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Research In Pharmaceutical Biotechnology Vol. 2(1), pp. 001-006, February, 2010 Available online at http://www.academicjournals.org/RPB Validation and stability indicating RP-HPLC method for
the determination of tadalafil API in pharmaceutical
formulations
B. Prasanna Reddy1*, K. Amarnadh Reddy2 and M. S. Reddy3
1Department of Quality control, Nosch Labs Pvt Ltd, Hyderabad-500072, A.P, India. 2 Department of AR and D, Aurigene Discovery Technologies Ltd, Bangalore, India. 3Department of Plant Pathology and Entomology, Auburn University, USA. The present study describes the development and subsequent of a stability indicating RP-HPLC method
for the analysis of tadalafil. The samples separated on an Inertsil C18, (5 µ , 150 mm x 4.6 mm i.d) by
isocratic run using acetonitrile and phosphate buffer as mobile phase), with a flow rate of 0.8 ml/min,
and the determination wavelength was 260 nm for analysis of tadalafil. The described method was
linear within range of 70 - 130 µg/ml (r2 = 0.999). The precision, ruggedness and robustness values were
also within the prescribed limits (< 1% for system precision and < 2% for other parameters). Tadalafil
was exposed to acidic, basic, oxidative and thermal stress conditions and the stressed samples were
analyzed by the proposed method. Chromatographic peak purity results indicated the absence of co-
eluting peaks with the main peak of tadalafil, which demonstrated the specificity of assay method for
estimation of tadalafil in presence of degradation products. The proposed method can be used for
routine analysis of tadalafil in quality control laboratories.

Key words: RP-HPLC, tadalafil, validation, stability indicating assay, forced degradation.


INTRODUCTION

Tadalafil hydro-2-methyl-6-[3,4-(methylenedioxy)phenyl] EXPERIMENTAL
pyrazino-[1’,2’:1,6]pyrido[3,4-b]indole-1,4-dione (Figure 1), is a phosphodiesterase type 5 inhibitor used in the Chemical and reagents
management of erectile dysfunction. It is not official y included in any of the pharmacopoeias. It is listed in the Tadalafil standard and API were provided from Smilax laboratories Merck Index (Budavari et al., 2001) and Martindle and limited. HPLC grade sodium dihydrogen phosphate (NaH2PO4), complete drug reference (Sean et al., 2002). There are disodium hydrogen phosphate (Na2HPO4), acetonitrile, hydrogen peroxide and sodium hydroxide were procured from Merck Ltd. several (Cheng et al., 2005) methods for determination of High pure water was prepared by using Mil ipore Mil i Q plus tadalafil such as HPLC-EIMS (Zhu et al., 2005) and capil ary electrophoresis methods (Aboul-Enein, 2005) and by HPLC (Aboul, 1994). The present work was designed to develop a simple, precise and rapid HPLC instrumentation and conditions
analytical LC procedure, which would serve as stability indicating assay method for analysis of tadalafil active A high performance liquid chromatograph system, with LC solutions data handling system, with an auto sampler was used for the analysis. The data was recorded using LC 2010 solutions software. The samples separation was performed on a Shimadzu Inertsil C (5 µ, 150 mm x 4.6 mm, Japan) with the mobile phase consisting of acetonitrile and phosphate buffer (pH 7.0) with a ratio of 60: 40 (v/v) *Corresponding author. E-mail: [email protected] Tel: at ambient temperature. The flow rate was kept at 0.8 ml/min and the determination wavelength was 262 nm.
Figure 1. Structure of Tadalafil.

Standard and sample preparation

Robustness of method was investigated by varying the chromatographic conditions such as change of flow rate (± 10%), Mobile phase
organic content in mobile phase (± 2%), wavelength of detection (± Mix 700 ml of acetonitrile to the buffer, the mobile phase was 5%) and pH of buffer in mobile phase (± 0.2%). Robustness of the sonicated for 15 min and then it was filtered through 0.45 µm developed method was indicated by the overal %RSD between the
Limit of detection (LOD)

Standard solution

The standard was dissolved with mobile phase to 0.5 mg/ml. The
The detection limit is determined by the analysis of samples with test samples were dissolved with mobile phase. With the optimized the known concentrations of analyte and by establishing the chromatographic conditions, a steady baseline was recorded, the minimum level at which the analyte can be reliably detected. The standard solution was injected and the chromatogram was recorded. This procedure was repeated for the sample solution. Forced degradation studies
Limit of quantitation (LOQ)
Tadalafil was al owed to hydrolyze in different strengths of base The quantitation limit is determined by the analysis of sample of (0.005 N and 0.05 N NaOH), acid (0.05 N, 0.5 N and 1 N HCl) and known concentration of analyte and by establishing the minimum hydrogen peroxide (30%, 10, 3 and 1%). Tadalafil was also studied level at which the analyte can be quantified with acceptable for its thermal degradated at 80, 100, 120°C for 1 h respectively. An accurately weighed 50 mg of tadalafil API was dissolved in 1 ml of respective base (NaOH), acid (HCl) or hydrogen peroxide and kept for specified period after which the volume was made up to 50 ml with water: acetonitrile (70:30, v/v). Five mil iliters of the above solution were diluted with water: acetonitrile (70:30, v/v) to get a where S: Slope of the calibration curve, : Average standard concentration of 100 ppm. Blank was also treated in same way. Validation
Linearity was determined by injecting different concentration of sample solutions (50 - 150 µg/ml). For system precision, standard solution (100 µg) was injected to six replicates injections to check Chromatographic conditions
%RSD (relative standard deviation) and for method precision six time samples were prepared and each of those were injected in In order to separate tadalafil API, phosphate buffer- duplicate. Mean of al of these values gives to assay value. To establish the within-day (intra-assay) and between-day (inter- acetonitrile mixtures were used as the mobile phase. assay) accuracy and precision of the method, tadalafil was assayed Satisfactory resolution was obtained using the mobile on one day and three separate days. Intra-assay and inter-assay phase system of acetonitrile/phosphate buffer (70:30, v/v, pH 7.0) at a flow rate of 0.8 ml/min with UV detection at
Figure 2. Chromatogram of Tadalafil API.
Table 1. Results of force degradation studies of Tadalafil.
Stress condition/duration/solution
Degradation %
Alkaline degradation (0.005 N NaOH, 1 hr) Alkaline degradation (0.05 N NaOH, 2 hr) Oxidative degradation (30 % H2O2, 80°C for 10 min) Thermal degradation (Solid sample, 80°C, 1 hr) Thermal degradation (Solid sample, 100°C, 1 hr) Thermal degradation (Solid sample, 120°C, 1 hr) 260 nm. Under these conditions tadalafil with the stable under the applied stress conditions like heat, acid retention time of tadalafil was 2.88 min. Figure 2 showed and alkaline and oxidative degradation states. a typical chromatogram obtained under these conditions Linearity
Forced degradation studies
The calibration curve showed good linearity in the range of 50 - 150 µg/ml, for tadalafil API with correlation co- During the study it was observed that upon treatment of efficient (R2) of 0.9998 (Figure 4). A typical calibration tadalafil with different strengths of base (0.005 N and curve has the regression equation of y = 23646x 0.05 N NaOH), acid (0.05 N, 0.5 N and 1 N HCl) and hydrogen peroxide (30, 10, 3 and 1%) the degradation was observed only with the higher strengths (0.05 N NaOH), acid (1 N HCl) and hydrogen peroxide (30%), Precision
where as with the lower strengths of alkali (0.005 N NaOH), acid (0.05 N and 0.05 N HCl) and hydrogen The results of system precision (% RSD = 0.26), method peroxide (1, 3 and 10%) no degradation was observed precision (% RSD = 0.10) are found within the prescribed (Table 1).Further it is important to note that from the limit of ICH guidelines (% RSD < 1%, and % RSD < 2 % chromatograms (Figure 3a to c), it is evident that respectively in case of system precision and method although the degraded peaks are observed. The tadalafil
Figure 3.
Chromatograms of (a) Base hydrolyzed-degraded sample (b) Thermal degraded sample and (c) Acid hydrolyzed

Intra-assay and Inter-assay
Method robustness
The intra and inter-day variation of the method was Influence of smal changes in chromatographic conditions carried out and the high values of mean assay and low such as change in flow rate (± 10%), organic content in values of standard deviation and %RSD (%RSD < 2%) mobile phase (± 2%), wavelength of detection (± 5%) and within a day and day to day variations for tadalafil pH of buffer in mobile phase (± 0.2%) studied to revealed that the proposed method is precise in (Table 2 determine the robustness of the method are also in favor (Table 4, %RSD < 2%) of the developed RP-HPLC
Figure 4.
Linearity curve for Tadalafil.
Table 2. Intra-assay precision data of proposed RP-HPLC method
Mean (% w/w)
Table 3. Inter-assay precision data of proposed RP-HPLC method.
Mean (% w/w)
Table 4. The influence of changes in chromatographic
parameters on RP-HPLC analysis of Tadalafil API Change in parameter
method for the analysis of tadalafil API. REFERENCES
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Abdul-Fattah AM, Bhargava HN (2002). A new high performance liquid Chromatography (HPLC) method for the analysis of halofantrine (HF) The minimum concentration level at which the analyte in Pharmaceuticals. J. Pharm. Biomed. Anal. 29: 901-908. can be reliably detected (LOD) and quantified (LOQ) Avarez-Lueje A, Pujol S, Squel a JA, Nunez Vergara LJ (2003). A Selective HPLC method for determination of lercanidipine in tablets. were found to be 0.05 and 0.5 µg/ml, respectively. Bebawy L (2003). I. Spectrophotometric and HPLC determination of linezolid in the Presence of its alkaline-induced degradation products Specificity and stability in analytical solution
and in pharmaceutical Tablets. Anal. Lett. 36: 1147-1161. Budavari S, Eds (2001): In, The Merck Index, 13th Edn, Merck & Co., The results of specificity indicated that the peak was pure Cheng CL, Chou CH (2005). J. Chromatogr. B Analyt. Technol. Biomed. in presence of degraded sample. It is important to mention here that the tadalafil API was stable in solution ICH (1996). Validation of Analytical Procedures: Methodology, in: Proceeding of the International Conference on Harmonization, The results of linearity, precision, inter and intra- ICH (2002).Guideline on Analytical method Validation, in: Proceeding assays, method robustness, LOD, LOQ and specificity of International Convention on quality for the pharmaceutical industry, and stability in analytical solution established the ICH (1993). Stability testing of new drug substances and procedure in: validation of the developed RP-HPLC assay for the Proceeding of the International conference on Harmonization, Lambropouls J, Spanos GA, Lozaridis (1999). V.N. Method development and Validation for the HPLC assay (potency and related substances) for 20 mg paroxetine tablets. J. Pharm. Biomed. DISCUSSION
Sean C, Sweetman Eds. (2002). In., Martindale, The Complete Drug The present study is the first time report on stability Reference, 34th Edn., The Pharmaceutical Press: London p. 875. indicating assay of tadalafil in presence of degradation The United States Pharmacopoeia” 26th ed. (2003). US Pharmacopoeia products by HPLC. In this method isocratic elution Zhu X, Xiao S, Chen B, Zhang F, Yao S, Wan Z, Yang D, Han H (2005). method is selected for the analysis of tadalafil API J. Chromatogr A., eb25;1066(1-2): 89-95. because it gave better base line separation and peak width, which is suitable for the routine analysis of tadalafil. The developed method was validated as per ICH guidelines (ICH, 1996) and its updated international Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance varies with time under the influence of a variety of environmental factors such as temperature, humidity and light, and enables recommendation of storage conditions, retest periods, and shelf life to be established (Avarez-Lueje et al., 2003; Abdul-Fattah et al., 2002; Lambropouls et al., 1999; Bebawy, 2003). The assay of tadalafil API (Active Pharmaceutical Ingredient) in stability test sample needs to be determined using stability indicating method, as recommended by the International Conference on Harmonization (ICH)

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