Home Medicine Comparing the effect of isotopically labeled or structural analog internal standards on the performance of a LC-MS/MS method to determine ciclosporin A, everolimus, sirolimus and tacrolimus in whole blood
Article
Licensed
Unlicensed Requires Authentication

Comparing the effect of isotopically labeled or structural analog internal standards on the performance of a LC-MS/MS method to determine ciclosporin A, everolimus, sirolimus and tacrolimus in whole blood

  • Henar Valbuena EMAIL logo , Maria Shipkova , Sophie-Maria Kliesch , Simon Müller and Eberhard Wieland
Published/Copyright: August 19, 2015
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 54 Issue 3

Abstract

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used for analysis of immunosuppressive drugs. This study investigated whether replacing analog internal standards (ANISs) with isotopically labeled internal standards (ILISs) has an impact on the performance of a LC-MS/MS method for the quantification of tacrolimus (TAC), sirolimus (SIR), ciclosporin A (CsA) and everolimus (EVE) in whole blood.

Methods: Following hemolysis, protein precipitation, and extraction with either ANISs (ascomycin, desmethoxy-rapamycin, CsD), or ILISs (TAC-13C,D2; SIR-13C,D3; CsA-D12; EVE-D4), samples were centrifuged and injected into a LC-MS/MS device equipped with a C18 reversed phase column. The effect of the two ISs on the linearity, precision, accuracy, trueness, matrix effects, and carryover was investigated by using the same patient-, proficiency testing-, and quality control samples. Statistical analysis of agreement between results includes a standard random effects model and Passing-Bablok regression.

Results: Within-day imprecision was <10%, between-day <8%, and trueness 91%–110% for all the analytes with both ISs. No carryover or matrix effects were observed. The median accuracy was −2.1% for CsA, 9.1% for EVE, 12.2% for SIR, and −1.2% for TAC with the ILISs; and −2% for CsA, 9.8% for EVE, 11.4% for SIR, and 0.2% for TAC with the ANISs. Results of patient and proficiency testing samples were not statistically different.

Conclusions: Although ILISs are generally considered superior to ANISs, they may not be always essential. When optimizing a LC-MS/MS method other factors must be also considered.

Keywords: immunosuppressant monitoring; internal standard; isotopes; liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Corresponding author: Henar Valbuena, Department of Clinical Chemistry, Vall d’Hebron University Hospital. Barcelona, Spain, Fax: +34 933868198, E-mail:

Acknowledgments

Henar Valbuena was awarded with an Exchange Programme Fellowship from Fundación José Luis Castaño to complete her scientific education and be able to take part in this project.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Jusko WJ, Thomson AW, Fung J, McMaster P, Wong SH, Zylber-Katz E, et al. Consensus document: therapeutic monitoring of tacrolimus (FK-506). Ther Drug Monit 1995;17:606–14.10.1097/00007691-199512000-00011Search in Google Scholar

2. Yatscoff RW, Boeckx R, Holt DW, Kahan BD, LeGatt DF, Sehgal S, et al. Consensus guidelines for therapeutic drug monitoring of rapamycin: report of the consensus panel. Ther Drug Monit 1995;17:676–80.10.1097/00007691-199512000-00022Search in Google Scholar

3. Kahan BD, Shaw LM, Holt D, Grevel J, Johnston A. Consensus document: Hawk’s Cay meeting on therapeutic drug monitoring of cyclosporine. Clin Chem 1990;36:1510–6.10.1093/clinchem/36.8.1510Search in Google Scholar

4. MacDonald AS. Rapamycin in combination with cyclosporine or tacrolimus in liver, pancreas, and kidney transplantation. Transplant Proc 2003;35:201S–8S.10.1016/S0041-1345(03)00231-8Search in Google Scholar

5. Srinivas TR, Meier-Kriesche H-U. Minimizing immunosuppression, an alternative approach to reducing side effects: objectives and interim result. Clin J Am Soc Nephrol 2008;3:S101–16.10.2215/CJN.03510807Search in Google Scholar

6. McAlister VC, Gao Z, Peltekian K, Domingues J, Mahalati K, MacDonald AS. Sirolimus-tacrolimus combination immunosuppression. Lancet 2000;355:376–7.10.1016/S0140-6736(99)03882-9Search in Google Scholar

7. Seger C, Tentschert K, Stöggl W, Griesmacher A, Ramsay SL. A rapid HPLC-MS/MS method for the simultaneous quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human blood samples. Nat Protoc 2009;4:526–34.10.1038/nprot.2009.25Search in Google Scholar PubMed

8. Grote-Koska D, Czajkowski S, Klauke R, Panten E, Brand K, Schumann G. A candidate reference measurement procedure for Cyclosporine A in whole blood. Accred Qual Assur 2014;19:147–57.10.1007/s00769-014-1048-5Search in Google Scholar

9. Koster RA, Alffenaar J-WC,Greijdanus B, Uges DR. Fast LC-MS/MS analysis of tacrolimus, sirolimus, everolimus and cyclosporin A in dried blood spots and the influence of the hematocrit and immunosuppressant concentration on recovery. Talanta 2013;115:47–54.10.1016/j.talanta.2013.04.027Search in Google Scholar PubMed

10. Annesley TM, McKeown DA, Holt DW, Mussell C, Champarnaud E, Harter L, et al. Standardization of LC-MS for therapeutic drug monitoring of tacrolimus. Clin Chem 2013;59:1630–7.10.1373/clinchem.2013.209114Search in Google Scholar PubMed

11. Korecka M, Shaw LM. Review of the newest HPLC methods with mass spectrometry detection for determination of immunosuppressive drugs in clinical practice. Ann Transplant 2009;14:61–72.Search in Google Scholar

12. Buchwald A, Winkler K, Epting T. Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA. BMC Clin Pharmacol 2012;12:2.10.1186/1472-6904-12-2Search in Google Scholar PubMed PubMed Central

13. Meinitzer A, Gartner G, Pilz S, Stettin M. Ultra fast liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of cyclosporin A, tacrolimus, sirolimus, and everolimus in whole blood using deuterated internal standards for cyclosporin A and everolimus. Ther Drug Monit 2010;32:61–6.10.1097/FTD.0b013e3181c49a00Search in Google Scholar PubMed

14. O’Halloran S, Ilett KF. Evaluation of a deuterium-labeled internal standard for the measurement of sirolimus by high-throughput HPLC electrospray ionization tandem mass spectrometry. Clin Chem 2008;54:1386–9.10.1373/clinchem.2008.103952Search in Google Scholar PubMed

15. Heideloff C, Payto D, Wang S. Comparison of a stable isotope-labeled and an analog internal standard for the quantification of everolimus by a liquid chromatography–tandem mass spectrometry method. Ther Drug Monit 2013;35:246–50.10.1097/FTD.0b013e318283403eSearch in Google Scholar PubMed

16. Vogeser M, Seger C. Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory. Clin Chem 2010;56:1234–44.10.1373/clinchem.2009.138602Search in Google Scholar PubMed

17. Stokvis E, Rosing H, Beijnen JH. Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun Mass Spectrom 2005;19:401–7.10.1002/rcm.1790Search in Google Scholar PubMed

18. Annesley TM, Clayton L. Simple extraction protocol for analysis of immunosuppressant drugs in whole blood. Clin Chem 2004;50:1845–8.10.1373/clinchem.2004.037416Search in Google Scholar PubMed

19. Streit F, Armstrong VW, Oellerich M. Rapid liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of sirolimus, everolimus, tacrolimus, and cyclosporin A in whole blood. Clin Chem 2002;48:955–8.10.1093/clinchem/48.6.955Search in Google Scholar

20. European Medicines Agency. Guideline on bioanalytical method validation. 2011; Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. Accessed: 19 Mar 2015.Search in Google Scholar

21. Korecka MA, Patel R, Shaw LM. Evaluation of performance of new, isotopically labeled internal standard ([13c2d4]RAD001) for everolimus using a novel high-performance liquid chromatography tandem mass spectrometry method. Ther Drug Monit 2011;33:460–3.Search in Google Scholar

22. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49:1041–4.10.1373/49.7.1041Search in Google Scholar PubMed

23. Taylor PJ, Franklin ME, Graham KS, Pillans PI. A HPLC-mass spectrometric method suitable for the therapeutic drug monitoring of everolimus. J Chromatogr B Analyt Technol Biomed Life Sci 2007;848:208–14.10.1016/j.jchromb.2006.10.029Search in Google Scholar PubMed

24. Carstensen B. Comparing clinical measurement methods a practical guide. Chichester, West Sussex: John Wiley & Sons, 2010.10.1002/9780470683019Search in Google Scholar

25. Carstensen B, Simpson J, Gurrin LC. Statistical models for assessing agreement in method comparison studies with replicate measurements. Int J Biostat 2008;4:Article 16.10.2202/1557-4679.1107Search in Google Scholar PubMed

26. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307–10.Search in Google Scholar

27. Analytical Services International. Analitical Services International [Internet]. Available from: http://www.bioanalytics.co.uk/index.html. Accessed: 20 Feb 2015.Search in Google Scholar

28. Carstensen B, Gurrin L, Ekstrom C, Figurski M. MethComp: functions for analysis of agreement in method comparison studies. R package version 1.22.2. 2015; Available from: http://CRAN.R-project.org/package=MethComp. Accessed: 20 Feb 2015.Search in Google Scholar

29. R Core Team. R: A language and environment for statistical computing. Viena: R Foundation for Statistical Computing, 2015. Available from: http://www.R-project.org/. Accessed: 20 Feb 2015.Search in Google Scholar

30. Shaw LM, Annesley TM, Kaplan B, Brayman KL. Analytic requirements for immunosuppressive drugs in clinical trials. Ther Drug Monit 1995;17:577–83.10.1097/00007691-199512000-00006Search in Google Scholar PubMed

31. Taylor PJ, Brown SR, Cooper DP, Salm P, Morris MR, Pillans PI, et al. Evaluation of 3 internal standards for the measurement of cyclosporin by HPLC-mass spectrometry. Clin Chem 2005;51:1890–3.10.1373/clinchem.2005.055160Search in Google Scholar PubMed

32. Becker S, Thiery J, Ceglarek U. Evaluation of a novel commercial assay for the determination of cyclosporine A, tacrolimus, sirolimus, and everolimus by liquid chromatography-tandem mass spectrometric assay. Ther Drug Monit 2013;35:129–32.10.1097/FTD.0b013e318274827dSearch in Google Scholar PubMed

Supplemental Material:

The online version of this article (DOI: 10.1515/cclm-2015-0519) offers supplementary material, available to authorized users.

Received: 2015-6-4
Accepted: 2015-6-30
Published Online: 2015-8-19
Published in Print: 2016-3-1

©2016 by De Gruyter

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. The way of prostate cancer diagnostics
  4. Review
  5. Statistical methods used in the calculation of geriatric reference intervals: a systematic review
  6. Opinion Paper
  7. The side effects of translational omics: overtesting, overdiagnosis, overtreatment
  8. Genetics and Molecular Diagnostics
  9. Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis
  10. General Clinical Chemistry and Laboratory Medicine
  11. Patient pools and the use of “patient means” are valuable tools in quality control illustrated by a bone-specific alkaline phosphatase assay
  12. Long-term stability of glucose: 96-h study using Terumo Glycaemia tubes
  13. Glucose is stable during prolonged storage in un-centrifuged Greiner tubes with liquid citrate buffer, but not in serum and NaF/KOx tubes
  14. Croatian laboratories have a good knowledge of the proper detection and management of hemolyzed, icteric and lipemic samples
  15. Fetal exposure to ethanol: relationship between ethyl glucuronide in maternal hair during pregnancy and ethyl glucuronide in neonatal meconium
  16. Comparing the effect of isotopically labeled or structural analog internal standards on the performance of a LC-MS/MS method to determine ciclosporin A, everolimus, sirolimus and tacrolimus in whole blood
  17. Relationship between matrix metalloproteinase-9 and oxidative stress in drug-free male schizophrenia: a case control study
  18. Comparison of functional fibrinogen (FF/CFF) and FIBTEM in surgical patients – a retrospective study
  19. Reference Values and Biological Variations
  20. Assessment of serum free light chain levels in healthy adults immediately after marathon running
  21. Pharmacokinetics of a novel dosing regimen of oral melatonin in critically ill patients
  22. Cancer Diagnostics
  23. An epidemiology-based model as a tool to monitor the outbreak of inappropriateness in tumor marker requests: a national scale study
  24. Comparative analysis of prostate cancer specific biomarkers PCA3 and ERG in whole urine, urinary sediments and exosomes
  25. Infectious Diseases
  26. Comparative evaluation of the Aptima HIV-1 Quant Dx assay and COBAS TaqMan HIV-1 v2.0 assay using the Roche High Pure System for the quantification of HIV-1 RNA in plasma
  27. Evaluating the use of procalcitonin in an asymptomatic, HIV-infected antiretroviral therapy-naïve, South African cohort
  28. Diabetes
  29. Early prediction of gestational diabetes: a practical model combining clinical and biochemical markers
  30. Letters to the Editors
  31. Highly-trained dogs’ olfactory system for detecting biochemical recurrence following radical prostatectomy
  32. More on the accuracy of the Architect enzymatic assay for hemoglobin A1c and its traceability to the IFCC reference system
  33. Assessing quality from an accuracy-based HbA1c proficiency survey
  34. HbG-Honolulu interferes with some cation-exchange HPLC HbA1c assays
  35. Between analyser differences in chloride measurements and thus anion gap cause different interpretations of the acid-base balance
  36. Non-albumininuric proteinuria: a urinary tubular marker in the diagnosis of diabetic kidney disease
  37. International Normalized Ratio (INR) testing: analytical and clinical performance of four point-of-care devices versus central laboratory instrumentation analysis
  38. Genetic screening of the makorin ring finger 3 gene in girls with idiopathic central precocious puberty
  39. Practical approach for medical validation of therapeutic drug monitoring results
  40. Marked elevation of procalcitonin in a patient with a drug related infusion reaction to rituximab
https://doi.org/10.1515/cclm-2015-0519
Keywords for this article
immunosuppressant monitoring; internal standard; isotopes; liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. The way of prostate cancer diagnostics
  4. Review
  5. Statistical methods used in the calculation of geriatric reference intervals: a systematic review
  6. Opinion Paper
  7. The side effects of translational omics: overtesting, overdiagnosis, overtreatment
  8. Genetics and Molecular Diagnostics
  9. Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis
  10. General Clinical Chemistry and Laboratory Medicine
  11. Patient pools and the use of “patient means” are valuable tools in quality control illustrated by a bone-specific alkaline phosphatase assay
  12. Long-term stability of glucose: 96-h study using Terumo Glycaemia tubes
  13. Glucose is stable during prolonged storage in un-centrifuged Greiner tubes with liquid citrate buffer, but not in serum and NaF/KOx tubes
  14. Croatian laboratories have a good knowledge of the proper detection and management of hemolyzed, icteric and lipemic samples
  15. Fetal exposure to ethanol: relationship between ethyl glucuronide in maternal hair during pregnancy and ethyl glucuronide in neonatal meconium
  16. Comparing the effect of isotopically labeled or structural analog internal standards on the performance of a LC-MS/MS method to determine ciclosporin A, everolimus, sirolimus and tacrolimus in whole blood
  17. Relationship between matrix metalloproteinase-9 and oxidative stress in drug-free male schizophrenia: a case control study
  18. Comparison of functional fibrinogen (FF/CFF) and FIBTEM in surgical patients – a retrospective study
  19. Reference Values and Biological Variations
  20. Assessment of serum free light chain levels in healthy adults immediately after marathon running
  21. Pharmacokinetics of a novel dosing regimen of oral melatonin in critically ill patients
  22. Cancer Diagnostics
  23. An epidemiology-based model as a tool to monitor the outbreak of inappropriateness in tumor marker requests: a national scale study
  24. Comparative analysis of prostate cancer specific biomarkers PCA3 and ERG in whole urine, urinary sediments and exosomes
  25. Infectious Diseases
  26. Comparative evaluation of the Aptima HIV-1 Quant Dx assay and COBAS TaqMan HIV-1 v2.0 assay using the Roche High Pure System for the quantification of HIV-1 RNA in plasma
  27. Evaluating the use of procalcitonin in an asymptomatic, HIV-infected antiretroviral therapy-naïve, South African cohort
  28. Diabetes
  29. Early prediction of gestational diabetes: a practical model combining clinical and biochemical markers
  30. Letters to the Editors
  31. Highly-trained dogs’ olfactory system for detecting biochemical recurrence following radical prostatectomy
  32. More on the accuracy of the Architect enzymatic assay for hemoglobin A1c and its traceability to the IFCC reference system
  33. Assessing quality from an accuracy-based HbA1c proficiency survey
  34. HbG-Honolulu interferes with some cation-exchange HPLC HbA1c assays
  35. Between analyser differences in chloride measurements and thus anion gap cause different interpretations of the acid-base balance
  36. Non-albumininuric proteinuria: a urinary tubular marker in the diagnosis of diabetic kidney disease
  37. International Normalized Ratio (INR) testing: analytical and clinical performance of four point-of-care devices versus central laboratory instrumentation analysis
  38. Genetic screening of the makorin ring finger 3 gene in girls with idiopathic central precocious puberty
  39. Practical approach for medical validation of therapeutic drug monitoring results
  40. Marked elevation of procalcitonin in a patient with a drug related infusion reaction to rituximab
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2026 De Gruyter Brill
Downloaded on 23.1.2026 from https://www.degruyterbrill.com/document/doi/10.1515/cclm-2015-0519/html
Scroll to top button