Presenter Bio: I work as a principal clinical scientist in Glasgow Royal Infirmary.
I specialise in Endocrinology with a particular interest in LC-MS/MS and have many years experience in developing, validating and implementing methods for routine and research purposes.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction: The two major metabolically active oestrogens in the non-pregnant population are oestrone (E1) and oestradiol (E2). Accurate measurement of E2 at low concentrations is important in a variety of clinical situations including inborn errors of metabolism, disorders of puberty, post-menopausal women, monitoring aromatase inhibitor treatment, and in men. Measuring oestrogens at very low concentrations remains an analytical challenge. Many published methods involve complex and time-consuming sample preparations, large sample volumes and chemical derivatisation. Here we present a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure serum E1 and E2 in the low pmol/L range.
Methods: 250 µL of serum was spiked with diluent containing deuterated internal standards (d4-E1 and d5-E2), loaded onto a Biotage ISOLUTE® supported liquid extraction (SLE) 96-well plate and eluted using dichloromethane. This process was automated using the Biotage® Extrahera™. Samples were evaporated to dryness, reconstituted in 50% methanol and injected onto a Waters® Acquity UPLC™ BEH phenyl column. E1 and E2 were separated under isocratic conditions using methanol and water containing 0.02 mM ammonium fluoride with a run time of 3.5 minutes. Detection was performed using the Waters Xevo® TQ-S instrument in negative ion mode with transitions 269>145 (quantifier) and 269>159 (qualifier) for E1 and 271>183 (quantifier) and 271>145 (quantifier) for E2.
Results: The functional limit of detection was 3.7 pmol/L for E1 and 18.5 pmol/L for E2 and the calibration curve was linear up to 2570 pmol/L for both. The intra-assay and inter-assay precision, expressed as percentage CV, was < 15% at 22 pmol/L, 111 pmol/L and 554 pmol/L for E1 and E2. Accuracy was evaluated using BCR®-576 certified reference material (CRM) with a bias of 3.8% for E2. The average recovery for E2 was 105%. The LC-MS/MS E2 assay was compared with an Abbott Architect i2000SR chemiluminescent immunoassay (n=70) with r2 of 0.91.
Conclusions: We have developed and validated a fit for purpose LC-MS/MS assay for the simultaneous measurement of serum E1 and E2 in men, children and menopausal women. The concentration of ammonium fluoride in the aqueous mobile phase was critical in achieving an improved functional limit of detection, and avoidance of an interfering peak. It has high throughput owing to the readily automatable sample extraction and uses a relatively small sample volume with no requirement for chemical derivatisation. This assay offers significant advantages in terms of sensitivity when compared to our current commercial immunoassay. We hope to further improve the functional limit of detection, as recommended in The Endocrine Society position statement, following the purchase of a Waters Xevo® TQ-XS instrument.
= Discovery stage.
= Translation stage.
= Clinically available.
