MSACL 2024 Abstract

Optimized Extraction Protocol for Analysis of 2,3-Dinor 11β-Prostaglandin F2α in Urine Kayla Moehnke, Jennifer Kemp, Michelle R. Campbell, Ravinder Singh, Anne Tebo, Anthony Maus Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Kayla Moehnke, M.S. in MLS; B.S. in MLS (Presenter) Mayo Clinic

Presenter Bio: I have been employed at the Mayo Clinic, in Rochester, MN for 15 years. I obtained a bachelor's degree in Medical Laboratory Science and subsequent master's degree of the same discipline, both from the University of North Dakota. I am board certified for the American Society of Clinical Pathology. My current academic rank is Instructor of Laboratory Medicine and Pathology. I have been practicing mass spectrometry for 3 years.

Problem
Historically, the measurement of 2,3-dinor 11β-Prostaglandin F2α (BPG) in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) proved challenging due to interferences impacting peak integration, as well as fragment ion ratio discrepancies, both leading to challenges in reporting patient results. Currently, our laboratory method utilizes an ethyl acetate liquid-liquid extraction (LLE), followed by LC-MS/MS. Due to the aforementioned challenges, the opportunity to improve various aspects of the current method by investigating alternative enrichment methods has been identified.
Method Information
• Prepare 1 mL urine sample, calibrator, QC, blank
• Add 25 mcL internal standard, deuterium-labeled 2,3-dinor 11β-Prostaglandin F2α (Cayman Chemical, 9000603)
• Add 50 mcL 50% acetic acid
• Add 1 mL ethyl acetate
• Vortex 1 minute (x3)
• Centrifuge at 3000 rpm for 10 minutes
• Transfer 500 mcL of top ethyl acetate layer to deep well plate
• Dry plate under nitrogen at 50°C for 30 minutes
• Reconstitute with 135 mcL of 40% methanol
Troubleshooting Steps
Optimization of a new BPG sample preparation procedure required several months of experimentation as we strategically evaluated one variable at a time. Some variables were carried through the project to a newly optimized sample preparation procedure, while others were abandoned.
A. A direct comparison of the current ethyl acetate LLE to a solid-phase extraction (SPE) (Agilent, A3967010) was performed, using a protocol developed with a similar analyte, precondition plate, sample transfer, wash, and collect eluate. Initial SPE experiments demonstrated improved signal-to-noise, but interfering peaks continued to be observed.
B. The addition of a third wash step to the SPE protocol was attempted to see if further cleanup would reduce the interference peaks. The initial SPE protocol had two wash steps after the sample transfer; wash #1 500 mcL of water and wash #2 500 mcL of 100% methanol. Variable mixtures and concentrations of methanol, acetic acid, acetonitrile, dichloromethane (DCM), isopropanol, and ammonium hydroxide were evaluated as a wash #3. The addition of a third wash step did not show a significant improvement. For example, observations included low signal-to-noise, poor peak shape, or no signal at all.
C. Modifications to the current wash #2 (methanol) and elution buffer (1% acetic acid in methanol) in the SPE protocol were evaluated to further reduce peak interferences. The optimization of both the wash #2 and elution buffer identified a new combination that improved signal intensity and peak shape. The new combination included a change to 30% methanol for wash #2 and to 1% acetic acid in 75% methanol for the elution buffer. Despite this improvement, some fragment ion ratios continued to give discordant results, including linearity discrepancies.
D. Reversed phase SPE (8E-S100-AGB, Phenomenex) was evaluated, as an alternative chemistry for purification. While using the established SPE protocol, we evaluated methanol, DCM, and isopropanol as options for wash #2. Overall, the reversed phase SPE did not resolve ion ratio discordance and instead appeared to worsen the discrepancies. Therefore, reversed phase SPE was not explored any further.
E. Supported liquid extraction (SLE) was evaluated. Both diatomaceous (Biotage, 820-0400-P01) and synthetic plates (Agilent, 5610-2004) were evaluated using manufacturer-suggested protocols. This included the evaluation of multiple elution buffers such as methyl tert-butyl ether, ethyl acetate, DCM, and n-butyl-chloride. The SLE extractions proved to be less appropriate for BPG extraction. We observed low and variable recovery.
F. Finally, the current extraction methods were directly compared to variations of a SPE method, working through combinations of the most promising wash and elution buffers. The current LLE method was directly compared to SPE, with results favorable for SPE. The SPE method was then compared to a SPE method with DCM as wash #2. Finally, the wash #3 and elution buffer were optimized by comparing, wash #3 (methanol vs 20% methanol + 1% acetic) and the elution buffer (1% acetic acid in methanol vs 1% acetic acid in 80% methanol). Collectively, improved results, regarded as a decrease in interfering peaks, better ion ratio agreement and improved linearity, were observed with the optimized SPE method using methanol for wash #2, 20% methanol+ 1% acetic acid for wash #3, and 1% acetic acid in 80% methanol as the elution buffer.
Outcome
• Prepare 250 mcL urine sample, calibrator, QC
• Add 50 mcL internal standard, deuterium-labeled 2,3-dinor 11β-Prostaglandin F2α
• Add 50 mcL 1N NaOH
• Add 1 mL water
• Precondition SPE plate, with positive pressure manifold
o 500 mcL methanol, ~1 psi
o 500 mcL water, ~1 psi
• Transfer 1250 mcL sample into SPE plate
o 5 psi, 3-5 minutes
• Wash SPE plate
o 500 mcL water, 4 psi, 3 minutes
o 500 mcL methanol, 3 psi, 3 minutes
o 500 mcL 20% methanol + 1% acetic acid, 3 psi, 3 minutes
• Elute with 80% methanol + 1% acetic acid + 1 mcg/mL estriol (x2)
o 100 mcL, <1 psi, 30 seconds
o 100 mcL, <1 psi, 3 minutes
• Add 300 mcL water to eluate
Conclusion
As an inference-prone analyte, BPG proves difficult to isolate from urine samples and accurately measure. Troubleshooting the extraction variables led to the successful development of a SPE method that enriches BPG from urine while minimizing interferences. When isolated, each optimized variable marginally improved assay performance. Synergistically, a combination of the extraction medium and optimized reagent chemistries provided the best chromatographic performance with reduction of interferences, improving linearity and maintaining validation acceptance criteria.