Validated HPLC method for determination of PAT-5A, an insulin sensitizing agent, in rat plasma

A high performance liquid chromatographic method for the determination of PAT-5A (a potent insulin sensitizer) using DRF-2095 (a thiazolidinedione) as internal standard (I.S.) is described. A 1:1 v/v ethylacetate and dichloromethane solvent mixture was used for extraction of PAT-5A from plasma. A Kromasil KR100-5C18-250A, 5 microm, 4.6 x 250 mm SS column was used for the analysis. Mobile phase consisting of sodium dihydrogen phosphate (pH 4.0, 0.05 M) and methanol mixture (25:75, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector set at 345 nm. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. Using this method the absolute recovery of PAT-5A from rat plasma was > 90% and the limit of quantification was 0.05 microg/ml. The intra-day relative standard deviation (RSD) ranged from 2.19 to 4.98% at 1.0 microg/ml, 1.05 to 3.68% at 10.0 microg/ml and 3.14 to 5.08% at 50 microg/ml. The inter-day RSD were 1.6, 2.24 and 1.54% at 1, 10 and 50 microg/ml, respectively. The method was applied to measure the plasma concentrations of PAT-5A in pharmacokinetic and bioavailability studies in male Wistar rats.     

Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection

A simple and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of ciprofloxacin. Separation of ciprofloxacin and anthranilic acid (internal standard) was achieved on a Kromasil 100, C(18), 5 microm (250 x 4.6 mm i.d.) reversed-phase column, using fluorescence detection with lambda(exc)=300 nm and lambda(emi)=458 nm. The mobile phase consisted of acetonitrile-methanol-acetate buffer (pH 3.60; 0.05 M) (10:30:60 v/v/v) containing 1% v/v acetic acid. The analysis was performed in less than 9 min, with a flow rate of 0.8 ml min(-1). A rectilinear relationship was observed for concentrations between 0.005 and 1.0 microg ml(-1) of ciprofloxacin in aqueous standard solutions and serum and the detection limit was 20 pg injected on-column. The intra- and inter-day relative standard deviation (n=8) ranged from 1.6 to 2.6% and from 1.9 to 4.8%, respectively, calculated at three concentration levels of standard solutions. Direct measurements of ciprofloxacin in pharmaceutical preparations and in serum, after precipitation of proteins, were performed with high precision and accuracy. The application of the method to urine samples involved a solid-phase extraction treatment of the samples using C(18) cartridges. The linear working range in urine extended from 0.05 to 2.0 microg ml(-1) and the detection limit was 0.2 ng injected on-column.     

Improved RP-HPLC method to determine biapenem in human plasma/urine and its application to a pharmacokinetic study

Existing methods to determine biapenem (CAS 120410-24-4), a carbapenem, either lacked sensitivity/reproducibility or had no internal standard as a control. Here an improved reversed-phase high-performance liquid chromatographic (RP-HPLC) method was established in human plasma and urine. After adding p-aminobenzoic acid as the internal standard to plasma or urine, plasma samples were ultra-filtrated and urine samples were diluted directly. Chromatographic separations were carried out on a 4.6 mm x 150 mm column with acetonitrile-0.1 mol/l sodium acetate (2:98, v:v; pH 4.38 or 4.00) as mobile phase and UV detection at 300 nm. The extraction recovery was 91.51% for biapenem at the concentration level of 5 microg /ml in human plasma. The linear quantification range of the method was 0.1 to approximately 50 microg /ml for plasma and urine, with linear correlation coefficients greater than 0.998. The intra-day and inter-day relative standard deviations (R.S.D.) for biapenem at low, middle and high levels in human samples were less than 12.51% for plasma and less than 7.05% for urine. The RP-HPLC method was successfully applied to pharmacokinetic studies, in which healthy subjects received multiple doses of biapenem (300 mg, i.v., b.i.d., for 5 continuous days). The pharmacokinetic results are presented.     

A simple HPLC method using a microbore column for the analysis of doxorubicin

Doxorubicin is one of the most potent anti-tumor agents generally used in the treatment of bone cancer. A simple and sensitive HPLC method was developed and validated for the assay of doxorubicin. The method used a C18 Luna microbore column (50 x 1 mm) with a fluorescent detector (505 nm Ex. and 550 nm Em.). The mobile phase consisted of water-acetonitrile-acetic acid (80:19:1, v/v/v, pH 3.0) and the flow rate was 0.1 ml min(-1). Daunomycin was used as the internal standard. This isocratic system required a 10-min run-time, giving a detection limit of 0.02 ng (0.035 pmol per injection). Standard curves were linear over the concentration range of 0.01-0.1 microg ml(-1). Relative standard deviations (R.S.D.) for the within-day, day-to-day precision, and the accuracy measurement for the assay were less than 4.0, 3.2, and 4.1%, respectively. This HPLC method was used to study the in vitro release characteristics of doxorubicin from implantable drug delivery system.     

Determination of roxithromycin in rat lung tissue by liquid chromatography-mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method for the determination of roxithromycin in rat lung tissue is described. Liquid-liquid extraction was adopted for sample preparation with recoveries from 72.5 to 76.9% at levels of 0.1, 5.0 and 20.0 microg/ml. Chromatographic separation was performed on a C18 column using a mixture of methanol, water and formic acid (80:20:1, v/v/v) as mobile phase delivered at a flow rate of 0.5 ml/min. Positive selected ion monitoring (SIM) mode was used for the quantification of roxithromycin at m/z 837.7 and clarithromycin (internal standard) at m/z 748.7. The linearity was obtained over the concentration range of 0.05-20.0 microg/ml and the lower limit of quantification was 0.05 microg/ml. For each QC level of roxithromycin, the intra- and inter-day precisions relative standard deviation (R.S.D.) were less than 4.1 and 7.5%, respectively, and accuracy (RE) was +/-10.0%. The proposed LC-MS method has been successfully used for the determination of roxithromycin in rat lung tissue after oral administration of roxithromycin formulations to 44 SD rats. The present study demonstrates that the concentration of roxithromycin in rat lung tissues can be significantly increased by ambroxol when they are formulated in combination.     

High-performance liquid chromatographic determination and pharmacokinetic study of vitexin-2'-O-rhamnoside in rat plasma after intravenous administration

A simple and specific high-performance liquid chromatographic (HPLC) method was developed for the pharmacokinetic study of vitexin-2'-O-rhamnoside (VOR) in rat after intravenous administration. The plasma samples were deproteinized with methanol after addition of internal standard (i.s.) hesperidin. HPLC analysis was performed on a Diamonsil ODS C18 analytical column, using acetonitrile-0.3% formic acid (20:80, v/v) as the mobile phase with UV detection at 270 nm. The standard curve was linear over the range of 0.1070-21.41 microg/mL in rat plasma. The average extraction recovery of VOR was 97.9+/-3.1%, and the relative standard deviations (R.S.D.s) of the intra- and inter-day precisions were no more than 7.4 and 8.5%, respectively. The lower limit of quantification (LLOQ) was 0.1070 microg/mL. The AUC of VOR was proportional to the dose after intravenous administration of 15, 30, 60 and 120 mg/kg body weight, and the elimination half-life (t1/2beta), systemic clearance (Cl) and apparent volume of distribution (Vc) were not significantly different among the four doses, and all the results indicated that the pharmacokinetics of VOR in rat obeyed first-order kinetics.     

Simultaneous LC determination of tizanidine and rofecoxib in tablets

A reverse phase high performance liquid chromatographic method to determine tizanidine (TZ) and rofecoxib (RF) in combination is proposed and applied to the pharmaceuticals. This method allows the determination of 0.1-0.5 microg/ml of TZ and 1.2-6.0 microg/ml of RF along with 10 microg/ml of nimesulide (internal standard), in a mobile phase consisting of 1% (v/v) triethylamine (pH adjusted to 2.5 using dilute orthophosphoric acid):acetonitrile in the ratio 55:45% (v/v). Detection wavelength of 303 nm and flow rate of 0.8 ml/min were fixed for the study. The limit of detection (LOD) for TZ and RF were found to be 10 and 1 ng/ml, respectively. The limit of quantification (LOQ) for TZ and RF were found to be 80 and 12 ng/ml, respectively. The amount of drug present in the tablet and the recovery studies were also carried out. The % R.S.D. of recovery studies for TZ and RF were found to be 0.0673 and 0.0146, respectively. The method is validated for accuracy, precision, ruggedness and robustness.     

Bioavailability of salvianolic acid B in conscious and freely moving rats

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. The aim of this study was to apply an automated blood sampling system coupled to a simple liquid chromatographic system to determine the bioavailability of salvianolic acid B in stress-free rats. The plasma sample (25 microl) was vortex-mixed with 50 microl of internal standard solution (chloramphenicol 10 microg/ml in acetonitrile) to achieve protein precipitation. Salvianolic acid B in the rat plasma was separated using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of acetonitrile-methanol-20mM NaH(2)PO(4) (adjusted to pH 3.5 with H(3)PO(4)) (20:10:70 v/v/v) containing 0.1mM 1-octanesulfonic acid, and the flow-rate of 1 ml/min. The UV detection wavelength was 286 nm. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.5-200 microg/ml. Intra- and inter-assay precision and accuracy of salvianolic acid B fell well within the predefined limits of acceptability (<15%). The plasma sample of salvianolic acid B was further identified by LC-MS/MS in the negative ion mode using mass transition m/z 358.2 to the product ion m/z 196.9. After salvianolic acid B (100mg/kg, i.v.; 500 mg/kg, p.o.) was given in conscious and freely moving rats, the AUC were 5030+/-565 and 582+/-222 min microg/ml for intravenous (100 mg/kg) and oral (500 mg/kg) doses, respectively. The oral bioavailability of salvianolic acid B in freely moving rats was calculated to be 2.3%.     

Development and validation of a liquid chromatographic method for determination of enantiomeric purity of citalopram in bulk drugs and pharmaceuticals

A simple, rapid and robust LC method for enantiospecific separation and determination of citalopram in drugs and pharmaceuticals was developed using UV and polarimetric detectors connected in series. Baseline separation with resolution > or = 3.0 was achieved within 20 min on Chiralcel OD-H (250 mm x 4.6 mm) 5 microm column using a mobile phase containing of n-hexane:2-propanol:triethylamine (TEA) (95:05:0.1 v/v/v) at a flow rate of 1.0 ml/min at 25 degrees C. Effects of 2-propanol, triethylamine and temperature on enantioselectivity and resolution of the enantiomers were evaluated. Clopidogrel hydrogen sulphate was used as an internal standard (IS) for quantitative determinations using UV detector at 240 nm. Polarimetric detector was used for identification of enantiomers. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 1.3 microg/ml respectively for both the enantiomers. The linearity of the method was in the range of 50-600 microg/ml with r2 > 0.9999. The inter- and intra-day assay precision was less than 0.63% (%R.S.D.) and recoveries were in the range 99.38-100.41%. The method was validated and found to be suitable for determination enantiomeric purity of citalopram in bulk drugs and pharmaceutical formulations.     

Quantification and 24-hour monitoring of mycophenolic acid by high-performance liquid chromatography in Japanese renal transplant recipients

We developed a rapid, simple, and sensitive high-performance liquid chromatography method with UV detection for the quantitative determination of mycophenolic acid (MPA) in human plasma. MPA and the internal standard (naproxen) were separated using a mobile phase of 0.04 M H(3)PO(4)-acetonitrile-methanol (3:3:4 v/v/v) over a CAPCELL PAK C18 MG column. A flow-rate of 0.5 ml/min was used at ambient temperature and sample detection was carried out at 254 nm. The assay required only 100 microl of plasma and involved liquid-liquid extraction, which gave high recovery (>94%). The lower limit of quantification for MPA was 0.05 microg/ml. Inter- and intra-day coefficients of variation were less than 9.6% and accuracies were within 9.3%. Additionally, we validated this method in 24-hour monitoring of plasma MPA concentrations after mycophenolate mofetil (MMF) morning and evening administration in 40 Japanese renal transplant recipients with 1.5 g/day MMF. The time to reach the maximum (11.7 microg/ml) and second peak (4.5 microg/ml) of MPA after morning 0.75 g MMF administration was 2.6 h and 9.0 h, and time to reach maximum (10.5 microg/ml) after evening 0.75 g administration was 4.0 h.     

Optimized procedure for lamotrigine analysis in serum by high-performance liquid chromatography without interferences from other frequently coadministered anticonvulsants

The authors have developed a simple isocratic high-pressure liquid chromatographic (HPLC) assay for the simultaneous determination of lamotrigine and other frequently coadministered antiepileptic drugs in serum samples. Lamotrigine extraction was performed on a reversed-phase Oasis HBL preparation column. The eluates containing butalbital as internal standard were separated with a 7-microm Chromsystems C18 250 x 4.0 mm I.D. reversed-phase column at a temperature of 40 degrees C using a mobile phase consisting of pH 3.8 phosphate-acetonitrile buffer (55:45, v/v), at a flow rate of 0.8 mL/min. Ultraviolet detection was carried out at 210 nm. Measurement of the peak:height ratio allowed quantitative determination of the samples. The method was linear over a concentration range of 0.2 to 20 microg/mL for lamotrigine. Recovery was >90%. Within-day and between-day coefficients of variation ranged from 1.8% to 6.7%. The mean lamotrigine concentration was 8.01 +/- 5.63 microg/mL. After studying sera from 130 patients treated with lamotrigine the authors confirmed that associated antiepileptic therapy affected the serum lamotrigine levels, which were significantly higher in patients under valproic acid treatment.     

Development and validation of an accurate HPLC method for the quantitative determination of picroside II in tablets

A simple, sensitive and accurate high performance liquid chromatographic method (HPLC) with UV detection was developed and validated to determine picroside II in a new tablet formulation with paeoniflorin as internal standard. Chromatographic separation was achieved on an Agilent XDB C18 column (250 x 4.6 mm I.D., 5 microm) using a mobile phase consisting of acetonitrile-water-acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 ml/min. The UV detection wavelength was set at 265 nm. Linear calibration curves were obtained in the concentration range of 0.10-100 microg/ml with the limit of quantification (LOQ) of 0.10 microg/ml. The within- and between-run precisions in terms of % relative standard deviation (RSD) were lower than 5.7% and 6.3%, respectively. The accuracy in terms of % relative error (RE) ranged from -2.3% to 5.0%. This validated method was successfully applied to the determination of the content of picroside II in a new tablet formulation.     

HPLC法测定丙戊酸的血药浓度

目的：建立快速柱前衍生-高效液相色谱法测定丙戊酸（VPA）血药浓度的方法。方法：选择ω-溴苯乙酮为衍生试剂,环己烷羧酸为内标,Hypersil ODS-C18柱（250mm×4．6mm,5μm）为分析柱;流动相为甲醇-水（75：25,V／V）,检测波长为248nm,流速为1．0ml／min。血样经酸化后以二氯甲烷提取。结果：内标环己烷羧酸和VPA的保留时间分别为8．0min和12．8min,线性范围为25～200μg／ml,平均回收率为100．61％,日内、日间RSD均小于4．19％。结论：本法快速、简便、准确,适合临床常规监测需要。     

Determination of chlorogenic acid in rat plasma by high performance chromatography after peritoneal administration of compound Daqingye injection

A simple and sensitive high performance liquid chromatographic method has been developed for the determination of chlorogenic acid (3-O-caffeoyl-D-quinic acid) in rat plasma and applied to its pharmacokinetic study in rats after peritoneal administration of compound Daqingye injection. Plasma samples are extracted with perchloric acid. HPLC analysis of the chlorogenic acid is performed on a C(18) reversed-phase column using methanol-water (80: 20, v/v, pH 2.8) as mobile phase with UV detector set at 327 nm. The standard curves are linear in the range of 0.200-10.0 microg/ml (r=0.9982). The inter- and intra-day precision (relative standard deviation) was less than 9% and the accuracy (relative error) was less than 10%. The limit of quantitation was 0.200 microg/ml. The plasma concentration of chlorogenic acid shows a C(max) of 7.53+/-0.52 microg/ml at 13.33+/-4.00 min with a t(1/2) of 59.10+/-5.42 min.     

Determination of colchicine in mouse plasma by high performance liquid-chromatographic method with UV detection and its application to pharmacokinetic studies

A simple and sensitive high performance liquid chromatography method with UV detection was described for the determination of colchicine (COL) in mouse plasma. After single-step deproteinization by acetonitrile using berberine hydrochloride as an internal standard (I.S.), solutes were separated on a Diamonsil C(18) column (250 mm x 4.6 mm I.D., 5 microm particle size) (Dikma), using acetonitrile-0.15% phosphoric acid solution (27:73, v/v) as mobile phase (flow-rate 1.0 ml/min); wavelength of the UV detector was set at 350 nm. No interference from any endogenous substances was observed during the elution of COL and internal standard (I.S., berberine hydrochloride). The retention times for COL and I.S. were 11.23 min and 8.82 min, respectively. The limit of quantification was evaluated to be 1.5 ng/ ml and the limit of detection was 0.5 ng/ml. The method was used in the study of pharmacokinetics of COL after intravenous injection (i.v.) and intraperitoneal injection (i.p.). The result indicated that COL disappears from the plasma according to a three compartment open model.     

Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz

A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.     

Simultaneous determination of HIV-protease inhibitors lamivudine and zidovudine in pharmaceutical formulations by micellar electrokinetic chromatography

A micellar electrokinetic chromatographic (MEKC) method for the simultaneous separation and determination of lamivudine (LMV) and zidovudine (ZDV) in pharmaceutical formulation has been developed. Factors that affect the separation, such as buffer pH, surfactant concentration (sodium dodecyl sulfate, SDS), organic solvents and applied voltage were optimized. Buffer consisting of 12.5 mM sodium tetraborate decahydrate and 15 mM boric acid adjusted at pH 10.8, containing 90 mM SDS and 5% (v/v) acetonitrile (ACN) was found to be suitable for the separation of the drugs. p-Aminobenzoic acid (PABA) was used as internal standard (I.S.). Detection of analytes and I.S. was performed at a wavelength of 210 nm. It was observed that both the drugs and I.S. were migrated within 20 min at the applied voltage of +10 kV. Validation of the method was performed in terms of linearity, accuracy, precision, limit of detection (LOD) and quantification (LOQ). An excellent linearity was obtained in the concentration range 10-80 microg/ml for LMV and 10-100 microg/ml for ZDV. The detection limits for LMV and ZDV were found to be 2.5 and 2.0 microg/ml, respectively. The optimized method was applied to the simultaneous determination of LMV and ZDV in pharmaceutical formulation and human plasma (spiked) samples. Recovery of both the drugs in tablet dosage form and spiked drugs in plasma were > or =99.72% (relative standard deviation (R.S.D.)< or =1.84%) and > or =80.4% (R.S.D.< or =5.4%), respectively. In the electropherogram no interfering peaks were observed in the region of analytes and I.S. due to inactive ingredients in the tablets and matrices in plasma.     

Sensitive LC determination of piroxicam after in vitro transdermal permeation studies

A direct, very sensitive, simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of piroxicam, with tenoxicam as internal standard, has been developed and validated. Samples were chromatographed on a 5 microm Scharlau C(18) column. The mobile phase was a mixture of acetonitrile-acetic acid 4% (pH 2.8) (45:55, v/v). Detection was at 354 nm and the run time was 7 min. The limit of detection was 0.025 microg/ml. The detector response was found to be linear in the concentration range 0.05-9 microg/ml. This HPLC assay has been applied to measure the 'in vitro' percutaneous permeation of piroxicam through abdominal hairless rat skin, using Franz-type diffusion cells, in order to obtain the concentration-time profiles of piroxicam.     

Determination of desoxyepothilone B in nude mice plasma by liquid-liquid extraction and reversed-phase high-performance liquid chromatography

A novel reversed-phase high-performance liquid chromatographic (HPLC) method has been established for the determination of a newly developed anti-cancer agent desoxyepothilone B (dEpo B) in nude mice plasma. The sample preparation involved deproteination of 200 microl of plasma sample first, followed by liquid-liquid extraction of the resultant supernatant with chloroform. The compound taxol was used as the internal standard. Chromatographic separations were carried out on a 250 mm x 4.6 mm Zorbax SB-phenyl column with acetonitrile-0.25% orthophosphoric acid (50/50, v/v) as mobile phase and UV detection at 250 nm. For dEpo B and taxol at the concentration level of 10 microg/ml in nude mice plasma, the absolute extraction recoveries were 85.3 and 87.2%, respectively. The linear quantification range of the method was 0.1-100 microg/ml in nude mice plasma with linear correlation coefficients greater than 0.999. The within-day and between-day relative standard deviations (R.S.D.s) for dEpo B at 0.5, 2.5 and 10 microg/ml levels in nude mice plasma fell in the range of 2.8-4.8 and 1.5-4.6%, and the within-day and between-day recoveries were in the range of 96.5-101.7 and 97.7-101.2%, respectively.     

Rapid and simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by high-performance liquid chromatography

A simple and reproducible high-performance liquid chromatographic method was developed for simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in human plasma. The method entailed injection of the samples after deproteination with perchloric acid and subsequent neutralizing. Primidone was used as internal standard. Chromatography was performed on a C(18) column (250 mm x 4.6 mm, 5 microm) under isocratic elution with 50 mM aqueous sodium dihydrogen phosphate-acetonitrile-triethylamine (100:25:0.5, v/v), pH 5.9. Detection was made at 240 nm and analyses were run at a flow-rate of 1.5 ml/min at a temperature of 35 degrees C. The recovery was 83.4, 88.5 and 98.2% for TMP, SMX and internal standard, respectively. The precision of the method was 2.6-9.8% over the concentration range of 0.125-2 microg/ml for TMP and 0.39-50 microg/ml for SMX. The limit of quantification (LOQ) in plasma was 0.125 and 0.39 microg/ml for TMP and SMX, respectively. The method was used for a bioequivalence study.