S. Sharma et al.

/ Journal of Pharmacy Research 2011,4(5),1538-1540

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

A Validated Densitometric Method for Duloxetine Hydrochloride in pharmaceutical dosage form

M.C. Sharma 1, S. Sharma *, A.D. Sharma 2 * Department of Chemistry Chodhary Dilip Singh Kanya Mahavidyalya, Bhind (M.P) India 1 School of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore (M.P) 452001, India 2 Shri Aurobindo Institute of Pharmacy Ujjain Road Indore (452003), India

Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011 ABSTRACT

A rapid and simple high performance thin layer chromatography method with densitometry 295 nm was developed and validated for simultaneous determination of Duloxetine Hydrochloride from pharmaceutical preparation. Separation was performed on aluminium-backed silica gel 60F254 HPTLC plates as stationary phase and using a mobile phase comprising of ethyl Acetate: carbon tetrachloride: Methanol: toluene: Glacial acetic acid (2:1.2:0.5:3.5:0.5:1.0 v/v/v/v/v) respectively. After development, plates were observed under UV light. The detector response was linear in the range of 12.2 to 20.1 g/spot for Duloxetine Hydrochloride. Both the drugs were subjected to stress test conditions like acid/ alkali/ neutral hydrolysis, oxidation, dry heat treatment and photo degradation. The spots for product of degradation were well resolved from the spot of respective drugs. The linear regression analysis data for the calibration plots showed good linear relationship with coefficient of regression value, r2= 0.9996. The method was validated in accordance with ICH guidelines. There was no chromatographic interference from capsule excipients. Developed method was validated in terms of linearity, accuracy, precision, repeatability and specificity Key words:Duloxetine Hydrochloride, TLC Densitometric, Stability indicating.

INTRODUCTION
Duloxetine hydrochloride, (+)-(S)-N-methyl-gamma- (1-naphthyloxy)-2 thiophenepropylamine hydrochloride1, although the exact mechanisms of the antidepressant and central pain inhibitory action of duloxetine in humans are unknown, the antidepressant and pain inhibitory actions are believed to be related to its potentiation of serotonergic and noradrenergic activity in the CNS. Preclinical studies have shown that duloxetine is a potent inhibitor of neuronal serotonin and nor epinephrine reuptake and a less potent inhibitor of dopamine reuptake 2. A survey of literature revealed that the following analytical methods were reported for determination of duloxetine hydrochloride and its metabolites by spectrofluorimetric method3 and chromatographic methods viz; high performance liquid chromatography4-9 and tandem mass spectrometry detector10, HPTLC11. Author of the article and his research team has developed a HPTLC Method development different pharmaceutical dosage form 12-16. The present work describes the development of a simple, precise and accurate method for Duloxetine hydrochloride and their degradation products in bulk drugs and marketed formulation. MATERIALS AND METHOD A CAMAG HPTLC system comprising of CAMAG Linomat IV semiautomatic sample applicator, CAMAG TLC scanner 3, CAMAG twin trough chamber(10 x 10 cm), CAMAG CATS 4 software, Hamilton syringe (100L) were used during the study. Tablets were purchased from local market. Acetonitrile and Water of HPLC grade (E.Merck India Ltd.) were used for preparing the mobile phase. Duloxetine Hydrochloride dosage form was purchased from a local pharmacy. HPTLC method and chromatographic conditions: The samples were spotted in the form of bands of width of 8 mm with space between bands of 5 mm, with a 100 L sample syringe (Hamilton, Bonaduz, Switzerland) on precoated silica gel aluminium plate 60 F254 (10 10) with 250 m thickness (E. MERCK, Darmstadt, Germany) using a CAMAG Linomat 4 sample applicator (Switzerland). The slit dimensions 6 mm 0.45 mm and scanning speed of 20 mm/sec was employed. The linear ascending development was carried out in 10 cm 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland) using mobile phase ethyl Acetate: carbon tetrachloride: Methanol: toluene: Glacial acetic acid (2:1.2:0.5:3.5:0.5:1.0 v/v/v/v/v). The optimized chamber saturation time for mobile phase was 20 min. The length of chromatogram run was 9 cm and development time was approximately 15 min. TLC plates were dried in a current of air with the help of a hair drier. Densitometric scanning was performed on CAMAG thin layer chromatography scanner 3 at 295 nm for all developments operated by WINCATS software version. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200 to 400 nm. Selection of Detection Wavelength After chromatographic development bands were scanned over the range of 200-400 nm and the spectra were overlain. It was observed that both the drug showed considerable absorbance at 251 nm. So, 251 nm was selected as the wavelength for detection. Linearity of detector response Aliquots of working standard (50 g/ml) solution (1, 2, 3,4,5,6 L) of Duloxetine were spotted as sharp bands on the precoated TLC plate, using Camag linomat IV semiautomatic applicator under nitrogen stream. The plate was developed under chromatographic conditions mentioned above. The plate was removed from the chamber and dried in hot air dryer. Preparation of standard solution: Duloxetine was accurately weighed and transferred to 50 ml volumetric flask and volume was made up to the mark with methanol to give a standard stock solution of 5 mg/ml. The aliquots (0.5 to 5 ml) of stock solution were transferred to 10 ml volumetric flasks and the volume of each was adjusted to 10 ml with methanol to obtain working standard solution containing 5, 10, 15, 20, 25, 30,35,40,45 and 50 g/ml of Duloxetine respectively. Preparations of sample solution Twenty tablets were accurately weighed and average weight was calculated. Accurately weighed quantity of tablet powder equivalent to 20 mg of the drug was transferred to 50 ml volumetric flask. To it 15 ml of methanol was added and shaken for 10 min and the volume was adjusted up to the mark with methanol and then filtered through Whatmann filter paper no.41. This solution is used as the sample solution. On the HPTLC plates spots of the standard and sample were applied. The plates were developed and after development the bands of the drugs were scanned at 295 nm. The peak height and area of the standard and sample bands were compared to obtain the concentration. Calibration curve for Duloxetine: Standard solutions of Duloxetine (20 l each) were applied in triplicate on TLC plate. The plate was developed in solvent system composed of Methanol: Ammonia (8:0.2 v/v).up to distance of 8 cm. Composed of development, the plates were dried in hot air and scanned at 295 nm. The peak areas were recorded. Calibration curve of Duloxetine was obtained by plotting peak area vs concentration of Duloxetine applied. Stress degradation of Duloxetine17 At ambient temperature 0.5 mg of drug was weighed separately and dissolved in a volumetric flask of 25 mL with, ammonia. To 2 mL of this solution, 0.5 mL of 0.1 N NaOH was added and final volume was made up to 10mL with ammonia. 200 ng of sample was spotted at time 0, 20, 40 min with a reagent blank spotted in adjoining track. Hydrolytic condition Accurately weighed 0.5 mg of drug was separately dissolved in 25 mL of ammonia, then to 2 mL of this solution, 5 mL of 0.1 N HCl was added and final volume was made up to 50 mL with ammonia, final solution was refluxed for 2 hr and 6 hr. 200 ng of each sample was spotted against the standard solution after appropriate dilution. Oxidative Degradation: Accurately weighed 1.0 mg of drug was separately dissolved in 25 mL of ammonia. Then to 5 mL of this solution, 1 mL of 30% H2O2 was added and final volume was made up to 50mL with ammonia. Final solution was refluxed for 3 hr. 200 ng each sample was spotted. The standard solution in methanol was also spotted after appropriate dilution. Degradation by dry Heat Accurately weighed 5 mg of drug was separately transferred to the 25 mL of volumetric flask, and was kept in oven at 100 0C for 1 hr, after that sample was appropriately diluted to spot 200ng of each drug.

*Corresponding author. S. Sharma Department of Chemistry, Chodhary Dilip Singh Kanya Mahavidyalya, Bhind (M.P), 477001, India E-mail:drssharma@rediffmail.com

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Validation of method: The method was validated as per the ICH18 guidelines in terms of linearity, accuracy and specificity, intra-day and inter-day precision, repeatability of measurement of peak area as well as repeatability of sample application. Precision (Repeatability) The precision of the method was verified by repeatability and intermediate precision studies. Repeatability studies were performed by analysis of three different concentrations (200, 400 and 600 ng spot -1 Duloxetine) of the drug in six times on the same day. The intermediate precision of the method was checked by repeating studies on three different days. Additionally, the developed HPTLC method was checked by separation studies on the mixture of reaction solutions on a different chromatographic system on a different day. Intra-day and inter-day precision The intra-day precision was determined by analyzing standard solutions of Duloxetine in range 200, 400 and 600ng/band for three times on the same day while inter-day precision was determined by analyzing corresponding standards on three different days over a period of one week. Robustness studies Robustness studies were carried out by examining the effect of small, deliberate variation of the analytical conditions on the peak areas of the drug. Factors varied were volume of mobile phase ( 0.70 %), time from application to development (5, 15, 30 and 60 min) and from development to scanning (0, 30, 60, and 90 min). One factor at a time was changed to study the effect. The robustness of the method was checked at amount of 300 ng/band. Linearity Accurate quantities from working standard solutions (5, 10, 15, 20, 25, 30,35,40,45 and 50 g/ml) were applied to the TLC plate to give bands containing 200600 ng spot -1 of Duloxetine. Each amount was applied five times and the plate was developed, using the previously described optimized mobile phase, and scanned. The calibration curves were constructed by plotting peak areas versus concentrations. Recovery Studies Accuracy of the method was determined by standard addition method in which the known amount of standard Duloxetine solutions were added to pre analyzed capsule solution. These amounts corresponded to 80, 100 and 120 % of the amounts claimed on the label. The amounts of Duloxetine were estimated by applying these values to the regression equation of the calibration curve. Limit of Detection (LOD) and Limit of Quantification (LOQ) The LOD and LOQ were separately determined based on the calibration curves. The standard deviation of the y intercepts and slope of the regression lines were used. In order to determine LOD and LOQ, concentrations in the lower part of the linear range of the calibration curve were used. Stock solution of Duloxetine (0.5 mg mL-1) was prepared and different volume of stock solution in the range 200 to 600 ng spot -1 were spotted in triplicate. The amount Duloxetine by spot versus average response (peak area) was graphed and the equation for this was determined. The standard deviations (S.D.) of responses were calculated. The average of standard deviations was calculated (A.S.D.). Detection limit was calculated by (3.3 A.S.D.) / b and quantification limit was calculated by (10A.S.D.) / b, where b corresponds to the slope obtained in the linearity study of method. Solution Stability The stability of standard solutions was tested after 0, 6, 12, 24, 48 and 72 h of storage. The stability of the solutions was determined by comparing peak area percentage and peak purity at 500 ng spot -1. Specificity The specificity of the method was ascertained by analyzing standard drugs and sample. The spots of Duloxetine in samples were confirmed by comparing the Rf and spectra of the spots with that of standard. The peak purity of Duloxetine was assessed by comparing the spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) positions of the spot. Analysis of the marketed formulation Weigh and finely powder not less than 20 capsules. Transfer blend equivalent to 20 mg of for the assay of marketed formulation, 5 mL of the marketed sample solution was pipette out using a volumetric pipette and transferred to a 10 mL of to 100 mL volumetric flask, extracted with methanol, sonicated for 30 min and diluted to mark with the same solvent. The resulting solution was filtered, through 0.45 m filter. The solution (2. 0 g/ml,) was spotted for assay of Duloxetine. The spots at R f 0.32 for Duloxetine were observed in the densitogram extracted from capsules. RESULTS AND DISCUSSION Use of pre-coated silica gel HPTLC plates with Methanol: Ammonia (8:0.2 v/v) resulted in good separation of the drug. The correlation of coefficient (r2) obtained was 0.9986 for Duloxetine.That means a good linear relationship was observed between the concentration range 12.2 to 20.1 g/spot for Duloxetine. The system suitability experiment was carried out before the determination of Duloxetine in unknown samples. The coefficient of variation was

Table 1. Method validation parameters

Parameters Linearity range Correlation coefficient Limit of detection (LOD) Limit of quantification (LOQ) Accuracy/Recovery Precision (%CV) Repeatability of application (n=6) Repeatability of measurement (n=6) Inter day (n=3) Intra day (n=3) Specificity Results 200 600 ng/spot 0.9993 41ng/spot 67 ng/spot 100.19% 0.85 0.25 0.643-1.32 0.283-0.986 Specific

Table 2 -Recovery Study

Duloxetine hydrochloride Drug %Amount added 20 80 100 120 Found in(g/ml) 20.09 19.98 20.01 %recovery 100.10 99.99 100.01

Table 3 . Result of Assay of Tablet Formulation

Duloxetine hydrochloride Amount (mg/capsule) 20 Mean +SD Amount found (mg/tablet) 20.06 19.98 19.92 0.98 0.326

Table 4- Result of stability formulation

Experiment Photolytic (2-Days in Sun Light) Thermal (4-Days in 110C) Alkali Deg (0.1 NaOH 1ml) Acid Deg (0.1 HCl 1ml) Oxidising Agent (30% H2 O2 ) percencentage degradation 3.76 16.32 38.76 72.41 38.76

less than 2% for replicate measurements of the same sample. The method was validated for precision (repeatability and reproducibility), accuracy, and linearity (sensitivity). Limit of detection and limit of quantification were recorded. Instrumental precision was checked by repeated scanning of same spot of Duloxetine six times and was expressed as coefficient of variance (% CV). The repeatability of the method was affirmed by analyzing 400 ng /spot of standard solution of Duloxetine individually (n=6) and was expressed as coefficient of variance (% CV). Reproducibility of the method was studied by analyzing aliquots of standard solutions of Duloxetine (200, 400 and 600 ng spot -1 Duloxetine) on the same day (intra-day precision) and on different days (inter-day precision) and the results were expressed as % CV. Accuracy of method was tested by performing recovery studies at three different levels for Duloxetine viz. after addition of 50, 75 and 100 % drug in the sample and estimated as described above. Limit of detection (LOD) and limit of quantification (LOQ) of Duloxetine was found to be 41 ng/spot and 67 ng/spot, respectively. Proposed method for the quantification of Duloxetine from capsule dosage forms was found to be simple, specific, sensitive, accurate and precise and may be used for routine quality control. CONCLUSION The developed HPTLC procedure was precise, specific and accurate. Statistical analysis indicated that the method was reproducible and selective for the analysis of Duloxetine capsules without interference from excipients. This methodology may also be applied to the

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study of degradation kinetics and for its determination in plasma and other biological fluids. This study is a typical example of assay, established following the recommendations of ICH guidelines. ACKNOWLEDGEMENT We are grateful to Prof.Abhay Kumar Singhai Department of Pharmaceutical Sciences Dr.Hari.Singh.Gaur.SagarUniversity Sagar (M.P) India, for given valuable suggestion. REFERENCE
1. 2. 3. Martindale, The complete drug reference, 34th Edn, 291. The pharmacological basis of therapeutics by Goodman and Gilmans. 11th edn. 436-450. Prabu, S.L., Shahnawaz, S., Dinesh Kumar, C., Shirwaikar, A. Spectrofluorimetric method for determination of duloxetine hydrochloride in bulk and pharmaceutical dosage forms, Indian Journal Pharmaceutical sciences, 70(4), 502-503(2008). Johnson, J.T., Oldham, S.W., Lantz, R.J., DeLong, A.F. High performance liquid chromatographic method for the determination of duloxetine and desmethyl duloxetine in human plasma, Journal of Liquid Chromatography and Related Technologies, 19, 1631-41(1996). Pankaj, S., Mariappan, T.T., Banerjee, U.C. High performance liquid chromatographic method for the simultaneous estimation of the key intermediates of duloxetine,Talanta, 67, 975-978(2005). Waldschmitt, C., Vogel, F., Maurer,C., Hiemke, C. Measurement of Duloxetine in Blood Using High-performance Liquid Chromatography With Spectrophotometric Detection and Column Switching, Therapeutic Drug monitoring, 29(6), 767-772(2007). Sinha, V.R., Anamika Kumria, R., Bhinge, J.R. Stress Degradation Studies on Duloxetine Hydrochloride and Development of an RPHPLC Method for its Determination in Capsule Formulation,Journal Chromatography Science, 47(7), 589-593(2009). Mercolini, L., Mandrioli, R., Cazzolla, R., Amore, M., Raggi,MA. HPLC analysis of the novel antidepressant duloxetine in human plasma after an original solid-phase extraction procedure,Journal of Chromatography B, 856 (1-2), 81-87(2007). Lakshmana Prabu, S., Shanawaz, S., Karthik, A., Dinesh Kumar, C., Vasatharaju, S.G. High performance liquid chromatography method for the quantification of duloxetine in rat plasma, ARS Pharm, 49(4), 283-292(2008). 10. Stonin, D.K., Mcculloch, J.D., Fengjiun, K., Knadler, M.P. Development and validation of a liquid chromatography: tandem mass spectrometric method for the determination of the major metabolites of duloxetine in human plasma, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 852(1-2), 582-589(2007). Dhaneshwar, S.S., Deshpandem P., Patil, M., Vadnerkar, G., Dhaneshwar, S.R. Development and validation of a HPTLC method for Estimation of Duloxetine Hydrochloride in Bulk Drug and in Tablet Dosage Form, Indian Journal Pharmaceutical Sciences, 70(2):233-6(2008). Sharma, M. C., Sharma ,S., Kohli, D. V., Sharma, A. D. A validated HPTLC method for determination of simultaneous estimation Rosuvastatin Calcium and Ezetimibe in pharmaceutical solid dosage form, Archives of Applied Science Research, 2 (1) 1-7(2010). Sharma, M. C., Sharma ,S., Kohli, D. V.HPTLC method development and validation for the estimation of Atorvastatin Calcium and Pioglitazone Hydrochloride in pharmaceutical combined tablet dosage form,Annals of Biological Research, 1 (1), 124-129(2010). Sharma,S.,Sharma,M.C.Development and Validation of An HPTLC Method for Determination of Oseltamivir phosphate in pharmaceutical dosage form, Indian Drugs ,47(11),68-72(2010). Sharma, M. C., Sharma, S., Kohli, D. V., Sharma, A. D.Validated TLC Densitometric method for the quantification of Torsemide and Spironolactone in bulk drug and in tablet dosage form, Der Pharma Chemica, 2(1), 121-126(2010). Sharma ,S., Sharma, M. C., Kohli, D.V., Sharma, A. D. Development and Validation of TLCDensitometry Method for Simultaneous Quantification of Montelukast Sodium and Levocetirizine Dihydrochloride Pharmaceutical Solid dosage form, Der Pharmacia Lettre , 2 (1) 489-494(2010). Sharma, M.C., Sharma ,S. Development and Validation of Densitometric Method for Metronidazole and Tetracycline hydrochloride in capsule Dosage form. Int.J. Pharm. Tech. Resea, 3(2), 1169-1173(2011). Sharma, M.C., Sharma ,S. Validated Densitometric Method for the Quantification of Lamotrigine in Dosage Form.. Int.J. Pharm. Tech. Resea, 3(2), 1174-1178 (2011). Sharma, M.C., Sharma ,S. Development and Validation of TLC Densitometric Method for Gatifloxacin in Pharmaceutical Formulations. Int.J. Pharm. Tech. Resea, 3(2), 1179-1185 (2011). Stability Testing of New Drug Substances and Products Q2 (R1), ICH Harmonized Tripartite Guideline, Nov 2005. ICH Harmonized Tripartite Guidelines on Validation of Analytical procedures: Text and Methodology Q2 (R1), Current step 4 version, Geneva, 2005.

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 5. May 2011

1538-1540