Author(s):
Ajay I. Patel, Kishna Ram, Swati Guttikar, Amit Kumar J. Vyas, Ashok B. Patel, Nilesh K. Patel, Vikash Trivedi
Email(s):
aipvk84@gmail.com , sundeshakishan555@gmail.com , swati.guttikar@veedacr.com , amitvyas77@gmail.com , abp2038@gmail.com , nkpatel99@gmail.com , vikas.trivedi@veedacr.com
DOI:
10.52711/0974-360X.2021.00515 
Address:
Dr. Ajay I. Patel1, Kishna Ram*1, Ms. Swati Guttikar2, Dr. Amit Kumar J. Vyas1, Dr. Ashok B. Patel1, Dr. Nilesh K. Patel1, Dr. Vikash Trivedi2
1B. K. Mody Government Pharmacy College, Rajkot, Gujrat (India).
2Veeda Clinical Research Pvt. Ltd., Ahmedabad, Gujrat (India).
*Corresponding Author
Published In:
Volume - 14,
Issue - 6,
Year - 2021
ABSTRACT:
A rapid, sensitive, accurate and precise high-performance liquid chromatography–tandem mass spectrometric (HPLC– MS/MS) method equipped with Electro Spray Ionization (ESI) source, operating in the Positive ion and Multiple Reaction Monitoring (MRM) mode was developed and validated for the estimation of Phenytoin in human plasma using Phenytoin D10 as an internal standard. Liquid-liquid extraction (LLE) technique was used to extract analyte and ISTDs from 0.2mL human plasma. The analytical separation was carried out in a reverse phase liquid chromatography by using C18 (150 x 4.6mm, 5µm) column, and 2mM Ammonium Acetate in water (pH 6.3): Methanol (30:70% v/v) mobile phase at 1mL/min in isocratic mode. Analytes were monitored in multiple reactions monitoring (MRM) mode using the respective [M+H]? ions, m/z 253.10? 182.30 for Phenytoin and m/z 263.30 ? 192.20 for the internal standard, respectively. The Phenytoin and Phenytoin D10 retained at about 2.50 and 2.46 minutes respectively with total run time of 4 minute. The response of the LC-ESI-MS/MS method for both analyte and ISTD was linear over the dynamic range of 60-12000ng/mL with correlation coefficient r2 = 0.9963. The Accuracy was well within the accepted limit of ± 20% at lower limit of quantification (LLOQ) and ± 15% at all the other concentrations in the linear range. Recovery of drug and ISTD was 87.52% and 86.42%, respectively. This method was fully validated for all the validation parameters and Stability studies. After optimization, the Bioanalytical method was validated according to USFDA, EMA and ANVISA guidelines for Bioanalysis.
Cite this article:
Ajay I. Patel, Kishna Ram, Swati Guttikar, Amit Kumar J. Vyas, Ashok B. Patel, Nilesh K. Patel, Vikash Trivedi. Rapid, Sensitive and Simple LC-MS/MS Method Development and Validation for Estimation of Phenytoin in Human Plasma by using Deuterated Internal Standard. Research Journal of Pharmacy and Technology. 2021; 14(6):2937-4. doi: 10.52711/0974-360X.2021.00515
Cite(Electronic):
Ajay I. Patel, Kishna Ram, Swati Guttikar, Amit Kumar J. Vyas, Ashok B. Patel, Nilesh K. Patel, Vikash Trivedi. Rapid, Sensitive and Simple LC-MS/MS Method Development and Validation for Estimation of Phenytoin in Human Plasma by using Deuterated Internal Standard. Research Journal of Pharmacy and Technology. 2021; 14(6):2937-4. doi: 10.52711/0974-360X.2021.00515 Available on: https://www.rjptonline.org/AbstractView.aspx?PID=2021-14-6-5
REFERENCES:
1. Pallavi A, Prasad K, Srinivas K, Siva Sanker RY, Sathyanarayana B. Validation of HPLC-MS method in positive ion mode for estimation of phenytoin in human plasma using phenytoin D10 as internal standard. Asian Journal of Chemistry. 2017; 29(8): 1845-1852
2. Tripathi KD. Antiepileptic Drugs. Essential of Medical Pharmacology. Jaypee Brothers Medical Publishers, New Delhi. 2019; 8th ed: pp 438-451.
3. Rang HP, Ritter JM, Flower RJ and Henderson G. Antiepileptic Drugs. Rang and Dales’ Pharmacology, Elsevier Publication, 2016; 8th ed: pp 546-558.
4. Uttam G, Judy P, Clinton F, Trang N, Angela M. A simple isotope diluted electrospray ionization tandem mass spectrometry method for the determination of free phenytoin. Ther Drug Monit. 2013; 35(6): 831-835.
5. Roy SMN, Yetal SM, Vaidya VV, Joshi SS. Determination and quantification of phenytoin in human plasma by liquid chromatography with electrospray ionization tandem mass spectrometry. E-Journal of Chemistry. 2008; 5(1): 169-176.
6. Sofiya B, James CO, Alan PB, Thomas JO. Determination of free level of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2000; 23: 573-579.
7. Rampalli SS, Palani S. Rapid, sensitive and simple LC-MS/MS method development and validation for estimation of phenytoin in human plasma. Asian Journal of Chemistry. 2017; 29(10): 2305-2310
8. Baldo MN, Hunzicker GA, Altamirano JC, Murguia MC, Hein GJ. Saliva as a noninvasive biological sample to compare bioavailability of phenytoin formulation by LC-MS/MS. International Journal of Pharmaceutical Science and Research. 2015; 6(9): 3752-3760.
9. Yi Zang, Nitin M, Nageshwer RB, Michael LC, Bernd M. A tandem mass spectrometry assay for the simultaneous determination of Acetaminophen, Caffeine, Phenytoin, Ranitidine and Theophylline in small volume pediatric plasma specimens. Clinica Chimica Acta. 2008; 398: 105-112.
10. Raphael H, Stefan K, Stefan FM. Development and validation of an LC-MS/MS and comparison with GC-MS method to measure phenytoin in human brain dialysate, blood, and saliva. Journal of Analytical Methods in Chemistry. 2018; 1-14.
11. Kadi AA, Kassem MG, Makeen HA, Alhazmi HA. Simultaneous determination of phenytoin and lamotrigine in human plasma using hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry. Digest Journal of Nanomaterials and Biostructures. 2013; 8(3): 1113-1122.
12. Bhatti MM, Hanson GD, Schultz L. Simultaneous determination of phenytoin, carbamazepine and 10,11-carbamazepine epoxide in human plasma by high performance liquid chromatography with ultraviolet detection. Journal of Pharmaceutical and Biomedical Analysis, 1998; 5: 1233-1240.
13. Michael J, Maozhi L, Kelly D. Simultaneous rapid HPLC determination of Phenytoin and its prodrug Fosphenytoin in human plasma and ultra filtrate. Journal of Chromatography B. 1997; 693: 407-414.
14. Jesse F, Sheril A, Mariana B. A novel HPLC method for determination of phenytoin in human plasma. Journal of Pharmaceutical Research International. 2018; 22(6): 1-7.
15. Andrea B, Anral T, Grorge H, Viola H. Determination of Phenytoin in human plasma by molecular imprinted solid phase extraction. Journal of Chromatography A. 2001; 930: 31-38.
16. Sergio L, Daniele R, Lidiane M. Determination of phenytoin in human plasma by a validated liquid chromatography method and its application to a bioequivalence study. Latin American Journal of Pharmacy. 2009; 28(2): 247-253.
17. European Medicine Agency, “Guideline on bio analytical method validation”, July 2011, https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf
18. Anjana V, Ashok P, Ajay P, Amit V, Nilesh P. Sample Preparation in Bioanalysis: A Review. International Journal of Scientific and Technology Research. 2016; 5(5): 6-10.
19. Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Centre for Drug and Research (CDER), May 2018. https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs
20. ANVISA, Guide for validation of analytical and bioanalytical methods. May 29. 2003.
21. Bhole RP, Jagtap SR, Chadar KB, Zambare YB. Liquid Chromatography-Mass Spectrometry Technique-A Review. Research Journal of Pharmacy and Technology. 2020; 13(1): 505-516.