INTRODUCTION:

Thalidomide chemically is 1(H)-isoindole-1,3 (2H)-dione, 2(2,6-dioxo-3-piperidinyl) and official in USP. Thalidomide is a well known hypnotic drug, renowned for its human embryonic mal development Properties [1]. Thirty years of its withdrawal from the pharmacopoeia, Thalidomide, has recently been re-admitted as a treatment of graft-verse-host disease and leprosy because of its immune modulating and anti-inflammatory properties [2]. Thalidomide has been reported to be beneficial in treatment of oral aphthous ulcers [3-6]. Literature survey reveals few analytical methods for the determination of Thalidomide by HPLC in plasma and in blood from pharmaceutical preparation [7,8]. Other different methods have been reported for its estimation including HPLC in API [9,10,11]. This describes a fast, practical and precise method for the analysis of Thalidomide in solid dosage form. The aim of the work was to develop a simple, accurate and cost effective HPLC method for solid dosage form of Thalidomide.

EXPERIMENTAL:

Chemical and Material

Thalidomide capsules and Thalidomide reference standard were supplied by NATCO Pharmaceuticals, Hyderabad. Acetonitrile, Di Methyl Formamide, o-Phosphoric acid used were of HPLC grade. HPLC grade water was prepared using Millipore Purification System (Millipore, Molseheim, France Model Elix-10).

Instrument:

Analysis by HPLC was performed using an isocratic system consisting of a pump (Waters 600), autosampler (waters 717), UV detector (waters 486). The system was connected with the help of a millenenium 32 software in a computer system for data connection and processing. The analytical column used was xterra Rp C₁₈ (150 × 4.6mm).

Chromatographic Conditions:

The mobile phase was the mixture of Acetonitrile: Di Methyl Formamide: Water (6:1:3, v/v) and pH was adjusted to 2.5 with o-phosphoric acid (1:100, v/v) and was filtered through 4.5µm membrane filter. The flow rate was maintained at 1.0ml/min/ the injection volume was 20µl and detected at the wave length of 297nm at ambient temperature.

Standard and Sample Preparation:

25mg of Thalidomide reference standard was accurately weighed and dissolved in 25ml volumetric flask with Di Methyl Formamide and made up the volume with Di Methyl Formamide. To added 5ml of o-phosphoric acid (1:100, v/v) to it and dilute it with water and made up to volume to obtain a standard stock solution having a concentration of 100µg/ml. The sample solution was also prepared in the same manner and with the same concentration. 20µl of the solution were injected.

RESULT AND DISCUSSION:

Method Development:

System suitability test was carried out on freshly prepared standard stock solution of Thalidomide to check various parameters.

System suitability results were as follow:

Retention Time 2.17

Tailing Factor 1.55

Theoretical Plates 1400

Calibration Range 25-200µg

Figure 1Retention Time of Thalidomide

Method Validation:

The describe method has been validated for the assay of major components of bulk drug using following parameters [12,13].

Parameter	Thalidomide
Linearity Range (µg/ml)	25-200
Correlation Coefficient (r)	0.9986
Limit of Detection (LOD) (µg/ml)	0.374
Limit of Quantitation (LOQ) (µg/ml)	0.113

Specificity and Selectivity:

Specificity and selectivity were studied for examination of the presence of interfering endogeneous components, a reference solution containing Thalidomide was prepared and was compared with blank. Result indicates that the retention time of Thalidomide at 2.17 and none of the impurities were interfering in its assay. The results of assay were complied in Table No. 1

Table No. 1 Specificity of Method:

S. No.	Bulk Drug
S. No.	Actual Amount Claim (in mg.)	Found (in mg.)	% Claim
1 2 3	25.1 25.1 25.0	25.2 25.0 25.1	101.2 99.92 100.56
		Mean	100.58

Linearity:

Linearity was studied by preparing standard solution sets of different concentration level. The linearity range was found to be 25-200µg/ml. calibration curves containing the standard of 25µg/ml to 200µg/ml were used for the determination of the linearity of the Thalidomide.

Figure 2 Linearity curve of Thalidomide

Accuracy:

Accuracy was determined by recovery studies of Thalidomide, known amount oof Thalidomide reference standard was added it to preanalyzed sample and subjected them to the proposed HPLC method. Results of the recovery study were shown in Table No. 2. The study was carried out at three different concentration levels.

TABLE No. 2 Recovery Study of Thalidomide

Label Claim (mg)/capsule

Amount Added (mg)

Amount recovered* (mg)

% Recovered

Thalidomide 100mg

5.0

10.1

15.1

20.1

30.3±0.04

35.4±0.05

40.1±0.01

45.3±0.07

100.5

100.1

99.25

99.48

Mean

99.83

*Each value is mean deviation of three determinations

Precision:

Precision was studied to find out intraday and interday variation in test methods of thalidomide in the concentration ranges of 20µg/ml to 200µg/ml. for three times on the same day and later day. Precision was determined by analyzing corresponding standard daily for a period of three days. The %RSD in case of intraday and interday was found to be 0.37 and 0.56 respectively.

Stability:

Stability of reagents, mobile phase, standard and sample solutions were studied for 48 hours and compared with the freshly prepared solutions and was found to be stable.

CONCLUSION:

An HPLC method with UV detection was developed and was validated for the determination of Thalidomide drug substance. The method was found to be specific, accurate and sensitive. Therefore this method is suitable alternative to the current USP HPLC procedure. Additionally the method offers the advantage of being quantitative and automated.

REFERENCES:

1. Mellin GW, Katzenstoin M The Saga of Thalidomide- Neuropathy to Embryopathy, with Case Reports of Congenital Anomalies. New English Journal of Medicine. 267 (23); 1962: 1184-1193.

2. Czejka MJ anf Koch HP Determination of thalidomide and its major metabolites by high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications. 413; 1987: 181–187

3. Youle M Clarbour J Farthing C Connolly M Hawkins D Staughton R and Gazzard B Treatment of resistant aphthous ulceration with thalidomide in patients positive for HIV antibody. British Medical Journal 432 (1); 1989: 298.

4. Gorin I Vilette B Gehanno P and Escande JP Thalidomide in hyperalgic pharyngeal ulceration of AIDS. Lancet 335 (8701); 1990: 1343.

5. Georghiou PR, Kemp RJ. HIV-associated esophageal ulcers treated with thalidomide. Medical Journal of Australia. 152; 1990:382-383.

6. Radeff B Kuffer R Samson J. Recurrent aphthous ulcer in patient infected with human immunodeficiency virus: successful treatment with thalidomide. Journal of American Acadamy of Dermatol. 23; 1990:523-525.

7. Li J Jaworsky MS and Stirling DI The determination of a potential impurity in Thalidomide drug substance and product by HPLC with indirect UV detection. Journal of Pharmaceutical and Bioedical Analysis. 31(1); 2003: 19-27.

8. Toraño JS Verbon A Guchelaa HJ Quantitative determination of thalidomide in human serum with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and ultraviolet detection. Journal of Chromatography B: Biomedical Sciences and Applications. 734(2); 1999: 203-210.

9. Singh RM Shivraj Mathur SC Singh GN Sharma PK and Gupta SK Method development and its validation of Drug Thalidomide in API usinf RP-HPLC method. Journal of Indian Council of Chemist. 22(2); 2006: 25-27.

10. Singh S Singh UK Singh RM Singh GN Mathur SC Saini PK Yadav A Gupta V and Duggal D Development and validation of a RP-HPLC method for estimation of prulifloxacin in tablet dosage form. Indian Journal of Pharmaceutical Science. 73(5); 2011: 557.

11. Chen TL Vogelsang GB Petty BG Brundrett RB Noe DA Santos GW and Colvin OM Plasma pharmacokinetics and urinary excretion of thalidomide after oral dosing in healthy male volunteers.Drug Metabolism Disposition 17; 1989: 402-405

12. Shabir GA (2003) Validation of HPLC methods for pharmaceutical analysis: Understanding the differences and similarities between validation requirements of the U.S. Food and Drug Administration, the U.S. Pharmacopoeia and the International Conference on Harmonization. J Chromatogr A. 987; 57.

13. International Conference on Harmonisation, (1996) Guidance for Industry In: Q2B Validation on Analytical Procedures. Methodology Switzerland IFPMA, 1.

Received on 13.02.2013 Accepted on 15.03.2013

Asian J. Pharm. Ana. 3(1): Jan.-Mar. 2013; Page 17-19