INTRODUCTION:

Thalidomide is chemically known as 2-(2, 6-dioxopiperidin-3-yl)-1H-isoindole-1, 3(2H)-dione. The molecular formula is C₁₃H₁₀N₂O_4.The molecular weight is 258.23 g/mol. Thalidomide is used for a number of conditions including erythema nodosum leprosum, multiple myeloma (in combination with dexamethasone), and various other cancers, for some symptoms of HIV/AIDS, Crohn's disease, sarcoidosis, graft-versus-host disease, rheumatoid arthritis and a number of skin conditions that have not responded to usual treatment.[1] The bacterium that causes tuberculosis is related to leprosy. Thalidomide may be helpful in some cases where standard TB drugs and are not sufficient to resolve severe inflammation in the brain [2, 3]. A few analytical methods were reported for the determination of Thalidomide by HPLC in plasma and in blood from pharmaceutical preparation [4, 5].

Other different methods have been reported for its estimation including HPLC in API [6-10]. Hence no method has been reported for the determination of thalidomide along with its impurities. The aim of the work was to develop a simple, accurate and cost effective HPLC method for the determination of Thalidomide and its impurities. This method was developed and validated as per ICH guidelines [11].

MATERIALS AND METHODS:

Chemicals and Reagents:

The Samples of Thalidomide and its impurities were obtained from Fortunee Laboratories (P) Ltd, Kakinada, and Andhra Pradesh, India. All other analytical reagents such as Ammonium formate, acetonitrile, potassium dihydrogen orthophosphate, hydrochloric acid, sodium hydroxide and hydrogen peroxide (30%) were obtained from Merck specialty chemicals, Mumbai, India. Milli Q’ water is used for the preparation of Solutions. Figure-1 expressed the structure of thalidomide and its impurities.

Figure 1:- Structure of thalidomide and its impurities

Instrument specification:

This research has been performed on Agilent make HPLC 1100 instrument. It has binary gradient pump, photo diode array detector (UV), column oven with range of 25°C to 60°C with auto injector. The modules are G1310A isocratic pump with solvent cabinet; G1314A variable wavelength detector (VWD) with standard flow cell (10 mm path length, 14 µl volume, 40 bar maximum pressure) and G2220AA 2D-Value Solution Chem Station.

METHODS:

Chromatographic specification:

The instrument works with a mobile phase of potassium dihydrogen orthophosphate and Acetonitrile in the ratio of 80:20 (v/v) and a column used Develosil ODS UG-5 column (150mm X 4.6 mm X 5um particle size).The flow rate was maintained at 0.700ml with a detecting wavelength of 297nm.

Impurities standard Stock Solutions:

Weigh accurately about 25 mg of each impurity standards of N-acetyl-Glucosamine, 5- Hydroxymethyl-2-Furaldehyde, Pyrazine, 2- Furaldehyde and Pyrrole-2-Carboxaldehyde then transfer into individual each 50 ml volumetric flasks, add 35 ml of diluent into each flasks, sonicate for 10 minutes to dissolve the material and make up the volume up to mark with diluent and mix.

Diluted Standard solution (Resolution solution):

From the impurities standard stock solution, transfer each 5 ml of solution into 100 ml volumetric flask and make up the volume up to mark with diluent and mix well.

Sample Preparation:

Weigh accurately not less than 20 tablets note down the weight. Then calculate average weight. Crush the 20 tablets in to fine powder with mortar pestle, then transfer an accurately weighed quantity equivalent to 500 mg of Glucosamine Hydrochloride into a 100 ml Volumetric flask, add 70 ml of diluent, sonicate for 30 minutes with intermediate shaking by maintaining sonicator bath temperature below at 28°C. Cool it, make up the volume up to the mark with diluent and mix well. Centrifuge the Test solution by closing the centrifuge tube tightly with stopper or Para film (to avoid solvent evaporation) at 3000 RPM for 10 minutes, use supernatant solution for filtration. Filter the supernatant solution through 0.45 µm PVDF filter. Inject the clear filtrate solution into HPLC.

Note: The sample solution should be freshly prepared and injected immediately.

Procedure:

Separately inject equal volumes of (about 10 µl) of one injection of diluent as blank, five replicate injections of diluted Standard solution, and one injection of Sample solution into the chromatograph, and record the chromatograms and measure the peak responses. Developed method were validated by following parameters linearity, accuracy, precision, LOD, LOQ and stability testing.

RESULTS AND DISCUSSION:

Method development

System suitability:

Standard solution was prepared as per test procedure and injected into the HPLC system as per test method. Evaluated the system suitability parameters and Summarized in the table 1 given below.

Table-1: System suitability results

System suitability parameters		Observed value	Acceptance criteria
Theoretical Plates	Thalidomide	5758	Should be NLT 2000
	Impurity-A	6154
	Impurity-B	5199
%RSD	Thalidomide	0.1	Should be NMT 5.0
	Impurity-A	0.6
	Impurity-B	0.1
Tailing factor	Thalidomide	1.2	Should be NMT 2.0
	Impurity-A	1.1
	Impurity-B	1.1

Method validation:

The described method has been validated for thalidomide and its impurities using the following parameters

Linearity:

Linearity was established by plotting a graph between concentration versus peak area and the correlation coefficient was determined. A series of solutions of thalidomide impurities with concentrations ranging from LOQ% to 200% of the target concentration (0.5%) prepared and injected into the HPLC system. Table 3 and figure 2, 3, 4 expressed the linearity data of thalidomide and its impurities.

Figure 2: Thalidomide linearity graph

Figure 3: Thalidomide impurity A linearity graph

Figure 4: Thalidomide impurity B linearity graph

Table 3: Linearity data

Parameter	Thalidomide	Impurity a	Impurity b
Linearity range µg/ml	0.03-4.29	0.031-4.1	0.03-4.31
Correlation coefficient	0.99922	0.99924	0.99913
Limit of detection µg/ml	0.0001	0.0002	0.0003
Limit of quantification µg/ml	0.0005	0.0005	0.0012

Limit of Quantitation and Limit of Detection:

A study to establish the Limit of Detection and Limit of Quantification of Thalidomide impurities was conducted.

Limit of Detection and Limit of Quantitation were established based on signal to noise ratio. A series of injections of blank solution were injected and average noise was calculated. Limit of Detection for each impurity was established by identifying the concentration which gives signal to noise ratio about 3. Limit of Quantitation was established by identifying the concentration which gives signal to noise ratio about 10. Table 4 expressed the LOD and LOQ data of thalidomide and its impurities.

Table 4: LOD and LOQ Values

Name	% of Impurity		Signal to Noise Ratio
Name	LOD	LOQ	LOD	LOQ
Thalidomide	0.0001	0.0005	3.5	9.8
Impurity-A	0.0002	0.0005	3.6	10.1
Impurity-B	0.0003	0.0012	3.0	9.3

Precision:

Stock solutions of impurities were prepared. Six replicates of the drug product were spiked with impurity stock solutions, such that each preparation consists of each known impurity at its specification level (0.2%). Sample solutions were analyzed as per method. The amount of each known impurity in percent in each replicate, the average, and its %RSD were calculated.

Accuracy:

A study of accuracy of thalidomide impurities from spiked samples of test preparation was conducted. Samples were prepared in triplicate at each level by spiking test preparation with LOQ, 50%, 80%, 100%, 150% and 200% of target concentration of thalidomide impurities. The individual values, the % recovery, the % relative standard deviation for % recovery of samples at each concentration level are reported.

Stability:

Solution for 48hrs and compared with freshly prepared solutions and was found to be stable. Stability was observed for mobile phase, reagents, standard and sample.

CONCLUSION:

A Simple and sensitive HPLC method for the determination of thalidomide and its impurities has been successfully developed and validated. The proposed method is simple, accurate, precise and highly sensitive .Hence this method can be used for routine analysis in pharmacy.

REFERENCES:

1. "Thalidomide” The American Society of Health -System pharmacists. Retrieved June 2014.

2. Buonsenso. D, Serranti. D, Valentine’s.” Management of central nervous system tuberculosis in children/; light and shade”. European review for medical and pharmacological sciences 14(10): 845-53, 2010.

3. Van Toorn R, Solomons R. “Update on the diagnosis and management of tuberculous meningitis in children “. Seminars in pediatric neurology 21(1):12-8, 2014.

4. 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 Biomedical Analysis. 31(1): 19-27, 2003.

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

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

7. Singh S Singh UK Singh RM Singh GN Mathur SC Saini PKY adav 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); 557, 2011.

8. 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; 402-405, 1989.

9. Shabir GA. 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

10. International Conference on Harmonization. J Chromatogr A. 987; 57, 2003.

11. Shiv Kumar Gupta, Babita Kumar and Pramod Kumar Sharma. Development and validation of a RP-HPLC method for estimation of Thalidomide in solid dosage form. Asian Journal of Pharmaceutical Analysis. 3(1); 17-19, 2013.

12. International Conference on Harmonization, (1996). Guidance for Industry in Q2B Validation on Analytical Procedures.

Received on 18.04.2016 Accepted on 02.05.2016

Asian J. Pharm. Ana. 2016; 6(2): 115-118.

DOI: 10.5958/2231-5675.2016.00017.X

Observed value

Acceptance criteria