Gas chromatography-mass spectrometry for quantitating valproic acid in blood serum

Background. Generating a sensitive and selective method to quantitate valproic acid (VPA) for therapeutic drug monitoring, using as low as 50 µl of blood serum sample, is highly relevant for pediatric patients, for whom biomaterial collection can be challenging.

Objective: To develop and validate a method to quantitate VPA level in blood serum using gas chromatography-mass spectrometry (GC-MS) without derivatization, added with phenylacetic acid as an internal standard (IS), for therapeutic drug monitoring, and to comparatively analyze results obtained using developed method and immunochemical approach.

Material and methods. Extraction of VPA and IS was performed using liquid-liquid extraction with ethyl acetate, after preacidification of blood serum sample with 1 M hydrochloric acid. The lower limit of quantification was 5 µg/ml, with calibration range 5–200 µg/ml. The study employed a GC-MS system consisting of Agilent 5977B SQ single quadrupole MS-detector equipped with a high-efficiency source for electron impact ionization, and Agilent 7890 gas chromatograph (both – Agilent Technologies, USA). Analytes were quantitated by using electron impact ionization in selected ion monitoring mode. For VPA, the characteristic fragment ion with mass-to-charge ratio (m/z) 102 was used, and for IS – most intense characteristic fragment ion with m/z 91. Chromatographic separation was carried out on HP-5MS Ultra Inert column (30 m × 250 µm × 0.25 µm) (Agilent Technologies, USA).

Results. A comparative analysis of the results obtained in determining VPA level in blood serum samples using GC-MS and immunochemical method demonstrated a high degree of correlation, thereby verifying feasibility of the developed method for therapeutic drug monitoring.

Conclusion. The developed GC-MS method is characterized by high sensitivity and selectivity and can be recommended for introduction into clinical practice for therapeutic drug monitoring of VPA concentration as an alternative to immunochemical approach.

1. Belousov Yu.B., Leonova M.V., Shteinberg L.L., et al. Therapeutic drug monitoring of anticonvulsants in clinical practics. Epilepsia i paroksizmal'nye sostoania / Epilepsy and Paroxysmal Conditions. 2013; 5 (3): 6–16 (in Russ.).

2. Kirpichenko A.A. Modern place of valproates in the treatment of mental and behavioral disorders. Medical News. 2022; 5: 34–8 (in Russ.).

3. Raspopova N.I. Use of valproic acid in complex therapy of postcovid mental disorders (clinical case). Current Therapy of Mental Disorders. 2024; 2: 68–76 (in Russ.). https://doi.org/10.21265/PSYPH.2024.31.31.007.

4. Zhang C.Q., Li H.Y., Wan Y., et al. Efficacy, safety, and retention rate of extended-release divalproex versus conventional delayed-release divalproex: a meta-analysis of controlled clinical trials. Front Pharmacol. 2022; 13: 811017. https://doi.org/10.3389/fphar.2022.811017.

5. Romanov A.S., Sharakhova E.F. Marketing research of the national antiepileptic drug market. Journal of Siberian Medical Sciences. 2022; 6 (4): 48–60. https://doi.org/10.31549/2542-1174-2022-6-4-48-60.

6. Pylaeva O.A., Mukhin K.Yu. Safety and tolerability of antiepileptic therapy: emphasis on the use of valproates (literature review). Russian Journal of Child Neurology. 2019; 14 (3): 37–60 (in Russ.). https://doi.org/10.17650/2073-8803-2019-14-3-37-58.

7. Rifai N. Tietz textbook of laboratory medicine. Saunders; 2022: 1584 pp.

8. Leonova M.V., Ivzhits M.A., Tischenkova I.F., Shteinberg L.L. Therapeutic drug monitoring of anticonvulsants in children in clinical practice. Epilepsia i paroksizmal'nye sostoania / Epilepsy and Paroxysmal Conditions. 2017; 9 (1): 26–34 (in Russ.). https://doi.org/10.17749/2077-8333.2017.9.1.026-034.

9. Rubricator of Сlinical Recommendations. Epilepsy and status epilepticus in adults and children. 2022. Available at: https://cr.minzdrav.gov.ru/preview-cr/741_1 (in Russ.) (accessed 16.01.2026).

10. Patsalos P.N., Spencer E.P., Berry D.J. Therapeutic drug monitoring of antiepileptic drugs in epilepsy: a 2018 update. Ther Drug Monit. 2018; 40 (5): 526–48. https://doi.org/10.1097/FTD.0000000000000546.

11. Shaikh A.S., Liu H., Li Y., et al. Therapeutic drug monitoring of valproic acid. Pak J Pharm Sci. 2018; 31 (4): 1773–6.

12. Abaimov D.A., Sariev A.K., Noskova T.Yu., et al. Modern technologies in therapeutic drug monitoring (review). Epilepsia i paroksizmal'nye sostoania / Epilepsy and Paroxysmal Conditions. 2013; 5 (2): 31–41 (in Russ.).

13. Vlase L., Popa D.S., Muntean D., Leucuta S. A new high-throughput LC-MS/MS assay for therapeutic level monitoring of valproic acid in human plasma. Sci Pharmaceut. 2008; 76: 663–71. https://doi.org/10.3797/scipharm.0808-12.

14. Malygin A.S., Popov N.S., Demidova M.A., Kudrayshova M.N. Chromatography-tandem MASS spectrometry (HPLC-MS/MS) for the detection of valproic acid and its metabolites in blood plasma. Epilepsia i paroksizmal'nye sostoania / Epilepsy and Paroxysmal Conditions. 2018; 10 (2): 35–42 (in Russ.). https://doi.org/10.17749/20778333.2018.10.2.035-042.

15. Blume de Oliveira J., Pereira dos Santos B., Arbo M. Valproic acid extraction methods in human samples for gas chromatography analysis: a review. Drug Anal Res. 2024; 8 (1): 3–12. https://doi.org/10.22456/2527-2616.138963.

16. Abaimov D.A., Sariev A.K., Tyurin I.A., et al. Application of gas chromatography/mass spectrometry to therapeutic monitoring of valproic acid. Khimiko-farmatsevticheskii zhurnal. 2012; 46 (11): 36–40 (in Russ.).

17. Xia Y., Long J.Y., Shen M.Y., et al. Switching between LC-ESI-MS/ MS and EMIT methods for routine TDM of valproic acid in pediatric patients with epilepsy: what clinicians and researchers need to know. Front Pharmacol. 2021; 12: 750744. https://doi.org/10.3389/fphar.2021.750744.

18. Bioanalytical Method Validation. Guidance for Industry. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM). 2018. Available at: https:// www.fda.gov/files/drugs/published/Bioanalytical-Method-ValidationGuidance-for-Industry.pdf (accessed 12.01.2026).

19. Guideline on bioanalytical method validation. European Medicines Agency. 2011. Available at: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf (accessed 12.01.2026).

20. Istomin D.S., Zolotavina M.L. Immunochemiluminescent analysis as an alternative to the tandemic HPLC-massspectrometry method for the determination of valproid acid. Russian Journal of Biological Physics and Chemisrty. 2020; 5 (2): 345–8 (in Russ.).

21. Meyer M.R., Welter J., Weber A.A., Maurer H.H. Development, validation, and application of a fast and simple GC–MS method for determination of some therapeutic drugs relevant in emergency toxicology. Ther Drug Monit. 2011; 33 (5): 649–53. https://doi.org/10.1097/FTD.0b013e3182305409.

22. Leis H.J., Windischhofer W., Rechberger G.N., Fauler G. Synthesis of [18O2]valproic acid and its use as an internal standard for the quantitative measurement by gas chromatography–electron ionization mass spectrometry. J Chromatogr B. 2003; 784 (1): 69–75. https://doi.org/10.1016/S1570-0232(02)00755-9.

23. Davis С. Statistical testing in practice with StatsDirect: research without tears or algebra. Llumina Press; 2013: 124 pр.

24. Giavarina D. Understanding Bland Altman analysis. Biochem Med. 2015; 25 (2): 141–51. https://doi.org/10.11613/BM.2015.015.

25. Saveliev L.I., Tsvirenko S.V. Comparison of methods for measuring analytes using patient samples (general principles of conducting research and analyzing results). Yekaterinburg: Ural State Medical University; 2023: 138 pp. (in Russ.).