Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.     

Development of a gradient reversed-phase HPLC method for the determination of sodium ferulate in beagle dog plasma

A gradient reversed-phase HPLC assay has been developed to determine sodium ferulate (SF) in beagle dog plasma with tinidazole as an internal standard. Chromatographic separation was made on a C(18) column using 0.5% acetic acid and acetonitrile (80:20, v/v) as mobile phase. UV detection was performed at 320 nm. The calibration curve for SF was linear in the range of 0.05-10 microg/ml, and the achieved limit of quantification (LOQ) was 51.4 ng/ml. The results of linearity, within- and between-day precision, and accuracy demonstrate that this method is reliable, sensitive and sufficient for in vivo beagle dog pharmacokinetic (PK) studies of SF.     

Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to determine S-(-) and R-(+) enantiomers of esmolol in human plasma was developed. The method involved liquid-liquid extraction of esmolol from human plasma, using S-(-)-propranolol as the internal standard, and employed 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The derivatized products were separated on a 5-microm reversed-phase C18 column with a mixture of acetonitrile/0.02 mol/L phosphate buffer (pH 4.5) (55:45, v/v) as mobile phase. The detection of esmolol derivatives was made at lambda=224 nm with UV detector. The assay was linear from 0.035 to 12 microg/ml for each enantiomer. The analytical method afforded average recoveries of 94.8% and 95.5% for S-(-)- and R-(+)-esmolol, respectively. For each enantiomer, the limit of detection was 0.003 microg/ml and the limit of quantification for the method was 0.035 microg/ml (RSD<14%). The reproducibility of the assay was satisfactory.     

Determination of scopoletin in rat plasma by high performance liquid chromatographic method with UV detection and its application to a pharmacokinetic study

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for determination of scopoletin in rat plasma using psoralen as internal standard. Chromatographic separation was achieved on a C(18) column using methanol and distilled water (49:51, v/v) containing 0.05% (v/v) phosphoric acid as mobile phase. The UV detector was set at 345 nm. The calibration curve was linear over the range of 0.165-9.90 microg/ml with a correlation coefficient of 0.9994. The recovery for plasma samples of 0.165, 1.32 and 6.60 microg/ml was 93.2%, 95.9% and 95.5%, respectively. The RSD of intra- and inter-day assay variations was less than 6.7%. This HPLC assay is a precise and reliable method for the analysis of scopoletin in pharmacokinetic studies.     

Development and validation of a liquid chromatographic method for Casiopeina IIgly in rat plasma

A sensitive and specific liquid chromatographic method using solid-phase extraction with Sep-pak cartridges has been developed for the determination of Casiopeina IIgly and validated over the linear range 2.5-50 microg/ml in rat plasma. The analysis was performed on a Symetry C(18) (5 microm) column with a Phenomenex C(18) precolumn. The mobile phase was methanol-water (58:42, v/v). The column effluent was monitored at 273 nm. The results showed that the assay is sensitive at 2.5 microg/ml. Maximum intra-day coefficient of variation was 11.47%. The recovery based upon addition of internal standard to rat plasma was 80.98%. The method was used to perform preclinical pharmacokinetic studies in rat plasma and was found to be satisfactory.     

Bioanalysis and pharmacokinetics of chitosan ester in rabbit serum by HPLC with postcolumn fluorescence derivatization

Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol-water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 degrees C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5-100 microg/ml. The lower limit of detection (LLOD) was found to be 1.0 microg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.     

Development and validation of an ion-pair liquid chromatographic method for the quantitation of sodium cromoglycate in urine following inhalation

An ion-pair liquid high-performance chromatography method with solid-phase extraction for measuring urinary concentrations of sodium cromoglycate following inhalation has been developed and validated. Sodium cromoglycate was extracted from urine on a 100-mg phenyl cartridge (Isolute, Jones Chromatography) and then quantified on a 25-cm C₈ Spherisorb 5 μm stationary phase with a mobile phase of methanol-0.045 M phosphate buffer-0.05 M dodecyl triethyl ammonium phosphate (550:447.6:2.4, v/v) pH 2.3, at 0.85 ml min⁻¹ using nedocromil sodium as an internal standard and UV detection at 238 nm. The inter- and intra-day reproducibilities were 8.33 and 13.63%, respectively, at 0.25 mg l⁻¹. The limit of determination for sodium cromoglycate was 0.25 mg l⁻¹ (with a signal-to-noise ration of greater than 10:1). Following oral and inhaled administration of 20 mg of sodium cromoglycate to eight healthy volunteers, the mean and S.D. of sodium cromoglycate excreted in the urine at 0.5, 1 and 24 h post-dose were 0.02, 0.05 and 0.33%, and 0.16, 0.30 and 1.55% of the dose, respectively. The urinary recovery of sodium cromoglycate at 0.5 and 1 h following inhalation can therefore be used to compare the amount of drug reaching the respiratory tract using different sodium cromoglycate inhaled products or inhalation methods.     

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.     

A LC-MS/MS method to quantify the novel cholesterol lowering drug ezetimibe in human serum, urine and feces in healthy subjects genotyped for SLCO1B1

Ezetimibe (Ezetrol) is a novel cholesterol lowering drug which disposition is not fully understood in man. We developed a selective and high-sensitive assay to measure serum concentration-time profiles, renal and fecal elimination of ezetimibe in pharmacokinetic studies. Ezetimibe glucuronide, the major metabolite of ezetimibe was determined by enzymatic degradation to the parent compound. Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-hydroxychalcone as internal standard and liquid chromatography coupled via an APCI interface with tandem mass spectrometry (LC-MS/MS) for detection. The chromatography (column XTerra) MS, C(18), 2.1 mm x 100 mm, particle size 3.5 microm) was done isocratically with acetonitrile/water (60/40, v/v; flow rate 200 microl/min). The MS/MS analysis was performed in the negative ion mode (m/z transition: ezetimibe 408-271, internal standard 223-117). The validation ranges for ezetimibe and total ezetimibe were as follows: serum 0.0001-0.015 microg/ml and 0.001-0.2 microg/ml; urine and fecal homogenate 0.025-10 microg/ml and 0.1-20 mg/ml, respectively. The assay was successfully applied to measure ezetimibe disposition in two subjects genotyped for the hepatic uptake transporter SLCO1B1.     

Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis determination method with UV detection of grepafloxacin and clinafloxacin has been developed. The separation was performed in 35 mM borate-35 mM phosphate buffer solution (pH 8.6), containing 6% (v/v) of acetonitrile. Analyses were realised using fused-silica capillaries (57 cm length x 75 microm I.D.) and the operating conditions were: 15 kV applied voltage, 30 degrees C and detection at 279 nm. Piromidic acid was used as an internal standard. The linear concentration range of application was 1.0-120.0 microg ml(-1) for both compounds, with a detection limit of 0.2 microg ml(-1) for grepafloxacin and 0.3 microg ml(-1) for clinafloxacin. The analysis yielded good reproducibility (RSD between 3.37 and 1.74%). It was applied to the determination of grepafloxacin and clinafloxacin in human and rat urine samples. The method was validated using HPLC as a reference method. Recovery levels were between 94.5 and 103%.     

Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.     

Determination of mitiglinide in rat plasma by high-performance liquid chromatography with UV detection

A selective and sensitive high-performance liquid chromatography method has been developed and validated for determination of mitiglinide (MGN) in rat plasma using 2-(4-biphenylyl) propionic acid (BPA) as internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and 0.02 mol/l KH(2)PO(4) buffer (pH 4.0) (45:55, v/v) as mobile phase delivered at 1.0 ml/min. The UV detector was set at 210 nm. The assay was linear over the range 0.1-20 microg/ml for MGN. The average extraction recoveries of MGN and BPA from rat plasma were 98.6 and 97.4%, respectively. The developed method has been applied to the pharmacokinetic study of MGN in rats.     

Measurement of amoxicillin in plasma and gastric samples using high-performance liquid chromatography with fluorimetric detection

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue which is applicable to low concentrations of amoxicillin (<1 microg mL(-1)) or small sample volumes. Amoxicillin was converted, via an internal rearrangement, to form a fluorescent product which was subsequently recovered using liquid-liquid extraction. A Kromasil ODS 3 microm (150 x 3.2 mm I.D.) column was maintained at 40 degrees C and used with a mobile phase consisting of methanol-water (55:45, v/v). Fluorimetric detection was at an lambda(ex) of 365 nm and an lambda(em) of 445 nm. The limits of quantitation for amoxicillin were 0.1 microg mL(-1) for gastric juice aspirate (500 microL), 0.5 microg mL(-1) for plasma (50 microL) and 0.075 microg g(-1) for gastric tissue (250 mg). The method was linear up to at least 15 microg mL(-1) in gastric juice aspirate, up to 200 microg mL(-1) in plasma and up to 100 microg g(-1) in gastric tissue, with inter- and intra-day RSDs being less than 19%. The assay has been applied to the measurement of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.     

A simple and rapid liquid chromatography method for simultaneous determination of zidovudine and nevirapine in plasma

We describe a simple, fast, isocratic, reversed-phase high performance liquid chromatographic method for simultaneous determination of plasma zidovudine and nevirapine with UV detection at 260 nm. The method involves liquid-liquid extraction with ethyl acetate and using 3-isobutyl 1-methyl xanthine as internal standard. The system requires a C(18) column (150 mm x 4.6 mm I.D.) and a mobile phase composed of potassium dihydrogen phosphate (15 mM; pH 7.5) and acetonitrile in the ratio of 80:20 (v/v). The assay was linear from 0.025 to 10.0 microg/ml for zidovudine and 0.05 to 10.0 microg/ml for nevirapine. The intra- and inter-day variations were less than 10% for both the drugs. The method was specific and sensitive enough to allow quantification of zidovudine and nevirapine in concentrations observed clinically. The average recoveries of zidovudine and nevirapine from plasma were 95 and 94%, respectively. The method was applied to a pharmacokinetic study in HIV-infected patients who were receiving antiretroviral treatment with zidovudine and nevirapine containing regimens. The method spans the blood concentration range of clinical interest. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring in patients taking a combination treatment of zidovudine and nevirapine.     

Determination of paeoniflorin in rat hippocampus by high-performance liquid chromatography after intravenous administration of Paeoniae Radix extract

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of paeoniflorin in rat hippocampus was developed in this study. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Zorbax SB-C(18) column, a mobile phase of methanol-water (32:68, v/v), and detection by ultraviolet (UV) absorption at 233 nm. The lower limits of quantitation (LLQ) were 1 microg/ml for paeoniflorin. The calibration curve for paeoniflorin was linear (r = 0.9999) over the concentration range of 1-50 microg/ml. The coefficients of variation of intra- and inter-day assays were 7.00, 0.58, 1.46% and 5.48, 1.79, 1.70% at concentrations of 1, 10, 50 microg/ml, respectively. The recoveries of paeoniflorin from rat hippocampus were 98.28 +/- 2.14, 98.96 +/- 1.48, and 95.34 +/- 0.92 at concentrations of 1, 10 and 50 microg/ml, respectively. Stability studies showed that paeoniflorin was stable at temperatures of 2-8 degrees C in methanol for at least 20 days. The method was applied to determine the time course of paeoniflorin in rat hippocampus, following the administration of a 60 mg/kg i.v. dose of paeoniflorin in Paeoniae Radix extract to a male Wistar rat.     

Improvement and validation of an HPLC method for examining the effects of the MDR1 gene polymorphism on sparfloxacin pharmacokinetics

A rapid, simple, accurate, and precise reverse-phase high-performance liquid chromatography (HPLC) method for measuring sparfloxacin in human serum was improved, validated, and applied to determine the influence of polymorphisms in MDR1 (exons 12, 21, and 26) gene on sparfloxacin pharmacokinetics. Sparfloxacin and an internal standard, ciprofloxacin, were extracted from human serum by protein precipitation with dilution and analyzed on a Luna C(18) 5-microm column in a mobile phase of acetonitrile-0.035 M perchloric acid (28:72, v/v, adjusted to pH 2.0 with 0.015 M triethylamine) and UV detection at 300 nm. This analysis was performed at three different laboratories using the same quality control (QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum. The method showed linear responses over a concentration range of 0.05-2 microg/ml, with correlation coefficients of greater than 0.999 at the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. The mean absolute recovery for human serum was 98.8+/-5.7%. Sparfloxacin in human serum was stable during storage and the assay procedure. The lower limit of quantification using 0.2 ml of serum was 0.05 microg/ml, which was sensitive enough for pharmacokinetic studies. This method was used to study the pharmacokinetics of sparfloxacin in human volunteers, following a single oral administration of sparfloxacin (100 mg) two tablets at three different laboratories. MDR1 polymorphisms did not significantly (P < 0.01) affect the pharmacokinetic parameters (AUC and C(max)) of sparfloxacin.     

Determination of vertilmicin in rat serum by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

A procedure for the high-performance liquid chromatographic determination of vertilmicin in rat serum was described using pre-column derivatization. The serum proteins were precipitated with acetonitrile and vertilmicin in the supernatant was derivatized with 1-fluoro-2,4-dinitrobenzene. Etimicin was selected as the internal standard. The mobile phase consisted of methanol--20mM ammonium acetate (80:20, v/v), and flow-rate was 0.9 ml/min. Ultraviolet detection was set at 365 nm. The reaction products were chromatographed on a C(18) column kept at 40 degrees C. A good linearity was found in the range of 0.5-250 microg/ml. Both intra- and inter-day precisions of vertilmicin, expressed as the relative standard deviation, were less than 7.4%. Accuracy, expressed as the relative error, ranged from -0.1 to 3.6%. The mean absolute recovery of vertilmicin at three different concentrations was 92.5%. Serum volumes of 50 microl were sufficient for the determination of vertilmicin. The method was proved suitable for the pharmacokinetic study of vertilmicin in rats.     

Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 microM (0.13-39.44 microg/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 microM (0.26-60 microg/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus C(18) column, 5 microm, 150 x 4.6 mm, and a Waters C(18), 4 microm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6+/-0.5, 18.1+/-1.1 and 29.0+/-0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.