Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA

BMC Clinical Pharmacology, Dec 2012

Background Therapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated. From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards. Methods Plasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes. Results Low quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 μg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards. Conclusions The authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid.

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Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA

BMC Clinical Pharmacology Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA Armin Buchwald 0 Karl Winkler 0 Thomas Epting 0 0 Division of Clinical Chemistry, Department of Medicine, University Medical Center Freiburg , Hugstetterstrasse 55, 79106 Freiburg , Germany Background: Therapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated. From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards. Methods: Plasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes. Results: Low quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 μg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards. Conclusions: The authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid. Background Therapeutic drug monitoring (TDM) of immunosuppressive drugs in organ-transplanted patients is vitally important to prevent intoxication or rejection due to incorrect dosage. New therapeutic regimens combine immunosuppressants with different intracellular targets to lower blood concentrations and prevent undesired side effects [ 1-3 ]. This practice requires a precise and accurate analytical method, especially for the lower ranges of concentrations. With regards to sensitivity immunoassays often fail to meet clinical needs, due to their restricted detection limits. Moreover, they are vulnerable to cross-reactions against pharmacologically inactive metabolites, resulting in limited specificity and possibly false results [ 4 ]. In order to minimize imprecision at low drug concentrations, elaborate sample preparation is required to separate the molecules of interest from the patient’s blood matrix molecules [ 5 ]. Any remaining matrix can adversely affect the efficiency of ionization and lead to erroneous results. Thus, matrix effects need to be identified and compensated by internal standards (IS). Since stable isotope labeled, structurally analogous isoforms are the most appropriate controls for matrix compensation [ 6,7 ], deuterated equivalents are about to replace the common IS ascomycin, cyclosporine D (CSD) [ 8-10 ] and carboxy butoxy ether of mycophenolic acid (MPAC). Cyclosporine A (CSA), tacrolimus (TAC), everolimus (EVE) and sirolimus (SIR) are measured in whole blood, whereas mycophenolic acid (MPA) is determined in plasma [ 11,12 ]. Several methods have been reported to measure these drugs using different techniques for sample preparation and high pressure liquid chromatographic (HPLC) schedules [ 8,13-15 ]. However, these applications lack either appropriate IS or MPA to complete the analytical spectrum. Moreover, the separation of the main MPA metabolite mycophenolic acid glucuronide (MPAG) is essential for mass spectrometric analysis because MPAG can undergo in-source fragmentation to MPA via loss of the glucuronic acid moiety [ 11 ], which in the case of coelution is determined as MPA. Generally, sample preparation consists of precipitation with a mixture of zinc-sulfate, organic solvent (methanol, acetonitrile or acetone) and IS, usually CSD, ascomycin and MPAC. The method proposed by Koster et al. for example precipitates proteins using zn-sulfate only for CSA and TAC, but not for SIR and EVE [ 16 ]. This approa (...truncated)


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Armin Buchwald, Karl Winkler, Thomas Epting. Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA, BMC Clinical Pharmacology, 2012, pp. 2, Volume 12, Issue 1, DOI: 10.1186/1472-6904-12-2