Volumetric absorptive microsampling meets electromembrane extraction for the first time: Case example of doxorubicin and its metabolite in whole blood samples

Adam Reguli; Hana Bavlovič Piskáčková; Olga Lenčová-Popelová; Petra Kollárová-Brázdová; Martin Štěrba; Stig Pedersen-Bjergaard; Petra Štěrbová-Kovaříková

doi:10.1016/j.aca.2024.343459

Volumetric absorptive microsampling meets electromembrane extraction for the first time: Case example of doxorubicin and its metabolite in whole blood samples

Adam Reguli, Hana Bavlovič Piskáčková, Olga Lenčová-Popelová, Petra Kollárová-Brázdová, Martin Štěrba, Stig Pedersen-Bjergaard, Petra Štěrbová-Kovaříková^*

^*Corresponding author for this work

Microscale Analytical Systems

Research output: Contribution to journal › Journal article › Research › peer-review

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Abstract

Background: Microsampling of biological fluids followed by innovative sample pre-treatment reflects trends in bioanalytical chemistry. Volumetric absorptive microsampling (VAMS) enables exact whole blood volume collection and reduces the impact of hematocrit on the assay. In animal studies, it complies with the 3R principles (refine, reduce, replace). It allows for a gentle bleeding technique and a reduction in the number of laboratory animals by enabling ethically acceptable repeated blood collection from a single animal. Treating VAMS tips with electromembrane extraction (EME) in 96-well format offers a smart combination of non-invasive, low-volume blood collection with effective, environmentally friendly sample clean-up. Results: This study introduces the first application of EME in 96-well format for direct isolation of analytes from 10 μL of whole blood collected onto a VAMS device. Doxorubicin, a clinically used anticancer drug also utilized in cancer/cardio-oncology research involving rodents, where microsampling offers important advantages, and its metabolite doxorubicinol, were selected as relevant analytes. The optimized EME yielded reproducible recoveries for both analytes regardless of hematocrit levels, different anticoagulants, or free multivalent ions in the sample. Compared to conventional VAMS tips treatment, EME reduced matrix effects, increased throughput, and an environmental friendliness of the extraction. The EME followed by the UHPLC-MS/MS assay was validated for both analytes in whole blood absorbed onto VAMS tips. The same protocol was implemented to treat plasma to determine the blood-to-plasma ratio of the analytes in the same experiments. The practical utility was demonstrated by analyzing real samples collected from the doxorubicin-treated nude mice. Significance: The study offers a novel assay combining whole blood microsampling and sample clean-up in microextraction scale for preclinical pharmacokinetic studies with doxorubicin in rodents and for pharmacokinetic/pharmacodynamic modeling. This advancement in bioanalytical chemistry promotes scalable environmentally friendly procedures compatible with the 3R ethical principles in animal studies. Moreover, the concept of direct VAMS tips treatment with EME may also be easily translatable to clinical settings.

Original language	English
Article number	343459
Journal	Analytica Chimica Acta
Volume	1335
Number of pages	12
ISSN	0003-2670
DOIs	https://doi.org/10.1016/j.aca.2024.343459
Publication status	Published - 2025

Bibliographical note

Funding Information:
VAMS tips were dropped into the donor well and the donor solution containing 1 \u03BCL of internal standards (13C-d3-DOX and 13C-d3-DOXol, concentration of 2 \u03BCM) was added. Then 3 \u03BCL of water immiscible organic solvent was dispensed into the outer part of PVDF membranes, attached to the wells of donor plate and acceptor plate was filled with acceptor solution. To initiate the extraction, a voltage was applied (tested range 20\u201335 V) and the process took 15\u201325 min while shaken at 850\u20131050 RPM. The following parameters were optimized during method development: volume (180\u2013235 \u03BCL) and composition of donor solution (buffer of pH 3, 200 mM formic acid), composition of supported liquid membrane (DEHPi, NPPE, ENB, 1-undecanol, 2-undecanone and different combinations of these solvents) and volume (50\u2013100 \u03BCL) of acceptor solution. The EME procedure was optimized using whole blood absorbed onto VAMS tips spiked at a concentration of 100 nM for both analytes.We thank Petra Kazimirova for skilled technical assistance during the in vivo experiments. This work was supported by the Czech Science Foundation (project no. 23-06558S), Charles University (GAUK project No. 232223 and SVV 260666) and the project \u201CNew Technologies for Translational Research in Pharmaceutical Sciences/NETPHARM\u201D (project ID CZ.02.01.01/00/22_008/0004607), co-funded by the European Union.

Funding Information:
We thank Petra Kazimirova for skilled technical assistance during the in vivo experiments. This work was supported by the Czech Science Foundation (project no. 23-06558S), Charles University (GAUK project No. 232223 and SVV 260666) and The project New Technologies for Translational Research in Pharmaceutical Sciences /NETPHARM, project ID CZ.02.01.01/00/22_008/0004607, co-funded by the European Union.

Publisher Copyright:
© 2024

Keywords

Anthracyclines
Doxorubicin
Doxorubicinol
Electromembrane extraction
Microextraction
Volumetric absorptive microsampling

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Reguli, A., Bavlovič Piskáčková, H., Lenčová-Popelová, O., Kollárová-Brázdová, P., Štěrba, M., Pedersen-Bjergaard, S., & Štěrbová-Kovaříková, P. (2025). Volumetric absorptive microsampling meets electromembrane extraction for the first time: Case example of doxorubicin and its metabolite in whole blood samples. Analytica Chimica Acta, 1335, Article 343459. https://doi.org/10.1016/j.aca.2024.343459

Volumetric absorptive microsampling meets electromembrane extraction for the first time: Case example of doxorubicin and its metabolite in whole blood samples. / Reguli, Adam; Bavlovič Piskáčková, Hana; Lenčová-Popelová, Olga et al.
In: Analytica Chimica Acta, Vol. 1335, 343459, 2025.

Research output: Contribution to journal › Journal article › Research › peer-review

Reguli, A, Bavlovič Piskáčková, H, Lenčová-Popelová, O, Kollárová-Brázdová, P, Štěrba, M, Pedersen-Bjergaard, S & Štěrbová-Kovaříková, P 2025, 'Volumetric absorptive microsampling meets electromembrane extraction for the first time: Case example of doxorubicin and its metabolite in whole blood samples', Analytica Chimica Acta, vol. 1335, 343459. https://doi.org/10.1016/j.aca.2024.343459

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title = "Volumetric absorptive microsampling meets electromembrane extraction for the first time: Case example of doxorubicin and its metabolite in whole blood samples",

abstract = "Background: Microsampling of biological fluids followed by innovative sample pre-treatment reflects trends in bioanalytical chemistry. Volumetric absorptive microsampling (VAMS) enables exact whole blood volume collection and reduces the impact of hematocrit on the assay. In animal studies, it complies with the 3R principles (refine, reduce, replace). It allows for a gentle bleeding technique and a reduction in the number of laboratory animals by enabling ethically acceptable repeated blood collection from a single animal. Treating VAMS tips with electromembrane extraction (EME) in 96-well format offers a smart combination of non-invasive, low-volume blood collection with effective, environmentally friendly sample clean-up. Results: This study introduces the first application of EME in 96-well format for direct isolation of analytes from 10 μL of whole blood collected onto a VAMS device. Doxorubicin, a clinically used anticancer drug also utilized in cancer/cardio-oncology research involving rodents, where microsampling offers important advantages, and its metabolite doxorubicinol, were selected as relevant analytes. The optimized EME yielded reproducible recoveries for both analytes regardless of hematocrit levels, different anticoagulants, or free multivalent ions in the sample. Compared to conventional VAMS tips treatment, EME reduced matrix effects, increased throughput, and an environmental friendliness of the extraction. The EME followed by the UHPLC-MS/MS assay was validated for both analytes in whole blood absorbed onto VAMS tips. The same protocol was implemented to treat plasma to determine the blood-to-plasma ratio of the analytes in the same experiments. The practical utility was demonstrated by analyzing real samples collected from the doxorubicin-treated nude mice. Significance: The study offers a novel assay combining whole blood microsampling and sample clean-up in microextraction scale for preclinical pharmacokinetic studies with doxorubicin in rodents and for pharmacokinetic/pharmacodynamic modeling. This advancement in bioanalytical chemistry promotes scalable environmentally friendly procedures compatible with the 3R ethical principles in animal studies. Moreover, the concept of direct VAMS tips treatment with EME may also be easily translatable to clinical settings.",

keywords = "Anthracyclines, Doxorubicin, Doxorubicinol, Electromembrane extraction, Microextraction, Volumetric absorptive microsampling",

author = "Adam Reguli and {Bavlovi{\v c} Pisk{\'a}{\v c}kov{\'a}}, Hana and Olga Len{\v c}ov{\'a}-Popelov{\'a} and Petra Koll{\'a}rov{\'a}-Br{\'a}zdov{\'a} and Martin {\v S}t{\v e}rba and Stig Pedersen-Bjergaard and Petra {\v S}t{\v e}rbov{\'a}-Kova{\v r}{\'i}kov{\'a}",

note = "Funding Information: VAMS tips were dropped into the donor well and the donor solution containing 1 \u03BCL of internal standards (13C-d3-DOX and 13C-d3-DOXol, concentration of 2 \u03BCM) was added. Then 3 \u03BCL of water immiscible organic solvent was dispensed into the outer part of PVDF membranes, attached to the wells of donor plate and acceptor plate was filled with acceptor solution. To initiate the extraction, a voltage was applied (tested range 20\u201335 V) and the process took 15\u201325 min while shaken at 850\u20131050 RPM. The following parameters were optimized during method development: volume (180\u2013235 \u03BCL) and composition of donor solution (buffer of pH 3, 200 mM formic acid), composition of supported liquid membrane (DEHPi, NPPE, ENB, 1-undecanol, 2-undecanone and different combinations of these solvents) and volume (50\u2013100 \u03BCL) of acceptor solution. The EME procedure was optimized using whole blood absorbed onto VAMS tips spiked at a concentration of 100 nM for both analytes.We thank Petra Kazimirova for skilled technical assistance during the in vivo experiments. This work was supported by the Czech Science Foundation (project no. 23-06558S), Charles University (GAUK project No. 232223 and SVV 260666) and the project \u201CNew Technologies for Translational Research in Pharmaceutical Sciences/NETPHARM\u201D (project ID CZ.02.01.01/00/22_008/0004607), co-funded by the European Union. Funding Information: We thank Petra Kazimirova for skilled technical assistance during the in vivo experiments. This work was supported by the Czech Science Foundation (project no. 23-06558S), Charles University (GAUK project No. 232223 and SVV 260666) and The project New Technologies for Translational Research in Pharmaceutical Sciences /NETPHARM, project ID CZ.02.01.01/00/22_008/0004607, co-funded by the European Union. Publisher Copyright: {\textcopyright} 2024",

year = "2025",

doi = "10.1016/j.aca.2024.343459",

language = "English",

volume = "1335",

journal = "Analytica Chimica Acta",

issn = "0003-2670",

publisher = "Elsevier",

}

TY - JOUR

T1 - Volumetric absorptive microsampling meets electromembrane extraction for the first time

T2 - Case example of doxorubicin and its metabolite in whole blood samples

AU - Reguli, Adam

AU - Bavlovič Piskáčková, Hana

AU - Lenčová-Popelová, Olga

AU - Kollárová-Brázdová, Petra

AU - Štěrba, Martin

AU - Pedersen-Bjergaard, Stig

AU - Štěrbová-Kovaříková, Petra

N1 - Funding Information: VAMS tips were dropped into the donor well and the donor solution containing 1 \u03BCL of internal standards (13C-d3-DOX and 13C-d3-DOXol, concentration of 2 \u03BCM) was added. Then 3 \u03BCL of water immiscible organic solvent was dispensed into the outer part of PVDF membranes, attached to the wells of donor plate and acceptor plate was filled with acceptor solution. To initiate the extraction, a voltage was applied (tested range 20\u201335 V) and the process took 15\u201325 min while shaken at 850\u20131050 RPM. The following parameters were optimized during method development: volume (180\u2013235 \u03BCL) and composition of donor solution (buffer of pH 3, 200 mM formic acid), composition of supported liquid membrane (DEHPi, NPPE, ENB, 1-undecanol, 2-undecanone and different combinations of these solvents) and volume (50\u2013100 \u03BCL) of acceptor solution. The EME procedure was optimized using whole blood absorbed onto VAMS tips spiked at a concentration of 100 nM for both analytes.We thank Petra Kazimirova for skilled technical assistance during the in vivo experiments. This work was supported by the Czech Science Foundation (project no. 23-06558S), Charles University (GAUK project No. 232223 and SVV 260666) and the project \u201CNew Technologies for Translational Research in Pharmaceutical Sciences/NETPHARM\u201D (project ID CZ.02.01.01/00/22_008/0004607), co-funded by the European Union. Funding Information: We thank Petra Kazimirova for skilled technical assistance during the in vivo experiments. This work was supported by the Czech Science Foundation (project no. 23-06558S), Charles University (GAUK project No. 232223 and SVV 260666) and The project New Technologies for Translational Research in Pharmaceutical Sciences /NETPHARM, project ID CZ.02.01.01/00/22_008/0004607, co-funded by the European Union. Publisher Copyright: © 2024

PY - 2025

Y1 - 2025

N2 - Background: Microsampling of biological fluids followed by innovative sample pre-treatment reflects trends in bioanalytical chemistry. Volumetric absorptive microsampling (VAMS) enables exact whole blood volume collection and reduces the impact of hematocrit on the assay. In animal studies, it complies with the 3R principles (refine, reduce, replace). It allows for a gentle bleeding technique and a reduction in the number of laboratory animals by enabling ethically acceptable repeated blood collection from a single animal. Treating VAMS tips with electromembrane extraction (EME) in 96-well format offers a smart combination of non-invasive, low-volume blood collection with effective, environmentally friendly sample clean-up. Results: This study introduces the first application of EME in 96-well format for direct isolation of analytes from 10 μL of whole blood collected onto a VAMS device. Doxorubicin, a clinically used anticancer drug also utilized in cancer/cardio-oncology research involving rodents, where microsampling offers important advantages, and its metabolite doxorubicinol, were selected as relevant analytes. The optimized EME yielded reproducible recoveries for both analytes regardless of hematocrit levels, different anticoagulants, or free multivalent ions in the sample. Compared to conventional VAMS tips treatment, EME reduced matrix effects, increased throughput, and an environmental friendliness of the extraction. The EME followed by the UHPLC-MS/MS assay was validated for both analytes in whole blood absorbed onto VAMS tips. The same protocol was implemented to treat plasma to determine the blood-to-plasma ratio of the analytes in the same experiments. The practical utility was demonstrated by analyzing real samples collected from the doxorubicin-treated nude mice. Significance: The study offers a novel assay combining whole blood microsampling and sample clean-up in microextraction scale for preclinical pharmacokinetic studies with doxorubicin in rodents and for pharmacokinetic/pharmacodynamic modeling. This advancement in bioanalytical chemistry promotes scalable environmentally friendly procedures compatible with the 3R ethical principles in animal studies. Moreover, the concept of direct VAMS tips treatment with EME may also be easily translatable to clinical settings.

AB - Background: Microsampling of biological fluids followed by innovative sample pre-treatment reflects trends in bioanalytical chemistry. Volumetric absorptive microsampling (VAMS) enables exact whole blood volume collection and reduces the impact of hematocrit on the assay. In animal studies, it complies with the 3R principles (refine, reduce, replace). It allows for a gentle bleeding technique and a reduction in the number of laboratory animals by enabling ethically acceptable repeated blood collection from a single animal. Treating VAMS tips with electromembrane extraction (EME) in 96-well format offers a smart combination of non-invasive, low-volume blood collection with effective, environmentally friendly sample clean-up. Results: This study introduces the first application of EME in 96-well format for direct isolation of analytes from 10 μL of whole blood collected onto a VAMS device. Doxorubicin, a clinically used anticancer drug also utilized in cancer/cardio-oncology research involving rodents, where microsampling offers important advantages, and its metabolite doxorubicinol, were selected as relevant analytes. The optimized EME yielded reproducible recoveries for both analytes regardless of hematocrit levels, different anticoagulants, or free multivalent ions in the sample. Compared to conventional VAMS tips treatment, EME reduced matrix effects, increased throughput, and an environmental friendliness of the extraction. The EME followed by the UHPLC-MS/MS assay was validated for both analytes in whole blood absorbed onto VAMS tips. The same protocol was implemented to treat plasma to determine the blood-to-plasma ratio of the analytes in the same experiments. The practical utility was demonstrated by analyzing real samples collected from the doxorubicin-treated nude mice. Significance: The study offers a novel assay combining whole blood microsampling and sample clean-up in microextraction scale for preclinical pharmacokinetic studies with doxorubicin in rodents and for pharmacokinetic/pharmacodynamic modeling. This advancement in bioanalytical chemistry promotes scalable environmentally friendly procedures compatible with the 3R ethical principles in animal studies. Moreover, the concept of direct VAMS tips treatment with EME may also be easily translatable to clinical settings.

KW - Anthracyclines

KW - Doxorubicin

KW - Doxorubicinol

KW - Electromembrane extraction

KW - Microextraction

KW - Volumetric absorptive microsampling

U2 - 10.1016/j.aca.2024.343459

DO - 10.1016/j.aca.2024.343459

M3 - Journal article

C2 - 39643313

AN - SCOPUS:85209753594

SN - 0003-2670

VL - 1335

JO - Analytica Chimica Acta

JF - Analytica Chimica Acta

M1 - 343459

ER -