Chromatographic Method For Simultaneous Determination Research Articles

Development and validation of a reversed-phase column liquid chromatographic method for simultaneous determination of two novel gliptins in their binary mixtures with Metformin

A new, simple, accurate, and precise liquid chromatographic method has been developed and validated for the determination of two novel dipeptidylpeptidase-4 (DPP-4) inhibitors; namely vildagliptin (VLG) and saxagliptin HCl (SXG) simultaneously in their binary mixtures with metformin HCl (MET). Chromatographic separation was achieved on an Inertsil® CN-3 column (250 mm x 4.6 mm, 5 µm). Isocratic elution using a mobile phase of potassium dihydrogen phosphate buffer pH (4.6) - acetonitrile (15:85, v:v) at a flow rate of 1 mL/min with UV detection at 208 nm was performed. The liquid chromatographic method was used for the simultaneous determination of either VLG, SXG and MET in the range of 5-200, 0.5-20 and 50-2000 μg/mL, respectively. The methods developed were 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.

A High-Performance Liquid Chromatographic Method for Simultaneous Determination of 21 Free Amino Acids in Tea

Free amino acids are closely related to the savory taste and beneficial effects of tea, and high-performance liquid chromatography (HPLC) is the most widespread analytical approach for simultaneous determination of free amino acids in tea. However, the reported HPLC methods have drawbacks such as long run times, low resolution, or poor efficiency. In this study, a special amino acid analysis column was used to separate and verify 21 amino acids including l-theanine, the predominant amino acid in tea. The mobile phases, including the sodium acetate and acetic acid concentration in buffer B, and the pH and concentration of sodium acetate in buffer A were optimized. The elution gradients were optimized using DryLab 2000 Plus software. In this way, an online o-phthaldialdehyde precolumn derivatization HPLC-fluorescence detection method was developed for simultaneous determination of 21 amino acids. Comparison to other HPLC methods for simultaneous determination of free amino acids in tea showed that our method is easy (automated derivatization), quick (30 min), inexpensive, and green (using a minimum of solution). It has good resolution (≥1.8) and high selectivity (interpark time ≥ 0.5 min). Free amino acids in six tea samples were analyzed. This work provides an HPLC method to simultaneously measure 21 amino acids in tea and potential in other food products.

Optimization of hydrophilic interaction liquid chromatographic method for simultaneous determination of cetylpyridinium chloride and benzocaine in lozenges

A new Hydrophilic Interaction Liquid Chromatography (HILIC) method was developed for simultaneous determination of cetylpyridinium chloride and benzocaine as active substances in lozenges. Method development included investigation of the influence of the critical chromatographic conditions such as: type of organic modifier, mobile phase strength, ionic strength and pH value of the buffer used in the mobile phase, as well as experimental determination of dominant mechanism of retention. The optimal chromatographic conditions were obtained using bare silica column and a mobile phase consisting of acetonitrile and ammonium formate (50 mM, pH = 4.0) in ratio 80 : 20 V/V. The method was validated in terms of specificity, linearity, accuracy and precision. The validation results indicate that the proposed HILIC method is suitable for quantitative analysis of cetylpyridinium chloride and benzocaine in lozenges.

Simultaneous Determination of Ritonavir and Atazanavir in Combined Tablet Dosage Form by HPTLC

A new, simple, sensitive, precise and accurate High-performance thin-layer chromatographic method for simultaneous determination of Ritonavir and Atazanavir in their combined tablet dosage form has been developed, validated and used for determination of the compounds in commercial pharmaceutical products. Chromatographic separation was achieved on aluminium plates precoated with silica gel 60 F254 as the stationary phase and chloroform: ethyl acetate: acetone (5:2:3, v/v/v) as a mobile phase. Densitometric measurements of their spots were achieved at 244 nm over the concentration ranges of 800-2800 ng spot-1 and 2400-8400 ng spot-1, with mean recoveries of 98.57 ± 0.35 and 99.16 ± 0.20 for ritonavir and atazanavir respectively. Limit of detection for ritonavir and atazanavir were found to be 300 ng spot-1 and 200 ng spot-1 respectively.

A Versatile High Performance Liquid Chromatography Method for Simultaneous Determination of Three Curcuminoids in Pharmaceutical Dosage forms

A simple, precise, isocratic, reverse phase high performance liquid chromatography (HPLC) method was developed for the rapid determination of three curcuminoids viz. Curcumin (C), Desmethoxycurcumin (DMC) and Bisdesmethoxycurcumin (BDMC) using a Agilent RP C 18 , 4.6 mm × 150 mm, 5 μm XDB column. The run time was 7 min. The influence of mobile phase composition, injection volume, mobile phase pH, flow rate, temperature, and detector wavelength on resolution was investigated. The method was validated according to the International Conference on Harmonization (ICH) guidelines for validation of analytical procedures with respect to precision, accuracy and linearity. The limit of detection and limit of quantitation were 0.015 and 0.050 µg/mL respectively. Linearity was from 0.05 to 15 µg/mL. Further, the proposed method was found to be reproducible and convenient for stability indicating analysis of curcumin in marketed formulation, polymeric nanoparticles and solubility studies.

Development and validation of HPTLC method for simultaneous determination of amlodipine besylate and metoprolol succinate in bulk and tablets

A simple, selective, precise high-performance thin-layer chromatographic method for simultaneous determination of amlodipine besylate and metoprolol succinate in bulk and pharmaceutical combined dosage form was developed and validated. The method employed HPTLC aluminum plates precoated with silica gel 60F-254 (10×10) as the stationary phase. The solvent system consisted of toluene:ethyl acetate:methanol:triethylamine (4:1:1:0.4 v/v/v). The system was found to give a compact spot for amlodipine besylate (Rf = 0.39±0.02) and metoprolol succinate (Rf = 0.59±0.02). Densitometric analysis of amlodipine besylate and metoprolol succinate was carried out in the absorbance mode at 254 nm. Linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9990±0.0013 with respect to peak area in the concentration range 400-1400 ng per spot for amlodipine besylate and r2 = 0.9993±0.0013 with respect to peak area in the concentration range 3800–13300 ng per spot for metoprolol succinate. The method was validated for precision, recovery and robustness. The limits of detection and quantitation were 39.99 and 121.20 ng per spot for amlodipine besylate and 234.31 and 710.03 ng per spot for metoprolol succinate, respectively. Statistical analysis proved that the method is selective, precise and accurate for the estimation of amlodipine and metoprolol.

Validated Chromatographic Methods for Simultaneous Determination of Amlodipine Besylate and Perindopril Arginine in Binary Mixtures and in Pharmaceutical Dosage Form

Two accurate, selective and precise chromatographic methods, namely TLC-densitometric method & RP-HPLC method & were developed and validated for the simultaneous determination of Amlodipine besylate and Perindopril Arginine in their binary mixtures. The developed TLC-densitometric method depends on separation and quantitation of the studied drugs on silica gel 60F254 TLC plates. Chloroform:methanol:deionized water:glacial acetic acid:triethylamine (10:7:5:0.3:0.2, by volume) was used as developing system and the separated bands were scanned at 208 nm. Linear relationship was obtained in the range 1-10 μg for both drugs. The developed RP-HPLC depends on quantitative chromatographic separation of the studied drugs on a C18 column using phosphate Buffer:acetonitrile (60:40, v/v), pH=4.6 as a mobile phase delivered at constant flow rate of 1 mL.min-1 with UV detection at 210 nm. Calibration curves for Amlodipine besylate and Perindopril Arginine were constructed over the concentration range of 1-50 μg.mL-1 for both drugs. The proposed methods were successfully applied for determination of the studied drugs in their bulk powder and in pharmaceutical formulations. The proposed methods were statistically compared to each other using student’s t test and F-test and no significant difference was found between them.

Stability-indicating reversed-phase liquid chromatographic method for simultaneous determination of losartan potassium and ramipril in tablets

A stability-indicating reversed-phase liquid chromatographic method has been developed and validated for simultaneous determination of losartan potassium and ramipril. Separations were achieved using a C18 column with mobile phase consisting of acetonitrile and (0.2% v/v, pH 2.5) aqueous trifluoroacetic acid (45:55, v/v) in isocratic mode at 1 ml/min flow rate. Column effluent was monitored at 210 nm using a UV detector. The method was validated for selectivity, linearity, accuracy, precision, sensitivity and robustness. Novel microwave-assisted forced degradation technique was employed for evaluation of selectivity. The method demonstrated excellent linearity for losartan potassium and ramipril with regression coefficients of 0.9999 and 0.9998, respectively. The linearity range was found to be 62.5-5000 ng/ml and 125-10,000 ng/ml with the mean percentage recoveries of 100.36% (±2.27) and 100.16% (±3.33) for losartan potassium and ramipril, respectively. In a robustness study, a full factorial design revealed that the analytical response remains unaffected by small variations in the critical chromatographic factors. The method was found to be sensitive with quantification limits of 44.30 and 79.93 ng/ml for losartan potassium and ramipril. The method was successfully employed for the determination of losartan potassium and ramipril in commercially available and in-house prepared tablets.

Development and Validation of HPTLC Method for the Simultaneous Estimation of Gemifloxacin Mesylate and Ambroxol Hydrochloride in Bulk and Tablet Dosage Form

A new simple high performance thin layer chromatography method for simultaneous determination of gemifloxacin mesylate and ambroxol hydrochloride in bulk and tablet dosage form were investigated. Chromatographic separation of the drugs was performed on aluminum plates precoated with silica ge160 F254 as a stationary phase and solvent system consisted of acetone:methanol:toluene:formic acid (4:2:3:1 v/v/v/v). Densitometric evaluation of the separated zones was performed at 252nm and the method was validated. The Rf values and drug content of gemifloxacin mesylate and ambroxol hydrochloride were 0.41±0.02, 0.68±0.02 and 100.1 %, 99.84 %. The calibration curves of peak area versus concentration, which was linear from 400–2200 ng/band for gemifloxacin mesylate, 100–550 ng/band for ambroxol hydrochloride and regression coefficient (r2) was greater than 0.99. LOD for gemifloxacin mesylate and ambroxol hydrochloride was 30 and 25 ng/band respectively, while LOQ was 100 and 70 ng/band respectively. The method was validated for linearity, accuracy, robustness as per International Conference on Harmonisation (ICH) guidelines. The study shows that the developed method is simple and accurate and it was found to be suitable for the simultaneous determination of gemifloxacin mesylate and ambroxol hydrochloride in bulk and pharmaceutical formulations.

Rapid simultaneous determination of organochlorine and pyrethroid pesticide residues in Lycium barbarum L. using gas chromatography with electron-capture detector

A fast gas chromatographic method for simultaneous determination of 50 organochlorine (OCP) and pyrethroid (PP) pesticides in Lycium barbarum L. is established. OCP and PP were extracted from L. barbarum L. using acetonitrile, cleaned with alumina neutral-florisil column, and eluted by mixed solvents of ethyl acetate and hexane (15 : 85, v/v). Selected pesticides were identified using HP-5 and DB1701 capillary dual column and detected by electron-capture detector. Quantitative analysis was performed using an external standard by HP-5 capillary column. Results show that recoveries were 70.3%–115.7%, and the relative standard deviations (RSDs) were 2.0%–14.4%. The limits of detection of the method were 0.0023–0.0075 mg kg−1, and the limits of quantity were 0.0076–0.0247 mg kg−1. The method can treat multiple samples simultaneously. The results are reproducible and satisfactory.

Validated High Performance Liquid Chromatographic Method for Simultaneous Determination of Phenytoin, Phenobarbital and Carbamazepine in Human Serum

A high performance liquid chromatographic (HPLC) method for the simultaneous determination of phenytoin (CAS 57-41-0), phenobarbital (CAS 50-06-6, phenobarbitone) and carbamazepine (CAS 298-46-4) is described. The serum was extracted with ethyl acetate, the dried extract was reconstituted in mobile phase and the aliquot was injected. The eluent drugs were detected at 230 nm. The mobile phase consisting of methanol: water: glacial acetic acid mixture (67:33:1, v/v/v) was used at a flow rate of 1 ml/min on C-18 column. The absolute recovery was above 96% of all the three drugs over a concentration range of 0.5-50.0 micrograms/ml. The inter-day and intra-day precision relative standard deviation (RSD) ranged from 0.79-5.13% and 0.11-6.81%, respectively. The method is simple, rapid, accurate and sensitive and is presently used for therapeutic drug monitoring in epileptic patients. The results obtained with this method showed very good clinical correlation.

Validated High Performance Liquid Chromatographic Method for Simultaneous Determination of Rosiglitazone, Cilostazol, and 3,4-Dehydro-cilostazol in Rat Plasma and its Application to Pharmacokinetics

A high performance liquid chromatographic (HPLC) method for simultaneous determination of rosiglitazone, CAS 122320-73-4, RSG), cilostazol (CAS 73963-72-1, CLZ) and its active metabolite 3, 4-dehydro-cilostazol (DCLZ), using pioglitazone (PIO) as internal standard (IS), in rat plasma is described. The plasma was extracted with methyl t-butyl ether, the dry extract was reconstituted in mobile phase and the aliquot was injected. The eluent drugs were detected by UV at dual wavelength of 226 nm (RSG and DCLZ) and 257 nm (CLZ). The mobile phase consisting of acetonitrile:potassium di-hydrogen phosphate buffer (35:65 v/v) was used at the flow rate of 1.2 ml/min on a reverse phase C18 column. The absolute recovery was above 90% of all analytes over the concentration range of 25-2500 ng/ml for RSG and CLZ and 20-2000 ng/ ml for DCLZ. The relative standard deviation (RSD) of the inter-day and intra-day precision ranged from 2.8 to 8.4% and 0.9 to 5.9%, respectively. The method is simple, rapid, accurate and sensitive and was applied to pharmacokinetic studies.

Optimization and validation of a fast ultrahigh‐pressure liquid chromatographic method for simultaneous determination of selected sulphonamides in water samples using a fully porous sub‐2 μm column at elevated temperature

High temperature in HPLC is considered a valuable tool helping to overcome the increase in the column backpressure when using small packing particles such as sub-2 μm, as it allows reduction in the mobile-phase viscosity. In this study, a fast analytical method based on HPLC-UV was developed using a sub-2 μm column at elevated temperature for the simultaneous determination of nine sulphonamides. Owing to the lower viscosity of the mobile phase, the separation could be achieved in 3 min at 60°C for all analytes. The effect of temperature, the organic modifier percentage and the flow rate on the retention time was studied. The method developed was used for the determination of selected sulphonamides in surface and wastewater samples. Sample preparation was carried out by solid-phase extraction on Oasis HLB cartridges. The method developed was validated based on the linearity, precision, accuracy, detection and quantification limits. The recovery ranged from 70.6 to 96 % with standard deviations not higher than 4.7%, except for sulphanilamide. Limits of detection ranged from 1 to 10 μg/L after optimization of all analytical steps. This method has the highest performance in terms of analytical speed compared with other published HPLC-UV methods for the determination of sulphonamides in water.

Determination of phenolic composition and antioxidant capacity of native red wines by high performance liquid chromatography and spectrophotometric methods

A reversed-phase high performance chromatographic method for simultaneous determination of 14 phenolic compounds in native red wines was developed in this study. The identified compounds contained gallic acid, (+)-catechin, 3,4-dihydroxybenzoic acid, chlorogenic acid, (−)-epicatechin, 4-hydroxybenzoic acid, syringic acid, caffeic acid, p-coumaric acid, rutin, resveratrol, myricetin, quercetin and kaempferol. The method includes liquid–liquid extraction of acidic pH with ethylacetate. The analysis used a Zorbax Eclipse XDB-C18 column (5μm, 4.6mm×250mm). The chromatographic separation of these compounds performed in a single run by using the mobile phase gradient elution of methanol water mixture (% 0.2 formic acid) at room temperature, with flow rate at 1mL/min. Detection was carried out by UV–vis and fluorescence detector. Each analysis required an equilibration period of 10min and a run time of 14min for completion. The optimized chromatographic method was carefully validated for precision and accuracy. Our findings indicated that the developed HPLC method was precise, accurate, specific and sensitive for simultaneous determination of phenolic compounds. Consequently, the described method was applied to the analysis of six wines from Malatya and Elazığ. Gallic acid was dominant phenolic acid in red wines. (+)-catechin, (−)-epicatechin and p-coumaric acid were the next most abundant phenolics. The highest level of trans-resveratrol among the red wines was found Buzbağı (Bogazkere–Öküzgözü). The red wines were analyzed for total polyphenol content (TP) by Folin–Ciocalteu (FC) method, using gallic acid as standard. Antioxidant activities (AA) of the red wines were measured using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. There was a very high correlation between AA and TP in all of the wines tested.

Development and validation of a liquid chromatography method for simultaneous determination of three process-related impurities: yeastolates, triton X-100 and methotrexate

Yeastolates, triton X-100 (TX-100) and methotrexate (MTX) are common process-related impurities (PRI) in cell-based bioproduction of many active biopharmaceuticals. In this study, a reverse phase high performance liquid chromatography (RP-HPLC) method coupled with ultraviolet (UV) detection was developed for simultaneous determination and quantitation of these impurities. The chromatographic separation was achieved using a Jupiter C4 column and analyses of yeastolates, TX-100 and MTX were monitored at 257, 280 and 302 nm, respectively. The method was further validated with respect to selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. The limits of quantitation for yeastolates, TX-100 and MTX were determined to be 27 ppm, 10 ppm and 41 ppb, respectively. Finally, the suitability of the method for analyses of recombinant human hyaluronidase (rHuPH20) in-process (viral inactivation, QFF, PS, APB and CHT filtered, final viral filtrate) and final manufacturing materials was demonstrated, and trace levels of yeastolates, TX-100 and MTX were reliably measured except for three matrices early in the purification process in which TX-100 was not accurately determined due to interfering effects.

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F254 as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (Rf 0.73) and pantoprazole (Rf 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min−1 for the separation of mosapride (tR 11.4) and pantoprazole (tR 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.

Development of a Green Chromatographic Method for Simultaneous Determination of Food Colorants

A green chromatographic method for the successful separation and determination of eight synthetic food colorants (Tartrazine E 102, Quinoline Yellow E 104, Sunset Yellow E 110, Carmoisine E 122, Ponceau 4R E 124, Allura Red E 129, Indigo Carmine E 132 and Brilliant Blue E 133) was developed. A C8 stationary phase was used and the mobile phase was a mixture of 50 mM phosphate buffer at pH 7 containing triton X-100 (0.25% v/v). The method was validated as regards its selectivity, linearity, precision, accuracy, limit of detection (LOD) and quantification (LOQ). LOD of colorants varied between 0.17 μg mL−1 in Allura Red and 1.91 μg mL−1 in Quinoline Yellow. In the case of LOQ, it was ranged from 0.52 in the Allura Red to 5.79 in the Quinoline Yellow. The method applicability was verified by the determination of colorants present in 22 samples. The 15 samples were only unicolor and the color concentration in these samples varied from 18.426 ± 0.100 to 610.390 ± 4.711 ppm. The method can be used successfully to the determination of binary and ternary color food and drug samples too. This method provides substantial green benefits without using organic solvents in extraction procedure and in both liquid and paper chromatographic methods.

An improved high-performance liquid chromatographic method for simultaneous determination of tocopherols, tocotrienols and γ-oryzanol in rice

An improved normal phase high performance liquid chromatographic (NP-HPLC) method was developed for simultaneous quantification of eight vitamin E isomers (α-, β-, γ- and δ-tocopherols and α-, β-, γ- and δ-tocotrienols) and γ-oryzanol in rice. A complete separation of all compounds was achieved within 25 min using an Inertsil ® CN-3, SIL-100A 5 μM (4.6 mm × 250 mm) column and an isocratic elution system of hexane/isopropanol/ethylacetate/acetic acid (97.6:0.8:0.8:0.8, v/v/v/v) at a flow rate varying from 0.7 to 1.5 mL min −1. A linear correlation coefficient ( r 2 > 0.99) and high reproducibility were obtained at concentrations ranging 0.05–10 μg mL −1 for vitamin E isomers and 0.5–500 μg mL −1 for γ-oryzanol. This method proved to be rapid, accurate and reproducible.

Simultaneous determination of withanolide A and bacoside A in spansules by high-performance thin-layer chromatography

The objective of this work was to develop and validate a simple, rapid, precise, and accurate high performance thin layer chromatography method for simultaneous determination of withanolide A and bacoside A in combined dosage form. The stationary phase used was silica gel G60F(254). The mobile phase used was mixture of ethyl acetate: methanol: toluene: water (4:1:1:0.5 v/v/v/v). The detection of spots was carried out at 320 nm using absorbance reflectance mode. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 800 ng/spot for withanolide A and 50 to 350 ng/spot for bacoside A. The limit of detection and limit of quantification for the withanolide A were found to be 3.05 and 10.06 ng/spot, respectively and for bacoside A 8.3 and 27.39 ng/spot, respectively. The proposed method can be successfully used to determine the drug content of marketed formulation.

Development of a high‐performance liquid chromatography method for simultaneous determination of pioglitazone and felodipine in pig serum: application to pharmacokinetic study

A simple and sensitive high-performance liquid chromatographic method was developed and validated for simultaneous estimation of pioglitazone and felodipine in pig serum. The present method consists of protein precipitation, extraction of analytes from pig serum into dichloromethane and separation using reversed-phase C(18) column. Nitrendipine was used as an internal standard and the eluent was monitored by UV detector at 240 nm. The mobile phase used was acetonitrile and 50 mm ammonium acetate buffer at a flow rate of 1 mL/min. The retention times for pioglitazone, felodipine and nitrendipine were found to be 5.12, 10.53 and 7.14 min, respectively. The intraday and inter-day coefficient of variation and percent error values of assay method were less than 7% and mean recovery was more than 94% for each analyte, and the method was found to be precise, accurate and specific during study. The method was successfully applied for pharmacokinetic study of pioglitazone and felodipine from bioadhesive buccal tablet after buccal administration to pigs. The C(Max) , T(Max) , and AUC(0-24) of pioglitazone and felodipine from buccal tablet were found to be 394.6 ng/mL, 5.6 h, 2624.2 ng h/mL and 44.4 ng/mL, 5.5 h, 275.8 ng h/mL, respectively.