MSACL 2019 EU Abstract

Quantitation of 17β-Estradiol and Estrone in Serum by LC-MS/MS

Geoffrey Rule(1), Ravi S. Orugunty, Linda Smith, Chau Radojicic, Uma Sreenivasan, Deanna Anderson, Cris Cruz, Ryan Carrell, Jessica Aurora, Candice Cua
(1) MilliporeSigma, 595 North Harrison Rd, Bellefonte, PA 16823 and 811 Paloma Drive, Suite A, Round Rock , Texas, 78665

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Geoffrey Rule (Presenter) Millipore Sigma >> POSTER (PDF)

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Introduction

We developed Certified Reference Material calibrators (CRMs) for quantitation of estradiol and estrone in serum. Calibrators are prepared in SigmatrixUltra™and certified to ISO Guide 34. The matrix is a purified recombinant human albumin (rHSA) solution free of endogenous interferences present in serum. Absence of endogenous interferences supports standardization and accurate measurement of analytes. Five calibrator levels were prepared gravimetrically at concentrations of 1000, 500, 100, 20 and 10 pg/mL in Sigmatrix Ultra™ from Cerilliant CRMs E-060 (Estradiol 1mg/mL) and E-075 (Estrone 1mg/mL). The product was certified by a combination of gravimetric mass measurements and LC-MS/MS analysis.
The LC-MS/MS method was validated and based on a published Primary Reference Measurement procedure.1 The method was used to quantitate estrogens in the CRMs for certification and stability studies. The method uses liquid-liquid extraction with reconstitution such that extract concentration is at the middle of a value assignment curve made in solvent. The extract is analyzed against the value assignment curve to measure concentration of estrogens in CRMs. The method provides accurate quantitation to 10 pg/mL of estradiol and estrone.
Method validation was performed for precision and accuracy across three days. BCR578 and BCR577 were used as higher order controls along with gravimetrically prepared in-house quality controls. Recovery, matrix factors and extract stability were established. Results for precision and accuracy of control samples were ± 10%.
CRMs were used as calibrators to measure the concentration of estrogens in serum samples provided by Center for Disease Control (CDC). This study was undertaken to show that CRMs will perform as calibrators and provide measurement of estrogen concentrations in serum samples. Measured estradiol values using these CRMs were within 10% of the stated values for the samples.

Results
The concentration of estrogens was quantitated in each CRM using a validated method. The accuracy for the lower two CRMs was within ± 15% and within ±10% for the rest of the CRMs. The assay had an analytical measurement range of 10 to 1000 pg/mL. Each CRM had CVs of < 20% and accuracies ranged from 100-113%. The CRMs were able to quantify estradiol in CDC serum samples with an accuracy of 90.9 and a CV of 1.9%.

Conclusion
Matrix based CRMs have been developed, manufactured and certified under ISO17034 and ISO/IEC17025 for quantitation of estradiol and estrone in serum. A sensitive and accurate LC-MS/MS method was developed and validated to support product certification. Stability studies are on-going. SigmatrixUltra™, a recombinant HSA serum diluent, was a reliable alternative to stripped serum as matrix for calibrator preparation. Performance of CRMs was demonstrated by analyzing estradiol in CDC serum samples.