Simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, respectively.     

Development of a plasma high-performance liquid chromatographic assay for LY303366, a lipopeptide antifungal agent, and its application in a dog pharmacokinetic study

An HPLC assay for plasma analysis of LY303366 (I), a semi-synthetic lipopeptide antifungal related to echinocandin B (ECB), was developed to support the selection and subsequent preclinical development of I. The method involved extraction of I from plasma with the aid of solid-phase extraction (SPE) cartidges followed by reversed-phase HPLC with UV detection at 300 nm. The method is simple, selective and is applicable to dog, rat, mouse and rabbit plasma. Validation studies using dog plasma showed that the values obtained for parameters of linearity, precision and accuracy were within acceptable limits. Based on analysis of 0.3 ml of plasma, the lower limit of quantitation was 20 ng/ml. The method has been successfully applied to determine the pharmacokinetic parameters of I in the dog following intravenous (i.v.) and oral administration. Compared to first generation ECB antifungal agents, the results of the i.v. dog study indicated a 50% reduction in clearance of the drug from plasma (0.1 l/h/kg) and an 18-fold increase in the volume of distribution at steady state (1.8 l/kg). When administered orally, compound I had an absolute bioavailability of 9%; however, plasma levels remained above the MIC for C. albicans (0.005 μg/ml) through 48 h. Given the excellent potency of I and its broad spectrum of activity relative to first generation ECB antifungal agents, the assay results for I indicate the potential for its use as a broad spectrum i.v. and oral antifungal agent.     

Determination of a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist (LY300164) and its N-acetyl metabolite in mouse,rat, dog,and monkey plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor antagonist LY300164 (compound I) and its N-acetyl metabolite (compound II) in plasma was developed. The assay utilized solid-phase extraction on a C₁₈ Bond Elut cartridge followed by reversed-phase HPLC with UV detection at 310 nm. The method exhibited a large linear range from 0.05 μg/ml to 50 μg/ml with an intra-sassay accuracy for compound I and compound II ranging from 89.0% to 114.5% and intra-assay precision ranging from 0.5 to 15.3% in mouse, rat, dog, and monkey plasma. The inter-assay accuracy of compound I and compound II was 93.3% to 101.8% and the inter-assay precision was 1.6% to 11.2% in dog plasma. The lower limit of quantitation was 0.05 μg/ml for compound I in plasma from all species tested. The lower limit of quantitation for compound II was 0.05 μg/ml in dog and monkey plasma and 0.1 μg/ml in mouse and rat plasma. Extracts of compound I and II from dog plasma were shown to be stable for 24 h at room temperature, and both compounds were stable when spiked into rat and monkey plasma frozen at −70°C for 27 days. The method has shown to be useful in the investigation of the pharmacokinetics of the parent compound (I) and metabolite (II) in preclinical studies.     

Determination of eltanolone in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of eltanolone in plasma has been developed. Plasma samples containing eltanolone were diluted with acetonitrile to precipitate plasma proteins, and derivatized with 2,4-dinitrophenylhydrazine before direct injection onto a C₁₈ column. The mobile phase was acetonitrile–water (70:30, v/v) containing 0.1% trifluoroacetic acid and detection was by UV absorbance at 367 nm. The quantitation limit was 0.020 μg/ml. The method has proven to be rapid, precise and sensitive in the range of concentrations found during and following intravenous anaesthesia.     

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column eluted with a phosphate buffer—acetonitrile—methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195–200 μg/ml, the propionic acid metabolite 0.195–50.0 μg/ml, the p-hydroxy metabolite 0.781 to 50.0 μg/ml, and the monocarbamate metabolite 0.098–50.0 μg/ml.     

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 μg/ml in plasma and 0.1 μg/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01–2.5 μg/ml, and for the metabolite 0.01–1 μg/ml. In urine, the range for both compounds was 0.1–10 μg/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.     

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.     

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.     

Sensitive and specific determination of clindamycin in human serum and bone tissue applying liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

A method for the quantification of clindamycin in human serum and in human bone tissue samples applying high-performance liquid chromatography with atmospheric pressure chemical ionization–mass spectrometry (APCI–MS) is presented. Lincomycin is used as the internal standard. Serum samples are prepared only by protein precipitation with acetonitrile. Bone tissue samples have to be crushed and homogenized in extraction buffer prior to analysis. The chromatographic separation is achieved on an RP-18 stationary phase with 0.02% trifluoroacetic acid in water 60%/acetonitrile 40% v/v as mobile phase. The limits of quantification are 0.1 μg/ml for serum samples and 0.1 μg/g for bone tissue samples. The coefficients of variation for the assays are 4.48 and 8.41% at the limit of quantification for serum and bone tissue samples, respectively. Bone tissue samples as small as 50 mg can be used.     

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.     

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.     

A HPLC method for the quantitative determination of N-(2-hydroxy-5-nitrophenylcarbamothioyl)-3,5-dimethylbenzamide in biological samples

A sensitive and simple HPLC method was developed for the determination of a novel compound, a potential anti-cancer drug, N-(2-hydroxy-5-nitrophenylcarbamothioyl)-3,5-dimethylbenzamide (DM-PIT-1), a member of the new structural class of non-phosphoinositide small molecule antagonist of phosphatidylinositol-3,4,5-trisphosphate-pleckstrin-homology domain interactions, in mouse plasma and tumor tissue homogenates. The chromatographic separation of DM-PIT-1 was achieved on C18 column using isocratic elution with acetonitrile-water (70:30) containing 0.1% formic acid (v/v). DM-PIT-1 was detected by UV absorbance at 320 nm and confirmed by LC-MS. The extraction of the DM-PIT-1 from the plasma and tumor tissue with methylene chloride resulted in its high recovery (70-80%). HPLC calibration curves for DM-PIT-1 based on the extracts from the mouse plasma and tumor tissue samples were linear over a broad concentration range of 0.25-20 μg/ml/g, with intra/inter-day accuracy of 95% and the precision of variation below 10%. The limits of detection and quantification were 0.1 ng and 0.2 ng, respectively. The described method was successfully applied to study the pharmacokinetics of the DM-PIT-1 following the parenteral injections of DM-PIT-1 entrapped in 1,2-disteratoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (PEG-PE) micelles.     

Measurement and pharmacokinetic analysis of unbound ceftazidime in rat blood using microdialysis and microbore liquid chromatography

To evaluate the biodisposition of ceftazidime in rat blood, a rapid and simple microbore liquid chromatographic technique together with a microdialysis sampling technique were developed. This method involves an on-line design for blood dialysate directly injected into a microbore liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol–acetonitrile–100 mM monosodium phosphoric acid (pH 3.0) (10:10:80, v/v/v) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. With the detection wavelength set at 254 nm, a good linear correlation was observed between the peak area and the ceftazidime concentration at 0.1 to 50 μg/ml (r=0.999). Microdialysis probes, being custom-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity, as well as intra-day and inter-day variabilities. This method was then applied to the pharmacokinetic profiling of ceftazidime in blood following intravenous 50 mg/kg administration to rats. The pharmacokinetics was calculated from the corrected data for dialysate concentrations of ceftazidime versus time. This method has been used to study ceftazidime pharmacokinetics in rats and has proven to be rapid and reproducible.     

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.     

Optimization of a solid-phase extraction method for determination of indapamide in biological fluids using high-performance liquid chromatography

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C(8) column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0-100.0 ng/ml for serum, and 50.0-500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.     

Development of a validated capillary electrophoresis method for enantiomeric purity control and quality control of levocetirizine in a pharmaceutical formulation

A chiral capillary electrophoresis method has been developed for the quantification of 0.1% of the enantiomeric impurity (dextrocetirizine) in levocetirizine and determination of both in pharmaceuticals using sulfated-β-cyclodextrins (CDs) as chiral selector. Several parameters affecting the separation were studied such as the type and concentration of chiral selectors, buffer composition and pH, organic modifier, mixtures of two CDs in a dual system, voltage, and temperature. The optimal separation conditions were obtained using a 50 mM tetraborate buffer (pH 8.2) containing 1% (w/v) sulfated-β-CDs on a fused-silica capillary. Under these conditions, the resolution of two enantiomers was higher than 3. To validate the method, the stability of the solutions, robustness (two level half fraction factorial design for 5 factors using 19 experiments [2(n-1)+3]), precision, linearity (dextrocetirizine 0.25-2.5 μg/ml, R(2) = 0.9994, y = 0.0375x + 0.0008; levocetirizine 15-100 μg/ml, R(2) = 0.9996, y = 0.0213x + 0.0339), limit of detection (0.075 μg/ml, 0.03% m/m), limit of quantification (0.25 μg/ml, 0.1% m/m), accuracy (dextrocetirizine 84-109%, levocetirizine 97.3-103.1%), filter effect, and different CD batches were examined. The validated method was further applied to bulk drug and tablets of levocetirizine.     

Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Liquid chromatographic determination of ketoconazole, a potent inhibitor of CYP3A4-mediated metabolism

A high-performance liquid chromatographic assay with UV detection has been developed for the determination of ketoconazole in human plasma. Quantitative extraction was achieved by a single solvent extraction involving a mixture of acetonitrile–n-butyl chloride (1:4, v/v). Ketoconazole and the internal standard (clotrimazole) were separated on a column packed with Inertsil ODS-80A material and a mobile phase composed of water–acetonitrile–tetrahydrofuran–ammonium hydroxide–triethylamine (45:50.2:2.5:0.1:0.1, v/v). The column effluent was monitored at a wavelength of 206 nm with a detector range set at 0.5. The calibration graph was linear in the range of 20–2000 ng/ml, with a lower limit of quantitation of 20.0 ng/ml. The extraction recoveries for ketoconazole and clotrimazole in human plasma were 93±9.7% and 83±10.0%, respectively. The developed method has been successfully applied to a clinical study to examine the pharmacokinetics of ketoconazole in a cancer patient.     

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.     

High-performance liquid chromatographic determination of spectinomycin in swine,calf and chicken plasma using post-column derivatization

An HPLC method for the determination of spectinomycin in swine, calf and chicken plasma at 0.1 μg/ml or higher is described. The clean-up is based upon ion-pair solid-phase extraction on a High Hydrophobic C₁₈ column treated with sodium dioctyl suflosuccinate. After elution with methanol, spectinomycin is chromatographed on a Spherisorb SCX column using 0.1 M sodium sulphate solution (pH 2.6)-acetonitrile (80:20, v/v) as mobile phase. Fluorescence detection is at an excitation wavelength of 340 nm and an emission wavelength of 460 nm after post-column oxidation with sodium hypochlorite followed by derivatization with o-phthaldialdehyde. Mean recoveries were 99 ± 2% (n = 6), 99 ± 2% (n = 7) and 104 ± 2% (n = 6) for swine, calf and chicken plasma, respectively, at the 0.1 μg/ml level.