MSACL 2022 Abstract

Interfering Peak in the Estradiol (E2U) LC-MS/MS Assay Mima Geere MD MS, Simone Arvisais-Anhalt MD, Deborah French PhD University of California, San Francisco, CA Mima Geere, MD (Presenter) UCSF >> POSTER (PDF) >> ADDL INFO (PDF)

Presenter Bio: Dr. Mima Geere is a Clinical Pathology trained Functional Medicine doctor who specializes in personalized medicine and nutrition and the use of advanced diagnostic testing in health and wellness for prevention.

Dr. Geere is passionate about creating a patient centered data driven healthcare experience. She advises startups in accelerating the growth and discovery of innovative approaches to health and wellness using technology and diagnostics.

Dr. Geere trained at UCSF in Clinical Pathology and Informatics, and has held positions as medical and product director of JumpstartMD and Genova Diagnostics. She was a staff physician at The Chopra Center and on faculty at The institute of functional medicine and most recently was CMO of a longevity startup.

She has a background and training in nutrition science, diagnostic testing and tech product management and has founded and led a tech startup ResultCare, a clinical decision support app for diagnostic testing. She is the founder of Mimansa, an online clinic that provides personalized nutrition and wellness through telemedicine.

Specialties: Functional Medicine, Pathology, Ayurveda
Public speaking, Medicine, Pathology, Exercise, Nutrition, Clinical Research, Human Gut Microbiome, Diabetes, Insulin Resistance, health and wellness

Title: Interfering peak in the Estradiol (E2U) LC-MS/MS Assay

1. Problem

Estradiol is measured by a highly sensitive LC-MS/MS assay and utilized by clinicians to investigate different processes and disorders in women including menstrual cycle regulation, reproductive function and infertility as well as amenorrhea and oligomenorrhea. It is used to evaluate pregnancy maintenance, precocious puberty, menopause and estrogen deficiency in men and women.

There is an interfering peak in the LC-MS/MS results that requires further evaluation and separation. It is unclear if this peak is due to underlying patient medications or disease characteristics. The interference peak only occurs in 1% of sample runs in a select group of 7 patients in multiple samples collected over the last 7 months.

2. Method Information

Estradiol is measured using ultra-fast liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Estradiol is extracted from serum using a mixture of hexane and ethyl acetate, dried under nitrogen and derivatized using dansyl chloride. The sample is injected into the LC-MS/MS system where it is eluted onto the analytical column with a gradient of water and organic solvent (methanol and acetonitrile). Addition of an internal standard (carbon 13 labeled estradiol) allows for quantification of estradiol compared with a six-point standard curve and three quality control samples are run in each batch.

Instruments: Sciex 5500 QTrap mass spectrometer and Shimadzu Prominence UFLC
Column: Phenomenex Kinetex Phenyl-Hexyl, 2.6 micron, 100x3mm
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: 0.1% formic acid in 70% methanol and 30% acetonitrile
Injection volume: 30 microL
Column Temperature: 40 degrees Celcius
Autosampler temperature: 15 degrees Celcius

3. Troubleshooting Steps

Variations to increase Selectivity:

Modify the LC gradient time program going across the run over 8 mins.
Current time program:

Time (minutes) Flow rate (mL/min) % mobile phase B
1.0 0.8 20
1.2 0.8 70
4.5 0.8 95
5.4 0.8 95
5.5 0.8 20
6.5 0.8 stop

Variations in Troubleshooting:

1. Change the Gradient (timing in the program) – try different variations as follows
a. Change the timing of the gradient – Change to a longer or more complex gradient.
b. Change the organic composition of the mobile phase

2. Mass spec option: Collect product ion spectra of estradiol and of the interfering peak using the QTrap with a collision energy spread. This might tell us if there are any significant fragments that differ between the two peaks.

3. Change the columns from pheylhexyl to C18

4. Outcome
Troubleshooting experimentation in process, to be determined.