Method development and validation of amlodipine and cilnidipine in human plasma by using liquid chromatography and tandem mass spectrometry


  • Muni Krishnaiah A Research Scholar, Department of Chemistry, JNTUA, Anantapur, Andhra Pradesh, India / Department of Science and Humanities, S.V. College of Engineering, Karakambadi road, Tirupati, Andhra Pradesh - 517507, India
  • Rama Chandraiah C Department of Chemistry, Sri Kalahastheeswara Institute of Technology, Srikalahasthi, Andhra Pradesh, India
  • Srinivas M Acubiosys Pvt. Ltd, IALA, Moula-Ali, Hyderabad, Telangana, India
  • Devanna N Department of Chemistry, College of Engineering, JNTUA, Anantapur, Andhra Pradesh, India


Amlodipine and Cilnidipine in K2EDTA plasma were determined by Liquid Chromatography and Tandem Mass Spectrometry. To improve LC separation and detection sensitivity for the LC-MS technique interface of contiguous stationary phases and LC gradients were enhanced. For separation Acetonitrile was used as eluent. Reversed phase C8 and C18, X-bridge columns were utilized. Telmisartan was used an Internal Standard in this process. For elution Telmisartan needs acidic PH but Amlodipine and Cilnidipine needs neutral PH, dilution with H2O for supernate get afterward extraction. The standards were separated in existence of Telmisartan as internal standard from Amlodipine and Cilnidipine by LC-MS/MS using the MRM transitions. The extracted ion chromatograms were considered from peak ratio of analyte to internal standard. Human plasma without analytes, and solutions containing analytes without human plasma were also analysed. To determine the stability analyte was kept in steady state at room temperature for 24 hrs in plasma. The instrument had operating in positive ion mode. The product ion mass spectra of Amlodipine were 409.1 → 237.7 and Cilnidipine was 493.2 → 117.1. Quadratic regression was used to generate calibration curve by the Analyst 1.5 software program. To obtain the linear response the concentration range is 1-1000ng/mL. For each analyte intra (n=6/batch, 3 days) and inter-batch (n=18) accuracy and precision values were obtained.


Amlodipine, Cilnidipine, LC-MS, Ve ion mode, Turbo spray


Download data is not yet available.


Adams, B. D., Browne, W. T. 1998. Amlodipine overdose causes prolonged calcium channel blocker toxicity. The American Journal of Emergency Medicine, 16(5):527–528.

Aruna, G., Bharathi, K., Prasad, K. 2017. Development and validation of bioanalytical hplc method for simultaneous estimation of cilnidipine and nebivolol in human plasma. International Journal of Pharmacy and Pharmaceutical Sciences, 9(10):253–253.

Bansal, S., Chauhan, D. K., et al. 2014. Blood pressure control and acceptability of Perindopril and its fixed dose combinations with Amlodipine or Indapamide, in younger patients with hypertension. Indian Heart Journal, 66(6):635–639.

Buchiya, F. V., Bhim, A. I., et al. 2015. Simultaneous Determination of Cilnidipine and Valsartan in Synthetic Mixture using Spectrophotometric Technique (Simultaneous Equation Method). Asian Journal of Pharmaceutical Analysis, 5(1):21–21.

Chandra, K. S., Ramesh, G. 2013. The fourth generation Calcium channel blocker: Cilnidipine. Indian Heart Journal, 65(6):691–695.

Dai, S. Y., Qiu, S. T., et al. 2013. Development and validation of an rp-hplc method for simultaneous determination of Ramipril and Amlodipine in tablets. Journal of Pharmaceutical Analysis, 3(6):440–446.

Fan, L., Yang, Q., et al. 2011. Dual actions of cilnidipine in human internal thoracic artery: Inhibition of calcium channels and enhancement of endothelial nitric oxide synthase. The Journal of Thoracic and Cardiovascular Surgery, 141(4):1063–1069.

Ghosh, S., Sircar, M. 2008. Calcium channel blocker overdose: experience with amlodipine. Indian journal of critical care medicine: peer-reviewed, official publication of Indian Society of. Critical Care Medicine, 12(4):190–193.

Kudumula, N., Prasad, Y. R. 2014. Development and validation of RP-HPLC Method for the Simultaneous Estimation of Chlorthalidone and Cilnidipine in Bulk and Combined tablet dosage form. Pharmacophore, 5(4):442–450.

Latha, M., Paul, A., Krishnankutty, B., Thomas, M. G., Thomas, M. D. 2011. Evaluation of Amlodipine Besylate in the Treatment of Isolated Systolic Hypertension in Indian Patients. Asian Journal of Pharmaceutical and Clinical Research, 4(1):80–82.

Lee, H. W., Seo, J. H., et al. 2008. Development of a liquid chromatography/negative-ion electrospray tandem mass spectrometry assay for the determination of cilnidipine in human plasma and its application to a bioequivalence study. Journal of chromatography B, 862(1-2):246–251.

Lee, K. R., Chae, Y. J., et al. 2012. Quantification of cilnidipine in human plasma by liquid chromatography-mass spectrometry. Journal of Liquid Chromatography & Related Technologies, 35(2):308–320.

Lei, B., Nakano, D., et al. 2012. N-type Calcium Channel Inhibition With Cilnidipine Elicits Glomerular Podocyte Protection Independent of Sympathetic Nerve Inhibition. Journal of Pharmacological Sciences, 119(4):359–367.

Mital, J. S., Patel, B., Paramar, A. 2014. Development and validation of RP-HPLC method for simultaneous estimation of Cilnidipine and Olmesartan medoxomil in their combined tablet dosage form. Int J Pharm Biosci, 4(1):157–60.

Narapusetti, A., Bethanabhatla, S. S., et al. 2015. Simultaneous determination of rosuvastatin and amlodipine in human plasma using tandem mass spectrometry: Application to disposition kinetics. Journal of Advanced Research, 6(6):931–940.

Pant, S., Pal, K. 2012. Development and Validation of a Simultaneous HPLC Method for Assay and Dissolution of Bisoprolol fumarate and Amlodipine besylate in Pharmaceutical Dosage. Research Journal of Pharmaceutical Dosage Forms and Technology, 4(1):62–66.

Porwal, P. K., Talele, G. S. 2017. Development of validated HPLC-UV method for simultaneous determination of Metformin, Amlodipine, Glibenclamide and Atorvastatin in human plasma and application to protein binding studies. Bulletin of Faculty of Pharmacy, Cairo University, 55(1):129–139.

Ravi, V. B., Inamadugu, J. K., et al. 2012. Simultaneous determination of telmisartan and amlodipine in human plasma by LC–MS/MS and its application in a human pharmacokinetic study. Journal of Pharmaceutical Analysis, 2(5):319–326.

Safhi, M., Nagaraj, M. Y. 2013. Development and validation of a Rapid Stability Indicating chromatographic determination of Cilnidipine in Bulk and Dosage form. Research Journal of Pharmacy and Technology, 6(3):296–299.

Sah, R., Arora, S. 2012. Development and validation of a HPLC analytical assay method for amlodipine besylate tablets: A Potent Ca channel blocker. Journal of Advanced Pharmacy Education & Research, 2(3):93–100.

Sakata, K., Shirotani, M., et al. 1999. Effects of Amlodipine and Cilnidipine on Cardiac Sympathetic Nervous System and Neurohormonal Status in Essential Hypertension. Hypertension, 33(6):1447–1452.

Shaalan, R. A., Belal, T. S., et al. 2017. Validated stability-indicating HPLC-DAD method of analysis for the antihypertensive triple mixture of amlodipine besylate, valsartan and hydrochlorothiazide in their tablets. Arabian Journal of Chemistry, 10:S1381–S1394.

Shah, D. A., Patel, D. V., et al. 2015. High-performance thin-layer chromatography method for estimating the stability of a combination of irbesartan and amlodipine besylate. Journal of Taibah University for Science, 9(2):177–186.

Tengli, A. R., Gurupadayya, B. M., Soni, N. 2013. Simultaneous estimation of hydrochlorothiazide, amlodipine, and losartan in tablet dosage form by RP-HPLC. International Journal of Chemical and Analytical Science, 4(1):33–38.

Uchida, R., Yamazaki, J., et al. 2001. State-Dependent Inhibition of L-type Ca2+ Channels in A7r5 Cells by Cilnidipine and Its Derivatives. Japanese Journal of Pharmacology, 85(3):260–270.



How to Cite

Muni Krishnaiah A, Rama Chandraiah C, Srinivas M, & Devanna N. (2021). Method development and validation of amlodipine and cilnidipine in human plasma by using liquid chromatography and tandem mass spectrometry. International Journal of Research in Pharmaceutical Sciences, 12(1), 620–630. Retrieved from



Original Articles