A new validated stability-indicating gradient RP-HPLC method for the determination of pemetrexed disodium and its process related substances

  • S. Hemchand
  • R. Ravi Chandra Babu
  • Mukthinuthalapati Mathrusri Annapurna


Pemetrexed disodium is used for the treatment of malignant pleural mesothelioma and lung cancer. In the present study a simple stability indicating RP-HPLC method was developed and validated for the determination of Pemetrexed disodium. The process related substances such as Dimer-1 impurity, Dimer-2 impurity, N-Methyl Pemetrexed, Pemetrexed diethyl ester, Alanine derivative of Pemetrexed, DMF derivative of Pemetrexed, Acid intermediate, Oxidation impurity and D-isomer were separated on gradient mode and quantified. Forced degradation studies were performed to prove the specificity. Hypersil BDS C18 100 x 4.6mm, 3µm was used for the separation (at 27°C) with mobile phase mixture consisting of (0.02M sodium dihydrogen phosphate with 0.1% HCOOH and pH 3.8 with dilute sodium hydroxide): Acetonitrile (40:60 v/v) (pH 3.8) with a flow rate of 1.2 mL/min. Methanol: water (1:1) was used as diluent and the eluted compounds were monitored at 240 nm. 0.5-1500 µg/mL with linear regression equation y = 20588x - 9294.1 (R2=0.9999). The degradation products observed during the forced degradation studies were well resolved from the drug peak and proving that the method is a stability-indicating method. The method was validated as per ICH guidelines.

Keywords: Pemetrexed disodium, RP-HPLC, gradient mode, Related substances, Stability indicating, Validation.


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1. Shih C, Chen VJ, Gossett LS, Gates SB, MacKellar WC, Habeck LL, Shackelford KA, Mendelsohn LG, Soose DJ, Patel VF, Andis SL, Bewley JR, Rayl EA, Moroson BA, Beardsley GP, Kohler W, Ratnam M, Schultz RM, LY221514, a pyrrolo [2,2-d] pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes, Cancer Research, 1997; 57(6):1116-1122.
2. Hanauske AR, Chen V, Paoletti P, Niyikiza C, Pemetrexed disodium: A novel antifolate clinically active against multiple solid tumors, Oncologist, 2001; 6(4):262-272.
3. Bischof M,Weber KJ, Blatter J, Wannenmacher M, Latz D, Interaction of Pemetrexed disodium (ALIMTA, multi targeted antifolate) and irradiation in vitro, International Journal of Radiation Oncology, Biology, Physics, 2002; 52(5):1281-1288.
4. Roland JWM, Robin Cornelissen, Rob JVK, Robert de Jonge, Ethan den Boer, Jan Lindemans, Theo ML, A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate Pemetrexed in plasma from lung cancer patients. Analytical and Bioanalytical Chemistry, 2010; 298(7-8):2942-2948.
5. Marcel PS, Sabine Visser, Evertvan Dijk, Joachim GJVA, Bruno HS, A new quantification method for assessing plasma concentrations of Pemetrexed and its polyglutamate metabolites, Journal of Pharmaceutical and Biomedical Analysis, 2016; 128(5): 1-8.
6. Laurent PR, Stephen JC, Michael B, James FB, Highly sensitive analysis of the antifolate pemetrexed sodium, a new cancer agent, in human plasma and urine by high-performance liquid chromatography, Journal of Chromatography B: Biomedical Sciences and Applications, 2001; 765(2): 125-140.
7. Vamsi Krishna G, Archana V, Rajeswari J, A new rapid stability indicating RP-PDA-UPLC method for the estimation of assay of Pemetrexed disodium-An anti-lung cancer drug from lyophilized parenteral formulation, Journal of Applied Pharmaceutical Science, 2017; 7(10): 121-127.
8. Ramulu K, Rao BM, Madhavan P, Lalitha Devi M, Srinivasu MK and Chandrasekhar KB, A validated chiral LC method for the determination of enantiomeric purity of Pemetrexed disodium on an amylose-based chiral stationary phase, Chromatographia, 2007; 65(2-4): 249-252.
9. Hemchand S, Ravi Chandra Babu R and Mathrusri Annapurna M, Enantiomeric separation and validation of D-isomer in Pemetrexed disodium–An anti-cancer agent using Chiral HPLC, Research Journal of Pharmacy and Technology, 2019; 12(2): 772-786.
10. Saravanan G, Suryanarayana MV, Jadhav MJ, Ravikumar M, Someswararao N and Acharyulu PVR. A stability-indicating LC assay method for Pemetrexed disodium. 2007, 66, 421-424.
11. Thahera Banu, Murali Balaram V, Mushraff Ali Khan M, Sharma JVC, Bhanu Teja B, Swaroop Kumar V and Habibuddin M, Validated reverse phase HPLC method for the determination of Pemetrexed disodium in pharmaceutical dosage forms, Oriental Journal of Chemistry, 2010; 26(4): 1225-1222.
12. Suresh Kumar A and Devendra Singh R, Development and validation of Pemetrexed by RP-HPLC method in bulk drug and pharmaceutical dosage forms, Indian Journal of Research in Pharmacy and Biotechnology, 2013; 1(4): 537-542.
13. Rakesh Gupta K, Balakumar C, Anjaneyulu V, Karpakavalli M, Lakshmi Narayanan B, Ranjith Kumar D, Kumar EP, Development and validation of RP-HPLC method for related substance of Pemetrexed disodium, International Journal of Pharmaceutical Sciences Review and Research, 2012; 15(2): 133-137.
14. Ankit DP, Shalin KP, Sen DJ and Patel CN, Development and validation of high performance liquid chromatographic and UV spectrophotometric method for estimation of Pemetrexed disodium in bulk drug and pharmaceutical formulation, International Journal of Drug Development & Research 2011; 2(2): 201-207.
15. Janaki Pathi P, Saifulla Khan P, Raveendra Reddy P and Appala Raju N, Visible spectrophotometric estimation of Pemetrexed disodium in pharmaceutical formulations, Journal of Pharmacy Research, 2011; 4(2): 524-525.
16. Umesh SD, Nagaraj PS, Suresh MT, Electrochemical oxidation and determination of an anticancer drug pemetrexed disodium, Asian Journal of Pharmaceutical and Clinical Research, 2017; 10(2): 492-496.
17. ICH Q2 (R1) Validation of analytical procedures: Text and methodology (2005).
18. ICH Q1A (R2) Stability testing of new drug substances and products 2002.
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Hemchand, S., Babu, R. R. C., & Annapurna, M. M. (2019). A new validated stability-indicating gradient RP-HPLC method for the determination of pemetrexed disodium and its process related substances. Journal of Drug Delivery and Therapeutics, 9(3-s), 588-610. https://doi.org/10.22270/jddt.v9i3-s.2918