HPLC-MS/MS method development and validation for the determination of tetradecapeptide in human plasma

Introduction. The number of peptide drugs being developed and registered has increased in recent years. Therefore, modern analytical approaches and methods are required to determine these substances in biological matrices during pharmacokinetic studies. Peptides are structurally intermediate between small molecules and biopolymers, making it difficult to develop methods for determining them using High Performance Liquid Chromatography with Tandem Mass Spectrometry (HPLC-MS/MS). Peptide derivatization can help achieve optimal chromatographic separation and increase method sensitivity.

Aim. To develop and validate a method for the determination of the tetradecapeptide (TDP) threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamayl-threonyl-valyl-glutamyl-arginyl-glutamyl-lysyl-glutamate in human plasma by HPLC-MS/MS.

Materials and methods. The determination of TDP in human plasma was performed by HPLC-MS/MS. Sample preparation included a combination of blood plasma protein precipitation with propionic acid solution in methanol, liquid-liquid extraction with chloroform, and peptide derivatization with propionic anhydride. Internal standard (IS) was threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamayl-threonyl-leucyl-glutamyl-arginyl-glutamyl-lysyl-glutamate. Chromatographic separation was performed in gradient mode, eluent A was 0.1 % formic acid solution in water, eluent B was 0.1 % formic acid in acetonitrile. Column: Waters XBridge C18, 4.6 × 50 mm, 5 µm. Ionization source was electrospray in positive mode. Multiple reaction monitoring (MRM) transitions for 4-substituted TDP propionate were: 681.30 → 73.95 m/z, 681.30 → 84.00 m/z, 681.30 → 101.90 m/z, 681.30 → 140.10 m/z, and for 4-substituted IS propionate: 686.00 → 74.10 m/z, 686.00 → 84.05 m/z, 686.00 → 102.00 m/z, 686.00 → 140.00 m/z.

Results and discussion. Validation of the developed method was carried out in accordance with the requirements of Eurasian Economic Union and the following parameters were determined: selectivity, matrix effect, calibration curve, accuracy and precision, recovery, lower limit of quantification, sample carryover, stability.

Conclusion. The method for the determination of TDP in human blood plasma by HPLC-MS/MS was developed and validated. The analytical range was 5.00–1000.00 ng/mL, allowing the method to be used to study TDP pharmacokinetics.

1. De la Torre B. G., Albericio F. Peptide Therapeutics 2.0. Molecules. 2020;25(10):2293. DOI: 10.3390/molecules25102293.

2. Torchilin V. Intracellular delivery of protein and peptide therapeutics. Drug Discovery Today: Technologies. 2008;5(2–3):e95–e103. DOI: 10.1016/j.ddtec.2009.01.002.

3. Al Shaer D., Al Musaimi O., Albericio F., de la Torre B. G. 2018 FDA Tides Harvest. Pharmaceuticals. 2019;12(2):52. DOI: 10.3390/ph12020052.

4. Al Shaer D., Al Musaimi O., Albericio F., de la Torre B. G. 2019 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals. 2020;13(3):40. DOI: 10.3390/ph13030040.

5. Al Musaimi O., Al Shaer D., Albericio F., de la Torre B. G. 2020 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals. 2021;14(2):145. DOI: 10.3390/ph14020145.

6. Al Shaer D., Al Musaimi O., Albericio F., de la Torre B. G. 2021 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals. 2022;15(2):222. DOI: 10.3390/ph15020222.

7. Al Musaimi O., Al Shaer D., Albericio F., de la Torre B. G. 2022 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals. 2023;16(3):336. DOI: 10.3390/ph16030336.

8. Kaspar A. A., Reichert J. M. Future directions for peptide therapeutics development. Drug Discovery Today. 2013;18(17–18):807–817. DOI: 10.1016/j.drudis.2013.05.011.

9. Apostolopoulos V., Bojarska J., Chai T.-T., Elnagdy S., Kaczmarek K., Matsoukas J., New R., Parang K., Paredes Lopez O., Parhiz H., Perera C. O., Pickholz M., Remko M., Saviano M., Skwarczynski M., Tang Y., Wolf W. M., Yoshiya T., Zabrocki J., Zielenkiewicz P., AlKhazindar M., Barriga V., Kelaidonis K., Mousavinezhad Sarasia E., Toth I. A Global Review on Short Peptides: Frontiers and Perspectives. Molecules. 2021;26(2):430. DOI: 10.3390/molecules26020430.

10. Ewles M., Goodwin L. Bioanalytical approaches to analyzing peptides and proteins by LC-MS/MS. Bioanalysis. 2011;3(12):1379–1397. DOI: 10.4155/bio.11.112.

11. Rauh M. LC-MS/MS for protein and peptide quantification in clinical chemistry. Journal of Chromatography B. 2012;883–884:59–67. DOI: 10.1016/j.jchromb.2011.09.030.

12. Fisher E. N., Melnikov E. S., Gegeckori V., Potoldykova N. V., Enikeev D. V., Pavlenko K. A., Agatonovic-Kustrin S., Morton D. W., Ramenskaya G. V. Development and Validation of an LC-MS/MS Method for Simultaneous Determination of Short Peptide-Based Drugs in Human Blood Plasma. Molecules. 2022;27(22):7831. DOI: 10.3390/molecules27227831.

13. Ding J.-S., Peng W.-X., Zhang Z.-H., Li H.-D., Jiang X.-H. Determination of octreotide in human plasma by HPLC-MS with solid-phase extraction and study on the relative bioavailability of domestic and imported octreotide injections. Yao Xue Xue Bao. 2004;39(7):542–545.

14. Sauter M., Uhl P., Burhenne J., Haefeli W. E. Application of triple quadrupole tandem mass spectrometry to the bioanalysis of collision-induced dissociation-resistant cyclic peptides – Ultra-sensitive quantification of the somatostatin-analog pasireotide utilizing UHPLC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis. 2021;194:113728. DOI: 10.1016/j.jpba.2020.113728.

15. Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015;72:4–15. DOI: 10.1016/j.peptides.2015.04.012.

16. Chen Y., Liang Y., Lv R., Xia N., Xue T., Zhao S. An immunological determination of somatostatin in pharmaceutical by sandwich ELISA based on IgY and polyclonal antibody. Microchemical Journal. 2019;145:532–538. DOI: 10.1016/j.microc.2018.11.019.

17. Oh H. S., Choi M., Lee T. S., An Y., Park E. J., Kim T. H., Shin S., Shin B. S. Pharmacokinetics and brain distribution of the therapeutic peptide liraglutide by a novel LC-MS/MS analysis. Journal of Analytical Science and Technology. 2023;14(1):19. DOI: 10.1186/s40543-023-00382-5.

18. Malm-Erjefält M., Bjørnsdottir I., Vanggaard J., Helleberg H., Larsen U., Oosterhuis B., van Lier J. J., Zdravkovic M., Olsen A. K. Metabolism and Excretion of the Once-Daily Human Glucagon-Like Peptide-1 Analog Liraglutide in Healthy Male Subjects and Its In Vitro Degradation by Dipeptidyl Peptidase IV and Neutral Endopeptidase. Drug Metabolism and Disposition. 2010;38(11):1944–1953. DOI: 10.1124/dmd.110.034066.

19. De Souza I. D., Queiroz M. E. C. Advances in sample preparation and HPLC–MS/MS methods for determining amyloid-β peptide in biological samples: a review. Analytical and Bioanalytical Chemistry. 2023;415(18):4003–4021. DOI: 10.1007/s00216-023-04631-9.

20. Hamman J. H., Enslin G. M., Kotzé A. F. Oral Delivery of Peptide Drugs. BioDrugs. 2005;19(3):165–177. DOI: 10.2165/00063030-200519030-00003.

21. Le Maux S., Nongonierma A. B., FitzGerald R. J. Improved short peptide identification using HILIC–MS/MS: Retention time prediction model based on the impact of amino acid position in the peptide sequence. Food Chemistry. 2015;173:847–854. DOI: 10.1016/j.foodchem.2014.10.104.

22. Doulou E., Kalomiraki M., Parla A., Thermos K., Chaniotakis N. A., Panderi I. Hydrophilic Interaction Liquid Chromatography Coupled with Fluorescence Detection (HILIC-FL) for the Quantitation of Octreotide in Injection Forms. Analytica. 2021;2(4):121–129. DOI: 10.3390/analytica2040012.

23. Kang L., Weng N., Jian W. LC-MS bioanalysis of intact proteins and peptides. Biomedical Chromatography. 2020;34(1): e4633. DOI: 10.1002/bmc.4633.

24. Böttger R., Hoffmann R., Knappe D. Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum. PLOS ONE. 2017;12(6):e0178943. DOI: 10.1371/journal.pone.0178943.

25. Powell M. F. Chapter 30. Peptide Stability in Drug Development: in vitro Peptide Degradation in Plasma and Serum. Annual Reports in Medicinal Chemistry. 1993;28:285–294.

26. Zee B. M., Garcia B. A. Discovery of lysine post-translational modifications through mass spectrometric detection. Essays in Biochemistry. 2012;52:147–63. DOI: 10.1042/bse0520147.

27. Lin S., Garcia B. A. Examining Histone Posttranslational Modification Patterns by High-Resolution Mass Spectrometry. Methods in Enzymology. 2012;512:3–28. DOI: 10.1016/B978-0-12-391940-3.00001-9.

28. Bonaldi T., Imhof A., Regula J. T. A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications. Proteomics. 2004;4(5):1382–1396. DOI: 10.1002/pmic.200300743.