INVESTIGATION OF RECENTLY ABUSED DRUGS IN DRIED BLOOD SPOTS USING FTA CARDS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY: VALIDATION AND APPLICATION TO REAL SAMPLES

Abstract

A robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the analysis of 19 illicit drugs and metabolites in whole blood dried blood spots (DBS) using FTA cards. Samples prepared using Ahlstrom Munksjö Gensaver™ Colorcards were extracted with methanol. Analytical separation of the analytes was maintained using an Agilent Poroshell column with mobile phase A (0.1% formic acid in water) and B (methanol). Multiple Reaction Monitoring (MRM) with positive ionisation on LC-MS/MS was used to develop the method. The method was validated and met the acceptance criteria with acceptable results for carry-over, linearity, specificity, sensitivity, accuracy, precision, matrix effect and recovery. The method was applied to positively reported whole blood samples from patients suspected of drug abuse. Good quantitative agreement was obtained between the DBS and whole blood methods. Application to real DBS samples showed that this method is a good alternative and useful technique for the detection of drugs of abuse in forensic toxicology and appears to provide a good alternative storage condition.

Keywords

• DBS
• LC-MS/MS
• drug abuse
• real samples
• illicit drugs

References

1. [1] Sadler Simões, S., Castañera Ajenjo, A., and Dias, M.J. (2018). Dried blood spots combined to an UPLC-MS/MS method for the simultaneous determination of drugs of abuse in forensic toxicology. J Pharm Biomed Anal, 5,147,634-644.
2. [2] Grüner, N., Stambouli, O., and Ross, R.S. (2015). Dried blood spots preparing and processing for use in immunoassays and in molecular techniques. J Vis Exp, 13(97), 52619.
3. [3] Quraishi, R., Jain, R., and Ambekar, A. (2013). The use of dried blood spot samples in screening drugs of abuse. Pharmacology & Pharmacy, 4(2), 152-159.
4. [4] Alfazil, A.A., and Anderson, R.A. (2008). Stability of benzodiazepines and cocaine in blood spots stored on filter paper. J Anal Toxico, 32(7), 511-515.
5. [5] Garcia Boy, R., Henseler, R., Mattern, R., and Skopp, G. (2008). Determination of morphine and 6-acetylmorphine in blood with use of dried blood spots. Ther Drug Monit, 30(6), 733-739.
6. [6] Beaudette, P., and Bateman, K.P. (2004). Discovery stage pharmacokinetics using dried blood spots, J Chromatogr B Analyt Technol Biomed Life Sci. 809(1), 153-158.
7. [7] Barfield, M., Spooner, N., Lad, R., Parry, S., and Fowles, S. (2008). Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies. J Chromatogr B Analyt Technol Biomed Life Sci, 870(1), 32-37.
8. [8] Spooner, N., Lad, R., and Barfield, M. (2009). Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method. Anal Chem, 81(4), 1557-1563.

9. [9] Dainty, T.C., Richmond, E.S., Davies, I., and Blackwell, M.P. (2012). Dried blood spot bioanalysis: an evaluation of techniques and opportunities for reduction and refinement in mouse and juvenile rat toxicokinetic studies. Int J Toxicol, 31, 4.
10. [10] Velghe, S., Troyer, R.D., and Stove, C. (2018). Dried blood spots in therapeutic drug monitoring and toxicology. Expert Opin Drug Metab Toxicol, 14, 1-3.
11. [11] Edelbroek, P.M., Van der Heijden, J., and Stolk, L.M. (2009). Dried blood spot methods in therapeutic drug monitoring: methods, assays, and pitfalls. Ther Drug Monit, 31, 327.
12. [12] McDade, T.W., Williams, S., and Snodgrass, J.J. (2007). What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography, 44, 899.
13. [13] Den Burger, J.C., Wilhelm, A.J., Chahbouni, A., Vos, R.M., Sinjewel, A., and Swart, E.L. (2012). Analysis of cyclosporin A, tacrolimus, sirolimus, and everolimus in dried blood spot samples using liquid chromatography tandem mass spectrometry. Anal Bioanal Chem, 404, 1803.
14. [14] Versace, F., Déglon, J., Lauer, E., Mangin, P., and Staub, C. (2013). Automated DBS extraction prior to HILIC/RP LC–MS/MS target screening of drugs. Chromatographia, 76(19-20), 1281–1293.
15. [15] Thomas, A., Deglon, J., Steimer, T., Mangin, P., Daali, Y., and Staub, C. (2010). On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed-phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites. J Sep Sci, 33(6-7), 873-879.
16. [16] Moretti, M., Freni, F., Tomaciello, I., Vignali, C., Groppi, A., Visonà, S.D., Tajana, L., Osculati, A.M.M., and Morini, L. (2019). Determination of benzodiazepines in blood and in dried blood spots collected from post-mortem samples and evaluation of the stability over a three-month period. Drug Test Anal, 11, 1403–1411.
17. [17] Moretti, M., Freni, F., Tomaciello, I., Vignali, C., Groppi, A., Visonà, S.D., Osculati, A.M.M., and Morini, L. (2019). Determination of antidepressants and antipsychotics in dried blood spots (DBSs) collected from post-Mortem samples and evaluation of the stability over a three-month period. Molecules, 24, 3636.
18. [18] Mercolini, L., Mandrioli, R., Sorella, V., Somaini, L., Giocondi, D., Serpelloni, G., and Raggi, M.A. (2013). Dried blood spots: liquid chromatography-mass spectrometry analysis of Δ9-tetrahydrocannabinol and its main metabolites. J Chromatogr A, 4, 1271(1), 33-40.
19. [19] Protti, M., Rudge, J., Sberna, A.E., Gerra, G., and Mercolini, L. (2017). Dried haematic microsamples and LC-MS/MS for the analysis of natural and synthetic cannabinoids. J Chromatogr B Analyt Technol Biomed Life Sci, 1044-1045, 77-86.
20. [20] Ambach, L., Redondo, A.H., König, S., and Weinmann, W. (2013). Rapid and simple LC-MS/MS screening of 64 novel psychoactive substances using dried blood spots. Drug Test Anal, 6(4), 367-375.
21. [21] Antelo-Domínguez, A., Cocho, J.A., Tabernero, M.J., Bermejo, A.M., Bermejo-Barrera, P., and Moreda-Piñeiro, A. (2013). Simultaneous determination of cocaine and opiates in dried blood spots by electrospray ionization tandem mass spectrometry. Talanta, 117, 235-241.
22. [22] Jantos, R., Veldstra, J.L., Mattern, R., Brookhuis, K.A., and Skopp, G. (2011). Analysis of 3,4-methylenedioxymethamphetamine: whole blood versus dried blood spots. J Anal Toxicol, 35(5), 269-273.
23. [23] Clavijo, C.F., Hoffman, K.L., Thomas, J.J., Carvalho, B., Chu, L.F., Driver, D.R., Hammer, G.B., Christians, U., and Galinkin, J.L. (2011). A sensitive assay for the quantification of morphine and its active metabolites in human plasma and dried blood spots using high performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem, 400(3), 715-728.
24. [24] Mommers, J., Mengerink, Y., Ritzen, E., Weusten, J., Van der Heijden, J., and Van der Wal, S. (2013). Quantitative analysis of morphine in dried blood spots by using morphine-d3 pre-impregnated dried blood spot cards. Anal Chim Acta, 774, 26-32.
25. [25] Saussereau, E., Lacroix, C., Gaulier, J.M., and Goulle, J.P. (2012). On-line liquid chromatography/tandem mass spectrometry simultaneous determination of opiates, cocainics and amphetamines in dried blood spots. J Chromatogr B Analyt Technol Bimed Life Sci, 885(886), 1-7.
26. [26] Sosnoff, C.S., Ann, Q., Bernert, J.T., Powell, M.K., Miller, B.B., Henderson, L.O., Hannon, W.H., Fernhoff, P., and Sampson, E.J. (1996). Analysis of benzoylecgonine in dried blood spots by liquid chromatography– atmospheric pressure chemical ionization tandem mass spectrometry. J Anal Toxicol, 20(3), 179–184.
27. [27] Thomas, A., Geyer, H., Schänzer, W., Crone, C., Kellmann, M., Moehring, T., and Thevis, M. (2012). Sensitive determination of prohibited drugs in dried blood spots (DBS) for doping controls by means of a benchtop quadrupole/Orbitrap mass spectrometer. Anal Bioanal Chem, 403(5), 1279-1289.
28. [28] Saracino, M.A., Marcheselli, C., Somaini, L., Pieri, M.C., Gerra, G., Ferranti, A., and Raggi, M.A. (2012). A novel test using dried blood spots for the chromatographic assay of methadone. Anal Bioanal Chem, 404(2), 503-511.
29. [29] Moretti, M., Visonà, S.D., Freni, F., Tomaciello, I., Vignali, C., Groppi, A., Tajana, L., Osculati, A.M.M., and Morini, L. (2018). A liquid chromatography-tandem mass spectrometry method for the determination of cocaine and metabolites in blood and in dried blood spots collected from postmortem samples and evaluation of the stability over a 3-month period. Drug Test Anal, 10, 1430–1437.
30. [30] Moretti, M., Manfredi, A., Freni, F., Previderé, C., Osculati, A.M.M., Grignani, P., Tronconi, L., Carelli, C., Vignali, C., and Morini, L. (2021). A comparison between two different dried blood substrates in determination of psychoactive substances in postmortem samples. Forensic Toxicol, 39, 385–393.
31. [31] Kacargil, C.U., Daglioglu, N., and Goren, I.E. (2020). Determination of illicit drugs in dried blood spots by lc–ms/ms method: validation and application to real samples. Chromatographia, 83(7), 885-892.
32. [32] Yeter, O., and Erol Öztürk, Y. (2019). Detection and quantification of 5F-ADB and its methyl ester hydrolysis metabolite in fatal intoxication cases by liquid chromatography-high resolution mass spectrometry. Forensic Sci Int, 302, 109866.
33. [33] Scientific Working Group for Forensic Toxicology. (2013). Standard practices for method validation in forensic toxicology. J Anal Toxicol, 37(7), 452-74.
34. [34] Timmerman, P., White, S., Globig, S., Lüdtke, S., Bruner, L., and Smeraglia, J. (2011). EBF recommendation on the validation of bioanalytical methods for dried blood spots. Bioanalysis, 3(14), 1567-1575.
35. [35] Matuszewski, B.K., and Constanzer, M.L. (2003). Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC – MS/MS. Anal Chem, 75, 3019–3030.
36. [36] Bland, J.M., and Altman, D.G. (1986). Statistical methods for assessing agreement between two methods of clinical measurement. Lancet, 1(8476), 307-310.
37. [37] Stockl, D., Dewitte, K., and Thienpont, L. (1998). Validity of linear regression in method comparison studies: is it limited by the statistical model or the quality of the analytical input data? Clin Chem, 44(11), 2340–2346.
38. [38] Dewitte, K., Fierens, C., Stockl, D., and Thienpont, L. (2002). Application of the Bland-Altman plot for interpretation of method- comparison studies: a critical investigation of its practice. Clin Chem, 48(5), 799–801.