Abstract
In this study, a sensitive, accurate and cost-effective ultra-performance liquid chromatography-tandem mass spectrometric method was developed using a new clean-up process for the quantification of leuprolide in rat plasma. Chromatography was carried on a HALO peptide ES-C18 column using water and acetonitrile (containing 1% acetic acid) as the mobile phase with a gradient elution. Electrospray ionization was applied and operated in the multiple reaction monitoring positive mode. Leuprolide and an internal standard (leuprolide-d5) were extracted by the liquid–liquid extraction with methylene chloride after protein precipitation by the new clean-up process. In the clean-up process, acetic acid was used for higher reproducibility, and then 10 μL of cleaned aqueous phase was finally injected into the UPLC–MS/MS system. The chromatograms showed good resolution, and no interfering peaks to plasma constituents were observed. The lower limit of quantification in rat plasma was 20 pg/mL for leuprolide. The calibration curves for leuprolide in rat plasma were linear over all the concentration range 20–10,000 pg/mL with a correlation coefficient of 0.991. The intra- and inter-day assay for precision and accuracy fulfilled the international guideline requirements. This validated method was successfully applied to characterize the pharmacokinetics of leuprolide in rats after an intravenous administration of 0.1 mg/kg. Our method allows laboratory researchers to simply determine leuprolide in plasma.
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References
Adjei A, Love S, Johnson E, Diaz G, Greer J, Haviv F, Bush E (1993) Effect of formulation adjuvants on gastrointestinal absorption of leuprolide acetate. J Drug Target 1:251–258
Alcala-Alcala S, Urban-Morlan Z, Aguilar-Rosas I, Quintanar-Guerrero D (2013) A biodegradable polymeric system for peptide-protein delivery assembled with porous microspheres and nanoparticles, using an adsorption/infiltration process. Int J Nanomedicine 8:2141–2151
Borghouts C, Kunz C, Groner B (2005) Current strategies for the development of peptide-based anti-cancer therapeutics. J Pept Sci 11:713–726
Bylda C, Thiele R, Kobold U, Volmer DA (2014) Recent advances in sample preparation techniques to overcome difficulties encountered during quantitative analysis of small molecules from biofluids using LC–MS/MS. Analyst 139:2265–2276
Fosgerau K, Hoffmann T (2015) Peptide therapeutics: current status and future directions. Drug Discov Today 20:122–128
Gallarate M, Battaglia L, Peira E, Trotta M (2011) Peptide-loaded solid lipid nanoparticles prepared through coacervation technique. Int J Chem Eng 2011:6
Hintzen F, Perera G, Hauptstein S, Muller C, Laffleur F, Bernkop-Schnurch A (2014) In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin. Int J Pharm 472:20–26
Hu Q, van Gaal EV, Brundel P, Ippel H, Hackeng T, Rijcken CJ, Storm G, Hennink WE, Prakash J (2015) A novel approach for the intravenous delivery of leuprolide using core-cross-linked polymeric micelles. J Control Release 205:98–108
Iqbal J, Shahnaz G, Perera G, Hintzen F, Sarti F, Bernkop-Schnurch A (2012) Thiolated chitosan: development and in vivo evaluation of an oral delivery system for leuprolide. Eur J Pharm Biopharm 80:95–102
Ismaiel OA, Zhang T, Jenkins RG, Karnes HT (2010) Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry. J Chromatogr B 878:3303–3316
Ito Y, Murano H, Hamasaki N, Fukushima K, Takada K (2011) Incidence of low bioavailability of leuprolide acetate after percutaneous administration to rats by dissolving microneedles. Int J Pharm 407:126–131
Ki MH, Lim JL, Ko JY, Park SH, Kim JE, Cho HJ, Park ES, Kim DD (2014) A new injectable liquid crystal system for one month delivery of leuprolide. J Control Release 185:62–70
Leitner JM, Mayr FB, Spiel AO, Firbas C, Savulsky C, Mis R, Corrado ME, Jilma B (2008) The pharmacokinetics and pharmacodynamics of a new sustained-release leuprolide acetate depot compared to market references. Int J Clin Pharmacol Ther 46:407–414
Lovgren U, Johansson S, Jensen LS, Ekstrom C, Carlshaf A (2010) Quantitative determination of peptide drug in human plasma samples at low pg/ml levels using coupled column liquid chromatography–tandem mass spectrometry. J Pharm Biomed Anal 53:537–545
MacNeill R, Evans E (2011) Evaluation of acetone as organic modifier in SPE for bioanalytical quantitative LC–MS/MS. Bioanalysis 3:291–300
Medeiros Mdos S, Turner AJ, Ljungqvist A, Folkers K (1991) Metabolic stability of the LHRH antagonist antide to cell-surface peptidases. Biochem Biophys Res Commun 176:25–30
Okada H, Inoue Y, Heya T, Ueno H, Ogawa Y, Toguchi H (1991) Pharmacokinetics of once-a-month injectable microspheres of leuprolide acetate. Pharm Res 8:787–791
Parekh JM, Sanyal M, Yadav M, Shrivastav PS (2014) Systematic evaluation of plasma phospholipids for reliable and precise determination of dronedarone and desbutyldronedarone by LC–MS/MS. Bioanalysis 6:2635–2650
Sofianos ZD, Katsila T, Kostomitsopoulos N, Balafas V, Matsoukas J, Tselios T, Tamvakopoulos C (2008) In vivo evaluation and in vitro metabolism of leuprolide in mice–mass spectrometry-based biomarker measurement for efficacy and toxicity. J Mass Spectrom 43:1381–1392
Thayer AM (2011) Improving peptides. Chem Eng News 89:13–20
Tombal B, Miller K, Boccon-Gibod L, Schroder F, Shore N, Crawford ED, Moul J, Jensen JK, Kold Olesen T, Persson BE (2010) Additional analysis of the secondary end point of biochemical recurrence rate in a phase 3 trial (CS21) comparing degarelix 80 mg versus leuprolide in prostate cancer patients segmented by baseline characteristics. Eur Urol 57:836–842
Ueno H, Matsuo S (1991) High-performance liquid chromatography followed by radioimmunoassay for the determination of a luteinizing hormone-releasing hormone analogue, leuprorelin, and its metabolite. J Chromatogr 566:57–66
US Food and Drug Administration, Department of Health and Human Services, Center for Drug Evaluation and Research, Guidance for Industry: Bioanalytical Method Validation, 2013
Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M (2010) Synthetic therapeutic peptides: science and market. Drug Discov Today 15:40–56
Zhan Y, Chen X, Zhao X, Zhong D (2009) Rapid and sensitive liquid chromatography–tandem mass spectrometry method for the determination of leuprolide in human serum. J Chromatogr B 877:3194–3200
Acknowledgements
All authors (D. -S. Lee, S. -H. Heo, S. -J. Kim, Y. -B. Lee, H. -Y. Cho) declare that they have no conflicts of interest. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A1A3053185).
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Lee, DS., Heo, SH., Kim, SJ. et al. Development of new clean-up method for UPLC–MS/MS analysis of leuprolide. Journal of Pharmaceutical Investigation 47, 531–540 (2017). https://doi.org/10.1007/s40005-016-0284-6
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DOI: https://doi.org/10.1007/s40005-016-0284-6