Abstract
To analyze Isosorbide Mononitrate in human plasma, a highly sensitive and accurate LC–MS/MS bioanalytical method was developed and validated using Isosorbide mononitrate-¹³C₆ as the internal standard. Validate a suitable analytical method for the estimation of unknown drug concentrations in plasma. It can be used for the quantitation of isosorbide mononitrate in K₂EDTA human plasma for bioequivalence and bioavailability studies. The method was found to be specific, sensitive, linear, precise, accurate, and reproducible for the extraction and analysis of isosorbide mononitrate in K₂EDTA human plasma samples over the investigated concentration range of 5.000 ng/mL to 1800.000 ng/mL using a processing volume of 0.100 mL. Chromatographic separation was achieved using a 2 mM ammonium acetate (acidified) mobile phase with acetonitrile containing 0.1% acetic acid in a ratio of 55:45 (v/v) at a flow rate of 0.5 mL/min. Detection was carried out using multiple reaction monitoring with transitions (m/z) of 249.90 → 59.00 for isosorbide mononitrate and 255.90 → 58.90 for the internal standard. The analytes were detected using tandem mass spectrometry (LC–MS/MS). The proposed method was comprehensively validated in terms of linearity, accuracy, precision, specificity, sensitivity, recovery, and stability, with results meeting the acceptance criteria. The developed and validated method was found to be simpler, faster, more specific, precise, and cost-effective compared with previously reported methods.
Data availability
Availability of data and materialThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Abbreviations
- ISMN:
-
Isosorbide mononitrate
- ISTD:
-
Internal standard
- LC–MS–MS:
-
Liquid chromatography mass spectrometry–mass spectrometry
- K2EDTA:
-
Dipotassium ethylene diamine tetra acetic acid
- (m/z):
-
Mass to charge ratio
- (cGMP):
-
Current manufacturing practices
- ESI:
-
Electron spray ionization
- HPLC:
-
High performance liquid chromatography
- UPLC–TMS:
-
Ultra performance liquid chromatography–tandem mass spectrometry
- MRM:
-
Multi reaction monitoring
- QC:
-
Quality control
- HQC:
-
Highest quality control
- MQC:
-
Median quality control
- LQC:
-
Lowest quality control
- LLOQ:
-
Lower limit of quantification
- ULOQ:
-
Upper limit of quantification
- % CV:
-
Percent coefficient variations
References
Silva, L. C. et al. Quantification of isosorbide5-mononitrate in human plasma by liquid chromatography- tandem mass Spectrometry using atmospheric pressure photoionization. J. Chromatogr. B 832, 302–306 (2006).
Patel, B. D. et al. Development and validation of a chiral LC-ESI-MS/MS method for simultaneous determination of the enantiomeric metabolites isosorbide 2-mononitrate and isosorbide 5-mononitrate of isosorbide dinitrate in rat and human plasma. J. Pharm. Biomed. Anal. 174, 396–405 (2019).
Jain, D. S. et al. An extensive study on ISMN acid adducts for quantification in human plasma using LC-ESIMS/MS and its application to bioequivalence sample analysis. Rapid Commun. Mass Spec. 20(19), 2921–2931 (2006).
Xu, B. B., Shuang-Li, Z., Huiyu, Z. & Tang, C. Bioequivalence Study of isosorbide mononitrate in human plasma using high performance liquid chromatography. Latin Am. J. Pharm. 33(1), 41–46 (2014).
Jalakam, S. P., Waghmode, J., Shinde, D., Pawar, P., Mane, G. An Ultra performance liquid chromatography- tandem mass spectrometric method for the determination of isosorbide 5- mononitrate in human plasma.
Kosoglou, T. et al. Pharmacokinetics of isosorbide-5-mononitrate after oral administration of an extended-release mononitrate formulation versus a standard dinitrate formulation. Clin. Ther. 17(2), 241–251 (1995).
Hohyum, K. et al. simultaneous separation and determination of isosorbide dinitrate and isosorbide mononitrate in human plasma by LC-MS-MS. Chromatographia 71(8), 595–602 (2010).
Karmalkar, H. S., Metku, M. M., Bagul, M. S., Nimkar, A. C. & Shah, R. D. LC determination of isosorbide-5-mononitrate in human plasma. Chromatographia 69, 1035–1039 (2009).
Pitt, J. Principle and application of LC-MS in clinical biochemistry: the clinical biochemist reviews. Clin. Biochem. Rev. 30(1), 19–34 (2009).
Dodda, S., Makula, A. & Kandhagatla, R. N. Development and validation of bioanalytical method for quantification of cycloserine in human plasma by liquid chromatography–tandem mass spectrometry: Application to pharmacokinetic study. Biomed. Chromatogr. https://doi.org/10.1002/bmc.4548 (2019).
Dodda, S., Makula, A., Polagani, S. R. & Kandhagatla, R. N. High sensitive LC–MS/MS method for estimation of eluxadoline in human plasma and its application to pharmacokinetic study. J. Pharm. Biomed. Anal. 165, 65–72. https://doi.org/10.1016/j.jpba.2018.11.056 (2019).
Padmabhushana, C. V., Sireesha, D. & Srinivasa, R. P. Development and validation of a bioanalytical method for estimation of prucalopride in human plasma using liquid chromatography-tandem mass spectrometry. J. Appl. Spectrosc. 92, 388–392. https://doi.org/10.1007/s10812-025-01924-y (2025).
Viritha, B., Padma, M., Srinivasa Rao, P. & Sireesha, D. Bioanalysis of bedaquiline in human plasma by liquid chromatography-tandem mass spectrometry: Application to pharmacokinetic study. J. Mass Spectr. Adv. Clin. Lab 31, 27–32. https://doi.org/10.1016/j.jmsacl.2024.01.001 (2024).
Dodda, S., Makula, A., Polagani, S. R. & Kandhagatla, R. N. Development and validation of bioanalytical liquid chromatography-tandem mass spectrometry method for the estimation of pentoxifylline in human plasma: Application for a comparative pharmacokinetic study. Eur. J. Mass Spectrum 25(4), 372–380. https://doi.org/10.1177/1469066718817929 (2019).
Thammana, M. A review on high performance liquid chromatography. Rese Rev. J. Pharm. Anal. 5(2), 22–28 (2016).
Jannetto, P. Liquid Chromatography Tandem Mass Spectrometry, Clinical Laboratory. Amer Assoc of Clinical Chem (2015).
Bruins, A. P., Niessen, W. M. A. Mass Spectrometry: Atmospheric Pressure Ionization Technique, 3rd edn. in Encyclopedia of Analytical Science (2019).
Hanold, K. A., Fischer, S. M., Cormia, P. H., Miller, C. E. & Syage, J. A. Atmospheric pressure photoionization: General properties for LC/MS. Anal. Chem. 76(10), 2842–2851 (2004).
Tijare, L. K., Rangari, N. T. & Mahajan, U. N. A Review on bioanalytical method development and validation. Asian J. Pharm. Clin Res. 9(9), 6–10 (2016).
Taleuzzaman, M., Gilani, S. J., Ali, S., Hafeez, A. & Nagarajan, K. Bio-analytical method validation—a review. Austin J. Anal. Pharm. Chem. 2(5), 1–5 (2015).
Deshpande, M. M., Kasture, V. S., Mohan, M. & Chavan, M. J. Bioanalytical method development and validation: A review. Recent Adv. Anal. Chem. https://doi.org/10.5772/intechopen.81620 (2019).
Moein, M. M., Beqqali, A. E. & Abdel-Rehim, M. Bioanalytical method development and validation: Critical concepts and strategies. J. Chrom. B 1043, 3–11 (2017).
Kirthi, A., Shanmugam, R., Shanti, P. M. & Jamal, B. D. A review on bioanalytical method development and validation by RP-HPLC. J. Glob. Trends Pharm Sci. 5(4), 2265–2271 (2014).
Ravi, S. P., Sai, G. A., Rachana, G., Srinivas, B. P. & Bhargavi, J. Bioanalytical method validation: a comprehensive review. Int. J. Pharm. Sci. Rev. Res. 56(1), 50–58 (2019).
Whitmire M. et al. LC-MS/MS bioanalysis method development, validation, and sample analysis: points to consider when conducting nonclinical and clinical studies in accordance with current regulatory guidance’s. J. Anal. Bio. Tech. 1(1) (2011).
Moosavi, S. M. & Ghassabian, S. Linearity of calibration curves for analytical methods: A review of criteria for assessment of method. Calibr. Valid. Anal. Methods: Sampl. Curr. Approaches https://doi.org/10.5772/intechopen.72932 (2018).
Ingle, A., Baheti, M. & Bhusari, K. Bioanalytical method validation and its importance in pharma research-a review. Int. J. Pharm. Chem. 7, 28–31 (2017).
Sonawane, L. V., Poul, B. N., Usnale, S. V., Waghmare, P. V. & Surwase, L. H. Bioanalytical method validation and its pharmaceutical application-a review. Pharm. Anal. Acta. 5(3), 288 (2014).
Saida, S., Sai, S. B. & Vijayaraja, S. Bioanalytical technique-an overview. Pharm. Tutor. 3(9), 14–24 (2015).
Sowjanya, P., Subashini, D. & Lakshmi, R. K. Analytical validation parameters, research and reviews. J. Pharm. Anal. 4(1), 5–74 (2015).
Acknowledgements
The authors gratefully acknowledge Amrutvahini college of Pharmacy, Sangamner (Maharashtra, India) for providing necessary facilities for carrying out this review and also grateful to all the staff and friends for their help and support.
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R.N.K. has Oversight and leadership responsibility for the literature review planning and execution, including mentorship external to the core team P.S.T. carried out HPLC method performed the validation parameters. H.U.C. & P.L.P. has contributing in grammatically molding and writing of manuscript SVA gave their scientific suggestion. All authors have read and approved the manuscript.
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Kachave, R.N., Thombare, P.S., Chikhale, H.U. et al. Bioanalytical method development and validation of isosorbide mononitrate in human plasma by using LC–MS/MS. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35330-x
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DOI: https://doi.org/10.1038/s41598-026-35330-x
