorodensitometric evaluation of its 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) derivative is presented. The aminoglycoside components separated on silica gel plates using chloroform–methanol–20% ammonium hydroxide (2.4:2.2:1.5, v/v/v) as the mobile phase were reacted with NB-Cl to yield highly fluorescent derivatives. The calibration curves of gentamicin in water, plasma and urine were linear in the range 40–200 ng. The mean values of intercept, slope and correlation coefficient were 16.82±0.473, 6.83±0.015 and 0.9968±0.0017 for standard curves in water, 17.35±0.375, 6.85±0.018 and 0.9941±0.0012 for standard curves in plasma and 14.35±0.286, 6.86±0.002 and 0.9933+0.0011 for standard curves in urine respectively. The analytical technique was validated for within-day and day-to-day variation. The results indicate that HPTLC, coupled with in situ fluorodensitometry, is a reliable and valuable technique for quantitative analysis of the bulk drug gentamicin and gentamicin from urine and plasma.