Potassium Dihydrogen Phosphate Buffer Research Articles

Simultaneous Estimation of Luteolin and Apigenin in Methanol Leaf Extract of Bacopa monnieri Linn by HPLC

Aims: To develop, validate and quantify the content of flavonoids luteolin and apigenin in aerial parts of methanol leaf extract of Bacopa monnieri (B. monnieri) by reverse phase liquid chromatography. Study Design: High Performance Liquid Chromatography Place and Duration of Study: Department of Pharmaceutical Analysis, KMCH College of Pharmacy, Coimbatore and Indian Pharmacopoeia Commission, Ghaziabad, India from 1.7.2010 to 30.6.2011. Methodology: Separation and quantification of flavonoids was performed on a C18 column (5μm, 200mm×4.6mm, id.) using potassium dihydrogen phosphate buffer (20mM, pH 3.5 adjusted with ortho phosphoric acid, v/v) and acetonitrile as mobile phase with a flow rate of 1ml/min. The column effluents were monitored at 348nm with column temperature kept at 30±1oC. Results: A validated method for simultaneous estimation of luteolin and apigenin was developed, where limits of detection and quantification was found to be 0.03 and 0.91μg/ml for luteolin, 0.041 and 0.13μg/ml for apigenin respectively. The percentage of Original Research Article British Journal of Pharmaceutical Research, 4(13): 1629-1637, 2014 1630 these phytoconstituents recovered was in the range of 98.07-99.71 (%RSD<2%). Conclusion: The developed validated HPLC method was found to be accurate, precise and robust and may be used for analysis of luteolin and apigenin in the extracts of B. monnieri.

HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency

We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100μL) pretreatment was based on simple deproteinization by 1.2M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4μm Hydro-RP, 150×4mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7mL/min. The UV detector was set at 267nm. The chromatographic run lasted 19min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0μg/mL. The limit of quantitation was 0.5μg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma.

Simultaneous estimation of levodopa and carbidopa by RP‐HPLC using a fluorescence detector: its application to a pharmaceutical dosage form

A reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous estimation of levodopa and carbidopa in bulk and pharmaceutical formulations. Chromatographic separation was achieved by using a C18 reverse-phase column and a mixture of an aqueous phase (10 mM potassium dihydrogen phosphate buffer, pH4.0) and methanol (90:10 v/v) as the mobile phase. Quantitative analysis of levodopa and carbidopa was performed using a fluorescence detector at an excitation wavelength of 280 nm and an emission wavelength of 310 nm. The method was linear between 5 and 500 ng/mL for both levodopa and carbidopa. The detection limits for levodopa and carbidopa were 0.30 and 0.60 ng/mL, respectively, whereas the quantitation limit was 0.80 ng/mL for levodopa and 1.2 ng/mL for carbidopa. The method demonstrated good and consistent recoveries (99.63-100.80% for levodopa and 98.97-100.94% for carbidopa) with low interday and intraday relative standard deviation. The validated method was successfully applied to quantify levodopa and carbidopa simultaneously in a pharmaceutical formulation. The method was found to be precise, sensitive and accurate for the simultaneous determination levodopa and carbidopa in bulk and pharmaceutical formulations.

HPLC and Densitometric TLC Methods for Simultaneous Determination of Gemifloxacin with Some Co-administered Drugs in Human Plasma

Two chromatographic methods have been developed for determination of gemifloxacin in human plasma with three co-administered drugs, theophylline, warfarin and omeprazole. First method depends on reverse phase high performance liquid chromatography. The plasma sample was extracted using acetonitrile. The method was linear over the concentration range 0.05 to 6 μg/mL, 0.25 to 8 μg/mL, 0.1 to 10 μg/mL and 0.1 to 6 μg/mL of gemifloxacin mesylate, theophylline, warfarin and omeprazole, respectively. The mobile phase used was prepared by mixing acetonitrile and 0.02 mol L-1 potassium dihydrogen phosphate buffer (pH adjusted to 2.5 using ortho phosphoric acid) in a ratio 10:90 with the addition of 1% TEA and flow rate 1 mL/min in isocratic mode and UV-detection at wavelength 254 nm. Second method depends on densitometric thin layer chromatography. The method was linear over concentration range 0.1 to 3 μg/ml, 0.5 to 6 μg/mL, 0.2 to 2.5 μg/mL and 0.1 to 1.5 μg/mL of gemifloxacin mesylate, theophylline, warfarin and omeprazole, respectively. The mobile phase was selected as mixture of dichloromethane, methanol and ammonia in the ratio of (7: 5.5: 3 v/v/v) for the development of plates. Densitometric analysis was carried out at wavelength 254 nm. The stability of gemifloxacin mesylate and the co-administered drugs in plasma was confirmed during three freeze–thaw cycles (−20°C).

Enantiomeric separation of sitagliptin by a validated chiral liquid chromatographic method

An enantioselective reversed phase high performance liquid chromatographic method was developed and validated. The enantiomers of sitagliptin were resolved on a Chiralcel OD-RH (150 mm × 4.6 mm, 5 μm) column using a mobile phase system containing 3 M potassium di-hydrogen phosphate buffer (pH 4.0)–methanol–acetonitrile (60:30:10, v/v/v). The resolution between the enantiomers was not less than 3.0. The pH value of buffer solution in the mobile phase played a key role in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was validated according to ICH guidelines. The calibration curve was found to be linear over the concentration range of 30–300 ng mL−1 (r2 = 0.9996). The limit of detection and limit of quantification of the (S)-enantiomer were found to be 8 and 30 ng mL−1, respectively, for 20 μL injection volume. The percentage recovery of the (S)-enantiomer ranged from 99.06 to 100.2 in bulk drug samples of sitagliptin. The sample solution and mobile phase were found to be stable at least for 48 h. The final optimized method was successfully applied to separate the (S)-enantiomer from sitagliptin and was proved to be reproducible, accurate and robust for the quantitative determination of the (S)-enantiomer in bulk drugs.

Development and Validation of a Method for Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in a Formulation by RP-HPLC

Present study was aimed to develop and validate a reverse-phase high-performance liquid chromatography method for simultaneous determination of sitagliptin phosphate and metformin hy-drochloride in a marketed formulation. The drug separation was performed on Hibar-240, Li-chrosphere-100 C18 ODS (250 × 4.6 mm, 5 μm) column, at a flow rate of 1 mL/min. The mobile phase used was a mixture of methanol: potassium di-hydrogen phosphate buffer at a ratio of 70:30 v/v. The detection was carried out at a wavelength of 266 nm. The retention times of sitagliptin phosphate and metformin hydrochloride were found as 6.1 and 4.9 min respectively. Linear calibration curves with good correlation coefficients were obtained over the concentration ranges of 10 - 50 μg/mL for sitagliptin and 20 - 100 μg/mL for metformin. The limit of detection was 0.016 and 0.14 μg/mL and the limit of quantification was 0.048 and 0.42 μg/mL for sitagliptin phosphate and metformin hydrochloride respectively. Validation of the method demonstrated system selectivity, specificity, linearity, accuracy and precision. The developed method was found useful in the simultaneous analysis of sitagliptin phosphate and metformin hydrochloride in formulation.

Determination of glibenclamide, metformin hydrochloride and rosiglitazone maleate by reversed phase liquid chromatographic technique in tablet dosage form

A simple, precise and accurate high performance liquid chromatography (HPLC) method was developed for the simultaneous estimation of metformin hydrochloride, rosiglitazone maleate, glibenclamide present in multicomponent dosage forms. Chromatography was performed on a 25 cm ? 4.6 mm i.d., 5-?m particle, C18 column with 78:22 (v/v) methanol: 20 mM potassium dihydrogen phosphate buffer as mobile phase at a flow rate of 1.0 ml/min and UV detection at 238 nm for metformin hydrochloride, rosiglitazone maleate, and glibenclamide. The total elution time was shorter than 9 min. This method was found to be precise and reproducible. This proposed method was successfully applied for the analysis of metformin hydrochloride, rosiglitazone maleate, glibenclamide as a bulk drug and in pharmaceutical formulation without any interference from the excipients.

Rapid determination of eight organic acids in plant tissue by sequential extraction and high performance liquid chromatography

A sequential extraction method was developed to determine different forms of oxalate and seven oxalate-metabolism-related organic acids (glyoxylic acid, tartaric acid, glycolic acid, malic acid, acetic acid, citric acid, succinic acid) in plant tissue. The ultra-pure water was used as the extraction medium to obtain water-soluble oxalic acid and the other seven water-soluble organic acids. After the extraction of the water-soluble organic acids, the residues were extracted by dilute hydrochloric acid successively to get the acid-soluble oxalate which entered the liquid phase. A Hypersil ODS column was used with 5 mmol/L potassium dihydrogen phosphate buffer solution (pH 2. 8) as the mobile phase. The diode array detector was set at 210 nm and the column temperature at 30 °C with the injection volume of 5 µL. The flow rate was controlled at different times which allowed a good and rapid separation of the organic acids and hydrochloric acid. Under these conditions, the linear ranges of the method were 1-2000 mg/L for oxalic acid, 25-2,000 mg/L for acetic acid, and 10-2,000 mg/L for glyoxylic acid, tartaric acid, glycolic acid, malic acid, citric acid and succinic acid, with the correlation coefficients of the eight organic acids ≥ 0. 9996. The average recoveries of the eight organic acids in leaves and roots were 93. 5%-104. 4% and 85. 3%-105. 4% with RSDs of 0. 15% -2.43% and 0. 31%-2. 9% (n=7), respectively. The limits of detection ranged from 1 to 10 ng (S/N=3). The results indicated that the method is accurate, rapid and reproducible for the determination of organic acids in plant samples.

A fast, stability-indicating, and validated liquid chromatography method for the purity control of lercanidipine hydrochloride in tablet dosage form.

A robust, sensitive, and stability-indicating rapid resolution liquid chromatography method for the simultaneous determination of process impurities and degradation products of lercanidipine hydrochloride in pharmaceutical dosage form was developed and validated. The chromatographic separation was performed on the Zorbax SB C18 [(50 × 4.6) mm] 1.8 μm column, using gradient elution of a potassium dihydrogen phosphate buffer (pH 3.5, 0.01 M) and acetonitrile. The flow rate was 1.0 ml/min and UV detection was performed at 220 nm. The method was further evaluated for its stability-indicating capability by hydrolytic, oxidative, thermal, thermal with moisture, and photolytic degradation studies. All acceptance criteria of the International Conference on Harmonization guidelines for validation were covered in the method validation. This method can be used for purity control during manufacture and real time stability studies. A shorter run time of 10 minutes and good solution stability for at least 48 hours allowed the quantification of more than 50 samples per day with comparatively lower costs than existing methods.

Development and validation of a reversed phase liquid chromatographic method for the determination of three Gliptins and Metformin in the presence of Metformin impurity (1-cyanoguanidine)

A simple and precise liquid chromatographic method has been developed and validated for the determination of either sitagliptin (STG), vildagliptin (VLG) or saxagliptin HCL (SXG) and metformin HCL (MET) in the presence of metformin degradation product, 1-cyanoguanidine (CGN). Chromatographic separation was achieved on a Symmetry ® cyanide column (150 mm × 4.6 mm, 5 μm). Isocratic elution using a mobile phase of potassium dihydrogen phosphate buffer (pH = 4.6) - acetonitrile (30:70, v : v ) at a flow rate of 1 mL/min with UV detection at 210 nm was performed. The LC method was used for the simultaneous determination of STG, VLG, SXG and MET in the ranges of 5-200, 5-200, 0.5-80.0 and 20-800 μg/mL, respectively. The results were statistically compared with the reference method for each drug using one-way analysis of variance (ANOVA). The method developed was satisfactorily applied to the analysis of the pharmaceutical formulations and proved to be specific and accurate for the quality control of the cited drugs in pharmaceutical dosage forms.

Simultaneous determination of sitagliptin and metformin in ternary mixture with sitagliptin acid degradation product

In this work, the acidic degradation product of sitagliptin phosphate monohydrate (STG) was synthesized, separated and its structure was elucidated. Additionally, two reversed-phase liquid chromatographic (RP-LC) methods have been developed for the determination of STG. The first method comprised the determination of STG in binary mixture with sitagliptin acid degradation product (SDP) in laboratory prepared mixtures, in plasma and in dosage form. This method was based on isocratic elution using a mobile phase consisting of potassium dihydrogen phosphate buffer (pH =4.6) - acetonitrile (30:70, v : v ) with fluorometric detection. The fluorometric detector was operated at 267 nm for excitation and 575 nm for emission. In the second method, the simultaneous determination of STG and metformin (MET) in the presence of SDP has been developed. In this method, the ternary mixture of STG, MET and SDP was separated using a mobile phase consisting of potassium dihydrogen phosphate buffer (pH = 4.6) - acetonitrile (15:85, v : v ) with UV detection at 220 nm. Chromatographic separation in the two methods was achieved on a Symmetry ® Waters C18 column (150 mm × 4.6 mm, 5 μm). The optimized methods were validated and proved to be specific, robust and accurate for the quality control of the cited drugs in pharmaceutical preparations.

Application of Validated Stability-Indicating HPLC Method in Stability Testing of Acetaminophen and Guaiphenesin Tablets

The aim of the work is to develop and validate the simple, fast and precise stability indicating high performance liquid chromatography method for the separation and quantification of acetaminophen and guaiphenesin in pharmaceutical dosage form. The quantification was carried out using Symmetry C18 (4.6x150 mm, 3.5 m) enhanced polar selectivity column and mobile phase comprised of potassium dihydrogen phosphate buffer pH 2.5 and methanol in proportion of ratio 65:35v/v. The flow rate was mL/min and the effluent was monitored at 228 nm. The retention time of acetaminophen and guaiphenesin were 2.6 and 4.6 min respectively. Linearity of acetaminophen and guaiphenesin were in the range of 50 to 90 µg/mL and 30 to 55 µg/mL respectively with correlation coefficients 0.999. The percentage recoveries were 99.80% and 99.85% for acetaminophen and guaiphenesin respectively. There was complete separation of degradation peaks and analyte peaks, which demonstrate the specificity of assay method in the presence of its degradation products; it can be employed as a stability indicating one. Due to simplicity, rapidity and accuracy of the proposed Stability Indicating High Performance Liquid Chromatography method, it is used for analysis of stability samples of Acetaminophen and Guaiphenesin in quality control laboratories.

High-Performance Liquid Chromatographic Method for Analysis of Emtricitabine in Rat Plasma: Method Development, Validation and Application to a Pharmacokinetic Study

A new reverse phase liquid chromatographic method for the investigation of emtricitabine in rat plasma was developed after oral administration to Wistar rats. The desired chromatographic separation was achieved on Phenomenex C18 column (250 mm × 4.6 mm I.D., 5 µm) column, under isocratic conditions using UV detection at 280 nm. The optimized mobile phase consisted of a mixture of 10 mM potassium dihydrogen phosphate buffer- (adjusted to pH 6.8) methanol-2% acetic acid in a ratio of (73 : 25 : 2, v/v/v) at a flow rate of 1 mL min−1. The system was found to produce sharp and well-resolved peaks for emtricitabine with retention time of 5.78 min. The linear regression analysis for the calibration curves showed a good linear correlation over the concentration range 0.050–3.0 µg mL−1, with determination coefficients, , exceeding 0.9970. The limits of detection (LOD) and quantitation (LOQ) were found to be 0.016 µg mL−1 and 0.049 µg mL−1, respectively. The method was successfully applied for the pharmacokinetic in rats. Emtricitabine concentration in plasma reached () was 1.357 µg mL−1 about 2 h after oral administration of 15 mg/kg/rat. The AUC0-24 was 12.175 µg mL−1* h and the apparent elimination half-life () was 8.153 h. This method was found to be suitable for examining emtricitabine concentration in rats, after oral administration of emtricitabine in a single dose.

In vivo pharmacokinetic applicability of a simple and validated HPLC method for orally administered trans-resveratrol loaded polymeric nanoparticles to rats

A new formulation of trans-resveratrol nanoparticles (t-RVT NPs) with potential stealth properties was prepared by nano-precipitation method in our laboratory. To investigate the pharmacokinetic of t-RVT NPs, a simple and rapid high performance liquid chromatographic method was developed for the quantification of trans-resveratrol in plasma of rats treated with t-RVT NPs. Catechin (0.2 μg/ml) was used as an internal standard (IS). The chromatographic separation was achieved on a reversed-phase C18 column using isocratic elution with methanol: 10 mM potassium dihydrogen phosphate buffer (pH 6.8): 3 % acetic acid solution (70:28:2, v/v/v) at a flow rate of 1 ml/min with a total run time of 6 min. The column effluent was monitored by UV detector at 306 nm. Calibration curve in plasma spiked with varying concentration of trans-resveratrol were linear over the concentration range of 0.010–3.2 μg/ml with correlation coefficient of (r2 > 0.9987). The system was found to construct sharp peaks for trans-resveratrol and IS with retention times of 3.82 and 5.49 min, respectively. The method was sensitive with a limit of quantification of 0.007 μg/ml. The method showing accuracy, precision (inter-day relative standard deviation (RSD) and intra-day RSD values <15.0 %. The method was applied for determining trans-resveratrol concentration in plasma after oral administration of 20 mg/kg of free trans-resveratrol and t-RVT NPs to rats. The plasma concentrations of trans-resveratrol and t-RVT NPs at 30 min and 10 h after oral administration were quantified as 0.1771 ± 0.38 and 5.64 ± 0.436 μg/ml. Results established selectivity and suitability of the method for pharmacokinetic studies of trans-resveratrol from t-RVT NPs.

Development and Validation of a Stability-Indicating RP-HPLC Method for the Estimation of Drotaverine Impurities in API and Pharmaceutical Formulation.

A sensitive, stability-indicating gradient RP-HPLC method with PDA detection has been developed for the simultaneous analysis of drotaverine impurities in active pharmaceutical ingredient (API) and pharmaceutical formulations. Efficient chromatographic separation was achieved on an XTerra RP18, 150 × 4.6 mm, 5 μm column using gradient elution at 230 nm detection wavelength. The optimized mobile phase consisted of a 0.02 M potassium dihydrogen orthophosphate buffer of pH 3.0 as solvent A and acetonitrile as solvent B. The flow rate of the mobile phase was 1.0 mL min−1 with a column temperature of 25°C. The method showed linearity over the range of 0.251–10.033 μg/mL, 0.231–9.995 μg/mL, 0.230–10.089 μg/mL, 0.334–10.011 μg/mL, and 0.324–10.050 μg/mL for impurities 1, 2, 3, 4, and drotaverine, respectively, with a correlation coefficient greater than 0.999. The relative retention times and relative response factors of impurities 1, 2, 3, 4 were 0.36, 0.90, 1.42, 1.55 and 1.04, 0.84, 1.10, 1.30, respectively. The drotaverine formulation sample was subjected to the stress conditions of acid, base, oxidative, thermal, humidity, and photolytic degradation. Drotaverine was found to degrade significantly in peroxide, base, and heat stress conditions. The degradation products were well-resolved from drotaverine and its impurities. The peak purity test results confirmed that the drotaverine peak was homogenous and pure in all stress samples and the mass balance was found to be more than 98%, thus proving the stability-indicating power of the method. The developed method was validated according to ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness.

QUANTITATIVE DETERMINATION OF MILNACIPRAN HCL IN RABBIT PLASMA BY HPLC AND ITS APPLICATION TO PHARMACOKINETICS STUDY

A simple, sensitive, and specific reverse phase high performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of Milnacipran HCl in rabbit plasma. Milnacipran HCl and internal standard (IS, Venlafaxine HCl) were extracted by protein precipitation method with chloroform. The separation was performed on a HiQ sil C18 column (250 mm × 4.6 mm i.d., 5 µm). The wavelength was set at 220 nm. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (0.0125 M) and acetonitrile (72:28%, v/v) with 0.20% triethylamine at a flow rate of 1.0 mL/min. The pH of the solution was adjusted to 3.65 with 0.1 M orthophosphoric acid. The calibration curve was linear over the concentration range 0.1–25 µg/mL. The intra-day and inter-day precision was ranged from 3.9 to 7.3% and 5.2 to 10.8%, respectively. Finally, this proposed method was successfully applied to rabbit pharmacokinetics study and yielded the most comprehensive data on systemic exposure of Milnacipran HCl to date.

VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF FEBUXOSTAT AND DICLOFENAC POTASSIUM IN BULK DRUG AND IN BI LAYER TABLET FORMULATION

A simple, rapid, accurate and precise isocratic reverse phase high performance liquid chromatography (RP - HPLC) method has been developed for simultaneous estimation of febuxostat and diclofenac potassium in combined dosage form by using phenomenex C18 column (statio nary phase) having dimension of 4.6 × 250 mm and particle size of 5 μm and mobile phase containing a mixtur e of 0.02 M potassium dihydrogen orthophosphate buffer (adjusted to pH 7 with sodium hydroxide): acetonitrile: methanol (35:9:56 v/v/v) at a flow rate of 1 ml/min and detection was carried out at 290 nm. The retention times of febuxostat and diclofenac pot assium were 6.01 ± 0.02 minutes and 7.10 ± 0.02 minutes respectively. The developed method was validated as per ICH guideline for specificity, linearity, accuracy and precision , limit of detection and limit of quantification. Linearity studies for the deve loped method were found in the range of 5 to 30 μg/ml for febuxostat and 12.5 to 75 μg/ml for diclofenac potassium respectively. The accuracy of the method was studied by recovery study and found to be in the range of 98 % - 102 %. The % RSD for intraday a nd inter day precision was found less than 2. The new RP - HPLC method was successfully applied to marketed formulation of Febuxostat and Diclofenac potassium without any interference from excipients.

A Rapid, Stability-Indicating RP-UPLC Method for the Simultaneous Determination of Fluticasone Furoate and Benzalkonium Chloride in a Pulmonary Drug Product

A stability-indicating reversed-phase ultra performance liquid chromatography (RP-UPLC) method was developed for the simultaneous determination of fluticasone furoate (FF) and benzalkonium chloride (BKC) in a pulmonary drug product. The desired chromatographic separation was achieved on the BEH C18, 1.7 µm (50 x2.1 mm) column, using isocratic elution at 215 nm detector wavelength. The optimized mobile phase consisted of 0.05 M potassium dihydrogen phosphate buffer and acetonitrile in the ratio of 45:55% v/v. The developed method separated FF and BKC within 5 minutes. The stability-indicating capability was established by forced degradation experiments. The developed RP-UPLC method was validated according to the International Conference on Harmonization (ICH) guidelines. This validated method was applied for the simultaneous estimation of FF and BKC in commercially available Furamist ® (Nasal spray formulation) sample. Furthermore, this method can be extended for individual estimation of FF and BKC in various commercially available pulmonary drug products.

Validation of RP-HPLC Method for Simultaneous Estimation of Cefixime Trihydrate and Clavulanate Potassium in Tablets

A simple, accurate, precise, rapid and economic HPLC method has been developed and validated for the simultaneous estimation of cefixime trihydrate and clavulanate potassium in tablet dosage forms. The chromatographic separation was achieved on a Hypersil C18 column (4.6 x 250 mm, 5 μm particle size). For HPLC method development, a mobile phase consisting of methanol: 30 mM potassium dihydrogen phosphate buffer pH 3.0 adjusted with orthophosphoric acid (30:70 v/v) was used at flow rate of 1.0 ml/min. The optimum wavelength selected was 278 nm. Under these chromatographic conditions, cefixime trihydrate and clavulanate potassium peaks were well resolved, with retention times of cefixime trihydrate and clavulanate potassium being 5.8266 and 3.2633 min, respectively. The proposed method was found to have excellent linearity in the concentration range 20-100 μg/ml with correlation coefficients r2 = 0.9946 and 0.9926, respectively. The method was validated for linearity, precision, LOD, LOQ and robustness. The proposed method was optimized and validated as per the ICH guidelines.

Development and application of an HPLC method for erlotinib protein binding studies.

The aim of the present study was to develop a simple and rapid reversed-phase high performance liquid chromatographic method with UV detection for erlotinib hydrochloride quantification, which is applicable for protein binding studies. Ultrafilteration method was used for protein binding study of erlotinib hydrochloride. For sample analysis a simple and rapid reversed-phase high performance liquid chromatographic method with UV detection at 332 nm was developed. The mobile phase was a mixture of methanol, acetonitril and potassium dihydrogen phosphate buffer (15:45:40 %v/v) set at flow rate of 1.3 ml/min. The run time for erlotinib hydrochloride was approximately 6 minutes. The calibration curve was linear over the range of 320-20000 ng/ml with acceptable intra- and inter-day precision and accuracy. The intra-day and inter-day precisions were less than 10% and the accuracies of intra and inter-day assays were within the range of 97.20-104.83% and 98.8-102.2% respectively. Based on the obtained results, a simple, accurate and precise reversed-phase isocratic HPLC method with UV detection has been optimized and validated for the determination of erlotinib hydrochloride in biological samples.