DEVELOPMENT AND VALIDATION OF A DRIED BLOOD SPOT LC-MS/MS ASSAY TO QUANTIFY GEMCITABINE IN HUMAN WHOLE BLOOD: A COMPARISION WITH AND WITHOUT CYTIDINE DEAMINASE INHIBITOR
Objective: The purpose of this paper is to develop and validate a LC-MS/MS method for the quantification of gemcitabine in whole human blood using dried blood spots.
Methods: Gemcitabine fortified blood samples without tetrahydrouridine were spotted (50 Âµl) onto the DBS cards and dried for 2h at ambient room temperature. 3 mm punched spots were extracted by acetonitrile: water (90:10v/v) containing carbamazepine as internal standard (IS). Analyte and IS were separated on BDS Hypersil C18,(100 X 4.6 mm, 5 Âµ) column using a mixture of methanol and 2 mM ammonium acetate buffer (65:35 v/v) at a flow rate of 0.5 mL/min. Detection involved API-4000 LC-MS/MS with electrospray ionization in the positive ion mode.
Results: The assay was validated over the concentration range of 5-5000 ng/ml. Intra and inters assay precision values (% CV) were less than 6.0% while the accuracy was withinÂ±15%. The mean recovery (%CV) of gemcitabine from DBS was â‰¥83.5% (â‰¤4.0). Hematocrit values ranging between 0.25 and 0.62 were within acceptable limit with accuracy 93.0-103.1% of nominal values and %CV of â‰¤6.5 across the LQC and HQC levels. Gemcitabine was stable on DBS cards for atleast 90days at room temperature.
Conclusions: A cytidine drug like gemcitabine exhibits ex vivo instability and rapidly converts to inactive metabolite in blood. All current published methods for stabilisation include tetrahydrouridine a cytidine deaminase inhibitor in the sample collection tubes. The proposed DBS method can be used as an alternative assay to conventional plasma analysis without adding enzyme inhibitor.
2. O'Sullivan A, Kocher HM. Pancreatic cancer. BMJ Clin Evidence 2007;11:409â€“37.
3. Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. Lancet 2004;363(9414):1049-57.
4. Abbruzzese JL, Grunewald R, Weeks EA, Gravel D, Adams T, Nowak B, et al. A phase I clinical, plasma, and cellular pharmacology study of gemcitabine. J Clin Oncol 1991;9(3):491-8.
5. Hilbig A, Oettle H. Gemcitabine in the treatment of metastatic pancreatic cancer. Expert Rev Anticancer Ther 2008;8(4):511-23.
6. Lorusso D, Di Stefano A, Fanfani F, Scambia G. Role of gemcitabine in ovarian cancer treatment. Annu Oncol 2006;17 Suppl 5:v188-94.
7. Dent S, Messersmith H, Trudeau M. Gemcitabine in the management of metastatic breast cancer: a systematic review. Breast Cancer Res Treat 2008;108(3):319-31.
8. Bellmunt J, Albiol S, de Olano AR, Pujadas J, Maroto P. Spanish oncology genitourinary G. Gemcitabine in the treatment of advanced transitional cell carcinoma of the urothelium. Annu Oncol 2006;17 Suppl 5:v113-7.
9. Plunkett W, Huang P, Xu YZ, Heinemann V, Grunewald R, Gandhi V. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin Oncol 1995;22(4 Suppl 11):3-1.
10. Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W. Action of 2',2'-difluorodeoxycytidine on DNA synthesis. Cancer Res 1991;51(22):6110-7.
11. 11. Freeman KB, Anliker S, Hamilton M, Osborne D, Dhahir PH, Nelson R, et al. Validated assays for the determination of gemcitabine in human plasma and urine using high-performance liquid chromatography with ultraviolet detection. J Chromatogr B Biomed Appl 1995;665(1):171-81.
12. Venook AP, Egorin MJ, Rosner GL, Hollis D, Mani S, Hawkins M, et al. Phase I and pharmacokinetic trial of gemcitabine in patients with hepatic or renal dysfunction: Cancer and leukemia group B 9565. J Clin Oncol 2000;18(14):2780-7.
13. Keith B, Xu Y, Grem JL. Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography. J Chromatogr B: Anal Technol Biomed Life Sci 2003;785(1):65-72.
14. Yilmaz B, Kadioglu YY, Aksoy Y. Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography. J Chromatogr B: Anal Technol Biomed Life Sci 2003;791(1-2):103-9.
15. Lin NM, Zeng S, Ma SL, Fan Y, Zhong HJ, Fang L. Determination of gemcitabine and its metabolite in human plasma using high-pressure liquid chromatography coupled with a diode array detector. Acta Pharmacol Sin 2004;25(12):1584-9.
16. Yilmaz B, Kadioglu YY, Aksoy Y. Investigation of the pharmacokinetics of gemcitabine and 2',2'-difluorodeoxyuridine in human plasma by liquid chromatography. Anal Biochem 2004;332(2):234-7.
17. Kirstein MN, Hassan I, Guire DE, Weller DR, Dagit JW, Fisher JE, et al. High-performance liquid chromatographic method for the determination of gemcitabine and 2',2'-difluorodeoxyuridine in plasma and tissue culture media. J Chromatogr B: Anal Technol Biomed Life Sci 2006;835(1-2):136-42.
18. Honeywell R, Laan AC, van Groeningen CJ, Strocchi E, Ruiter R, Giaccone G, et al. The determination of gemcitabine and 2'-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry. J Chromatogr B: Anal Technol Biomed Life Sci 2007;847(2):142-52.
19. Vainchtein LD, Rosing H, Thijssen B, Schellens JH, Beijnen JH. Validated assay for the simultaneous determination of the anti-cancer agent gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma by high-performance liquid chromatography with tandem mass spectrometry. Rapid Commun Mass Spectrom 2007;21(14):2312-22.
20. Xu Y, Keith B, Grem JL. Measurement of the anticancer agent gemcitabine and its deaminated metabolite at low concentrations in human plasma by liquid chromatography-mass spectrometry. J Chromatogr B: Anal Technol Biomed Life Sci 2004;802(2):263-70.
21. Bapiro TE, Richards FM, Goldgraben MA, Olive KP, Madhu B, Frese KK, et al. A novel method for quantification of gemcitabine and its metabolites 2',2'-difluorodeoxyuridine and gemcitabine triphosphate in tumour tissue by LC-MS/MS: comparison with (19)F NMR spectroscopy. Cancer Chemother Pharmacol 2011;68(5):1243-53.
22. Zhou J, Gao S, Zhang F, Jiang B, Zhan Q, Cai F, et al. Liquid chromatography-tandem mass spectrometry method for simultaneous determination of seven commonly used anticancer drugs in human plasma. J Chromatogr B: Anal Technol Biomed Life Sci 2012;906:1-8.
23. Bowen C, Wang S, Licea-Perez H. Development of a sensitive and selective LC-MS/MS method for simultaneous determination of gemcitabine and 2,2-difluoro-2-deoxyuridine in human plasma. J Chromatogr B: Anal Technol Biomed Life Sci 2009;877(22):2123-9.
24. Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 1963;32:338-43.
25. Edelbroek PM, van der Heijden J, Stolk LM. Dried blood spot methods in therapeutic drug monitoring: methods, assays, and pitfalls. Ther Drug Monit 2009;31(3):327-36.
26. Beaudette P, Bateman KP. Discovery stage pharmacokinetics using dried blood spots. J Chromatogr B: Anal Technol Biomed Life Sci 2004;809(1):153-8.
27. Barfield M, Spooner N, Lad R, Parry S, Fowles S. Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies. J Chromatogr B: Anal Technol Biomed Life Sci 2008;870(1):32-7.
28. Malm M, Lindegardh N, Bergqvist Y. Automated solid-phase extraction method for the determination of piperaquine in capillary blood applied onto sampling paper by liquid chromatography. J Chromatogr B: Anal Technol Biomed Life Sci 2004;809(1):43-9.
29. Kromdijk W, Mulder JW, Rosing H, Smit PM, Beijnen JH, Huitema AD. Use of dried blood spots for the determination of plasma concentrations of nevirapine and efavirenz. J Antimicrob Chemother 2012;67(5):1211-6.
30. Meesters RJ, van Kampen JJ, Reedijk ML, Scheuer RD, Dekker LJ, Burger DM, et al. Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots. Anal Bioanal Chem 2010;398(1):319-28.
31. Koal T, Burhenne H, Romling R, Svoboda M, Resch K, Kaever V. Quantification of antiretroviral drugs in dried blood spot samples by means of liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2005;19(21):2995-3001.
32. Li W, Zhang J, Tse FL. Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices. Biomed Chromatogr 2011;25(1-2):258-77.
33. Chen J, Hsieh Y. Stabilizing drug molecules in biological samples. Ther Drug Monit 2005;27(5):617-24.
34. D'Arienzo CJ, Ji QC, Discenza L, Cornelius G, Hynes J, Cornelius L, et al. DBS sampling can be used to stabilize prodrugs in drug discovery rodent studies without the addition of esterase inhibitors. Bioanalysis 2010;2(8):1415-22.
35. Bowen CL, Hemberger MD, Kehler JR, Evans CA. Utility of dried blood spot sampling and storage for increased stability of photosensitive compounds. Bioanalysis 2010;2(11):1823-8.
36. US DHHS, FDA and CDER. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Medicine; 2001. Available from: http://www/fda.gov/ cder/guidance/ index.htm [Last Accessed on 02 Mar 2015].