MSACL 2023 Abstract

Quantitation of Clinical Research Steroid Analytes from Blood Serum Utilizing Solid Phase Extraction Paired with LC-MS/MS

Mackenzie Freige, Stephanie J. Marin
Phenomenex, Inc., Torrance, CA

Mackenzie Freige, B.S. Life Sciences, B.A. Chemistry (Presenter) Phenomenex

Presenter Bio: Mackenzie received a Bachelor of Science degree in Life Sciences from Kansas State University and a Bachelor of Arts degree in Chemistry from Wichita State University. She has 5 years of experience in LC-MS/MS, GC-MS, and sample preparation. She has previously worked in labs in toxicology, environmental chemistry, and food science industries. She also has sales experience in the pharmaceutical industry.

Mackenzie joined Phenomenex in 2021 as an Application Scientist at PhenoLogix. She is based in Torrance, CA. She specializes in developing applications for clinical research and brings experience in method development and instrumentation. She focuses on helping customers with method development for HPLC, MS, and sample preparation. In her free time she practices yoga and enjoys reading.

Relevant Financial Disclosures (within past 24 months, reported on Dec 13, 2022) Salary Phenomenex, Inc., Torrance, CA Conflict mitigation NOT REQUIRED.

Quantitation of Clinical Research Steroid Analytes From Blood Serum Utilizing Solid Phase Extraction Paired With LC-MS/MS

INTRODUCTION: Steroid analysis for clinical research can require very low limits of detection which demand high recoveries from solid phase extraction (SPE) and very clean extracted samples. Labs desire high-throughput methods which consolidate analytes into one panel with fast chromatographic run times. Accurate quantitation makes it necessary to chromatographically separate steroids with the same m/z. Meeting these criteria can be challenging in a single LC-MS/MS method.

OBJECTIVES: Here, we present an effective sample cleanup method and LC-MS/MS analysis method for the quantitation of an 18-analyte steroid panel from serum.

METHODS: The (SPE) technique utilized a reversed phase Strata-X 30mg 96-well plate (Phenomenex, Torrance CA). A Core-Shell 2.6µm, 50x3 mm Kinetex C18 column (Phenomenex) was used for fast chromatographic separation. Detection employed a 7500 Triple Quadrupole LC-MS/MS equipped with an ESI source (Sciex, Framingham, MA).

RESULTS: Linearity is demonstrated for all analytes with an R squared value >0.992. The calibration curves prepared in steroid-free blood serum range from 10 pg/mL to 500 ng/mL, subject to unique cutoff requirements of the analytes of interest. LLOQ values range from 10 pg/mL to 5 ng/mL. The QC samples for 3 replicate extractions at 3 different levels showed relative standard deviation (RSD) below 15% and accuracy between 80-120%. The LC method with the use of a C18 column achieves chromatographic separation of all isomers with an 8-minute method.

CONCLUSION: The proposed sample prep method utilizing a 96-well plate SPE extraction and fast LC method resulted in a simple, rapid cleanup for identification and quantitation of the tested steroid panel analytes from blood serum. The SPE extraction method was robust, reproducible, and can be automated. This makes it a reliable method that combines accurate quantitation with high throughput.