Determination of thiopurine S -methyltransferase activity by hydrophilic interaction liquid chromatography hyphenated with mass spectrometry

Abstract

Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid chromatography hyphenated with mass spectrometry, Journal of Pharmaceutical and Biomedical Analysishttp://dx.doi.org/10. 1016/j.jpba.2017.05.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Highlights New HILIC-HPLC-MS method was developed for determination of TPMT activity  HILIC-HPLC-MS combination was approved by excellent performance parameters  HILIC-HPLC-MS was compared to traditional RP-HPLC-UV as well as advanced RP-HPLC-MS  Conventional approaches were surpassed in terms of selectivity, LOD, analysis time  HILIC-HPLC-MS is favorable for routine assay of 6-MMP/6-MP ratio in RBC lysates List of abbreviations6-MMP -6-methylmercaptopurine, 6-MP -6-mercaptopurine, 6-TG -6-thioguanine, AZA -azathioprine, Cl-P -6-chloropurine, DTT -dithiothreitol, IBD -inflammatory bowel diseases, RBC -red blood cells, SAM -S-adenosyl-L-methionine, TGN -thioguanine nucleotides, TPMT -thiopurine-S-methyl transferase. IndroductionThiopurine drugs, namely azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are immunosuppressive agents that are, due to their low cost and high effectiveness, widely used in the treatment of inflammatory bowel diseases (IBD), but also in other autoimmune diseases, some hemopoetic disorders and some solid organ transplant [1][2][3][4][5]. However, the occurrence of adverse drug reactions limits the use of these drugs. Up to 15 % of IBD patients discontinue this type of treatment due to adverse events that include nausea, skin reactions, pancreatitis, hepatotoxicity or myelotoxicity [6,7].Thiopurine drugs are prodrugs, so their biological activity is preceded by extensive metabolism. Briefly, after absorption, AZA is metabolized in the liver to 6-MP which then can be metabolized to active thioguanine nucleotides (TGN) and to inactive methylated product 6-methylmercaptopurine (6-MMP) by the thiopurine-S-methyl transferase (TPMT) enzyme [8]. TPMT (EC 2.1.1.67), a key enzyme involved in the thiopurine drug metabolism, is subject to common genetic polymorphism that leads to trimodial distribution of the TPMT activity in population [9]. Individuals with lower TPMT activity might have an increased risk of developing thiopurine-induced myelosuppression. On the contrary, individuals with high TPMT activity have lower concentrations of active TGN metabolites resulting in reduced therapeutic efficacy of the drug [10]. In addition, high TPMT activity leads to accumulation of the hepatotoxic methylated metabolites [11]. Therefore, some scientific committees advise to determine the TP...

Selecting a Structural Analog as an Internal Standard for the Quantification of 6-Methylmercaptopurine by LC-MS/MS

“…The results are in agreement with other methods using similar or identical compounds as IS for quantification of 6-MMP in cytolysed RBC via TPMT conversion utilizing MS instrumentation (34)(35)(36)(37). For example, 6-Cl-P has already been used ........................................................................................................... successfully as an SA-IS in a similar assay by Pecher et al in which they disclosed a new HILIC (hydrophilic interaction LC)-phase HPLC-MS method to phenotype TPMT activity (35). In addition, 2-CA was first suggested as an IS by Kalsi and colleagues who used 2-CA in a HLPC/tandem ion-trap mass detection method for the measurement of TPMT activity, although the method was never fully validated (36).…”

“…Although convention dictates that the SIL-IS, 6-MMP-d 3 is generally the preferred IS to use when quantifying 6-MMP, many of the SA-ISs examined in our studies performed as well as the SIL-IS (25,34). The results are in agreement with other methods using similar or identical compounds as IS for quantification of 6-MMP in cytolysed RBC via TPMT conversion utilizing MS instrumentation (34)(35)(36)(37). For example, 6-Cl-P has already been used ........................................................................................................... successfully as an SA-IS in a similar assay by Pecher et al in which they disclosed a new HILIC (hydrophilic interaction LC)-phase HPLC-MS method to phenotype TPMT activity (35).…”

“…The quantification of 6-MMP is a critical component of thiopurine S-methyltransferase (TPMT) enzyme activity testing in red blood cell (RBC); an important phenotyping test recommended by the US Food and Drug Administration for patients being considered for thipourine therapy (33). Many methods report analog IS for measuring TPMT enzyme activity utilizing MS instrumentation, making this an ideal assay for this study (34)(35)(36)(37). A cocktail solution that contained a mixture of the 9 structural analogs, which included a labeled structural isomer, were screened for acceptability as ISs.…”

Development and validation of a UPLC-UV method for the quantification of thiopurine methyltransferase enzyme activity in human erythrocytes

“…The retention time of 6-MMP was 1.89 min (Figure 1), and the total run time was 9 min. It represents a significant decrease from the run time of the previous HPLC method (40 min), and it is in line with the available LC-MS/MS methods (23)(24)(25)(26), with run times around 7 min.…”

“…In a second time, several HPLC methods with UV and fluorescence detection have been published (15)(16)(17)(18)(19)(20)(21)(22). More recently, the use of LC-MS/MS methods to measure TPMT activity has emerged (23)(24)(25)(26). Most methods are based on the enzymatic methylation of 6-MP by TPMT in red blood cell lysates with Sadenosyl-L-methionine (SAM) as methyl donor.…”