ABSTRACT
Title
A new hplc–uv validated method for therapeutic drug monitoring of deferasirox in thalassaemic patients.
Authors
F.M. Piccione1, S. De Francia1, E. Pirro1, D. Massano2, S. Racca1, F. Di Carlo1, A. Piga2.
1Laboratory of Clinical Pharmacology, Dept. of Clinical & Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy.
2Microcythemia Center, Dept. of Clinical & Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy.
1Laboratory of Clinical Pharmacology, Dept. of Clinical & Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy.
2Microcythemia Center, Dept. of Clinical & Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy.
Abstract
In the last years, many methods were described for the quantification of deferasirox (DFX) in human plasma. To this purpose Rouan et al. reported in 2001 the use of High Pressure Liquid Chromatography with Ultraviolet detection (HPLC-UV), and, based on this method, many papers studying DFX pharmacokinetic (PK) were published later (Piga 2006, Galanello 2008, Bruin 2008, Chirnomas 2009, Waldmeier 2010). More recently Liquid Chromatography tandem Mass Spectrometry (LC-MS)instrumentation has been used to this aim (Sechaud 2008, Chauzit 2010). Therapeutic drug monitoring (TDM) is an essential tool in the management of patients with different conditions and may be useful for thalassaemic patients. DFX plasma concentrations measurement, in fact, can give information to evaluate patient adherence to therapy, potential drugs interactions, treatment efficacy, and severe drug related adverse events (Alnaim 2007,Marquet 2008). The aim of this study has been to develop and validate an HPLC-UV method for DFX quantification in human plasma.
DFXchromatographic separation was achieved on a C18 reverse-phase5μ column at constant flow rate of 1.0ml/min. The eluate was monitored at 267nm, at 25°C. DFX extraction procedure from plasma was based on protein precipitation, followed by vortexing and 10 minutes centrifuging. 800μl of supernatant were then directly injected in HPLC for analysis. Internal standard (IS) used for quantification, added to each plasma aliquot before extraction procedure, wasimatinib, structurally similar to DFX.Mobile phase consisted of 40% solution (water-methanol-triethylamine), 20% methanol and 40% acetonitrile. For validation procedure DFX and IS were added to blank plasma, extracted and then analyzed; calibration curves (ranged from 20 to 0.156μg/ml)and quality controls (high, medium and low point of calibration range) were prepared and evaluated over 3 days to measure linearity, intra- and inter-day variability, accuracy and recovery, as requested by FDA international guidelines (2001).
Time of analytical run was chosen as 8 minutes, according to the substances retention times and their good separation. In spite of low wavelength employed no significant interference were observed for drugs detected. Over the concentration range regression analysis indicated a good linearity for DFX (r>0.999).Limit of quantification (LOQ) was set at 0.156μg/ml.
Quantification method developed for DFX assay is simple, fast and cheap: validated extraction and chromatographic conditions are reliably applicable. Furthermore method developed is sensitive because LOQ reported is lower than reported from Rouan. Parameters of this new method, then, are good to perform TDM and PK studiesof DFX.
REFERENCES
[1] Rouan MC. et al. Journal of Chromatography B, 755 (2001) 203–213.
[2] Piga A. et al. Haematologica,91 (2006) 873-80.
[3] Galanello R. et al. Journal of Clinical Pharmacology,48 (2008) 428-35.
[4] Bruin GJM. et al. Drug Metabolism and Disposition,36 (2008) 2523-38.
[5] Chirnomas D. et al. Blood,114 (2009) 4009-13.
[6] Waldmeier F. et al. Drug Metabolism and Disposition,38 (2010) 808-16.
[7] Sechaud R. et al. International Journal of Clinical Pharmacology and Therapeutics, 46 (2008) 102-8.
[8] Chauzit E. et al. Drug Monitoring, 32 (2010) 476-81.
[9] Alnaim L. Journal of Oncology Pharmacy Practice, 13 (2007) 207-221.
[10] Marquet P and Rousseau A. Bull Cancer, 95(2008) 903-909.
[11] Center for Drug Evaluation and Research of the U.S. Department of Health and Human Services Food and Drug Administration (2001).
DFXchromatographic separation was achieved on a C18 reverse-phase5μ column at constant flow rate of 1.0ml/min. The eluate was monitored at 267nm, at 25°C. DFX extraction procedure from plasma was based on protein precipitation, followed by vortexing and 10 minutes centrifuging. 800μl of supernatant were then directly injected in HPLC for analysis. Internal standard (IS) used for quantification, added to each plasma aliquot before extraction procedure, wasimatinib, structurally similar to DFX.Mobile phase consisted of 40% solution (water-methanol-triethylamine), 20% methanol and 40% acetonitrile. For validation procedure DFX and IS were added to blank plasma, extracted and then analyzed; calibration curves (ranged from 20 to 0.156μg/ml)and quality controls (high, medium and low point of calibration range) were prepared and evaluated over 3 days to measure linearity, intra- and inter-day variability, accuracy and recovery, as requested by FDA international guidelines (2001).
Time of analytical run was chosen as 8 minutes, according to the substances retention times and their good separation. In spite of low wavelength employed no significant interference were observed for drugs detected. Over the concentration range regression analysis indicated a good linearity for DFX (r>0.999).Limit of quantification (LOQ) was set at 0.156μg/ml.
Quantification method developed for DFX assay is simple, fast and cheap: validated extraction and chromatographic conditions are reliably applicable. Furthermore method developed is sensitive because LOQ reported is lower than reported from Rouan. Parameters of this new method, then, are good to perform TDM and PK studiesof DFX.
REFERENCES
[1] Rouan MC. et al. Journal of Chromatography B, 755 (2001) 203–213.
[2] Piga A. et al. Haematologica,91 (2006) 873-80.
[3] Galanello R. et al. Journal of Clinical Pharmacology,48 (2008) 428-35.
[4] Bruin GJM. et al. Drug Metabolism and Disposition,36 (2008) 2523-38.
[5] Chirnomas D. et al. Blood,114 (2009) 4009-13.
[6] Waldmeier F. et al. Drug Metabolism and Disposition,38 (2010) 808-16.
[7] Sechaud R. et al. International Journal of Clinical Pharmacology and Therapeutics, 46 (2008) 102-8.
[8] Chauzit E. et al. Drug Monitoring, 32 (2010) 476-81.
[9] Alnaim L. Journal of Oncology Pharmacy Practice, 13 (2007) 207-221.
[10] Marquet P and Rousseau A. Bull Cancer, 95(2008) 903-909.
[11] Center for Drug Evaluation and Research of the U.S. Department of Health and Human Services Food and Drug Administration (2001).