HPTLC DETECTION AND DIFFERENTIATION OF CARDIOVASCULAR DRUG ATORVASTATIN WITH CLOPIDOGREL AND ASPIRIN.

HPTLC  DETECTION  AND DIFFERENTIATION OF  CARDIOVASCULAR  DRUG  ATORVASTATIN  WITH  CLOPIDOGREL  AND  ASPIRIN.

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Pune -411 028 (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2012, 6-8, 1, Art. 1, 26^th Aug. 2012)

 

ABSTRACT

Atorvastatin drug helps in decreasing cholesterol and thus reduces the risk of heart attack. HPTLC, high performance thin-layer chromatography is described here  for the detection of atorvistatin.  HPTLC glass plates of the size of 10 cm x 10 cm,  pre-coated with silica gel 60F₂₅₄   as a stationary phase and solvent mixture Bexane : Acetone : Ethyl alcohol (8:1:1, v/v/v) as a mobile phase were preferred for it.  UV detection was carried at 254 nm. Atorvastatin was detected at R_f-values 0.25 ± 0.02. The LOD for these drugs was found to be 50 ng per spot. The solvent system used here nicely differentiates Atorvistatin from Aspirin and Clopidogrel having R_f-values  0.38 and 0.93, respectively.

KEY WORDS : Atorvastatin, clopidogrel, aspirin, cardiovascular, drug, HPTLC, UV, detection.

INTRODUCTION

Atorvastatin is a  drug of choice prescribed for cardiovascular disease. It minimizes chances of heart attack as it lowers amount of cholesterol in the blood. Panchal et. al. [1] has described use of HPLC and HPTLC methods for the determination of atorvastatin with ramipril in combined capsule dosage form. Atorvastatin was determined at 210 nm by HPTLC – densitometry. Easy and speedy HPTLC method  is described here for the detection of  Atorvistatin. Susceptibility of clopidogrel bisulphate to acids, alkali, oxidation and heat using HPTLC was explored by Himani et. al. [2]. Mitacos et. al. [3]  have validated clopidogrel using liquid chromatography in pharmaceutical preparations. Estimation of aspirin   has been explained in the literature using RP-HPLC [4,5,6], Rapid HPLC [7], HPTLC [8] and spectrophotometry [9]. Bexane : Acetone : Ethyl alcohol (8:1:1, v/v/v) as a mobile phase is used herein for the differentiation and detection of Atorvastatin, Clopidogrel and Aspirin.

EXPERIMENTAL

Equipments:

The following material / equipment was used for the experiment.

1. Merck, HPTLC glass plates pre-coated with silica gel 60 F 254 ( 10 cm x 10 cm).
2. Camag Development Chamber ( small size, twin type ).
3. Camag UV Viewing Chamber equipped with 254 nm and 366 nm wavelength exposure facility.

 

High Performance Thin-layer Chromatography (HPTLC):

Reagents: All reagents and solvents used were of analytical reagent grade.

95% ethyl alcohol and glass distilled water was used wherever required.

Standard Atorvistatin Solution : Atorvistatin Tablets IP (X’tor^®-10), each  containing Atorvistatin Calcium  powder equivalent to 10mg of  Atorvistatin  marketed by lpca Laboratories Ltd., Bharikhola, Sikkim, INDIA, were used for studies.

Standard Atorvistatin Solution (1mg/1ml) : Atorvistatin Calcium  powder equivalent to 10mg of  Atorvistatin  from Atorvistatin Tablets IP (X’tor^®-10) dissolved in small quantity of 95% of ethyl alcohol and made up to 10 ml with the 95% ethyl alcohol.

Standard Clopidogrel and Aspirin Solutions:

Clopitab A 150 capsules, each  containing clopidogrel bisulphate powder equivalent to 75 mg of clopidogrel and aspirin in tablet form, two tablets of 75 mg each (i.e.150 mg of aspirin), marketed by LUPIN LTD., MUMBAI, INDIA, were used for studies.

Clopidogrel Standard Solution ( 1mg/ml) : Clopidogrel bisulphate powder equivalent to 10 mg of clopidogrel was weighed from powder contents of capsule (Clopitab A 150), dissolved in small quantity of 95% of ethyl alcohol and made up to 10 ml with the 95% ethyl alcohol.

Aspirin Standard Solution ( 1mg/ml) : Two tablets of aspirin were separated out carefully, from the contents of  Clopitab A 150 capsule, finely powdered and quantity equivalent to 10 mg of aspirin was weighed, dissolved in small quantity of 95% ethyl alcohol which was further made up to 10 ml using the same.

PROCEDURE

Camag, HPTLC glass plate pre-coated   with silica gel 60 F 254 of the size of 10 cm x 10 cm was taken for the experiment. 0.05, 0.1, 0.2 and 0.5 μg quantities of Atorvistatin standard solution (1mg/1ml) were spotted on the HPTLC plate using micro capillaries. Further, 0.05, 0.1, 0.2 and 0.5 μg quantities of each of the clopidogrel and aspirin standard solutions were, also, spotted on the HPTLC plate. The HPTLC plate was eluted in a previously saturated small sized, twin trough Camag HPTLC chamber using 5 ml quantity of  solvent mixture Bexane : Acetone : Ethyl alcohol ( 8:1:1, v/v/v ) . The solvent was allowed to travel to a height of  5 cm. Then the HPTLC plate was taken out, air dried and viewed in a UV chamber at 254 nm wavelength.

RESULTS AND DISCUSSION

The developed and air dried HPTLC plate was then observed at wavelength 254nm using Camag UV viewing chamber. Atorvistatin showed its presence at at R^f-value 0.25±0.02 by fluorescence quenching phenomenon.  Aspirin showed its presence at Rf-value 0.38±0.02 and Clopidogrel  was found present at Rf-value 0.93 ±0.02. The chosen solvent system  showed very nice separation on HPTLC plate for cardiovascular drugs Atorvastatin, Aspirin and Clopidogrel thus differentiating these from each other. The LOD for these drugs is cal. 50 ng. However, the spot visualized for Clopidogrel has progressed towards the solvent front. Further, this system fails differentiate Carvedilol ( R_f =0.26 ) from Atorvastatin ( R_f =0.25±0.02  ) and hence the former drug has been ignored to get mentioned in detailed, herein.  Thus, HPTLC method is easier, simple, specific and speedy one to detect and differentiate cardiovascular drugs Atorvistatin, Aspirin and Clopidogrel, in combined formulations and biological material.

ACKNOWLEDGEMENT

Author’s thanks are due to Director, Directorate of Forensic Science Laboratories, Mumbai, State of Maharashtra, India.

REFERENCES

[1] Panchal S. J., Suhagia B. N.; JAOAC Int. Sept.-Oct 2010, 93(5), 1450-1457.

[2] Himani Agarwal, Niraj Kaul, Paradkar A. R. and Mahadik K. R.;  Talanta, 2003, 61(5), 581-589.

[3] Mitakos, A. and Panderi, I.; J. Pharm. Biomed. Anal., 2002, 28, 431-438.

[4] Gandhimathi, M., Ravi, T. K., Abraham, A and Thomas, R.;  J. Pharm. Biomed. Anal., 2003, 32, 1145-1148.

[5] Shah, D. A., Bhatt, K. K., Mehta, R. S., Shankar, M. B., Baldania, S. L. and Gandhi, T. R.; Ind. J. Pharm. Sci., 2007, 69(4), 571-574.

[6] Shah, D. A., Bhatt, K. K., Mehta, R. S., Shankar, M. B., Baldania, S. L. and Gandhi, T. R.; Ind. J. Pharm. Sci., 2007, 69(4), 546-549.

[7] Sawyer, M. and Kumar, V. J.; J. Chromatogr. Sc., 2003, 4, 393-397.

[8] Bernard Fried and Joseph Sharma; Practical Chromatography, A Multidisciplinary Approach, CRC Press, 1996, p-242.

[9] Sena, M. M. and Poppi, R. J.; J. Pharm. Biomed. Anal., 2004, 34, 27-34.