Bio-analytical method validation and its importance in pharma research - A review article

Bioanalytical method based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to give confidence in the results generated. It is the process used to establish that a quantitative analytical method is suitable for biomedical applications. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine is reliable and reproducible for the intended use. The present manuscript focuses on the consistent evaluation of the key bioanalytical validation parameters is discussed: accuracy, precision, sensitivity, selectivity, standard curve, limits of quantification, range, recovery and stability. These validation parameters are described, together with an example of validation methodology applied in the case of chromatographic methods used in bioanalysis, taking in account to the recent Food and Drug Administration (FDA) guidelines and EMA guid

A novel validated RP-HPLC method for the estimation of canagliflozin in bulk and pharmaceutical dosage forms

The objective of the present study was to develop a simple, specific and accurate reverse phase high performance liquid chromatographic method for the determination of Canagliflozin in bulk and pharmaceutical dosage forms. The method is optimized on an inertsil ODS-3(250.6mm, 5) column with a mobile phase combination of 0.02% Formic acid: Acetonitrile (40:60) at a flow rate 1.2ml/min and the eluents were monitored at 230nm. Under these LC conditions Canagliflozin peak was eluted at 4.4 min. The developed method was validated as per ICH guidelines. The calibration curve was linear over a concentration range of 10-50g/ml (R 2 =0.999) and the mean percentage assay was found to be 98.2. The statistical data proved that proposed method is accurate, precise and reproducible. The method which is LC-MS compatible can be adopted in the routine analysis of Canagliflozin in bulk and pharmaceutical dosage forms

Bioanalytical method validation of fenofibrate by hplc using human plasma

The bioanalytical method validation of fenofibrate was performed with all the basic parameter by HPLC using human plasma. This research paper describes a specific procedure for validation of Fenofibrate in human plasma. The mobile phase used was simple and column friendly and was sufficiently sensitive to quantify Fenofibrate in amounts as low as 0.095g/ml. This limit of quantitation (LOQ) was determined as the lowest concentration with a coefficient of variation lower than 20% and the total accuracy of the method ranged from 101.99 to 107.41%. The LOQ of this method is better than those of other reported methods. The calibration curve plotted concentration versus area and was linear from 0.095 g/ml to 19.924 g/ml, having r 2 greater than 0.98 during the course of validation. The above method is valid for the analysis of drug in human plasma. The method with slight modification could be used for the drug analysis in various dosage forms

RP-HPLC Method Development and Validation for Simultaneous Estimation of Duloxetin and Methylcobalamine in Combined Dosage Form

A simple, precise, accurate, simultaneous stability indicating RP-HPLC method for the estimation of DLU (Duloxetin) and MCB (Methylcobalamine) in combined dosage form was developed using Intersil-C18 (4.6 x 250mm, 5m) in an Isocratic mode with mobile phase comprising of Phosphate buffer (pH 4.5) The flow rate was 1 mL/ min and effluent was monitored at 255.0 nm. The retention times were found to be 5.32 min for DLU and 3.59 min for MCB. The assay exhibited a linear dynamic range of 20- 120 g/mL for DLU and 10- 60 g/mL for MCB. The calibration curves were linear (r 2 = 0.999 for DLU and r 2 = 0.999 for MCB) over the entire linear range. Mean % recovery was found to be 99.68 % for DLU and 100.3 % for MCB with % RSD was NMT 2 for both estimations which fully agrees with system suitability which is in good agreement with labeled amount of formulation. The % RSD for Intra- Day and Inter-Day Precision was NMT than 2 for both the drugs. The developed method was validated as per ICH guidelines

UV-Visible Spectrophotometric Method Development and Validation of Itraconazole in Bulk and Capsule Formulation

A simple, rapid, accurate and precise UV method was developed and validated for the estimation of Itraconazole in pharmaceutical dosage form. Spectroscopic method was carried out by using acidic ethanol as solvent. Itraconazole detection wavelength was set at 262nm for validation purpose linearity, accuracy, repeatability, precision, LOD, LOQ, and ruggedness parameters were studied. The linearity was found to be in the range of 2-12 g/ml

Brief review on analysis of Prazosin Hydrochloride

Prazosin is one of the alphaone adrenoreceptor blocker used in hypertension, benign prostatic hyperplasia, prostate cancer etc. The alpha blockers are relatively inexpensive and exert their effects quickly and Prazosin is the most commonly used alpha blocker. This review highlights various analytical methods for the determination of Prazosin hydrochloride in different matrices. Analytical methods reported are classified into four categories viz; spectrophotometry, chromatography, pharmacopoeial and other methods. The methods were described in terms of sensitivity (LOD and LOQ), linear range, principle and its applicability. This review also briefly highlights pharmacology of prazosin. This review is helpful for the researchers and scientists studying Prazosin hydrochloride in its analytical and pharmacological aspect

Stabilityindicating High Performance Thin Layer Chromatography /densitometry estimation of Formoterol fumarate dihydrate in bulk and capsules

Novel, simple, rapid and reliable High-Performance Thin-Layer Chromatographic (HPTLC) methods were developed and validated for the analysis of Formoterol Fumarate Dihydrate (FFD) in bulk and in in-house tablet formulation. HPTLC quantitation of FFD was done at UV detection of 281 nm and analysis was performed on (10 cm 10 cm) aluminium sheets precoated with silica gel 60-F 254 (E. Merck) as stationary phase and ethyl acetate: methanol: triethylamine (3.2:1.5:0.3 v/v ) as mobile phase. Quantitation by HPTLC method was performed over the concentration range of 400 - 900 ng/band. The HPTLC method resulted into compact and well resolved band for FFD at retention factor ( Rf ) of 0.51 0.3. Linear regression analysis data for calibration of HPTLC method represented good linear relationship with regression coefficient; r 2 = 0.999. The developed methods were validated for precision, robustness, ruggedness, accuracy, sensitivity as per guidelines laid by International Conference on Harmonisation (ICH). FFD was subjected to acid and alkali hydrolysis, oxidation, neutral, photo and thermal degradation. The drug undergoes degradation under acidic, basic, oxidation, and thermal conditions. This indicates that the drug is susceptible to acid, base, oxidation and thermal conditions. The degraded product was well resolved from the pure drug with significantly different R f value. Statistical analysis proved that the developed methods were precise, robust, sensitive and accurate and can be used effectively for the analysis of FFD in bulk and pharmaceutical formulations

Development and validation of a sensitive method for Levofloxacin in Gingival Crevicular Fluid by HPLC using UV - Visible detector

Increased interest in the clinical use of antibiotics for periodontal therapy required the development of a sensitive assay for the quantitation of levofloxacin in gingival crevicular fluid (GCF). The HPLC assay employs a C18 reversed-phase Hypersil BDS column with a mobile phase composed of methanol and phosphate buffer (pH 3.5). The chromatographic separation was monitored by a UV- Visible detector with an excitation wavelength of 290nm. The retention time of Levofloxacin and Ciprofloxacin were 5.55 min and 6.52min respectively. Levofloxacin was extracted from GCF collected on capillary tubes by addition of acetonitrile containing the internal standard ciprofloxacin, and phosphate buffer. The percentage mean extraction recovery of low, mid and high quality control samples was 89.53 0.91 % (Mean SD) for Levofloxacin and it was 91.2 2.2 % for Ciprofloxacin. The lowest limit of quantitation was 50 ng/ml, with a relative standard deviation of 2.56%. The interday and intraday precision at LLOQ was 3.20 0.80 (meanSD) and 3.505 0.84 (meanSD). The typical GCF volumes collected were 0.1-1 ml. The method was validated for the linear concentration range 50-1300 ng/ml of levofloxacin on the capillary tubes. This assay for levofloxacin was shown to be an accurate, precise and rugged method. The proposed method can be used for the estimation of Levofloxacin which was administered as in situ gels in periodontitis

Enantioselective method development and validation of proline by using high performance liquid chromatography

Chirality is a major concern in the modern pharmaceutical industry. The separation of chiral compounds has been of great interest because the majority of bioorganic and synthetic molecules are chiral. Aim of the present investigation was to develop a stereo specific, simple and precise normal phase high performance liquid chromatography (NP - HPLC) method for the separation and enantiopurity of Dextro (D) and Levo (L) enantiomers of proline (PRO) by using Lux 5m Amylose 1 LC column (250.6mm) by using n- Hexane: Iso propyl alcohol (IPA)as mobile phase in the ratios of 90:10 v/v at flow rate of 1.2 ml/ min. D and L forms of PRO was detected at 210nm with retention time of 8.1min and 9 min respectively with correlation coefficient (R 2 ) of 0.999.The method was validated with reference to International conference of harmonization (ICH) in terms of linearity, accuracy, precision (Inter - day and intra - day precision), limit of detection (LOD), limit of quantification (LOQ), stability of test solutions, specificity, system suitability, robustness and ruggedness

A validated stability-indicating HPLC assay method for Meclizine HCl in bulk drug

An isocratic reversed phase stability-indicating high-performance liquid chromatographic (HPLC) assay method was developed and validated for quantitative determination of Meclizine HCl in bulk drugs. An isocratic, reversed phase HPLC method was developed to separate the drug from the degradation products, using a Water Nova Pack C18 (250 x 4.6) mm, 5? column and the mobile phase containing 1.0 gm Sodium dihydrogen phosphate and 1.0 gm 1-octaneSulfonic acid salt in 1000ml water filter and mixed. Prepare a homogenous mixture of buffer, methanol and acetonitile. The detection was carried out at wavelength 264 nm. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, selectivity, robustness prove the stability indicating ability of the method