Kristina Lampič1, Jurij Trontelj2, Helena Prosen3, David Drobne4, Alenka Šmid2, Tomaž Vovk5. 1. Lek Pharmaceuticals d.d., Ljubljana, Slovenia; University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia; University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia. 2. University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia. 3. University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia. 4. University Medical Centre Ljubljana, Department of Gastroenterology, Ljubljana, Slovenia. 5. University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia. Electronic address: tomaz.vovk@ffa.uni-lj.si.
Abstract
BACKGROUND: Therapeutic drug monitoring of azathioprine metabolites is required for pharmacotherapy individualisation in patients with inflammatory bowel disease. Currently mainly hemolysates are used, requiring long sample preparation and showing limited analytes stability. Therefore, a quantitative LC-MS/MS method for determination of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in dried blood spot samples (DBS) was developed. METHODS: Analysis involves liquid extraction from 30 μL blood spot, hydrolysis and quantification with LC-MS/MS. RESULTS: Method met the validation criteria in terms of selectivity, linearity, accuracy, and precision in a range from 50 to 5300 pmol/8 × 108 Ery for 6-TG and from 260 to 5300 pmol/8 × 108 Ery for 6-MMP. Range can be increased to 8000 pmol/8 × 108 Ery. No matrix effect was observed and the recovery was >80%. DBS specific validation parameters were confirmed: spot homogeneity, no influence of blood spot volume (>30 μL) on 6 mm DBS disk, and absence of haematocrit effect. DBS samples were stable for at least one month at temperatures from -20 to 40 °C. Clinical validation confirmed that DBS method and routine clinical method with hemolysate samples give comparable results and enable similar clinical decisions. CONCLUSIONS: The newly developed DBS method is simple and presents an alternative to conventional methods for therapeutic drug monitoring of azathioprine metabolites.
BACKGROUND: Therapeutic drug monitoring of azathioprine metabolites is required for pharmacotherapy individualisation in patients with inflammatory bowel disease. Currently mainly hemolysates are used, requiring long sample preparation and showing limited analytes stability. Therefore, a quantitative LC-MS/MS method for determination of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in dried blood spot samples (DBS) was developed. METHODS: Analysis involves liquid extraction from 30 μL blood spot, hydrolysis and quantification with LC-MS/MS. RESULTS: Method met the validation criteria in terms of selectivity, linearity, accuracy, and precision in a range from 50 to 5300 pmol/8 × 108 Ery for 6-TG and from 260 to 5300 pmol/8 × 108 Ery for 6-MMP. Range can be increased to 8000 pmol/8 × 108 Ery. No matrix effect was observed and the recovery was >80%. DBS specific validation parameters were confirmed: spot homogeneity, no influence of blood spot volume (>30 μL) on 6 mm DBS disk, and absence of haematocrit effect. DBS samples were stable for at least one month at temperatures from -20 to 40 °C. Clinical validation confirmed that DBS method and routine clinical method with hemolysate samples give comparable results and enable similar clinical decisions. CONCLUSIONS: The newly developed DBS method is simple and presents an alternative to conventional methods for therapeutic drug monitoring of azathioprine metabolites.
Authors: Rihwa Choi; Mi Ryung Chun; Jisook Park; Ji Won Lee; Hee Young Ju; Hee Won Cho; Ju Kyung Hyun; Hong Hoe Koo; Eun Sang Yi; Soo-Youn Lee Journal: Ann Lab Med Date: 2021-03-01 Impact factor: 3.464