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.     

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.     

Individual Carotenoid Determinations in Human Plasma by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic technique is described to quantify beta carotene from alpha carotene and lycopene in human plasma. Total analysis time is 14.5 min. A reverse-phase column was employed with a mobile phase composed of 65% acetonitrile: tetrahydrofuran (90:10, v:v) in methanol. Use of the internal standard, beta-apo-8- carotenoic ethyl ester permitted a reliable way to quantify potential losses in plasma extractions. Plasma beta carotene levels obtained from subjects several days after supplement use were observed to increase three-fold or more.     

A Simple,Rapid, and Validated LC Method for the Estimation of Nimesulide in Human Serum and Its Application in Bioavailability Studies

Abstract

A new, simple, and rapid, sensitive reversed phase liquid chromatographic method was developed for the estimation of nimesulide in blood serum using a 60:40 mixture of acetonitrile and orthophosphoric acid (pH 3.0) as the mobile phase at 230 nm. The mobile phase and other chromatographic conditions were optimized to minimize interference from the serum matrix and at the same time provide sufficient sensitivity for the method to be adopted for in vivo studies of oral formulations of nimesulide. Acetonitrile was used to precipitate proteins from serum during sample preparation. Detector response was found to be linear in the region of 100–1000 ng/ml. The detection and quantitation limit, as per the error propagation theory, was found to be 50 ng/ml and 100 ng/ml, respectively. The linear equation obtained by the least square regression method was Area = 37.29 × Conc.(ng/ml)?1699.89 with a retention time of 3.97 ± 0.04 min. The results of the analysis were treated statistically, as per ICH guidelines for validation of analytical procedures, USP-2000. and by recovery studies. An internal standard was not employed in the method, as sample recoveries were in good agreement with their label claims. The results were found to be accurate, reproducible, and free from interference. The developed methods were further used for estimation of nimesulide for oral bioavailability of designed sustained release formulations of nimesulide.     

Development and validation of analytical method for estimation of bilastine in bulk and pharmaceutical (tablet) dosage form

BackgroundThe present work describe a simple, linear, precise, robust, accurate and selective HPLC method for estimation of Bilastine in bulk and tablet dosage form. Bilastine is a second generation antihistamine medication. Generally it is used for treatment of allergic rhinoconjunctivitis and urticaria (hives). Methanol: Acetonitrile: Phosphoric Acid Buffer pH 2.1 in proportion of (21:33:46) was used as mobile phase with flow rate 1.0 ​ml/min. The column used for the method development is 250 ​× ​4.6 mm ​× ​5 ​μm dimension.ResultIn the range of 5–25 ​μg/ml the linearity of Bilastine shows a correlation coefficient of 0.9981.ConclusionThe method was validated as per ICH guidelines for linearity, precision, accuracy, robustness.     

Liquid chromatography–tandem mass spectrometric assay for aliskiren,a novel renin inhibitor in micro‐volumes of human plasma: a pharmacokinetic application in healthy South Indian male subjects

This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of aliskiren in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from 100 μL of human plasma via liquid–liquid extraction using tert‐butyl methyl ether. The chromatographic separation was achieved on a C₁₈ column using a mixture of acetonitrile and 0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.10–1013 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 2.2 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing the productivity. The proposed method was found to be applicable to clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Simple Rapid Validated RP-LC Method for the Estimation of Flurbiprofen in Rabbit Serum and Aqueous Humor

Abstract

New reversed-phase liquid chromatographic methods, with UV detection, were developed for the quantitative estimation of flurbiprofen in rabbit blood serum and aqueous humor. The mobile phase and other chromatographic conditions were optimized to minimize interference from biological matrix and at the same time provide sufficient sensitivity for the method to be adopted for in vivo studies of ophthalmic formulations of flurbiprofen. Acetonitrile was used to precipitate proteins from serum or aqueous humor during sample preparation. A mobile phase of methanol: acetonitrile: phosphate buffer pH 5.6 (40:20:40) was employed with UV detection at 248 nm for estimation of drug in both the biological matrix. The retention time and asymmetry factor for the proposed method of estimation in serum and aqueous humor was found to be 3.1312±0.0101 min and 1.1310±0.0091 respectively. The linear regression equations obtained by least square regression method, were Area (µV sec) = 52.27 × Conc. (in ng/ml)–1618.70 in serum and Area (µV sec) = 61.79 × Conc. (in ng/ml) ? 783.24 in aqueous humor. The results of analysis were treated statistically, as per ICH guidelines for validation of analytical procedures, USP-2003, and by recovery studies. The results were found to be accurate, reproducible and free from interference. The developed methods were further used for estimation of flurbiprofen in rabbit serum and aqueous humor following single topical administration of in-house aqueous drop and market formulation to rabbit eye.     

Quick and Simple Simultaneous Determination for Some Amino Acids by Reversed-Phase HPLC with Uv Detection

Abstract

A simple and rapid high pressure liquid chromatographic method using an ultraviolet detector for simultaneous analysis of histidine, tyrosin and tryptophan, is presented.

Chromatographic separation is achieved on Spherisorb-5 RP-18 5μm reversed phase column and the mobile phase is the isocratic mixture of aceto-nitrile, methanol and water (5:30:65). the eluted amino acids are detected at 220 nm. the retention time is 1.55 min for histidine, 2.21 min for tyrosin and 2.80 min for tryptophan. the correlation of the integrated peak areas with the concentration of amino acids showed a linear relationship between 0.40 to 9.43 ppm for histidine, 0.24 to 22.6 ppm for tyrosin, and 0.20 to 12.8 ppm for tryptophan per 10μl injection.

Simultaneous analysis of histidine, tyrosin and tryptophan gave reproducible results with a mean coefficient of variation 1.93 pc for tryptophan, 2.29 pc for tyrosin and 3.51 pc for histidine and r² = 0.999. the proposed technique was applied to the analysis of these amino acids in urine samples.     

Determination of Ergotamine Tartarate and Cyclizine Hydrochloride in Pharmaceutical Tablets by Reverse Phase HPLC

Abstract

A high performance liquid chromatographic procedure is presented for the determination of ergotamine tartarate and cyclizine hydrochloride in pharmaceutical tablets. An aliquot of the sample is dissolved in methanol containing ethyl p-hydroxybenzoate as an internal standard and chromatographed on a 5 üm, C₁₈, Hibar pre-packed, Lichrospher (250 mm × 4.0 mm i.d.), column using a mobile phase of 0.01 M ammonium acetate in acetonitrile: water: triethylamine (35:64:1) solution, the pH was adjusted to 3.7 with glacial acetic acid. The method was tested for linearity, recovery, and specificity and was found to be fast, sensitive, accurate and reproducible with a total elution time of six min.     

Thin Layer Chromatographic Method for Rapid Quantification and Identification of Trimethoprim and Sulfamethoxazole in Pharmaceutical Dosage Forms

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of sulfamethoxazole (SMA) and trimethoprim (TMP) present in combined dosage forms were described. SMA and TMP were extracted with 90% acqueous methanol and the interfering and related contaminants were removed by thin layer chromatography (TLC) or high performance TLC (HPTLC) on silicagel plates using chloroform : isopropanol : diethylamine :: 10 : 6 : 1 (v/v) as mobile phase. Assay was done at the respective absorption maxima of the drugs by in situ densitometry and by spectroscopy after extracting the drugs from TLC plates with 90% acqueous ethanol. Results obtained by both the methods agreed well with those obtained by the method prescribed by the United States Pharmacopoeia XXI edition. Total time required for HPTLC and densitometric assay of 32 samples using 4 standards was 30 min. Probable source of errors in densitometric studies and their rectification was discussed.     

Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was 相似文献   

Routine high-performance liquid chromatographic determination of ascorbic acid in foods using L-methionine for the pre-analysis sample stabilization

The possibility of use of phosphoric acid (0.2% v/v, pH 2.1) in the mobile phase and co-existing compounds present in foods as the dissolving agent for the pre-analysis sample stabilization were examined for the routine determination of ascorbic acid (AA) in foods by high-performance liquid chromatography (HPLC) with electrochemical detection (ED). The applied potential was set at 400 mV versus an Ag/AgCl reference electrode. It was demonstrated that 0.2% v/v phosphoric acid was the useful mobile phase and l-methionine was the most effective dissolving agent for the pre-run sample stabilization of AA in foods after comparison with other amino acids and water-soluble vitamins. The proposed method was simple, rapid (retention time @ ca. 4 min), sensitive (detection limit: ca. 0.1 ng per injection (5 μl) at a signal-to-noise ratio of 3), highly selective and reproducible (relative standard deviation (R.S.D.); 2.5% (n=7), between-day R.S.D.: 3.7% (5 days)). The calibration graph of AA was linear in the range of 0.1-12.5 ng per injection (5 μl). Recovery of AA was over 90% by the standard addition method. Relationship between structure of compounds and the stability of AA was also examined.     

Triglyceride Assay by Amperometric Microbial Biosensor: Sample Hydrolysis and Kinetic Approach

ABSTRACT

A triglyceride assay based on triglyceride hydrolysis and glycerol detection was developed. Non-specific lipase isolated from Candida rugosa and intact Gluconobacter oxydans cells, containing membrane-bound glycerol dehydrogenase, were used to develop a biosensor. Two approaches were investigated: analysis of pre-hydrolysed samples and a kinetic approach. The sensor prepared from G. oxydans cells exhibited sensitive and fast response to glycerol: detection limit 20 μM (S/N=3), linear range up to 2 mM and response time 84 s (90% of steady-state). The triglyceride assay of pre-hydrolysed samples was based on a 20 min hydrolysis and determination of released glycerol by the biosensor. A calibration curve linear up to 12 mM was obtained for triolein samples. The kinetic approach was based on simultaneous glyceride hydrolysis and glycerol detection. Analysis time of 10 min, linear range up to 30 mM, and estimated detection limit of 50 μM were achieved using the kinetic approach. The kinetic triglyceride assay is not influenced by free glycerol present in a sample. Storage stability, expressed as a half life (50% of the initial response), was 7 days when trehalose was used as a stabiliser.     

Determination of Niacin in Cereal Samples by HPLC

Abstract

A liquid chromatographic method for the analysis of niacin in cereal products is presented. The sample is extracted in the presence of Ca(OH)₂, concentrated and purified on an anion exchange resin, and further cleaned up by oxidation with KMnO₄. Final quantitation is by HPLC with ultraviolet detection at 254 nm.

Using a μBondapak C₁₈ column, a mobile phase consisting of 5% methanol in water containing 0.005 M tetrabutyl ammonium phosphate, and a flow rate of 2.0 ml/min., niacin was found to have a retention time of about 7 minutes.     

A Simplex Optimized Colorimetric Method for Formaldehyde

Abstract

The development of a rapid and precise method for analyzing formaldehyde is described. By using an experiment design technique known as simplex optimization, the sensitivity for this procedure was maximized. The analysis requires under 2 minutes of the analyst's time per sample and gives an average coefficient of variation of 1.3 percent. The average sensitivity is 0.294 A/μg/ml and as little as 0.018 μg/ml can be detected with 90 percent confidence if triplicate analyses are performed.     

Determination of Pongamol and Karanjin in Karanja Oil By Reverse Phase Hplc

ABSTRACT

Pongamol and karanjin, naturally present in karanja (Pongamia glabra) oil possess potential UV A and UV B sunscreen activities, respectively. A reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for direct estimation of these constituents of karanja oil. This method of analysis requires no sample preparation of active ingredients. The analysis uses methanol, water and acetic acid in the ratio of 85: 13.5: 1.5 as mobile phase at a flow rate of 0.5 ml/ min on a Kromasil 100 C18 column, ex Tracer (250mm X46mm, particle size 5 microns) and dual wavelength detection at 350 and 300 nm. 2-ethylhexyl-p-methoxy cinnamate (Parsol MCX) was used as the internal standard. The identical UV spectrum of the chromatographic region (upslope, apex and downslope) confirmed that the analysis was free from substrate interference. The detection limits for pongamol and karanjin were found to be 0.1 μg/ml.     

High Performance Liquid Chromatographic Separation of Estradiol-17 α and -17/β in Biological Fluids; Application to Plasma,Milk and Urine of Cows

Abstract

A rapid HPLC technique was developed to separate estradiol epimers. In order to improve the sensitivity of the detection, a radioitmmunoassay was used.

Estrone, estradiol-17α and estradiol-17β were separated within 20 min using 10 ml of chloroform: acetone (90:10), as the mobile phase. The efficiency of the technique was assessed with ₃ steroids and the assay of collected fractions with antlsera specific to each estrogen. Using a non-specific radioimmunoassay, profiles of endogenous estrogens in different biological fluids (blood plasma, milk, urine) were obtained.

The efficiency of HPLC as a separation method and the high sensitivity of radioimmunoassay as a detector allows us to obtain profiles of estrogens from biological samples where steroid concentration is below lOOpg/ml.     

Analysis of Insulin by High Performance Liquid Chromatographic Method with Precolumn Derivatization with 4‐Chloro‐7‐Nitrobenzo‐2‐Oxa‐1,3‐Diazole

Abstract

A high performance liquid chromatographic method (HPLC) with precolumn derivatization and fluorescence detection for insulin was developed and applied for the quantification of insulin in spiked serum. To covalence couple with insulin, 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) was selected as fluorescent reagent. The optimal derivatization conditions were as follows: temperature 50; time 2 h, in the dark; 0.1 M phosphate buffer (pH 9.0). Analytical separation was carried out on a C18 column and the mobile phase including acetonitrile‐water containing 0.1% trifluoroacetic acid (TFA) (v/v∶ 30/70). The excitation/emission wavelengths were 470/540 nm. Under the conditions, the retention time and capacity factor of the adduct of insulin‐NBD were 10.03 min (flow rate 1 mL/min) and 3, respectively. The recovery of insulin in serum was 95.06% and the detection limit was 90 nM. In the investigated concentration ranges (0.46 µM～16.10 µM), R² was 0.9934, which indicated the potential for the application of NBD‐Cl derivatization to the analysis of insulin in the biological matrices, although with the shortcoming of long analytical time.