Divyabharathi Chepyala (Presenter)
National Taiwan University
Bio: My name is Divyabharathi Chepyala, and I am a third year Ph.D student at the School of Pharmacy, College of Medicine, at the National Taiwan University in Taiwan. I got my undergraduate degree from the Department of Pharmacy at Annamali University in India. I was major in Pharmacy. I started my graduate studies at the National Taiwan University as a Master’s student and subsequently I enrolled for the Ph.D program. Currently the focus of my graduate studies is in the field of pharmaceutical analysis.
Authorship: Divyabharathi Chepyala (1), Ching-Hua Kuo (1,2,3)*
(1) School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan. (2) The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan. (3) Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan
| Short Abstract Dried blood spot (DBS) is an important sampling technique, but individual variation of hematocrit (HCT) is the major sources of quantification errors for this technique. This study developed a potassium ion complexation (PIC) method to calibrate HCT on DBS samples using flow injection analysis electrospray ionization mass spectrometry. Extraction of potassium ion (K+) followed by complexation with 18-crown-6 aided the measurement of K+ from DBS samples which was used to calibrate HCT. Linearity, precision and accuracy tests were done to validate the method. We anticipate that the developed PIC method can be used to calibrate the HCT from DBS samples. |
Long Abstract
Introduction:
Dried blood spot (DBS) sampling technique is widely used in clinical analysis as a minimum invasive tool. However, the inter-individual variation of hematocrit (HCT) levels is one of the major sources of quantification errors when using the DBS sampling technique. Previously, potassium ion (K+) was demonstrated as a biomarker for HCT prediction using indirect potentiometry. Since liquid chromatography tandem mass spectrometry (LC-MS/MS) is widely used in clinical analysis, it is beneficial to develop an analytical method to estimate HCT using LC-MS/MS. In this study, we aimed to develop a novel strategy using a potassium ion complexation (PIC) method to calibrate HCT levels on DBS samples using flow injection analysis electrospray ionization mass spectrometry (FIA-ESI-MS).
Methods:
Three millimeter DBS spots were extracted with 50% methanol (MeOH) using a geno grinder. After extraction, the samples were subjected to centrifugation. Supernatant obtained from centrifugation was mixed with the complexing agent 18-crown-6 in 100% MeOH for potassium complexation. Two microliters of sample was injected through FIA with the mobile phase composed of 0.1% acetic acid buffer and MeOH (30/70). The flow rate was 0.3 mL min-1. Samples were analyzed through positive ionization mode.
Results:
The measured K+ levels were used to calibrate the HCT from DBS samples. Calibration curves were generated within the range of 30 to 55% HCT. The validation results revealed that the developed method is able to achieve an acceptable precision and accuracy. We applied the developed method to real patient samples to measure the HCT levels, and the results showed high correlation with results obtained from the typical hematology analyzer.
Conclusion:
We anticipate that the developed PIC method can be used to calibrate the HCT from DBS samples using mass spectrometry.
Novel Aspect:
A novel potassium ion complexation method (PIC) method was introduced in this study to calibrate HCT bias for DBS samples using flow injection analysis electrospray ionization mass spectrometry.
References & Acknowledgements:
Acknowledgments:
The authors appreciate the support from NTU Integrated Core Facility for Functional Genomics of National Research Program for Genomic Medicine of Taiwan and the Ministry of Science and Technology of Taiwan
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