Analytical and Bioanalytical Chemistry

, Volume 411, Issue 1, pp 181–191 | Cite as

Volumetric absorptive microsampling as an alternative sampling strategy for the determination of paracetamol in blood and cerebrospinal fluid

  • Lisa Delahaye
  • Evelyn Dhont
  • Pieter De Cock
  • Peter De Paepe
  • Christophe P. StoveEmail author
Research Paper


In the field of bioanalysis, dried matrix spot sampling is increasingly receiving interest, as this alternative sampling strategy offers many potential benefits over traditional sampling, including matrix volume-sparing properties. By using a microsampling strategy, e.g., volumetric absorptive microsampling (VAMS), the number of samples that can be collected from a patient can be increased, as a result of the limited sample volume that is required per sample. To date, no VAMS-based methods have been developed for the quantification of analytes in cerebrospinal fluid (CSF). The objective of this study was to develop and validate two LC-MS/MS methods for the quantification of paracetamol in dried blood and dried CSF, with both matrices sampled using VAMS. Both methods were fully validated based on internationally accepted guidelines. Paracetamol was chromatographically separated from its glucuronide and sulfate metabolites and no carry-over or unacceptable interferences were detected. The total precision (%RSD) was below 15% for all QC levels and accuracy (%bias) was below 7% (17% for the LLOQ of aqueous VAMS). The influence of the hematocrit on the recovery of blood VAMS samples appeared to be limited within the hematocrit range of 0.21 to 0.62. The blood VAMS samples were stable for 1 week if stored at 50 °C, and for at least 8 months when stored between − 80 °C and room temperature. The aqueous VAMS samples were stable for at least 9 months when stored between − 80 and 4 °C, and for 1 month when stored at room temperature. Application of the methods on external quality control material and analysis of patient samples demonstrated the validity and utility of the methods and provided a proof of concept for the analysis of CSF microsamples obtained via VAMS devices.

Graphical abstract


Volumetric absorptive microsampling Liquid chromatography-tandem mass spectrometry Alternative sampling strategies Cerebrospinal fluid Paracetamol 



Analysis of variance


Cerebrospinal fluid


Dried matrix spot


European Medicines Agency


External quality control


Electrospray ionization


Formic acid


US Food and Drug Administration




Internal standard


Liquid chromatography


Lower limit of quantification


Matrix effect


Multiple reaction monitoring


Tandem mass spectrometry


Quality control


Relative standard deviation


Upper limit of quantification


Volumetric absorptive microsampling



The authors wish to acknowledge Prof. Veronique Stove and her team for assistance with blood collection and hematocrit measurements and all volunteers who participated in the study.

Funding information

This study was supported by the Research Foundation – Flanders (G0E010916N).

Compliance with ethical standards

Approval for the clinical proof-of-concept part of this study was provided by the Ethics Committee of Ghent University Hospital (B670201629325). Informed consent has been obtained from the participants involved or their legal representatives.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1427_MOESM1_ESM.pdf (672 kb)
ESM 1 (pdf 671 kb)


  1. 1.
    Edelbroek PM, van der Heijden J, Stolk LML. Dried blood spot methods in therapeutic drug monitoring: methods, assays, and pitfalls. Ther Drug Monit. 2009;31(3):327–36.CrossRefGoogle Scholar
  2. 2.
    Stove CP, Ingels AS, De Kesel PM, Lambert WE. Dried blood spots in toxicology: from the cradle to the grave? Crit Rev Toxicol. 2012;42(3):230–43.CrossRefGoogle Scholar
  3. 3.
    De Kesel PM, Sadones N, Capiau S, Lambert WE, Stove CP. Hemato-critical issues in quantitative analysis of dried blood spots: challenges and solutions. Bioanalysis. 2013;5(16):2023–41.CrossRefGoogle Scholar
  4. 4.
    Wilhelm AJ, den Burger JC, Swart EL. Therapeutic drug monitoring by dried blood spot: progress to date and future directions. Clin Pharmacokinet. 2014;53(11):961–73.CrossRefGoogle Scholar
  5. 5.
    Velghe S, Capiau S, Stove CP. Opening the toolbox of alternative sampling strategies in clinical routine: a key-role for (LC-)MS/MS. TrAC Trends Anal Chem. 2016;84:61–73.CrossRefGoogle Scholar
  6. 6.
    Kok MGM, Fillet M. Volumetric absorptive microsampling: current advances and applications. J Pharm Biomed Anal. 2018;147:288–96.CrossRefGoogle Scholar
  7. 7.
    Denniff P, Spooner N. Volumetric absorptive microsampling: a dried sample collection technique for quantitative bioanalysis. Anal Chem. 2014;86(16):8489–95.CrossRefGoogle Scholar
  8. 8.
    Spooner N, Denniff P, Michielsen L, De Vries R, Ji QC, Arnold ME, et al. A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit. Bioanalysis. 2015;7(6):653–9.CrossRefGoogle Scholar
  9. 9.
    De Kesel PM, Lambert WE, Stove CP. Does volumetric absorptive microsampling eliminate the hematocrit bias for caffeine and paraxanthine in dried blood samples? A comparative study. Anal Chim Acta. 2015;881:65–73.CrossRefGoogle Scholar
  10. 10.
    Zimmer JSD, Christianson CD, Johnson CJL, Needham SR. Recent advances in the bioanalytical applications of dried matrix spotting for the analysis of drugs and their metabolites. Bioanalysis. 2013;5(20):2581–8.CrossRefGoogle Scholar
  11. 11.
    Hirtz C, Lehmann S. What is the potential of dried matrix spot sampling for cerebrospinal fluid analysis? Bioanalysis. 2015;7(22):2849–51.CrossRefGoogle Scholar
  12. 12.
    Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S. Paracetamol: new vistas of an old drug. Cns Drug Rev. 2006;12(3–4):250–75.CrossRefGoogle Scholar
  13. 13.
    Sharma CV, Long JH, Shah S, Rahman J, Perrett D, Ayoub SS, et al. First evidence of the conversion of paracetamol to AM404 in human cerebrospinal fluid. J Pain Res. 2017;10:2703–9.CrossRefGoogle Scholar
  14. 14.
    European Medicines Agency. Guideline on bioanalytical method validation 2011 [Available from:
  15. 15.
    U.S. Department of Health and Human Services Food and Drug Administration. Bioanalytical method validation. Guidance for industry 2018 [Available from:
  16. 16.
    Clinical and Laboratory Standards Institute. CLSI document EP05-A3: evaluation of precision of quantitative measurement procedures; approved guideline—Third Edition 2014.Google Scholar
  17. 17.
    Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75(13):3019–30.CrossRefGoogle Scholar
  18. 18.
    Winek CL, Wahba WW, Winek CLJ, Balzer TW. Drug and chemical blood-level data 2001. Forensic Sci Int. 2001;122(2–3):107–23.CrossRefGoogle Scholar
  19. 19.
    Velghe S, Stove CP. Volumetric absorptive microsampling as an alternative tool for therapeutic drug monitoring of first-generation anti-epileptic drugs. Anal Bioanal Chem. 2018;410(9):2331–41.CrossRefGoogle Scholar
  20. 20.
    Matuszewski BK. Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;830(2):293–300.CrossRefGoogle Scholar
  21. 21.
    Denniff P, Parry S, Dopson W, Spooner N. Quantitative bioanalysis of paracetamol in rats using volumetric absorptive microsampling (VAMS). J Pharm Biomed Anal. 2015;108:61–9.CrossRefGoogle Scholar
  22. 22.
    Li W, Doherty JP, Kulmatycki K, Smith HT, Tse FL. Simultaneous LC-MS/MS quantitation of acetaminophen and its glucuronide and sulfate metabolites in human dried blood spot samples collected by subjects in a pilot clinical study. Bioanalysis. 2012;4(12):1429–43.CrossRefGoogle Scholar
  23. 23.
    Abu-Rabie P, Denniff P, Spooner N, Chowdhry BZ, Pullen FS. Investigation of different approaches to incorporating internal standard in DBS quantitative bioanalytical workflows and their effect on nullifying hematocrit-based assay bias. Anal Chem. 2015;87(9):4996–5003.CrossRefGoogle Scholar
  24. 24.
    Xie I, Xu Y, Anderson M, Wang M, Xue L, Breidinger S, et al. Extractability-mediated stability bias and hematocrit impact: high extraction recovery is critical to feasibility of volumetric adsorptive microsampling (VAMS) in regulated bioanalysis. J Pharm Biomed Anal. 2018;156:58–66.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Lisa Delahaye
    • 1
  • Evelyn Dhont
    • 2
    • 3
  • Pieter De Cock
    • 2
    • 3
    • 4
  • Peter De Paepe
    • 3
    • 5
  • Christophe P. Stove
    • 1
    Email author
  1. 1.Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical SciencesGhent UniversityGhentBelgium
  2. 2.Department of Pediatric Intensive CareGhent University HospitalGhentBelgium
  3. 3.Heymans Institute of PharmacologyGhent UniversityGhentBelgium
  4. 4.Department of PharmacyGhent University HospitalGhentBelgium
  5. 5.Department of Emergency MedicineGhent University HospitalGhentBelgium

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