André, P.

Buclin, T.

Ceppi, F.

Choong, E.

Chtioui, H.

Da Silva Pereira Clara, J. A.

Decosterd, L. A.

Desfontaines, V.

Mercier, T.

Moradpour, D.

Nguyen, N.

English

Serveur académique lausannois

Development of an assay by liquid chromatography coupled to tandem mass spectrometry for thiopurines drugs in whole blood and application for their Therapeutic Drug MonitoringAS-SGAIMFK-2022-00218ObjectiveDevelopment and validation of in-house thiopurines assayCorrelations between concentrations of 6-TG and 6-MMP determined in blood collected on EDTA, versus on heparin with 0.02% (m/v) dithiothreitol (Hep-DTT) from external reference method, and versus DBS Methods34 patient samples. The DBS were obtained by spotting 20 µl of the Li-Hep/DTT samples on a Whatman FTA DMPK-B Card. Hematocrit and red blood cell were measured from Hep-DTT-blood to normalize the thiopurines levels aLinear regression, correlation and Bland-Altman test were performedConclusions Excellent concordance was found between the in-house and reference methods. The accuracy of our in-house method is strengthened by the good performance of our laboratory at the external QC program for thiopurines c 10-ml collecting tubes of heparin with addition DTT prepared by the external laboratory and used so far can be conveniently replaced by standard 2.6-ml EDTA tubes in our LC-MS/MS method DBS compared to both blood anticoagulant matrices provide lower thiopurines levels. At present, DBS approach for 6-TG, 6-MMP should be used for research purpose only Thiopurines DBS stability and potential adsorption on the solid support of DBS card will be further investigated.N. Nguyen1,2, J.A Da Silva Pereira Clara1,2, T. Mercier 1,2, V. Desfontaine1,2, F. Ceppi3 , D. Moradpour4,  H. Chtioui2 , P. André2, T. Buclin2, L.A. Decosterd 1,2, E. Choong 1,2. Context The cytotoxic and immunosuppressive thiopurine drugs 6-TG and 6-MMP, are mainly used in acute lymphocytic leukemia, disorders of immune regulation and inflammatory bowel disease. Quantification methods for their Therapeutic Drug Monitoring are usually reported in various blood matrices, but barely with Dried Blood Spots (DBS). The suitability of our in-house validated LC-MS/MS and the use of DBS are explored by a cross-validation with a well-established external assay 6-TG: 6-Thioguanine nucleotide, 6-MMP: 6-Methyl-mercaptopurine Affiliations. 1 Laboratory of Clinical Pharmacology and 2 Service of Clinical Pharmacology, Department of Laboratory Medicine and Pathology 3Service of Pediatric Hemato-Oncology 4 Service of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandReferences. a T. Dervieux, et al. Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC, Clin. Chem. 1998. b Dubinsky MC, et al.Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology. 2000. c KKGT: Kwaliteitsbewaking Klinische Geneesmiddelanalyse en Toxicologie, The Hague, The Netherlands.  http://kkgt.nl/?lang=en.  Thiopurine quantification with CHUV method. All patient samples were found to lie within the validated concentrations range of the assay(5-2’000 ng/ml for 6-TG, and 50-20’000 ng/ml for 6-MMP). EDTALi-Heparinate+ DTT100ul whole blood20ul whole blood/spot DBS(2h RT then -80°C till analysis)DTT : 1,4-dithio-D, L-thréitol100°C, 1hand/orNeutralizeCentrifuge2.6ml K3-EDTA10ml Heparinewith 2 mg DTTLC-MS/MSPerchloricacid 70%1 DBS spotValidation LC-MS/MS method Run: 5 minutes Chromatogram60%70%80%90%100%110%120%130%140%0 500 1000 1500 2000Accuracy [%]6-TG concentration in whole blood [ng/ml]6-TG : Accuracy Profil 60%70%80%90%100%110%120%130%140%0 5000 10000 15000 20000Accuracy [%]6-MMPD concentration in whole blood [ng/ml]6-MMPD : Accuracy Profil = mean recovery=  -tolerance intervals ( = 80%)= limits of acceptance (± 30%)EDTAHep-DTTDBSversusEDTAversusHep-DTTversusBland-Altman testsDBS6-TGN 6-MMP6-TGN 6-MMP6-TGN 6-MMP0 300 600 900 1200 1500 18000300600900120015001800Hep-DTT [pmol/8·108 RBC]EDTAorDBS[pmol/8·108 RBC]EDTADBSy=0.976x+32.2r2=0.93y=0.585+56.6r2=0.86Identity0 3000 6000 9000 12000 15000 180000300060009000120001500018000Hep-DTT [pmol/8·108 RBC]EDTAorDBS[pmol/8·108 RBC]EDTADBSy=1.009x+135.1r2=0.93y=0.736+51.4r2=0.88300 600 900 1200 1500 1800-40-20020406080%Difference3000 6000 9000 12000 15000 18000-40-20020406080%Difference300 600 900 1200 1500 1800-100-50050%Difference3000 6000 9000 12000 15000 18000-100-50050%DifferenceBland-Altman test (n = 34) with the percentage of difference versus average for 6-Thioguanine Nucleotide(6-TG, left panel) and 6-Methylmercaptopurine (6-MMP, right panel). The average percentage difference(continuous red line) and the 95% confidence interval (dotted black lines) are indicated for A. EDTA versusHeparin/DTT, B. DBS versus Heparin/DTT, and C. DBS versus EDTA..Correlation between blood levels of 6-Thioguanine Nucleotide (6-TGN, left panel), and 6-Methylmercaptopurine (6-MMP,right panel), measured in Heparin/DTT blood versus in EDTA-blood and versus DBS, n=34. The dotted lines representthe 95% confidence intervals of the linear regression which are indicated with red and blue lines for EDTA-blood andDBS, respectively. The identity line is indicated by a black line.300 600 900 1200 1500 1800-80-60-40-20020%DifferenceAverage [pmol/8·108 RBC]3000 6000 9000 12000 15000 18000-80-60-40-20020%DifferenceAverage [pmol/8·108 RBC]0 300 600 900 1200 1500 18000300600900120015001800EDTA [pmol/8·108 RBC]DBS [pmol/8·108 RBC]y=0.612x+30.48r2=0.95DBS0 3000 6000 9000 12000 15000 180000300060009000120001500018000EDTA [pmol/8·108 RBC]DBS[pmol/8·108 RBC]y=0.742x-97.7r2=0.98DBSABCAverage biais (%)95% limits of agreementn = 6, 18%n = 6, 18%n = 22, 65%n = 26, 76%n = 8, 24%Sub-therapeutic range a,bTherapeutic range for 6-TGN,No toxicity for 6-MMPToxicity (myelotoxicity for 6-TGN, hepatotoxicity for 6-MMP)6-TGN 6-MMPDosages of Thiopurines (n=34)6-TG 6-MMP0100020003000050001000015000200006- TG [pmol/8*108 RBC]6- MMP [pmol/8*108 RBC]Median (p50)Percentiles 25 and 75 (p25, p75)Linear regressions and correlationsEDTADBSversusEDTADBSversus6-TGN 6-MMP6-TGN 6-MMPHep-DTTversus0 300 600 900 1200 1500 18000300600900120015001800Hep-DTT [pmol/8·108 RBC]EDTAorDBS[pmol/8·108 RBC]EDTADBSy=0.976x+32.2r=0.96y=0.585+56.6r=0.920 3000 6000 9000 12000 15000 180000300060009000120001500018000Hep-DTT [pmol/8·108 RBC]EDTAorDBS[pmol/8·108 RBC]EDTADBSy=1.009x+135.1r=0.96y=0.736+51.4r=0.94300 600 900 1200 1500 1800-40-20020406080%Difference3000 6000 9000 12000 15000 18000-40-20020406080%Difference300 600 900 1200 1500 1800-100-50050%Difference3000 6000 9000 12000 15000 18000-100-50050%DifferenceBland-Altman test (n = 34) with the percentage of difference versus average for 6-ThioguanineNucleotide (6-TG, left panel) and 6-Methylmercaptopurine (6-MMP, right panel). The average percentagedifference (continuous red line) and the 95% confidence interval (dotted black lines) are indicated for A.EDTA versus Heparin/DTT, B. DBS versus Heparin/DTT, and C. DBS versus EDTA..Correlation between blood levels of 6-Thioguanine Nucleotide (6-TGN, left panel), and 6-Methylmercaptopurine(6-MMP, right panel), measured in Heparin/DTT blood versus in EDTA-blood and versus DBS, n=34. The dottedlines represent the 95% confidence intervals of the linear regression which are indicated with red and blue lines forEDTA-blood and DBS, respectively. The identity line is indicated by a black line.300 600 900 1200 1500 1800-80-60-40-20020%DifferenceAverage [pmol/8·108 RBC]3000 6000 9000 12000 15000 18000-80-60-40-20020%DifferenceAverage [pmol/8·108 RBC]0 300 600 900 1200 1500 18000300600900120015001800EDTA [pmol/8·108 RBC]DBS [pmol/8·108 RBC]y=0.612x+30.48r=0.98DBS0 3000 6000 9000 12000 15000 180000300060009000120001500018000EDTA [pmol/8·108 RBC]DBS[pmol/8·108 RBC]y=0.742x-97.7r=0.99DBSIdentity line95% confidence intervalsABC An excellent correlation between EDTA and hep-DTT results could be observed (slope 0.98, r=0.96 and 1.01, r=0.96, for 6-TG and 6-MMP r spectively). Despite a good corr lation between DBS and EDTA samples (r> 0.98), a significant bias is observed for 6-TG and 6-MMP with lower values in DBS (slopes of 0.61 and 0.74, respectively).  Similar correlations and biases are found for hep-DTT samples.

Development of an assay by liquid chromatography coupled to tandem mass spectrometry for thiopurines drugs in whole blood and application for their Therapeutic Drug Monitoring

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Development of an assay by liquid chromatography coupled to tandem mass spectrometry for thiopurines drugs in whole blood and application for their Therapeutic Drug Monitoring

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