Join us   Log in  

PHARMASPIRE - Volume 12, Issue 2, April - June, 2020

Pages: 55-60

Date of Publication: 14-Jun-2022

Print Article   Download XML  Download PDF

Development and validation of analytical method for simultaneous estimation of lutein, lycopene, and beta-carotene using reversed-phase high-performance liquid chromatography

Author: Harmandeep Singh, Gurpreet Singh, Rohit Bhatia

Category: Pharmaceutics


Background: Lutein, lycopene, and beta-carotene (MiNOXiV) drug combination is used for the treatment of eye diseases including age-related macular degeneration, cataracts, retinitis, retinitis pigmentosa, cancer of the prostate, breast, lung, bladder, ovaries, colon, and pancreas. Objective: The objective of the study was to develop and validate a high-performance liquid chromatographic method for the simultaneous determination of lutein, lycopene, and beta-carotene in tablet dosage forms. Materials and Methods: A Waters C18 column (50 mm × 4.6 mm, 5 μm) with mobile phase consisting of acetonitrile:butanee-1-ol:dichloromethane (70:30:5 [v/v])was used. The flow rate was 1.0 ml/min and effluents were monitored at 472 nm. Results: The retention time of lutein, lycopene, and beta-carotene in tablet formulation were found to be 7.2 min, 6.0 min, and 10.1 min, respectively. The method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantitation, precision, accuracy, linearity, ruggedness, and robustness. Conclusion: The method showed good reproducibility and recovery with % reflex sympathetic dystrophy <2. Hence, the proposed method was found to be simple, specific, precise, accurate, and linear. Hence, it can be applied for routine analysis of lutein, lycopene, and beta-carotene in pharmaceutical combined dosage forms.

Keywords: Lutein, lycopene, beta-carotene, validation


1. Analysis B. HPLC method for the simultaneous determination of beta-carotene, retinol and alpha-tocopherol in serum. J Pharm Biomed Anal 1988;6:853-7.

2. Thürmann PA, Schalch W, Aebischer JC, Tenter U, Cohn W. Plasma kinetics of lutein, zeaxanthin, and 3’ -dehydro-lutein after multiple oral doses of a lutein supplement. Am J Clin Nutr 2005;82:88-97.

3. Chandra-Hioe MV, Rahman HH, Arcot J. Lutein and β-carotene in selected asian leafy vegetables. J Food Chem Nanotechnol 2017;3:93-7.

4. Aguilar MI. Reversed-phase high-performance liquid chromatography. HPLC Pept Proteins 2004;251:9-22.

5. Ahuja S. 1-Overview of modern pharmaceutical analysis. Sep Sci Technol 2011;10:1-9.

6. Behera S, Ghanty S, Ahmad F, Santra S, Banerjee S. UV-visible spectrophotometric method development and validation of assay of paracetamol tablet formulation. Int J Pharm Sci Res 2012;3:4945-53.

7. Bose A. HPLC calibration process parameters in terms of system suitability test. Austin Chromatogr 2014;1:1-4.

8. Chan CC. Analytical Method Validation and Instrument Performance Verification. Hoboken: John Wiley & Sons; 2004.

9. Chatrabhuji P, Pandya C, Patel M. HPLC Method for Determination of APIs in Pharmaceutical Formulation. Morrisville: Lulu Press; 2002.

10. Chatwal G, Anand S. Instrumental Methods of Chemical Analysis: Analytical Chemistry. Bengaluru: Himalaya Publishing House; 1986.

11. Huber L. Validation of Analytical Methods. Germany: Agilent Technologies; 2010.

12. Bhatnagar-panwar M, Bhatnagar-Mathur P, Bhaaskarla VV, Dumbala SR, Sharma KK. Rapid, accurate and routine HPLC method for large-scale screening of pro-Vitamin A carotenoids in oilseeds. J Plant Biochem Biotechnol 2015;24:84-92.

13. Pathak A, Rajput SJ. Simultaneous derivative spectrophotometric analysis of doxylamine succinate, pyridoxine hydrochloride and folic acid in combined dosage forms. Indian J Pharm Sci 2008;70:513-7.

14. ICH. ICH Topic Q2 (R1) Validation of analytical procedures: Text and methodology. In: International Conference on Harmonization. Geneva: ICH; 2005. p. 17.

15. ICH. Guidance for industry: Q2B validation of analytical procedures: Methodology. In: International Conference on Harmonisation of Technical Requirements for Registration Tripartite Guideline. Geneva: ICH; 1996. p. 13.

16. Siddiqui MR, AlOthman ZA, Rahman, N. Analytical techniques in pharmaceutical analysis: A review. Arab J Chem 2017;10:S1409-21.