Ann Lab Med.  2018 May;38(3):255-260. 10.3343/alm.2018.38.3.255.

Evaluation of Stability of Thiopurine Metabolites Using a Validated LC-MS/MS Method

• Yoo IY
• Lee K
• Ji OJ
• Woo HI
• Lee SY

• KMID: 2403460
• DOI: http://doi.org/10.3343/alm.2018.38.3.255

Abstract

Measurement of thiopurine metabolites is helpful to monitor adverse effects and assess compliance in patients on thiopurine treatment. The purpose of this study was to develop and validate an analytical method for measurement of thiopurine metabolites, thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN), in RBCs. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 6-TGN and 6-MMPN and evaluated the stability of the thiopurine metabolites in RBC and whole blood states without any preprocessing at various storage conditions. The linear range was 0.1-10 µmol/L and 0.5-100 µmol/L for 6-TGN and 6-MMPN, respectively. The mean extraction recovery at the two concentrations was 71.0% and 75.0% for 6-TGN, and 102.2% and 96.4% for 6-MMPN. Thiopurine metabolites in preprocessed RBC samples were stable at 25℃ and 4℃ after storage for 4 hours and at −70℃ for up to 6 months. However, 6-TGN decreased by 30% compared with the initial concentration when stored at −20℃ for 180 days. In whole blood states, 6-TGN decreased by about 20% at four days after storage at 4℃. We validated a reliable LC-MS/MS method and recommend that the patient's whole blood sample be preprocessed as soon as possible.

Keyword

Thiopurine metabolites; 6-Thioguanine nucleotide; 6-Methylmercaptopurine nucleotide; Mass spectrometry; Stability

MeSH Terms

Compliance
Humans
Mass Spectrometry
Methods*
Nucleotides
Thioguanine
Nucleotides
Thioguanine

Figure

• 

  Fig. 1 Representative chromatograms of (A) 6-TGN, (B) 6-TG-13C215N (IS for 6-TG), (C) 6-MMPN, and (D) 6-MMP-d3 (IS for 6-MMP).Abbreviations: IS, internal standard; MRM, multiple-reaction monitoring; cps, counts per second.

• 

  Fig. 2 Short-term (A) and long-term (B) stability of 6-TGN and 6-MMPN at different temperatures. Each dot represents a measurement (N=4).Abbreviations: 6-MMPN, 6-methylmercaptopurine nucleotide; 6-TGN, 6-thioguanine nucleotide.

• 

  Fig. 3 Whole blood stability tests for (A) 6-TGN and (B) 6-MMPN.Abbreviations: see Fig. 2.

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