ENCLOSURE-I
6. BRIEF RESUME OF INTENDED WORK
6.1 Introduction
Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results which is an integral part of any good analytical practice.
Analytical methods need to be validated or revalidated.
· before their introduction into routine use;
· whenever the conditions change for which the method has been validated (e.g., an instrument with different characteristics or samples with a different matrix); and
· whenever the method is changed and the change is outside the original scope of the method.
Method validation has received considerable attention in the literature and from industrial committees and regulatory agencies.
· The U.S. FDA CGMP guidelines are specified in section 211.165 (e) methods to be validated: The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented. Such validation and documentation may be accomplished in accordance with Sec. 211.194(a). These requirements include a statement of each method used in testing the sample to meet proper standards of accuracy and reliability, as applied to the tested product. The U.S. FDA has also proposed an industry guidance for Analytical Procedures and Methods Validation.
· ISO/IEC 17025 consist of the validation of methods with a list of nine validation parameters. The ICH (4) has developed a consensus text on the validation of analytical procedures. The document includes definitions for eight validation characteristics. ICH also developed a guidance with detailed methodology.
· The U.S. EPA prepared a guidance for method’s development and validation for the Resource Conservation and Recovery Act (RCRA). The Association of official agricultural chemists (AOAC), the Environmental protection agency’s (EPA) and other scientific organizations provide methods that are validated through multi-laboratory studies.
6.2 Need of the study
“Process Validation is establishing documented evidence which provides a high degree of assurance that a specified process will consistently produce a product meeting its pre-determined specifications and quality characteristics.”
There are certain benefits of validation that the validation process will lead to quality improvement, in addition to better quality assurance. To optimize the process for maximum efficiency while maintaining the quality standards. Validation is considered to be an integral part of GMP’s essentially worldwide compliance with validation requirements which is necessary for obtaining approval to manufacture and to introduce new product. Oral dosage forms, among the most widespread drugs, have been particularly targeted by counterfeiters. World Health Organization emphasizes the need for development and distribution of screening methods explicitly targeted to counterfeit drugs for the simultaneous analysis of some of the most common and counterfeited essential oral dosage form. A full validation has to be performed in terms of linearity, precision, robustness and trueness; an assessment of uncertainty should be carried out exploiting these data. A wide linearity range has to be investigated considering the specific nature of counterfeit and sub-standard drugs, whose content of active substance may be rather far from the declared amount. A large span in robustness parameters has to be considered and a complete intermediate precision assessment conducted, envisaging the possibility of transferring the method to quality control laboratories, hopefully in developing countries. Finally, the method should be successfully applied to the analysis of oral dosage forms.
ENCLOSURE- II
6.3 Review of literature
Shewiyo DH et.al.,1 reported a pneumocystis carinii pneumonia (PCP) is often the ultimate mortal cause for immunocompromised individuals in HIV/AIDS patients. Currently, the most effective medicine for treatment and prophylaxis is co-trimoxazole, a synergistic combination of Sulfamethoxazole (SMX) and Trimethoprim (TMP). In order to ensure a continued availability of high quality co-trimoxazole tablets in poor countries, the Regulatory Authorities must perform quality control of these products. However, most pharmacopoeial methods are based on high-performance liquid chromatographic (HPLC) methods. Because of the lack of equipment, the Tanzania Food and Drugs Authority (TFDA) laboratory decided to develop and validate an alternative method of analysis based on the TLC technique with densitometric detection, for the routine quality control of co-trimoxazole tablets were separated on glass backed silica gel 60 F254 plates in a high-performance thin layer chromatograph (HPTLC). The mobile phase comprised of toluene, ethylacetate and methanol (50:28.5:21.5,). Detection wavelength was 254 nm. The Rf values were 0.30 and 0.61 for TMP and SMX, respectively. This method was validated for linearity, precision, trueness, specificity and robustness. The F-tests for lack-of-fit indicated that straight lines were adequate to describe the relationship between spot areas and concentrations for each compound. The percentage relative standard deviations for repeatability and time different precisions were 0.98 and 1.32, and 0.83 and 1.64 for SMX and TMP, respectively. Percentage recovery values were 99.00% ±1.83 and 99.66% ±1.21 for SMX and TMP, respectively. The method was found to be robust and was successfully applied to analyze co-trimoxazole tablet samples.
Ulu ST.,et.al2 developed a simple, rapid, accurate, precise and sensitive colorimetric method for the determination of Finasteride tablets. The proposed methods are based on the formation of ion-pair complexes between the examined drug with bromophenol blue (BPB), bromocresol green (BCG) and bromothymol blue (BTB), which can be measured at the optimum λ max. Beer’s law is obeyed in the concentration ranges 3.0–15.0, 3.0–15.0 and 5.0–20μg/mL with BPB, BCG and BTB, respectively. The detection limits of FIN was found to be 1.16 μg/mL for BPB, 1.17 for BCG, 1.76μg/mL for BTB. All the methods gave similar results and were validated for selectivity, linearity, precision and sensitivity. The proposed methods were directly and easily applied to the pharmaceutical preparation with accuracy, resulting from recovery experiments between 100.11 and 100.33% for BPB, 100.17 and 100.67% for BCG and 100.33 and 100.60% for BTB methods. The low relative standard deviation values indicate good precision and high recovery values indicate accuracy of the proposed methods. The proposed methods have been applied to the determination of drug in commercial tablets. Results obtained from the analysis of commercial preparations with the proposed methods are in good agreement with those obtained with the official HPLC method.
Tzanavaras PD et.al.,3 reported that high sample analysis rate is a key demand in modern pharmaceutical analysis, especially during new product development and validation of industrial-scale manufacturing process. The study reports a validated HPLC assay for the dissolution studies of nimesulide-containing tablets. Using a 50mm×4.6mm i.d. monolithic column and acetonitrile-phosphate buffer (pH 7.0; 10 mM) (34:66) as the mobile phase, the separation cycle was completed in 60 s at a flow rate of 4.0 ml min−1. The assay was validated in terms of selectivity against potential impurities of the active ingredient, detection and quantification limits, linearity, accuracy and inter-/intra-day precision. Results from the application of the HPLC method to the accelerated and long-term dissolution stability control of tablets are reported.
Jadhav AS et.al.,4 developed a chiral high performance liquid chromatographic method and validated for the enantiomeric resolution of Valacyclovir, l-valine 2-[(2-amino-1,6-dihydro-6-oxo-9h-purin-9-yl) methoxy] ethyl ester, an antiviral agent in bulk drug substance. The enantiomers of Valacyclovir were resolved on a Chiralpak AD (250mm×4.6 mm, 10µm) column using a mobile phase system containing n-hexane: ethanol: diethylamine (30:70:0.1). The resolution between the enantiomers was found not less than four. The presence of diethylamine in the mobile phase played an important role in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The limit of detection and limit of quantification of (d)-enantiomer were found to be 300 and 900 ng/ml, respectively, for 20 µL injection volume. The calibration curve showed excellent linearity over the concentration range of 900 ng/ml (LOQ) to 6000 ng/ml for (d)-enantiomer. The percentage recovery of (d)-enantiomer ranged from 97.50 to 102.18 in bulk drug samples of Valacyclovir. Valacyclovir sample solution and mobile phase were found to be stable for atleast 48hr. The proposed method was found to be suitable and accurate for the quantitative determination of (d)-enantiomer in bulk drugs substance. It can be also used to test the stability samples of Valacyclovir.
Snski R et.al.,5 developed nine accurate methods for determination of Amisulpride in tablets: reversed phase high pressure liquid chromatography (RP-HPLC), aqueous capillary electrophoresis (CE), non-aqueous CE, normal phase (NP) and reversed-phase (RP) high performance thin layer chromatography (HPTLC) with densitometry and video densitometry, and direct and derivative UV spectrophotometry and validated the method. The HPLC method was carried out using Nova-Pak C8 column and mobile phase consisted of acetonitrile–methanol phosphate buffer pH 4.50 (15:5:80) with flow rate 1mLmin−1and UV detection at 225 nm. The moclobemide was used as the internal standard. CE was performed using 75 µm × 82 cm fused silica capillary. Detection was carried out at 225 nm. For aqueous analysis, the 30mM phosphate buffer pH 6.00, 30 kV voltage and 30°C temperature were chosen, non-aqueous determination was performed with ammonium acetate 1mM in acetonitrile–methanol (1:1), 30 kV voltage and 25°C temperature. NP-HPTLC was carried out using HPTLC silica F254 plates, developed with hexane–ethanol–propylamine (5:5:0.1) through 9 cm distance. RP-HPTLC was developed with HPTLC RP8F254 plates, with mobile phase of tetrahydrofuran-phosphate buffer pH 3.50 (4:6), distance 4.5 cm. Both analyses were performed in horizontal chambers and scanned with densitometer at 275 nm or video densitometer at 254 nm. UV spectrophotometry was carried out in methanol, using 224 nm for direct assay and 258 nm for derivative assay. The precision and accuracy of all the methods were complexively compared. The highest accuracy was observed in RP-HPTLC.. The differences were not significant, so all the elaborated methods can be used in routine analysis.
Fazio TT et.al.,6 developed a cleaning validation as an integral part of current good manufacturing practices in any pharmaceutical industry. Azathioprine and several other pharmacologically potent pharmaceuticals are manufactured in same production area. Carefully designed cleaning validation and its evaluation can ensure that residues of azathioprine will not carry over and cross contaminate the subsequent product. The aim of this study was to validate simple analytical method for verification of residual azathioprine in equipments used in the production area and to confirm efficiency of cleaning procedure. The HPLC method was validated on a LC system using Nova-Pak C18 (3.9mm×150 mm, 4 μm) and methanol–water–acetic acid (20:80:1, v/v/v) as mobile phase at a flow rate of 1.0mL min−1. UV detection was made at 280 nm. The calibration curve was linear over a concentration range from 2.0 to 22.0 µgmL−1 with a correlation coefficient of 0.9998. The detection limit (DL) and quantitation limit were 0.09 and 0.29 µgmL−1, respectively. The intra-day and inter-day precision expressed as relative standard deviation (R.S.D.) were below 2.0%. The mean recovery of method was 99.19%. The mean extraction-recovery from manufacturing equipments was 83.5%. The developed UV spectrophotometric method could only be used as limit method to qualify or reject cleaning procedure in production area. The simplicity of spectrophotometric method makes it useful for routine analysis of azathioprine residues on cleaned surface and as an alternative to proposed HPLC method.
Raman NVVSS et.al.,7 developed two sensitive and selective liquid chromatographic methods for the assay of Voglibose (VB) and validated as per International Conference on Harmonization (ICH) guidelines. First method is based on the pre-column derivatization of VB followed by visible detection (LC–VD) and second method involves mass spectrometric detection (LC–MS). In LC–VD method, VB was derivatized with sodium metaperiodate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH). The derivatized color product of VB (DCPVB) was run through Novapak C18 (300×3.9mm) column using the mobile phase containing buffer (0.01M mixture of sodium di hydrogen orthophosphate and disodium hydrogen orthophosphate, pH 6.0) and acetonitrile in 35:65 v/v ratio. The eluted DCPVB was monitored at 667 nm. The fixation of optimum conditions in LC–VD method is described. DCPVB structure was confirmed by mass spectral analysis. In LC–MS method, VB was passed through Venusil XBPPH (150×4.6mm, 5µm) column using a 95:5 v/v mixture of 0.01% formic acid and methanol as mobile phase. The assay concentrations of VB in pure form and in tablets for LC–VD and LC–MS methods are 25 and 5 ng ml−1 , respectively.
Bianchini RM et.al.,8 developed a simple high performance liquid chromatographic method for the determination of process-related impurities in bulk drug of the central anticholinergic compound Pridinol mesylate. Spectroscopically characterized synthetic impurities were used as standards. The chromatographic separation was optimized employing an experimental design strategy, and was achieved on a C18 column with a mobile phase containing 50mM potassium phosphate buffer (pH 6.4), MeOH and 2 -propanol (20:69:11), delivered at a flow rate of 1.0mLmin−1. UV detection was performed at 245 nm. The optimized method was thoroughly validated, demonstrating to be selective, when the chromatogram was recorded with a diode-array detector and peak purities were evaluated (>0.9995). The method is robust and linear (r2 > 0.99) over the range 0.05–2.5% it is also precise, regarding repeatability and intermediate precision aspects and LOQ values for the impurities are below 0.01%. Method accuracy, evidenced by low bias of the results and analyte recoveries in the range of 99.1–102.7%, was assessed at five analyte concentration levels. The usefulness of the determination was also demonstrated through the analysis of different lots of pridinol mesylate bulk substance. The results indicate that the method is suitable for the quality control of the bulk manufacturing of pridinol mesylate drug substance.
Barmpalexis P et.al.,9 developed an isocratic reversed-phase high-performance liquid chromatography technique investigated for the separation of Nimodipine and impurities using statistical experimental design. Initially, a full factorial design was used in order to screen five independent factors: type of the organic modifier methanol or acetonitrile and concentration, column temperature, mobile phase flow rate and pH. Except pH, the rest examined factors were identified as significant, using ANOVA analysis. The optimum conditions of separation determined with the aid of central composite design were: (1) mobile phase: acetonitrile/H2O (67.5/32.5), (2) column temperature 40°C and (3) mobile phase flow rate 0.9 ml/min. The analysis was found to be linear, specific, precise, sensitive and accurate. The method was also studied for robustness and intermediate precision using experimental design methodology. Three commercially available nimodipine tablets were analyzed showing good percentage recovery and percentages RSD. No traceable amounts of impurities were found in all products.