Simultaneous determination of leflunomide and its active metabolite,A77 1726, in human plasma by high-performance liquid chromatography

The isoxazol derivative leflunomide [N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide] is an inhibitor of de novo pyrimidine synthesis used for the treatment of rheumatoid artrithis. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of leflunomide and its active metabolite, A77 1726, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile, water and formic acid (40/59.8/0.2, v/v), at a flow rate of 0.5 mL/min, and UV detection at 261 nm. The retention times for A77 1726, leflunomide and warfarin (internal standard) were 8.2, 16.2 and 12.2 min, respectively. The validated quantification range of the method was 0.05-100 micro g/mL for leflunomide and 0.1-100 micro g/mL for A77 1726. The developed procedure was applied to assess steady-state plasma concentrations of A77 1726 in patients with rheumatoid arthritis treated with 10 or 20 mg leflunomide per day.     

Simultaneous determination of oxycodone and its major metabolite, noroxycodone, in human plasma by high-performance liquid chromatography

Oxycodone (14-hydroxy-7,8-dihydrocodeinone) is a potent opioid receptor agonist. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of oxycodone and its major metabolite, noroxycodone, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile and phosphate buffer (8:92, v/v) at a flow rate of 1 mL/min, and UV detection at 205 nm. The retention times for oxycodone, noroxycodone and codein (internal standard) were 14.7, 13.8 and 10.2 min, respectively. The validated quantitation range of the method was 2-100 ng/mL for oxycodone and 10-100 ng/mL for noroxycodone. The developed procedure was applied to assess the pharmacokinetics of oxycodone and its metabolite following administration of a single 20 mg oral dose of oxycodone hydrochloride to one healthy male volunteer.     

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography

Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.     

Development and validation of reversed-phase column high-performance liquid chromatographic and first-derivative UV spectrophotometric methods for estimation of voriconazole in oral suspension powder

This research paper describes validated reversed-phase high-performance column liquid chromatographic (RP-HPLC) and first-derivative UV spectrophotometric methods for the estimation of voriconazole (VOR) in oral suspension powder. The RP-HPLC separation was achieved on Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using water-acetonitrile (40 + 60, v/v; pH adjusted to 4.5 +/- 0.02 with acetic acid) as the mobile phase at a flow rate of 1.4 mL/min and ambient temperature. Quantification was achieved with photodiode array detection at 255 nm over the concentration range of 0.1-1 microg/mL with mean recovery of 99.49 +/- 0.83% for VOR by the RP-HPLC method. Quantification was achieved with UV detection at 266 nm over the concentration range of 8-20 microg/mL with mean recovery of 99.74 +/- 0.664% for VOR by the first-derivative UV spectrophotometric method. These methods are simple, precise, and sensitive, and they are applicable for the determination of VOR in oral suspension powder.     

Validated RP-HPLC method for determination of permethrin in bulk and topical preparations using UV-vis detector

An isocratic reversed-phase high-performance liquid chromatographic method for the estimation of permethrin in raw materials and pharmaceutical topical preparations has been devised and validated. The chromatographic analysis was performed on a 5 μm particle C-18 Nucleosil (Macherey-Nagel, Germany) column (250 × 4.6 mm). Mobile phase consisted of methanol and 0.025 mM Phosphoric acid (85:15 v/v) at a flow rate of 1.5 mL/min. UV detection was performed at 272 nm and peaks were identified with retention times as compared with standards. The limit of detection was 1.782 μg/mL, while limit of quantitation was 48.0 μg/mL. The calibration was linear in a concentration range of 48.0-5000 μg/mL with correlation coefficient of 0.999978. Regression equation was absorbance =2833.23 × concentration(μg/mL) + 19.1045 with variance of the response variable, S(yx)(2), calculated to be 1.75328 (six degrees of freedom). The method was validated as per ICH guidelines and USP requirements and found advantageous for the routine analysis of the drug in pharmaceutical formulations and in pharmaceutical investigations involving permethrin.     

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.     

Determination of iguratimod in rat plasma by high performance liquid chromatography: method and application

A high-performance liquid chromatographic method with UV detection has been developed for the determination of iguratimod (T-614) in rat plasma. Plasma was precipitated with acetonitrile after the addition of the internal standard (IS), N-[4-(2-formylaminoacetyl)-5-methoxy-2-phenoxyphenyl]-methanesulfonamide. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase acetonitrile-acetic acid aqueous solution, pH 4.5 (40:60, v/v), at a flow rate of 1 mL/min, and the UV detection wavelength was set at 257 nm. The calibration curve was linear over the range 0.10-50.0 microg/mL, and the lower limit of quantification was 0.10 microg/mL. The intra- and inter-day relative standard deviations were all less than 11.5%. The method has been successfully applied to study the pharmacokinetics of iguratimod in rats. A single 10 mg/kg dose of iguratimod was given to the rats by intragastric administration. The mean maximum plasma concentration of iguratimod for the six rats was 14.5 microg/mL, and the mean elimination half-life of iguratimod was 4.0 h.     

A high-performance liquid chromatographic method for saikosaponin a quantification in rat plasma

A high-performance liquid chromatographic method with UV detection has been developed for the determination of saikosaponin a in rat plasma. Saikosaponin a and internal standard jujuboside A were isolated from plasma samples by solid-phase extraction. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 1 mL/min and UV detection was set at 205 nm. The standard curve for saikosaponin a was linear over the concentration range 0.25-10 microg/mL and the limit of detection was 0.05 microg/mL. The absolute recovery was greater than 82%. The precision and accuracy ranged from 3.05 to 9.59% and 95.61 to 110.00%, respectively. The validated method was used to determine saikosaponin a in plasma samples in a pharmacokinetic study of saikosaponin a administered to Sprague-Dawley rats.     

Stability indicating reversed-phase high-performance liquid chromatographic and thin layer densitometric methods for the determination of ziprasidone in bulk powder and in pharmaceutical formulations

Two sensitive and reproducible methods were developed and validated for the determination of ziprasidone (ZIP) in the presence of its degradation products in pure form and in pharmaceutical formulations. The fi rst method was based on reversed-phase high-performance liquid chromatography (HPLC), on a Lichrosorb RP C(18) column using water:acetonitrile:phosphoric acid (76:24:0.5 v/v/v) as the mobile phase at a fl ow rate of 1.5 mL min(-1) at ambient temperature. Quantification was achieved with UV detection at 229 nm over a concentration range of 10-500 micro g mL(-1) with mean percentage recovery of 99.71 +/- 0.55. The method retained its accuracy in presence of up to 90% of ZIP degradation products. The second method was based on TLC separation of ZIP from its degradation products followed by densitometric measurement of the intact drug spot at 247 nm. The separation was carried out on aluminium sheet of silica gel 60 F(254) using choloroform:methanol:glacial acetic acid (75:5:4.5 v/v/v) as the mobile phase, over a concentration range of 1-10 micro g per spot and mean percentage recovery of 99.26 +/- 0.39. Both methods were applied successfully to laboratory prepared mixtures and pharmaceutical capsules.     

Determination and pharmacokinetic study of chlorogenic acid in rat dosed with Yin-Huang granules by RP-HPLC

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was described for the determination of chlorogenic acid (CGA) in rat plasma using protocatechuic acid as internal standard (IS). CGA in plasma was extracted with acetonitrile, which also acted as deproteinization agent. Chromatographic separation was performed on a Kromasil C18 column with methanol-0.2 m acetic acid (pH 3.0, 25:75, v/v) as mobile phase at a flow-rate of 1.0 mL/min with an operating temperature of 30 degrees C and UV detection at 300 nm. The standard curve was found to be linear over the concentration ranges of 0.4-2.5 microg/mL and 2.5-40 microg/mL, and the limit of quantification (LOQ) was 0.4 microg/mL. The analytical precision and accuracy were validated by relative standard deviation (RSD) and relative error, which were in ranges 3.14-10.78% and -2.20-5.00%, respectively. The average recovery of CGA was 87.59%. The method was successfully applied to the pharmacokinetic study of CGA in Yin-Huang granules.     

Simultaneous HPLC-UV determination of ketamine, xylazine, and midazolam in canine plasma

An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.     

Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine-N-oxide in human plasma has been developed and validated. The isocratic HPLC assay uses a mobile phase consisting of an acetonitril-buffered aqueous solution containing 146 microL of triethylamine and 200 microL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydroxide solution (400:600, v/v) at a flow-rate of 0.8 ml/min and a Lichrospher 100 RP-18 reversed-phase column and UV detection at 215 nm. Doxepine was used as the internal standard. Mean recoveries for clozapine, norclozapine, clozapine-N-oxide and doxepine were 95%, 98%, 96% and 94%, respectively, whereas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1.8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concentration range of 100-1600 ng/mL was 0.9998 and 0.9997, respectively; for clozapine-N-oxide (20-200 ng/mL) it was found to be 0.9986. The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, norclozapine and clozapine-N-oxide, respectively.     

HPLC method for the simultaneous determination of atenolol and chlorthalidone in human breast milk

A high-performance liquid chromatographic method was optimized and validated for the determination of atenolol and chlorthalidone (CT) in human breast milk. The milk samples were extracted and purified using ACN and phosphoric acid for precipitation of proteins followed by removal of ACN and milk fats by extraction with methylene chloride. The samples were applied, after an extraction procedure, to a cyanide column using a mobile phase consisting of ACN/water (35:65 v/v) and buffered at pH 4.0 with flow rate of 1.0 mL/min. Quantitation was achieved with UV detection at 225 nm using guaifenesin as the internal standard. The effectiveness of protein precipitation and clean up procedure were investigated. The method was validated over the range of 0.3-20 microg/mL for atenolol and 0.25-5 microg/mL for CT.     

A solid phase extraction reversed-phase HPLC method for the simultaneous determination of methoprene, permethrin and selected metabolites in rat plasma and urine.

A method was validated and applied for the analysis of the insect growth regulator methoprene [Isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate], its metabolite methoprene acid, the insecticide permethrin [3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester], and two of its metabolites, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, in rat plasma and urine using solid-phase extraction and reversed-phase high performance liquid chromatography. The analytes were separated using gradient of 55-100% acetonitrile in water (pH 4.0) at a flow rate ranging between 0.6 and 1.0 mL/min over a period of 20 min, and UV detection at 210 and 254 nm. The retention times ranged from 7.3 to 18.4 min. The limits of detection ranged between 50 and 100 ng/ml, while limits of quantitation were 100-150 ng/mL. Average percentage recovery of five spiked plasma samples was 83.6 +/- 3.9, 80.1 +/- 5.4, 82.1 +/- 4.4, 83.7 +/- 3.9 and 83.1 +/- 4.7, and from urine 79.3 +/- 4.3, 82.0 +/- 5.4, 80.7 +/- 4.2, 78.9 +/- 5.7 and 83.9 +/- 4.5 for methoprene, methoprene acid, permethrin, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The method was linear and reproducible over the range of 100-1000 ng/mL. This method was applied to analyze the above chemicals and metabolites following their combined administration in rats.     

Determination of glucosamine and carisoprodol in pharmaceutical formulations by LC with pre-column derivatization and UV detection

A simple and reliable precolumn derivatization liquid chromatography method with ultraviolet detection has been developed and validated for the analysis of glucosamine (GS) in various dietary supplement formulations and raw materials. Additionally, the proposed method was used for analysis of carisoprodol (CR) found in ternary mixture with paracetamol (PR) and caffeine (CF). The linearity ranges were 1-100 μg/mL for GS, 1-150 μg/mL for CR, PR and CF. Derivatization was used with 1,2-naphthoquinone-4-sulphonic acid sodium salt in the presence of borate buffer. Chromatographic separation of GS-naphthoquinone derivative was achieved by using a mixture of acetonitrile and water (pH 7.3 adjusted with 0.1 M NaOH) in the ratio 10:90, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 280 nm. For PR, CF, and CR-naphthoquinone derivative, the chromatographic separation was achieved by using mixture of acetonitrile and 20 mM KH(2)PO(4) (pH 3.0 adjusted with phosphoric acid) in the ratio 20:80, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 275 nm. The limits of detection were 37.2, 35.9, 30.4 and 40.0 ng/mL for GS, CR, PR and CF, respectively.     

Sequential injection chromatographic determination of paracetamol, caffeine, and acetylsalicylic acid in pharmaceutical tablets

In this contribution, a new separation method for simultaneous determination of paracetamol, caffeine, acetylsalicylic acid, and internal standard benzoic acid was developed based on a novel reversed-phase sequential injection chromatography (SIC) technique with UV detection. A Chromolith Flash RP-18e, 25-4.6mm column (Merck, Germany) and a FIAlab 3000 system (USA) with an 8-port selection valve and a 5 mL syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-(0.01 M) phosphate buffer (10:90, v/v) pH 4.05, flow rate 0.6 mL min(-1). UV detection was at 210 and 230 nm. The validation parameters showed good results: linearity (r >0.999) for all compounds, detection limits in the range 0.3-0.8 microg mL(-1), repeatability (RSD) of peak heights between runs in the range 1.10-4.30% at three concentration levels and intra-day repeatability of the retention times in the range 0.28-0.43%. The analysis time was <6 min. The method was found to be applicable for the routine analysis of the active compounds paracetamol, caffeine, and acetylsalicylic acid in pharmaceutical tablets.     

RP-HPLC method for simultaneous estimation of lamivudine, tenofovir disoproxil fumarate and efavirenz in tablet formulation

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic method for the simultaneous determination of lamivudine, tenofovir disoproxil fumarate and efavirenz in bulk and tablet dosage form has been developed and validated. Chromatography was performed on a 150 mm × 4.6 mm i.d., 5-μm particle, Phenomenex Luna C₁₈ column with 30: 45: 25 (v/v/v) acetonitrile: methanol: water as mobile phase at a flow rate of 0.5 mL/min. UV detection was done at 258 nm; lamivudine, tenofovir disoproxil fumarate and efavirenz were eluted with retention times of 3.27, 4.58 and 10.90 min, respectively. The method was validated in accordance with ICH guidelines. Validation revealed the method is specific, rapid, accurate, precise, reliable and reproducible. Calibration plots were linear over the concentration ranges 1–6 μg/mL for lamivudine and tenofovir disoproxil fumarate and 2–12 μg/mL for efavirenz. Limits of detection were 0.05, 0.09 and 0.11 μg/mL and limits of quantification were 0.15, 0.28 and 0.34 μg/mL for lamivudine, tenofovir disoproxil fumarate and efavirenz, respectively. The high recovery and low coefficients of variation confirm the suitability of the method for the simultaneous determination of these three drugs in bulk and tablets.     

Validation of a liquid chromatographic method for determination of tacrolimus in pharmaceutical dosage forms

An accurate, simple, and reproducible liquid chromatographic method was developed and validated for the determination of tacrolimus in capsules. The analysis is performed at room temperature on a reversed-phase C18 column with UV detection at 210 nm. The mobile phase is methanol-water (90 + 10) at a constant flow rate of 0.8 mL/min. The method was validated in terms of linearity, precision, accuracy, and specificity by forced decomposition of tacrolimus, using acid, base, water, hydrogen peroxide, heat, and light. The response was linear in the range of 0.09-0.24 mg/mL (r2 = 0.9997). The relative standard deviation values for intra- and interday precision studies were 1.28 and 2.91%, respectively. Recoveries ranged from 98.06 to 102.52%.     

反相高效液相色谱法测定生物转化体系中的甘草酸

采用高效液相色谱法在Hypersil C18色谱柱(4.6 mm i.d.×250 mm,5 μm)上以甲醇-水-冰醋酸(70∶30∶1, 体积比)为流动相分离测定了甘草酸单铵盐生物(酶)转化体系中的甘草酸,流动相流速为1.0 mL/min,紫外检测波长254 nm。实验结果表明,该方法在进样量为0.2~20 μg时具有良好的线性;样品的加标回收率为98%～103％,相应的相对标准偏差为0.16％～1.58％。方法简便、快速、可靠。     

Determination of amlodipine in pharmaceutical dosage forms by liquid chromatography and ultraviolet spectrophotometry

A liquid chromatography (LC) method and an ultraviolet (UV) spectrophotometric method were developed and validated for quantitative determination of amlodipine in tablets and compounded capsules. The isocratic LC analyses were performed on an RP18 column using a mobile phase composed of 0.1% (v/v) ortho-phosphoric acid (pH 3.0) -acetonitrile (60 + 40, v/v) at a flow rate of 1.0 mL/min. The UV spectrophotometric method was performed at 238 nm. The analytical methods were validated according to International Conference on Harmonization Guidelines. The calibration graphs were linear [correlation coefficient (r) > 0.999] in the studied concentration range of 10-30 microg/mL for LC and 10-35 microg/mL for UV spectrophotometry. The relative standard deviation values for intraday and interday precision studies were less than 2%, and the accuracy was greater than 98% for both methods. The specificity of the LC method was proved using forced degradation. Statistical analyses showed no significant difference between the results obtained by the 2 methods. The proposed methods are precise and accurate and can be applied directly and easily to the oral pharmaceutical preparations of amlodipine.