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Simultaneous determination of three isomeric metabolites of tacrolimus (FK506) in human whole blood and plasma using high performance liquid chromatography-tandem mass spectrometry.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Jan 18; 830(2):330-41.JC

Abstract

An ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of three isomeric metabolites of tacrolimus (FK506), 13-O-demethylated (M1), 31-O-demethylated (M2) and 15-O-demethylated (M3) tacrolimus in human whole blood and plasma. These metabolites and the internal standards were extracted from biological matrix by methylbutyl ether (MTBE). Separation was achieved on a Genesis C(18) column with a gradient mobile phase elution. Ammonium-adduct ions formed by a Turbo Ionspray in positive ion mode were used to detect each analyte and internal standard. The MS/MS detection was by monitoring the fragmentation of 807.5-->772.4 (m/z) for M1, 807.5-->754.5 (m/z) for both M2 and M3, 795.5-->760.5 (m/z) for IS1 (FR298701) and 961.5-->908.5 (m/z) for IS2 (FR290198) on a triple quadrupole mass spectrometer (Sciex API 3000). The retention times were approximately 4.1 min for M1, 6.8 min for M2, 6.0 min for M3, and 3.9 min for IS1 and 6.4 min for IS2, respectively. The validated dynamic range was 0.2-20 ng/ml for all three metabolites based on a sample volume of 0.25-ml. The linearity of calibration curves for M1, M2, and M3 in both matrices had a correlation coefficient of >/=0.9984. In whole blood, validation data showed intra-batch (n=6) CVs of </=5.9% and REs between -4.9 and 3.6% and inter-batch (N=18) CVs of </=4.9% and REs between -3.5 and 1.5% for all three metabolites. In human plasma, validation data showed intra-batch (n=6) CVs of </=7.3% and REs between -5.1 and 7.6% and inter-batch (N=18) CVs of </=6.6% and REs between -0.3 and 4.7% for all three metabolites. Extraction recoveries were 72% for M1, 87% for M2, 69% for M3, 79% for IS1, and 74% for IS2 from blood; and 94% for M1, 96% for M2, 98% for M3, 92% for IS1, and 93% for IS2 from plasma. All three metabolites in human blood and plasma were stable for three freeze-thaw cycles, or 24-h ambient storage, or 12 months storage at approximately -80 degrees C. Extracted samples were stable for at least 50h at room temperature (RT). This method has been successfully used to analyze whole blood and plasma samples from human pharmacokinetic studies. Several key factors affecting the performance of the assay methods have also been addressed briefly.

Authors+Show Affiliations

Astellas Research Institute of America, Northwestern University Research Park, 1801 Maple Avenue, Evanston, IL 60201, USA. yu-luan.chen@us.astellas.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16318929

Citation

Chen, Yu-Luan, et al. "Simultaneous Determination of Three Isomeric Metabolites of Tacrolimus (FK506) in Human Whole Blood and Plasma Using High Performance Liquid Chromatography-tandem Mass Spectrometry." Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 830, no. 2, 2006, pp. 330-41.
Chen YL, Hirabayashi H, Akhtar S, et al. Simultaneous determination of three isomeric metabolites of tacrolimus (FK506) in human whole blood and plasma using high performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;830(2):330-41.
Chen, Y. L., Hirabayashi, H., Akhtar, S., Pelzer, M., & Kobayashi, M. (2006). Simultaneous determination of three isomeric metabolites of tacrolimus (FK506) in human whole blood and plasma using high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 830(2), 330-41.
Chen YL, et al. Simultaneous Determination of Three Isomeric Metabolites of Tacrolimus (FK506) in Human Whole Blood and Plasma Using High Performance Liquid Chromatography-tandem Mass Spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Jan 18;830(2):330-41. PubMed PMID: 16318929.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Simultaneous determination of three isomeric metabolites of tacrolimus (FK506) in human whole blood and plasma using high performance liquid chromatography-tandem mass spectrometry. AU - Chen,Yu-Luan, AU - Hirabayashi,Hideki, AU - Akhtar,Shahzad, AU - Pelzer,Mary, AU - Kobayashi,Masakazu, Y1 - 2005/11/28/ PY - 2005/09/07/received PY - 2005/11/04/revised PY - 2005/11/09/accepted PY - 2005/12/2/pubmed PY - 2006/2/18/medline PY - 2005/12/2/entrez SP - 330 EP - 41 JF - Journal of chromatography. B, Analytical technologies in the biomedical and life sciences JO - J Chromatogr B Analyt Technol Biomed Life Sci VL - 830 IS - 2 N2 - An ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of three isomeric metabolites of tacrolimus (FK506), 13-O-demethylated (M1), 31-O-demethylated (M2) and 15-O-demethylated (M3) tacrolimus in human whole blood and plasma. These metabolites and the internal standards were extracted from biological matrix by methylbutyl ether (MTBE). Separation was achieved on a Genesis C(18) column with a gradient mobile phase elution. Ammonium-adduct ions formed by a Turbo Ionspray in positive ion mode were used to detect each analyte and internal standard. The MS/MS detection was by monitoring the fragmentation of 807.5-->772.4 (m/z) for M1, 807.5-->754.5 (m/z) for both M2 and M3, 795.5-->760.5 (m/z) for IS1 (FR298701) and 961.5-->908.5 (m/z) for IS2 (FR290198) on a triple quadrupole mass spectrometer (Sciex API 3000). The retention times were approximately 4.1 min for M1, 6.8 min for M2, 6.0 min for M3, and 3.9 min for IS1 and 6.4 min for IS2, respectively. The validated dynamic range was 0.2-20 ng/ml for all three metabolites based on a sample volume of 0.25-ml. The linearity of calibration curves for M1, M2, and M3 in both matrices had a correlation coefficient of >/=0.9984. In whole blood, validation data showed intra-batch (n=6) CVs of </=5.9% and REs between -4.9 and 3.6% and inter-batch (N=18) CVs of </=4.9% and REs between -3.5 and 1.5% for all three metabolites. In human plasma, validation data showed intra-batch (n=6) CVs of </=7.3% and REs between -5.1 and 7.6% and inter-batch (N=18) CVs of </=6.6% and REs between -0.3 and 4.7% for all three metabolites. Extraction recoveries were 72% for M1, 87% for M2, 69% for M3, 79% for IS1, and 74% for IS2 from blood; and 94% for M1, 96% for M2, 98% for M3, 92% for IS1, and 93% for IS2 from plasma. All three metabolites in human blood and plasma were stable for three freeze-thaw cycles, or 24-h ambient storage, or 12 months storage at approximately -80 degrees C. Extracted samples were stable for at least 50h at room temperature (RT). This method has been successfully used to analyze whole blood and plasma samples from human pharmacokinetic studies. Several key factors affecting the performance of the assay methods have also been addressed briefly. SN - 1570-0232 UR - https://www.unboundmedicine.com/medline/citation/16318929/Simultaneous_determination_of_three_isomeric_metabolites_of_tacrolimus__FK506__in_human_whole_blood_and_plasma_using_high_performance_liquid_chromatography_tandem_mass_spectrometry_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1570-0232(05)00833-0 DB - PRIME DP - Unbound Medicine ER -