Analyzing Dried Blood Spots for Steroids with LC-MS/MS

A recent study (1) published in the journal Advanced Sample Preparation presented the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a driver for sample preparation from dried blood spot (DBS) samples to quantify the steroids testosterone (T), 17α-hydroxyprogesterone (17-OHP), progesterone (P), and cortisol (C), simultaneously. This method uses both standard electroporation cuvettes, as well as a novel custom vertical electric field setup. The findings, backed by computational modeling, reveal that a 10V DC application for 180 s will draw out double the amount of the previously mentioned steroids from sample collection cards when using the authors’ proprietary device as opposed to any standard collection methods which are solvent-based. The study presents the application of electric field for metabolomic sample preparation, as well as a discussion pertaining to a unique device which eliminates the electric double layer effect in the process.

A similar study, authored by two of the same authors, John Murphy and Anubhav Tripathi (2), discuss the establishment of a DBS extraction method using the synergistic action of electrophoretic and diffusive transport mechanisms to extract genomic DNA (gDNA) through the DBS matrix. The method (dubbed "Electro-DBS" by its creators) reduces extraction time from 40 min to 5 min, negating the need for heat, shaking, and DNA purification steps, yet still maintaining the yield and quality of gDNA extracted. The authors discovered that the electrophoretic transport speeds up the elution of gDNA, allowing the diffusive transport of polymerase chain reaction (PCR) inhibitors to be minimized because of the reduced elution time. Overall, this work added mechanistic insight into the extraction of DNA from DBS and developed a technique ideal for automation and point of care.

For decades, the article states, DBS have been used for the screening of newborns multiomics analysis (genomics, proteomics, and metabolomics) using LC-MS/MS. These techniques can provide reliable diagnostic source material to access patient samples from remote locations in Nepal and Bangladesh without having practiced phlebotomists and advanced technologies involved, due to analytes collected in the DBS papers being relatively stable and DBS papers being simple to move. However, although DBS has a role as a minimally invasive sample source, limitations exist regarding the possibility of widely using it for bioanalytical assays; reasons for this include sample preparation, and extraction efficiency. Although a variety of sample preparation methods are available to analysts (including solid-phase extraction, liquid-liquid extraction, andcombination reactions), it is difficult to perform steroid extraction from a DBS sample.

Although the analysis of steroids with LC-MS/MS for clinical application occurs often, it has become standard practice to perform method optimization and validation, as it is instrument specific, and there are lab-to-lab variabilities. The authors, therefore, chose a simple sample preparation process (protein precipitation), thus ensuring adaptability of automation utilized in the future. To eliminate the presence of any native steroids, the authors tested triplicates of blank whole blood, and observed consistently that there were no peaks at the expected retention times (RT) of the analytes. Because blood is a complicated matrix, they found it difficult to obtain uniform dispersion of T, 17-OHP, P, and C across a sample; thus, several iterations of the calibrators needed to be prepared and analyzed for reproducibility and consistency before analysts could begin other testing.

Dried blood spots on a fiber filter for laboratory analysis. © MarekPhotoDesign.com stock.adobe.com

References

1. Fariha, R.; Rothkopf, E.; Murphy, J.; Walters, N.; Okoh, O. D.; Lawandy, N. M.; Tripathi, A. Electric Field-Assisted Dried Blood Spot Sample Preparation for Analysis of Steroids Using LC-MS/MS. Adv. Samp. Prep. 2024, 100115. DOI: 10.1016/j.sampre.2024.100115

2. Lee, K.; Murphy, J.; Tripathi, A. (2022). Electro-DBS: A Simple Method to Rapidly Extract Genomic DNA from Dried Blood Spots. Anal. Chem. 2022, 94. DOI: 10.1021/acs.analchem.2c02021