Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

A Simple and Rapid Method for the Separation of the (R)- and (S)-Enantiomers of the Tetrahydroisoquinoline Alkaloid Salsolinol by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic technique for the separation of the optical isomers of salsolinol is described. The simple and rapid method allows the direct resolution of the enantiomers without derivatization. A complete separation (baseline resolution) of (R)-(+)-salsolinol and (S)-(-)-salsolinol could be achieved on a Chiral=Si 100 ß-cyclodextrin column using water mixed with 10% methanol (v/v) and 0.05% trifluoroacetic acid (v/v) as mobile phase. Analyses carried out at a flow rate of 1.0 ml/min were accomplished in less than 12 minutes.     

High Performance Liquid Chromatographic Determination of Pirenzepine Dihydrochloride in Its Pharmaceutical Formulation

Abstract

A high-performance liquid chromatographic procedure for the determination of pirenzepine dihydrochloride as a bulk material and in its tablet dosage form (Gastrozepin^R) is presented. Normal phase liquid chromatography has been performed on a Micro-pack Si-10 column using ammonium hydroxide (28–30% NH₃) in methanol (0.75: 99.25% v/v) as mobile phase at a flow rate of 2 ml/min. Clobazam has been used as internal standard with retention times of 1.9 and 2.8 minutes for clobazam and pirenzepine dihydrochloride, respectively at 254 nm. Analytical calibration yields a linear relationship between 5 and 25 μg/ml, with correlation coefficient of 0.999. Tablets each labelled to contain 25 mg pirenzepine dihydrochloride give mean percentage found of 99.98 ± 0.4. A plot of logarithm of concentration against time for a solution in 6 N hydrochloric acid gives a straight line with a slope of - 0.197 day^?1. The proposed method is, therefore, a stability indicating method.     

Development and validation of the HPLC method for the analysis of trimetazidine hydrochloride in bulk drug and pharmaceutical dosage forms

A simple, economic, selective, precise, and accurate high-performance liquid chromatographic (HPLC) method for the analysis of trimetazidine hydrochloride in both bulk drug and pharmaceutical formulations was developed and validated in the present study. The mobile phase consisted of water: methanol: triethylamine (75: 25: 0.1 v/v/v), and pH 3.3 was adjusted with orthophosphoric acid. This system was found to give a sharp peak of trimetazidine hydrochloride at a retention time of 3.375 ± 0.04 min. HPLC analysis of trimetazidine hydrochloride was carried out at a wavelength of 232 nm with a flow rate of 1.0 mL/min. The linear regression analysis data for the calibration curve showed a good linear relationship with a regression coefficient of 0.997 in the concentration range of 5–90 μg/mL. The linear regression equation was y = 35362x − 8964.2. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.6 and 10.9 μg/mL, respectively. The developed method was employed with a high degree of precision and accuracy for the analysis of trimetazidine hydrochloride. The developed method was validated for accuracy, precision, robustness, detection, and quantification limits as per the ICH guidelines. The wide linearity range, accuracy, sensitivity, short retention time, and composition of the mobile phase indicated that this method is better for the quantification of trimetazidine hydrochloride. The text was submitted by the authors in English.     

Simultaneous Determination of Tinidazole and Furazolidone in Suspension by HPTLC and HPLC

Abstract

High performance thin layer chromatographic (HPTLC) and high performance liquid chromatographic (HPLC) methods were developed for the simultaneous determination of Tinidazole and Furazolidone in suspension.

In the HPTLC method the separation of Tinidazole and Furazolidone was carried out on silica gel 60F₂₅₄ HPTLC glass plate using chloroform:methanol:ammonia (9:1:0.1 v/v) as a mobile phase. Rf values obtained were 0.63 and 0.79 for Furazolidone and Tinidazole respectively. Densitometric evaluation was done at 335 nm. Linearity was obtained within the concentration range 10–50 μg/ml and 3.5–17.5 μg/ml for Tinidazole and Furazolidone respectively.

The second method is based on high performance liquid chromatography on a reversed phase column (μ Bondapak C₁₈) using a mobile phase comprised of water: acetonitrile: triethylamine (80:20:0.1 v/v) adjusted to pH = 3.0 with dil. phosphoric acid. Retention times were 5.24 and 7.82 min for Tinidazole and Furazolidone respectively at a flow rate of 1.5 ml/min. Detection was done at 335 nm. Linearity was obtained within the concentration range 30–180 μg/ml and 10.5–63 μg/ml for Tinidazole and Furazolidone resp.     

A comparative study of the separation of oleanolic acid and ursolic acid in Prunella vulgaris by high-performance liquid chromatography and cyclodextrin-modified micellar electrokinetic chromatography

A high-performance liquid chromatographic (HPLC) method and a cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method were developed to separate and determine oleanolic acid (OA) and ursolic acid (UA) in Prunella vulgaris. HPLC separations were carried out on a Hedera ODS C18 column with methanol -H₂O- acetic acid (85:15:0.3, v/v/v) as mobile phase at a flow-rate of 0.8 ml min^?1. CD-MEKC analysis was performed on a CL1030 capillary electrophoresis system with a 6% (v/v) methanol solution (pH = 9.0) containing 10 mM disodium tetraborate, 10 mM sodium dihydrogen phosphate, 50 mM sodium dodecylsulfate (SDS), 15 mM 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) as background electrolyte. The analytical results of HPLC and CD-MEKC were compared with each other. CD-MEKC has better analytical efficiency for two components, and the analytical time (15 min) was shorter than that of HPLC (35 min).     

Simultaneous Determination of Diazepam and its metabolites in Plasma by High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic method (HPLC) for the simultaneous determination of diazepam and its three active metabolites, nordazepam, oxazepam and temazepam, in plasma was proposed. The compounds were isolated by solid-phase extraction. The chromatographic mobile phase was metanol-water (55:45, v/v) at a flow rate of 1 mL/min. UV detection was performed concurrently at 240 and 254 nm.     

Determination of Unbound Ampicillin in Rat Blood by Microdialysis and Liquid Chromatography

ABSTRACT

An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been applied for continuous monitoring of unbound ampicillin in rat blood. A microdialysis probe was inserted into the jugular vein/right atrium of Sprague-Dawley rats, and doses of 100 and 200 mg/kg ampicillin were then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Isocratic elution of ampicillin was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of methanol-100 mM monosodium phosphoric acid (25:75, v/v, pH 5.5). The wavelength of the UV detector was set at 230 nm. The calibration curves from 0.25 to 50 μg/ml were linear with correlation coefficients of 0.995. The method provides a simple technique for rapid analysis of unbound ampicillin in rat blood for used in pharmacokinetic study.     

Partial least squares model and design of experiments toward the analysis of the metabolome of Jatropha gossypifolia leaves: Extraction and chromatographic fingerprint optimization

A major challenge in metabolomic studies is how to extract and analyze an entire metabolome. So far, no single method was able to clearly complete this task in an efficient and reproducible way. In this work we proposed a sequential strategy for the extraction and chromatographic separation of metabolites from leaves Jatropha gossypifolia using a design of experiments and partial least square model. The effect of 14 different solvents on extraction process was evaluated and an optimized separation condition on liquid chromatography was estimated considering mobile phase composition and analysis time. The initial conditions of extraction using methanol and separation in 30 min between 5 and 100% water/methanol (1:1 v/v) with 0.1% of acetic acid, 20 μL sample volume, 3.0 mL min^?1 flow rate and 25°C column temperature led to 107 chromatographic peaks. After the optimization strategy using i‐propanol/chloroform (1:1 v/v) for extraction, linear gradient elution of 60 min between 5 and 100% water/(acetonitrile/methanol 68:32 v/v with 0.1% of acetic acid), 30 μL sample volume, 2.0 mL min^?1 flow rate, and 30°C column temperature, we detected 140 chromatographic peaks, 30.84% more peaks compared to initial method. This is a reliable strategy using a limited number of experiments for metabolomics protocols.     

Simultaneous Determination of Atenolol,Rosuvastatin, Spironolactone,Glibenclamide and Naproxen Sodium in Pharmaceutical Formulations and Human Plasma by RP‐HPLC

A rapid and sensitive high‐performance liquid chromatographic method was developed and validated for the simultaneous determination and quantification of atenolol, rosuvastatin, spirnolactone, glibenclamide and naproxen sodium in bulk drugs, pharmaceutical formulations and in human plasma in the presence of internal standard (flurbiprofen). Chromatograms were developed with methanol and water (80:20, v/v) solvent system on a Purospher start, C₁₈ (5 μm, 250 × 4.6 mm) column and pH was adjusted to 3.40 with ortho‐phosphoric acid. Mobile phase was pumped with a flow rate of 0.90 mL/minute with 235 nm UV detection. Standard curves were linear over the concentration range 0.25‐30 μg/mL^?1. The coefficients of variation (C.V.%), were < 3% and LOD and LOQ were <0.0154 & 0.06 for inter‐ and intra‐day, respectively. The method was applied to drug interaction studies of atenolol with rosuvastatin, spironolactone, glibenclamide and naproxen to illustrate the scope and application of the methods to manage four different therapeutic classes of drugs, as they are co‐administered.     

Reversed-Phase High-Performance Liquid Chromatographic Determination of Nicotine in Pesticide Formulations

Abstract

A simple and rapid reversed-phase high-performance liquid chromatographic procedure is described for the determination of nicotine in liquid formulations. Samples are diluted with methanol, and naphthalene is added as the internal standard. Peak height ratios obtained from injections of standard and sample filtrates are used for quanti-tation. An eluting solvent of 0.05M (NH₄)₂ HPO4 (pH 7.5) -methanol (40/60, v/v) at a flow of 2 mL/min gives retention times of 3.13 and 6.88 min respectively for nicotine and naphthalene. Sample analysis can be completed in approximately one hour by the method described as compared to 1.5 days required by the Official AOAC gravimetric method (6.176–6.177).     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

Determination of pKa values of nonsteroidal antiinflammatory drug‐oxicams by RP–HPLC and their analysis in pharmaceutical dosage forms

In this study, pK_a values were determined by using the dependence of the capacity factor on the pH of the mobile phase for four ionizable substances, namely, tenoxicam, piroxicam, meloxicam, and naproxen (I.S.). The effect of the mobile phase composition on the ionization constant was studied by measuring the pK_a at different ACN concentrations, ranging from 30 to 40%. The adequate condition for the chromatographic determination of these compounds in pharmaceutical dosage forms was established based on the different retention behaviors of the species. An octadecylsilica Nucleosil C18 column (150×4.6 mm, 5 μm) was used for all the determinations. The chromatographic separation of oxicams was carried out using acetonitrile (ACN)/water at 35% v/v, containing 65 mM phosphoric acid and UV detection at a wavelength of 355 nm. The method developed was successfully applied to the simultaneous determination of these drug compounds in laboratory‐prepared mixtures and their commercial pharmaceutical dosage forms. Each analysis requires no longer than 12 min.     

High-Performance Liquid Chromatographic Determination of Atenolol in Tablets

Abstract

A reversed phase high-performance liquid chromatographic method was developed for the determination of atenolol in four oral 100 mg atenolol preparations.

An aliquot of the sample is dissolved in a mobile phase consisting of 0.0612 M potassium hydrogen phosphate - isopropanol-tetrahydrofuran (84:10:6) v/v). The pH was adjusted to 6.7 with phosphate buffer. Nicotinamide was used as internal standard and chromatographed on a Pinkerton column ISRP (GFF-S5–80) 5 μm, 150 × 4.6 mm i.d. The applied column is convenient for the assay at least 90 samples of atenolol without degrading column performance. The detection was performed at 272 nm. The retention time for atenolol was 5.07 min.

The proposed HPLC method was found to be suitable for the rapid and precise routine analysis of atenolol in tablets.     

Determination of Selected Quinolones and Fluoroquinolones by Use of TLC

Abstract

There are many different methods of quinolones determination. The most often used method of quinolones analysis is liquid chromatography. In this work some selected quinolones (cinoxacin, pipemidic acid) and fluoroquinolones (ofloxacin, pefloxacin) were separated with thin-layer chromatography (TLC). The two different mobile phases were used as follows: buffer solution (pH = 5.5)-methanol, 40:10 (v/v) and acetonitrile-water-acetic acid, 6:40:4 (v/v/v), respectively, for quinolones and fluoroquinolones. The following chromatographic parameters were calculated for these separations: R_F, ?R_F, R_M, and R_S. The possibility of qualitative determination of cinoxacin, pipemidic acid, ofloxacin, and pefloxacin using TLC was shown.     

Stability Indicating Hplc Method for the Determination of Sparfloxacin (Spar)

ABSTRACT

A rapid, sensitive and stability indicating method for the determination of sparfloxacin (SPAR) by RP - HPLC has been developed on a Merck RP - Select B (5 μm; 12.5 cm x 4.0 mm) column using a mobile phase of water: acetonitrile: triethylamine (80 : 20 : 0.2 v/v) pH of which was adjusted to 2.6 with orthrophosphoric acid. The flow rate was 1 ml / min. and the detection was carried out at 304 nm using Waters 486 variable wavelength detector. The retention time for SPAR was 7.2 min. Linearity range was from 8 - 1000 ppm. The method showed good precision and accuracy when applied to two brands of tablets containing SPAR. In alkaline media SPAR is stable where as it undergoes degradation in acidic and oxidising conditions generating different degradation products the nature of which is required to be established. The proposed method nicely separates the degraded products from SPAR and hence can be used as stability indicating method for the assay of SPAR.     

A Rapid and Simple High Pressure Liquid Chromatographic Method for Pharmacokinetic Study of Ciprofloxacin in Human Serum

Abstract

A rapid, accurate and sensitive method has been developed for the quantitative determination of ciprofloxacin, a new second-generation quinolone carboxylic acid antimicrobial agent, with high in vitro activity against a wide range of Gram- negative pathogens and Gram-positive cocci.

A Lichrosorb RP-18 250 x 4.0 mm, 5 μm analytical column was used with an eluting system consisting of a mixture of CH₃CN-CH₃OH-Citric acid 0.4 M (1:3:6 v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in a limit of detection of 0.2 ng per 20 μl injection. For the quantitative determination theophylline was used as chromatographic internal standard at a concentration of 1.56 ng/μl. A rectilinear relationship was observed up to 20 ng/μl. Analysis time was less than 6 min. The statistical evaluation of the method was examined performing intra-day (n=8) and inter-day calibration (n=8) and was found to be satisfactory with highly accurate and precise results. The method was applied to the direct determination of ciprofloxacin in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of ciprofloxacin in spiked samples was 98 ± 4% over the range of 0.5–5 mg/μl.     

纤维素-三（4-甲基苯甲酸酯）手性固定相拆分5种芳基丙酸类药物对映体及其制剂的含量测定

采用纤维素-三（4-甲基苯甲酸酯）（CTMB）手性固定相,利用反相色谱法研究了氟比洛芬、普拉洛芬、布洛芬、萘普生、洛索洛芬5种芳基丙酸类手性药物的色谱拆分行为。考察了流动相组成、酸碱添加剂及柱温对上述5种药物对映体分离的影响,并通过热力学研究及对映体结构分析对CTMB固定相的手性拆分机理进行了探讨。结果表明,除萘普生采用乙腈-0.1%（v/v）甲酸溶液外,以甲醇-0.1%（v/v）甲酸水溶液为流动相可使普拉洛芬、洛索洛芬、氟比洛芬和布洛芬的对映体间的分离度均大于1.5,CTMB固定相对这5种芳基丙酸类药物的手性拆分能力依次为普拉洛芬>洛索洛芬>氟比洛芬>布洛芬>萘普生。在各自的优化色谱条件下,将方法应用于上述5种药物制剂的含量测定,结果令人满意。