Method Development on Analysis Cyclophosphamide and 4-Hydroxycyclophosphamide in Dried Blood Spot and Its Application in Breast Cancer Patients
Objective: To develop the method for simultaneous analysis of cyclophosphamide and 4-hydroxycyclophosphamide in Dried Blood Spot (DBS) using Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry and its application in breast cancer patients for therapeutic drug monitoring.
Methods: Sample preparation used protein precipitation with methanol and acetonitrile (2:1 v/v). The separation was conducted using 1.7μm (2.1 x 100 mm) Waters AcquityTM UPLC C18 column, mobile phase consists of 0.01% formic acid and methanol (50:50 v/v) with isocratic elution, column temperature 30oC, flow rate 0.3 mL/min and hexamethylphosphoramide used as internal standard. Analysis was performed by a triple quadrupole mass spectrometry with positive ion mode of Electrospray Ionization. Cyclophosphamide was detected at m/z 260,968>139,978, 4-hydoxycyclophosphamide at m/z 338,011>224,979, and hexamethylphosphoramide at m/z 180,17>92,08. The method was applied to quantify cyclophosphamide and 4-hydroxycyclophosphamide in Dried Blood Spot of breast cancer patients. Blood samples were collected at 2 and 4 hours after cyclophosphamide administration for therapeutic drug monitoring.
Results: The method was linear in the range of 50 – 30.000 ng/mL for cyclophosphamide and 10 – 1000 ng/mL for 4-hydroxycyclophosphamide. Lower Limit of Quantification (LLOQ) concentration of cyclophosphamide was 50 ng/mL and 4-hydroxycyclophosphamide was 10 ng/mL. Accuracy and precision within-run and between-run met the requirements with% diff and% CV not exceeding ± 15% and not more than ± 20% for Lower Limit of Quantification (LLOQ) concentration. The results from dried blood spot samples of cancer patients showed that the level of cyclophosphamide was in the range of 6045,980 ng/mL to 37024,403 ng/mL and 4-hydroxycyclophosphamide was in the range 33,155 ng/ml to 246,362 ng/ml.
Conclusion: The developed method met the requirements of all validation parameters under the Guideline on Bioanalytical Method Validation by the European Medicines Agency in 2011. Method can be applied on dried blood spot of cancer patients and the results showed that cyclophosphamide and 4-hydroxycyclophosphamide was detected on 17 samples of breast cancer patients. This can be one of the parameters for therapeutic drug monitoring.
2. Chabner BA, et al. Cytotoxic Agent. L Brunton, B Knollmann, B Chabner, editor. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 12th ed. New York: McGraw Hill. p. 1677-1730.
3. Veal GJ, et al. Cyclophosphamide pharmacokinetics and pharmacogenetics in children with B-cell non-Hodgkin’s lymphoma. European Journal of Cancer. 2016; 55: 56-64
4. Kalhorn T, et al. Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography–mass spectrometry. Journal Of Chromatography B.2006; 835(1-2): 105-113. http://dx.doi.org/10.1016/j.jchromb.2006.03.022.
5. Harahap Y, Samuel C, Andalusia R, Syafhan NF. Analysis of 4-Hydroxycyclophosphamide in Cancer Patients Plasma for Therapeutic Drug Monitoring of Cyclophosphamide. International Journal Of Pharmacy And Pharmaceutical Sciences. 2016; 8(9):194-200.
6. Ekhart C, Gebretensae A, Rosing H, Rodenhuis S, Beijnen JH, dan Huitema ADR. Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC–SM/SM). Journal of Chromatography B. 2007; 854: 345–349.
7. Sadagopan N, Cohen L, Roberts B, Collard W, Omer C. Liquid chromatography–tandem mass spectrometric quantitation of cyclophosphamide and its hydroxy metabolite in plasma and tissue for determination of tissue distribution. Journal Of Chromatography B. 2001; 759(2): 277-284.
8. Hussain S, Shaikh T. Ultra High Performance Liquid Chromatography (UPLC): a New Trend in Analysis. World Journal of Pharmaceutical Research. 2016; 5(3): 387–394.
9. Bai F, Fraga CH, Tagen M, Schaiquevich P, Hagedorn N, Stewart CF. Liquid chromatography–tandem mass spectrometric quantitation of cyclophosphamide and its hydroxy metabolite in plasma and tissue for determination of tissue distribution. Journal of Chromatography B. 2009; 759: 277-284.
10. Baumann F, Lorenz C, Jaehde, U, Preiss, R. Determination of cyclophosphamide and its metabolites in human plasma by high-performance liquid chromatography–mass spectrometry. Journal Of Chromatography B: Biomedical Sciences And Applications. 1999; 729(1-2): 297-305. http://dx.doi.org/10.1016/s0378-4347(99)00178-4.
11. Kalhorn T, et al. Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography–mass spectrometry. Journal Of Chromatography B. 2006; 835(1-2):105-113. http://dx.doi.org/10.1016/j.jchromb.2006.03.022.
12. Ministry of Health Indonesia. InfoDATIN : Pusat Data dan Informasi Kementerian Kesehatan Republik Indonesia. 2015. Jakarta. p.3.
13. European Medicines Agency. (2011). EMEA, Guideline on Bioanalytical Method Validation, Sciences Medicines Health. London : An Agency of the European Union.
14. Almeida AM, Castel-Branco M, Falcao A. Linear regression for calibration lines revisited: weighting schemes for bioanalytical methods. Elsevier, Journal of Chromatography B. 2002; 774 (2) : 215-222.
15. Comments G, et al. The Role of Derivatization in Chromatography. 2015; 307–331. https://doi.org/10.1016/B978-0-444-54319-6.00009-8.
16. Mess J, Taillon M, Côté C. Dried blood spot on-card derivatization?: an alternative form of sample handling to overcome the instability of thiorphan in biological matrix. Biomedical Chromatography. 2012. https://doi.org/10.1002/bmc.2745.
17. Torres LM, et al. A New Method to Quantify Ifosfamide Blood Levels Using Dried Blood Spots and UPLC-MS/MS in Paediatric Patients with Embryonic Solid Tumours. Plos One. 2015; 1-13.
18. Joy MS, et al. Cyclophosphamide and 4- hydroxycyclophosphamide pharmacokinetics in patients with glomerulonephritis secondary to lupus and small vessel vasculitis. Br J Clin Pharmacol. 2012; 74/3: 445–455.
19. Anderson L, Ludeman S, Colvin O, Grochow L, Strong J. Quantitation of 4-hydroxycyclophosphamide/aldophosphamide in whole blood. Journal Of Chromatography B: Biomedical Sciences And Applications. 1995; 667(2), 247-257. http://dx.doi.org/10.1016/0378-4347(95)00036-i.
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