Development of the dried blood spot preparation protocol for comprehensive evaluation of the hematocrit effect

Chih-Ning Cheng; Yu-Fong Peng; Ju-Yu Chen; Guan-Yuan Chen; Te-I Weng; Ching-Hua Kuo

doi:10.1016/j.aca.2022.340650

Development of the dried blood spot preparation protocol for comprehensive evaluation of the hematocrit effect

Anal Chim Acta. 2023 Jan 25:1239:340650. doi: 10.1016/j.aca.2022.340650. Epub 2022 Nov 21.

Authors

Chih-Ning Cheng¹, Yu-Fong Peng¹, Ju-Yu Chen², Guan-Yuan Chen³, Te-I Weng⁴, Ching-Hua Kuo⁵

Affiliations

¹ School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.
² School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan; Forensic and Clinical Toxicology Center, National Taiwan University Hospital, Taipei, Taiwan; Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
³ Forensic and Clinical Toxicology Center, National Taiwan University Hospital, Taipei, Taiwan; Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
⁴ Forensic and Clinical Toxicology Center, National Taiwan University Hospital, Taipei, Taiwan; Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; Department of Emergency Medicine, National Taiwan University Hospital, Taipei, Taiwan.
⁵ School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan; The Metabolomics Core Laboratory, Centers of Genomic and Precision Medicine, National Taiwan University, Taiwan; Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan. Electronic address: kuoch@ntu.edu.tw.

PMID: 36628747
DOI: 10.1016/j.aca.2022.340650

Abstract

The application of dried blood spots (DBS) has gradually increased in different fields because of its several advantages. The hematocrit (Hct) effect is one major analytical challenge that may affect the quantification accuracy of DBS samples and should be investigated when developing a novel DBS method. However, previous studies usually overlooked the Hct-related distribution bias when evaluating the Hct effect. This study aimed to propose an effective DBS preparation protocol for the comprehensive evaluation of the Hct effect. We selected voriconazole and posaconazole as the demonstration drugs. Fifteen microliters of the blood samples were spotted on DBS cards followed by whole spot extraction. An LC-MS/MS method was first developed to quantify voriconazole and posaconazole in DBS samples. The quantitation accuracy for both azole drugs was within 93.5%-111.7%, except for the accuracies of posaconazole at the LLOQ, which were less than 119%. The intra- and interday precision were below 11%. The validated LC-MS/MS method was used to develop the DBS preparation protocol for evaluating the Hct effect. Three critical parameters that may affect the observed Hct effect were investigated. The results showed that using the solid-state of the target analytes, spiking the target analytes before preparing different Hct levels, and allowing enough equilibrium time after spiking target analytes can provide a more holistic Hct effect evaluation. The validity of the proposed new protocol was verified by conversion factors obtained from 71 paired DBS and plasma samples. Conversion factors calculated by clinical samples were consistent with the Hct effect evaluated by manually prepared DBS samples. This new DBS preparation protocol eliminated the common pitfalls in studying the Hct effect and offered a comprehensive strategy to assess the Hct effect for further DBS studies.

Keywords: Azole drugs; Distribution bias; Dried blood spots; Hematocrit effect.

MeSH terms

Chromatography, Liquid / methods
Dried Blood Spot Testing* / methods
Hematocrit
Reproducibility of Results
Tandem Mass Spectrometry* / methods
Voriconazole

Substances

Voriconazole