• Yahdiana Harahap Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia.
  • Dimas Agus Putera Hardijanto Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia.
  • Delly Ramadon Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia.




5-fluorouracil, 6-mercaptopurine, 6-thioguanine, Dried blood spot, Hematocrit, Liquid chromatography tandem-mass spectrometry


Objective: This study aimed to determine the effects of the method of internal standard addition, spotting volume, paper type, and sample storage
temperature on 6-mercaptopurine, and 6-thioguanine on liquid chromatography tandem-mass spectrometry (LC-MS/MS) bioanalysis methods using
dried blood spot (DBS).
Methods: Blood samples were spotted on CAMAG DBS paper and a Perkin Elmer 226 sample collection device (paper) and extracted into methanol
containing 5-fluorouracil as an internal standard. The separation was performed on a water acquity ultra high-performance LC BEH Amide 1.7 μm
(2.1 mm×100 mm) column with a mobile phase of 0.2% formic acid in water - 0.1% formic acid in acetonitrile methanol with gradient elution at a
flow rate of 0.2 mL/min.
Results: The step at which the internal standard was added (blood, spot on DBS card, or extraction solution) affected the chromatogram. Differences
in paper types and blood volumes significantly affected (p<0.05) the percent coefficient of variation, whereas differences in blood hematocrit
significantly affected the peak area ratio.
Conclusion: The method of internal standard addition affected the chromatograms in this study. The best chromatogram was observed when
the Internal Standard was added to the extracting solution. The card type also affected the analysis, so it is recommended to use the same card during
sample analysis.


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Déglon J, Thomas A, Mangin P, Staub C. Direct analysis of dried

blood spots coupled with mass spectrometry: Concepts and biomedical

applications. Anal Bioanal Chem 2012;402:2485-98.

Evans C, Arnold M, Bryan P, Duggan J, James CA, Li W, et al.

Implementing dried blood spot sampling for clinical pharmacokinetic

determinations: Considerations from the IQ consortium microsampling

working group. AAPS J 2015;17:292-300.

Koster RA, Botma R, Greijdanus B, Uges DR, Kosterink JG, Touw DJ,

et al. The performance of five different dried blood spot cards for the

analysis of six immunosuppressants. Bioanalysis 2015;7:1225-35.

Denniff P, Spooner N. The effect of hematocrit on assay bias when using

DBS samples for the quantitative bioanalysis of drugs. Bioanalysis


Liang X, Li Y, Barfield M, Ji QC. Study of dried blood spots technique

for the determination of dextromethorphan and its metabolite

dextrorphan in human whole blood by LC-MS/MS. J Chromatogr B

Analyt Technol Biomed Life Sci 2009;877:799-806.

Prentice P, Turner C, Wong MC, Dalton RN. Stability of metabolites in

dried blood spots stored at different temperatures over a 2-year period.

Bioanalysis 2013;5:1507-14.

World Health Organization. Flame Retardants: Tris (2-Butoxyethyl)

Phosphate, Tris(2-Ethylhexyl) Phosphate and Tetrakis (Hydroxymethyl)

Phosphonium Salt, Environmental Health Criteria 218. Geneva: World

Health Organization; 2000. p. 1-130.

Weisz A, Milstein SR, Scher AL, Hepp NM. Colouring agents in

cosmetics: Regulatory aspects and analytical methods. In: Analysis of

Cosmetic Products. Ch. 7. 2nd ed. Amsterdam: Elsevier; 2018. p. 123‑57.

Ruiz NM, Perez MT, Diaz HM, Izquierdo M, Blanco M, Machado LY,

et al. Determination of HIV-1 viral load on dried blood spot specimens.

Biotechnol Apl 2014;31:146-9.

Lafreniere NM, Shih SC, Abu-rabie P, Jebrail MJ, Spooner N,

Wheeler AR. Multiplexed extraction and quantitative analysis of

pharmaceuticals from DBS samples using digital microfluidics.

Bioanalysis 2014;6:307‑18.

Spooner N, Ramakrishnan Y, Barfield M, Dewit O, Miller S. Use of

DBS sample collection to determine circulating drug concentrations

in clinical trials: Practicalities and considerations. Bioanalysis


Abu-Rabie P. Direct analysis of DBS: Emerging and desirable

technologies. Bioanalysis 2011;3:1675-8.



How to Cite

Harahap, Y., Hardijanto, D. A. P., & Ramadon, D. (2018). FACTORS AFFECTING LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY ANALYSIS OF 6-MERCAPTOPURINE AND 6-THIOGUANINE IN DRIED BLOOD SPOTS. International Journal of Applied Pharmaceutics, 10(1), 200–202. https://doi.org/10.22159/ijap.2018.v10s1.44



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