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Abstract Introduction
Estradiol and estrone are the two main biologically active estrogens. Of the two, estradiol is more biologically potent, and found at higher concentration in premenopausal women. Because of this, estradiol measurements are an important part of monitoring reproductive health and assessing menopausal status. Immunoassays of moderate sensitivity are adequate for measuring estradiol in premenopausal women but require large sample volume and still may not adequately detect male or postmenopausal female serum concentrations which can be as low as 20 pg/mL. An estradiol measurement may be required in diagnosing males with estrogen producing neoplasms, or for monitoring estrogen levels in a post-menopausal woman; in these cases, an LC-MS/MS technique would be employed.
Highly sensitive LC-MS/MS assays can quantify estradiol at sub pg/mL levels by extracting it from a serum sample, which requires that the patient have blood drawn by a phlebotomist, usually in a clinical setting. This requisite in-person sampling can be a barrier to care for many who live in underserved regions, so an at home self-collect sampling option would be a great benefit for ease of access to diagnostic testing.
Dried blood spots have long been used in such testing as the diagnosis of phenylketonuria in newborns, infectious disease testing, vitamin D3 testing, and increasingly other hormones and biomarkers. As a sample type, dried blood spots (DBS) have key advantages over other types, including reduced risk of bacterial contamination, lower levels of hemolysis, increased sample stability, and most importantly: the capacity for the patient to self-collect. Despite these advantages, dried blood spots assays are limited by sample volume requiring a highly sensitive method to quantify low levels of estradiol in dried blood spots.
Objective
To achieve the sensitivity required to quantify estradiol at male and post-menopausal levels in dried blood spot, while maintaining a high-throughput 96 well plate format, a combination of sample cleanup techniques, analyte derivatization and optimized instrumentation will be leveraged.
Methods
Dried blood spot patient samples were extracted using solid phase extraction and derivatized to enhance ionization prior to injection. Samples were injected on an HPLC (Schimadzu SIL-40 series) coupled with a triple quadrupole mass spectrometer (Sciex 6500+).
Results
Contrived quality control samples were successfully measured at 20 pg/mL.
Conclusions
Using high throughput sample cleanup technologies combined with the latest industry standards in instrumentation, A sensitive, high throughput, and automatable diagnostic test can be achieved for self-collected dried blood spot samples. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
