Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations

Wiwattanakul, Supaporn; Prommas, Santirhat; Jenjirattithigarn, Nuttawut; Santon, Siwalee; Puangpetch, Apichaya; Pakakasama, Samart; Anurathapan, Usanarat; Sukasem, Chonlaphat

doi:10.1016/j.jpba.2017.07.039

Cited by 8 publications

(8 citation statements)

References 26 publications

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Section: Discussionsupporting

confidence: 86%

“…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).…”

Section: Discussionmentioning

confidence: 85%

“…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.…”

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confidence: 99%

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

Smith

Merrigan

Johnson-Davis

2018

The Journal of Applied Laboratory Medicine

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Background When choosing an analog internal standard (IS) in a quantitative LC-MS/MS assay, careful selection and thorough verification are important for developing an accurate quantitative assay. The IS is a critical component in quantitative mass spectrometry because it is used to normalize results by compensating for variations in sample preparation and instrument performance. Here we present the results of our investigation in the selection process for a structural analog IS (SA-IS) to be used in the quantification of 6-methylmercaptopurine (6-MMP) in cytolysed red blood cell (RBC). Methods A cocktail solution of 9 SA-ISs including the isotopically labeled structural isomer and the 6-MMP stable isotope-labeled IS (SIL-IS) was spiked into cytolysed RBC controls and patient samples. Linearity, accuracy, sensitivity, precision, run stability, method comparison, and reinjection reproducibility experiments were performed. Ion suppression was also assessed by T-infusing the cocktail solution. Results All analogs were linear from 100 to 1200 ng/mL 6-MMP with acceptable precision and sensitivity by use of a spiked blank lysate. Method comparison plots of 6-MMP concentrations in patient samples had excellent agreement for 2 of the SA-ISs (i.e., the isotopically labeled structural isomer and an SA-IS with an added methyl group) when compared to the SIL-IS. Halogen-substituted analogs (i.e., Cl and Br) also met the criteria as an acceptable IS. However, 2 of the selected SA-ISs having substituted amine moieties showed unacceptable performance, with ≥15% bias when compared to the SIL-IS. Conclusion There are many parameters to consider when determining if an analog will be a good IS choice, and the approaches highlighted in this article can be applied to the selection of SA-IS in the development of other LC-MS/MS assays.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 85%

mentioning

confidence: 99%

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

Smith

Merrigan

Johnson-Davis

2018

The Journal of Applied Laboratory Medicine

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“…Thiopurine S-methyltransferase enzyme activity assay. To evaluate the effect of 5-ASA and N-Ac-5-ASA presenting in the blood of each patient on TPMT, TPMT activity was investigated using the method reported by Wiwattanakul et al 24 with slight modifications. Briefly, 105 μL 0.1 mol/L of phosphate buffer (pH 7.4), 10 μL 4 mmol/L of S-adenosyl methionine (SAM), 10 μL 50 mmol/L of 6-mercaptopurine, and 25 μL 1 mg/L of 6-MMP-d3 (internal standard) were added to 100 μL of red blood cell (RBC)-saline mixture.…”

Section: -Aminosalicylicmentioning

confidence: 99%

Differential effects of mesalazine formulations on thiopurine metabolism through thiopurine S‐methyltransferase inhibition

Morikubo

Ozaki

Okabayashi

et al. 2021

J of Gastro and Hepatol

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Background and Aim: Thiopurines are often used in combination with mesalazine for the treatment of ulcerative colitis (UC). Mesalazine formulations are delivered to the digestive tract by various delivery systems and absorbed as 5-aminosalicylic acid (5-ASA). 5-ASA is known to inhibit thiopurine S-methyltransferase (TPMT) activity and to affect thiopurine metabolism. There have been no studies comparing TPMT inhibition by multimatrix mesalazine (MMX) with other formulations. We investigated the difference in TPMT inhibition by different mesalazine formulations and prospectively confirmed the clinical relevance. Methods: Plasma concentrations of 5-ASA, N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA), and TPMT activities were measured in UC patients receiving various mesalazine formulations (time-dependent or pH-dependent mesalazine or MMX) as monotherapy. Patients already on both time-dependent or pH-dependent mesalazine and thiopurines switched their mesalazine to MMX, examining 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP) 0 and 8 weeks after switching. Clinical relapse after switching was also monitored for 24 weeks. Results: Plasma 5-ASA and N-Ac-5-ASA levels were significantly higher in patients receiving time-dependent mesalazine (n = 12) compared with pH-dependent mesalazine (n = 12) and MMX (n = 15), accompanied by greater TPMT inhibition. Prospective switching from time-dependent mesalazine to MMX decreased 6-TGN levels, increased those of 6-MMP, and increased 6-MMP/6-TGN ratios. Furthermore, this resulted in significantly more relapses than switching from pH-dependent mesalazine to MMX. Conclusions: Time-dependent mesalazine has higher plasma 5-ASA and N-Ac-5-ASA levels and greater TPMT inhibition than MMX. Therefore, switching from time-dependent mesalazine to MMX may lead to an increase of 6-MMP/6-TGN, which may reduce the clinical effectiveness of thiopurines, warranting close monitoring after switch.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Illamola

Echaabi

Mazeron

et al. 2019

Journal of Chromatography B

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Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations

Cited by 8 publications

References 26 publications

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

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

Differential effects of mesalazine formulations on thiopurine metabolism through thiopurine S‐methyltransferase inhibition

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

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