Simultaneous determination of anti-diabetic drugs

A novel reverse phase, isocratic HPLC method is described to separate five anti-diabetic drugs i.e., glimepiride, metformin, sitagliptin, rosiglitazone and pioglitazone. Nucleosil C18 analytical column was used as stationary phase, while mobile phase consisted of acetonitrile:phosphate buffer: methanol (40/40/20, v/v) pH 2.0. Effluent was monitored at a flow rate 1 mL/min and detected at wavelength of 240 nm. This research produced excellent chromatography over a wide concentration range of 25-10000 ng/mL. Sepprated and well resolved quantifiable peaks were obtained and test results were linear in this range. Correlation coefficient of more than 0.9990 was witnessed as well as Low %RSD values i.e., maximum 2.0% documented excellent precision of the method. Good recoveries from pharmaecutical (99-101%), urine and plasma samples (>96%) in a range of concentrtion granted very good linearity, accuracy and precision. The projected method has satisfactory applications in quality control of these molecules as well as quantification of these molecules in urine and plasma samples.

Development, Validation and Application of RP-HPLC Method: Simultaneous Determination of Antihistamine and Preservatives with Paracetamol in Liquid Formulations and Human Serum.

In this article we describe development and validation of stability indicating, accurate, specific, precise and simple Ion-pairing RP-HPLC method for simultaneous determination of paracetamol and cetirizine HCl along with preservatives i.e. propylparaben, and methylparaben in pharmaceutical dosage forms of oral solution and in serum. Acetonitrile: Buffer: Sulfuric Acid (45:55:0.3 v/v/v) was the mobile phase at flow rate 1.0 mL min(-1) using a Hibar(®) Lichrosorb(®) C18 column and monitored at wavelength of 230nm. The averages of absolute and relative recoveries were found to be 99.3%, 99.5%, 99.8% and 98.7% with correlation coefficient of 0.9977, 0.9998, 0.9984, and 0.9997 for cetirizine HCl, paracetamol, methylparaben and Propylparaben respectively. The limit of quantification and limit of detection were in range of 0.3 to 2.7 ng mL(-1) and 0.1 to 0.8 ng mL(-1) respectively. Under stress conditions of acidic, basic, oxidative, and thermal degradation, maximum degradation was observed in basic and oxidative stress where a significant impact was observed while all drugs were found almost stable in the other conditions. The developed method was validated in accordance with ICH and AOAC guidelines. The proposed method was successfully applied to quantify amount of paracetamol, cetirizine HCl and two most common microbial preservatives in bulk, dosage form and physiological fluid.

Pregabalin and tranexamic Acid evaluation by two simple and sensitive spectrophotometric methods.

This paper demonstrates colorimetric visible spectrophotometric quantification methods for amino acid, namely, tranexamic acid and pregabalin. Both drugs contain the amino group, and when they are reacted with 2,4-dinitrophenol and 2,4,6-trinitrophenol, they give rise to yellow colored complexes showing absorption maximum at 418 nm and 425 nm, respectively, based on the Lewis acid base reaction. Detailed optimization process and stoichiometric studies were conducted along with investigation of thermodynamic features, that is, association constant and standard free energy changes. The method was linear over the concentration range of 0.02-200 µgmL(-1) with correlation coefficient of more than 0.9990 in all of the cases. Limit of detection was in range from 0.0041 to 0.0094 µgmL(-1) and limit of quantification was in the range from 0.0137 to 0.0302 µgmL(-1). Excellent recovery in Placebo spiked samples indicated that there is no interference from common excipients. The analytical methods under proposal were successfully applied to determine tranexamic acid and pregabalin in commercial products. t-test and F ratio were evaluated without noticeable difference between the proposed and reference methods.

Development of new method for simultaneous analysis of piracetam and levetiracetam in pharmaceuticals and biological fluids: application in stability studies.

RP-HPLC ultraviolet detection simultaneous quantification of piracetam and levetiracetam has been developed and validated. The chromatography was obtained on a Nucleosil C18 column of 25 cm×0.46 cm, 10 µm, dimension. The mobile phase was a (70:30 v/v) mixture of 0.1 g/L of triethylamine and acetonitrile. Smooth flow of mobile phase at 1 mL/min was set and 205 nm wavelength was selected. Results were evaluated through statistical parameters which qualify the method reproducibility and selectivity for the quantification of piracetam, levetiracetam, and their impurities hence proving stability-indicating properties. The proposed method is significantly important, permitting the separation of the main constituent piracetam from levetiracetam. Linear behavior was observed between 20 ng/mL and 10,000 ng/mL for both drugs. The proposed method was checked in bulk drugs, dosage formulations, physiological condition, and clinical investigations and excellent outcome was witnessed.

Dual Wavelength RP-HPLC Method for Simultaneous Determination of Two Antispasmodic Drugs: An Application in Pharmaceutical and Human Serum.

A reverse phase stability indicating HPLC method for simultaneous determination of two antispasmodic drugs in pharmaceutical parenteral dosage forms (injectable) and in serum has been developed and validated. Mobile phase ingredients consist of Acetonitrile : buffer : sulfuric acid 0.1 M (50 : 50 : 0.3 v/v/v), at flow rate 1.0 mL/min using a Hibar µ Bondapak ODS C18 column monitored at dual wavelength of 266 nm and 205 nm for phloroglucinol and trimethylphloroglucinol, respectively. The drugs were subjected to stress conditions of hydrolysis (oxidation, base, acid, and thermal degradation). Oxidation degraded the molecule drastically while there was not so much significant effect of other stress conditions. The calibration curve was linear with a correlation coefficient of 0.9999 and 0.9992 for PG and TMP, respectively. The drug recoveries fall in the range of 98.56% and 101.24% with 10 pg/mL and 33 pg/mL limit of detection and limit of quantification for both phloroglucinol and trimethylphloroglucinol. The method was validated in accordance with ICH guidelines and was applied successfully to quantify the amount of trimethylphloroglucinol and phloroglucinol in bulk, injectable form and physiological fluid. Forced degradation studies proved the stability indicating abilities of the method.

Development and validation of new assay method for the simultaneous analysis of diltiazem, metformin, pioglitazone and rosiglitazone by RP-HPLC and its applications in pharmaceuticals and human serum.

Simple, sensitive, rapid, and accurate high-performance liquid chromatographic (HPLC) method is developed and validated for the simultaneous determination of diltiazem, metformin, pioglitazone, and rosiglitazone hydrochloride in raw materials, their pharmaceutical formulations, and human serum. In HPLC, all the above drugs were chromatographed using acetonitrile-methanol-water (30:20:50, v/v, pH 2.59 ± 0.02) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The separation is carried out on a Hiber, 250-4.6 RP-18 column, equipped with a UV-vis detector at 230 nm. All the antidiabetic drugs eluted at different retention time and each showed a good resolution from diltiazem. The method is successfully applied to pharmaceutical formulations because no chromatographic interferences from the tablet excipients are found. The method is found to be linear, accurate, and precise with apposite detection and quantification limit. Suitability of the method for the quantitative determination of the drugs is proven by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines. The validation results, together with statistical treatment of the data, demonstrated the reliability of this method.

Development and validation of stability-indicating HPLC method for the simultaneous determination of paracetamol, tizanidine, and diclofenac in pharmaceuticals and human serum.

A method is described for the simultaneous determination of paracetamol, tizanidine, and diclofenac in mixtures. The method was based on HPLC separation of the three drugs followed by UV detection at 254 nm. The separation was carried out on a Hypersil ODS, C18 (250 x 4.6 mm id, 10 microm particle size) column using the mobile phase aqueous 0.2% ammonium carbonate-methanol (60 + 40, v/v) at a flow rate of 1 mL/min. The linear regression analysis data were used for the regression curve in the range of 170-10 000 ng/mL for paracetamol, 120-10 000 ng/mL for tizanidine, and 20-10 000 ng/mL for diclofenac. No chromatographic interference from tablet excipients was found. In order to check the selectivity of the proposed method, degradation studies were carried out using hydrolysis (acid, basic, and neutral), thermolysis, and oxidation. The developed method, after being validated in terms of precision, robustness, recovery, LOD, and LOQ, was successively applied to the analysis of pharmaceutical formulations and human serum.

Simultaneous determination of piracetam and its four impurities by RP-HPLC with UV detection.

A simple and rapid high-performance liquid chromatographic method for the separation and determination of piracetam and its four impurities, 2-oxopyrrolidin-1-yl)acetic acid, pyrrolidin-2-one, methyl (2-oxopyrrolidin-1-yl)acetate, and ethyl (2-oxopyrrolidin-1-yl)acetate, was developed. The separation was achieved on a reversed-phase C(18) Nucleosil column (25 cm x 0.46 cm, 10 microm). The mobile phase is composed of an aqueous solution containing 0.2 g/L of triethyl amine-acetonitrile (85:15, v/v). The pH of the mobile phase was adjusted to 6.5 with phosphoric acid at a flow rate of 1 mL/min at ambient temperature and UV detection at 205 nm. The developed method was found to give good separation between the pure drug and its four related substance. The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 50-10,000 ng/mL, 25-10,000 ng/mL, 45-10,000 ng/mL, 34-10,000 ng/mL, and 55-10,000 ng/mL, respectively, with r(2) = 0.9999. The method was validated for precision, accuracy, ruggedness, and recovery. The minimum quantifiable amounts were found to be 50 ng/mL of piracetam, 25 ng/mL of 2-oxopyrrolidin-1-yl)acetic acid, 45 ng/mL of pyrrolidin-2-one, 34 ng/mL of methyl (2-oxopyrrolidin-1-yl)acetate, and 55 ng/mL of ethyl (2-oxopyrrolidin-1-yl)acetate. Statistical analysis proves that the method is reproducible and selective for the estimation of piracetam as well as its related substance. As the method could effectively separate the drug from the related substances, it can be employed as a stability-indicating one. The proposed method shows high efficiency, allowing the separation of the main component piracetam from other impurities.

Simultaneous determination of metformin, cimetidine, famotidine, and ranitidine in human serum and dosage formulations using HPLC with UV detection.

A new, simple, and reliable reversed-phase high-performance liquid chromatographic method has been developed and validated for the simultaneous determination of metformin (Metf), cimetidine (Cimt), famotidine (Famt), and ranitidine (Rant) in their synthetic mixtures and tablet formulations. These drugs were separated on a Purospher Star RP18 endcapped (250 mm × 4.6 mm i.d.) column packed with 5-µm particles. The mobile phase, optimized through an experimental design, consisted of methanol-water-triethylamine (20:80:0.05), whose pH was adjusted to 3.0 with phosphoric acid (85%) pumped at a flow rate of 1.0 mL/min. UV detection was performed at 229 nm. The method was validated in the sample concentration range of 5-25 µg/mL for all the drugs, where it demonstrated good linearity with r = 0.9998, 0.9979, 0.9997, and 0.9987 (n = 6), respectively. For independent 100% level samples, the intra-day and inter-day precision was in the range i.e. < 2.0 for all the drugs. The method demonstrated robustness, resisting to small deliberate changes in pH, flow rate, and composition (organic:aqueous ratio) of the mobile phase. The limit of detection values were 0.071, 0.116, 0.134, and 0.110 µg/mL, while the limit of quantitation were 0.217, 0.352, 0.405, and 0.368 µg/mL for Metf, Cimt, Famt, and Rant, respectively. The applicability of the method was demonstrated by determining the drug content in pharmaceutical formulations, where it exhibited good performance.

Simultaneous determination of gliquidone, fexofenadine, buclizine, and levocetirizine in dosage formulation and human serum by RP-HPLC.

In the present paper, a simultaneous method has been developed and validated for estimation of gliquidone in the presence of H(1)-receptor antagonists (fexofenadine hydrochloride, buclizine hydrochloride, and levocetirizine dihydrochloride) using reversed-phase high-performance liquid chromatographic technique. A good chromatographic separation between these drugs was achieved using a mobile phase containing methanol-water (80:20 v/v) at pH 3.5 with a flow rate of 1.0 mL/min; and detection was performed at 230 nm with a UV detector. Validation of the method was performed in terms of linearity, accuracy, precision, and limit of detection and quantification. The linearity of the calibration curves for gliquidone, fexofenadine hydrochloride, buclizine hydrochloride, and levocetirizine dihydrochloride were found to be 0.338-50 microg/mL (r = 0.9964), 5-50 microg/mL (r = 0.9956), 0.325-50 microg/mL (r = 0.9967), and 0.553-50 microg/mL (r = 0.9950), respectively. There was no significant difference between the amount of drug spiked in serum and the amount recovered, and serum did not interfere in simultaneous estimation. Thus, the proposed method is suitable for the simultaneous analysis of active ingredients in tablet dosage forms and human serum.

Spectrophotometric determination of gabapentin in pharmaceutical formulations using ninhydrin and pi-acceptors.

Simple, rapid and sensitive spectrophotometric procedures are developed for the analysis of gabapentin in pure form as well as in their pharmaceutical formulations. The methods are based on the reaction of gabapentin as n-electron donor with ninhydrin and pi-acceptors namely, 2,3,5,6-tetrachloro-1,4-benzoquinone, chloranilic acid, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, tetracyanoethylene and 7,7,8,8-tetracyanoquinodimethane. The obtained complexes were measured at 568, 230, 314, 304, 335 and 439 nm for ninhydrin, chloranil, Chloranilic acid, DDQ, TCNE and TCNQ respectively. The proposed procedures could be successfully applied to the determination of gabepentin with good recovery; percent ranged from 99.3 to 100.7 The association constants and free energy changes using Benesi-Hildebrand plots are also studied.

In vitro hypoglycemic activity of methanolic extract of some indigenous plants.

Pakistan is rich in medicinally important plants and has ancient herbal treatment methods. Present work is based on the study of six indigenous plants Eugenia jambolana, Lawsonia inermis, Momordica charantia, Morus alba, Nigella sativa and Trigonella foenum graecum which show the inhibitory effect of glucose utilization, and are in use as hypoglycemic agents of varying degree in traditional system of medicine. The glucose uptake activity of (methanolic extracts) of these plants was tested in vitro and glucose was estimated by glucose oxidase method. The results in three different media revealed that, hypoglycemic activity is more prominent in neutral and basic media as compared to acidic medium.

Optimization of levofloxacin analysis by RP-HPLC using multivariate calibration technique.

A rapid and sensitive reverse phase high performance liquid chromatographic (RP-HPLC) method for the analysis of levofloxacin from bulk materials, dosage formulations and human serum is described. This isocratic method employs, a Nucleosil, C18 (10 microm, 25 cm x 0.46 cm) column with a mobile phase of water and acetonitrile (6:5), where in phosphoric acid was used to adjust the pH to 2.9 and propylparaben as an internal standard. Optimization of levofloxacin analysis was carried out using multivariate calibration technique and detector response was recorded at five different wave lengths. A linear response (r > 0.9999) was observed in the range of 40 to 10000 ng ml(-1). The method shows good recoveries, intra and inter-day relative standard deviations were less than 1.2 %. Validation parameters as specificity, accuracy and robustness were also determined. The method can conveniently be used for analysis of levofloxacin pharmacokinetic levels in human serum and pharmaceutical formulations.

Spectrophotometric methods for the simultaneous analysis of meclezine hydrochloride and pyridoxine hydrochloride in bulk drug and pharmaceutical formulations.

Three new spectrophotometric procedures for the simultaneous determination of pyridoxine hydrochloride and meclezine hydrochloride are described. The first method depends on the application of simultaneous equation to resolve the interference due to spectral overlapping. The analytical signals were measured at 231 and 220 nm. Calibration graphs were established for 1 to 20 microGmL(-1) for pyridoxine hydrochloride and 0.5 to 10 microGmL(-1) for meclezine hydrochloride in binary mixture. In the second method, the determination of pyridoxine hydrochloride and meclezine hydrochloride was performed by measuring the absorbances at 290 and 235 nm in the simple absorbance spectra of their mixture. In third method a yellowish orange complex of pyridoxine hydrochloride was formed with ferric chloride, which absorbs in the visible region with lambda(max) at 445 nm. Calibration curve of complex formation range was conducted in between 20 to 250 microGmL(-1). These methods were validated with respect to accuracy, precision, linearity, limit of detection and quantification. Regression analysis of Beer's plot showed good correlation in a general concentration range of 1 to 20 microGml(-1) with correlation coefficient (r = 0.9999 and 0.9999; CV < 0.858) for pyridoxine hydrochloride, whereas meclezine hydrochloride concentration range 0.5 to 10 microGmL(-1) with correlation coefficient (r = 0.9998 and 0.9998; CV < 0.826). These methods can be readily applied, without any interference from the excipients. The suggested procedures were successfully applied to the determination of these compounds in synthetic mixtures and in pharmaceutical preparations, with high percentage of recovery, good accuracy and precision.

Quantitation of buclizine hydrochloride in pharmaceutical formulations and human serum by RP-HPLC.

An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 230 nm has been developed for the determination of buclizine hydrochloride in human serum and dosage formulation. Methylparaben was successfully used as an internal standard. Good chromatographic separation between buclizine and internal standard peaks was achieved by using a stainless steel analytical column Nucleosil, C18 (10 microm, 25 cm x 0.46 cm). The system was operated at room temperature using a mobile phase consisting of acetonitrile-water (1:1) (pH 2.6) with phosphoric acid 85% at a flow rate of 2 ml/min. The calibration curve for buclizine hydrochloride in human serum was linear over the tested concentration range of 10, 3, 1.5, 0.5, 0.15, 0.05, and 0.025 microg/ml with a correlation coefficient of 0.9999. The intra- and inter-run precision and accuracy results were 98.07 to 100.34. The proposed method was validated for selectivity, linearity, accuracy, and precision. The method was found to be suitable for the quality control of buclizine hydrochloride in bulk drug as well as in human serum.

Determination and quantification of cetirizine HCl in dosage formulations by RP-HPLC.

A simple, sensitive, reliable and rapid HPLC method for the determination of cetirizine hydrochloride using hyoscine butyl bromide as an internal standard has been developed. The chromatographic system consisted of Shimadzu LC-10 AT VP pump, SPD-10 AV VP with UV/visible detector and a CBM-102 Bus Module integrator. Separation was achieved on the U Bondapak 125 A C18 10 microm column at room temperature. The samples were introduced through an injector valve with a 10 microl sample loop. Acetonitrile-water (1:1 v/v) was used as mobile phase, with flow rate 2 ml/minute. pH was adjusted to 2.9 with phosphoric acid. UV detection was performed at 205 nm. The results obtained showed a good agreement with the declared content. Recovery values for cetirizine hydrochloride were 99.19-100.82%. The proposed method is reliable rapid, precise, selective and may be used for the quantitative analysis of cetirizine HCl, in presence of hyoscine butyl bromide as internal standard. The method was valid was for the determination in raw materials, bulk drug and formulations. The limit of quantification was 5-30 nano grams, while the limit of detection was 0.4 nano grams.

In vitro availability of trimethylphloroglucinol and its degradation product from dosage formulations by RP-HPLC.

A simple, sensitive, reliable, and rapid high-performance liquid chromatographic method for the simultaneous determination of phloroglucinol and trimethyl phloroglucinol has been developed. Acetonitrile-water (1:1 v/v) was used as mobile phase, with flow rate 2 ml/minutes. pH was adjusted to 3 with phosphoric acid. U.V detection was performed at 242 nm. The results obtained showed a good agreement with the declared content. Recovery values for phloroglucinol were from 99.91% to 100.62% and recovery values for trimethylphloroglucinol were from 98.44% to 100.04%. The proposed method is rapid, accurate, and selective, it may be used for the quantitative analysis of phloroglucinol and trimethylphloroglucinol. The method was found to be specific, accurate, precise and reliable for the determination of phloroglucinol and trimethylphloroglucinol in the form of raw materials, in bulk drugs and formulation. It was possible to determine both phloroglucinol and trimethylphloroglucinol in the concentration range of 5 nano gram to 30 nano grams. The detection limit of both phloroglucinol and trimethylphloroglucinol were 0.4 nano gram.

A new RP-HPLC method for analysis of meloxicam in tablets.

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination and quantification of meloxicam has been developed. The chromatographic system consisted of a Shimadzu LC-10 AT VP pump, SPD-10 AV VP UV/ visible detector, and a CBM-102 Bus Module integrator. Separation was achieved on the micro Bondapak 125 A C18 10 microm column at room temperature. The sample was introduced through an injector valve with a 10 microl sample loop. Methanol:water (70:30 v/v) was used as mobile phase, with flow rate 2 ml/minutes. pH was adjusted to 2.6 with phosphoric acid. U.V detection was performed at 230 nm. The results obtained showed a good agreement with the declared content. Recovery values of meloxicam in tablets (in Melfax 15 mg tablets) were from 99.27 % to 100.06 %. Piroxicam was used as an internal standard. The proposed method is rapid, accurate and selective; it may be used for the quantitative analysis of meloxicam from raw materials, in bulk drugs and other dosage formulations.

A new RP-HPLC method for analysis of mebeverine hydrochloride in raw materials and tablets.

A simple, sensitive, selective, reliable, least time consuming and rapid high-performance liquid chromatographic method for the determination and quantification of mebeverine hydrochloride using hyoscine butylbromide as internal standard has been developed. The chromatographic system consisted of a Shimadzu LC-10 AT VP pump, SPD-10 AV VP UV visible detector, and a CBM-102 Bus Module integrator. Separation was achieved on the micro Bondapak 125 a C18 10microm column at room temperature. The samples were introduced through an injector valve with a 10 microl sample loop. Acetonitrile-water (1:1 v/v) was used as mobile phase, with flow rate 1.7 ml/minutes. pH was adjusted to 2.9 with phosphoric acid. U.V detection was performed at 205 nm. The results obtained showed a good agreement with the declared content. Recovery values of mebeverine hydrochloride were from 99.80% to 100.13%. The proposed method is rapid, accurate, and selective and may be used for the quantitative analysis of mebeverine hydrochloride. The method was found to be specific, accurate, precise and reliable for the determination and quantification of mebeverine hydrochloride in form of raw materials, in bulk drugs and formulation. It was possible to determine all of them in the concentration range of 5-30 nano grams. The detection limit of mebeverine hydrochloride was 0.4-nano gram.