“Continental Drift” and Varying Vitamin D Results Dependent Upon Methanol Reagent Lot Joshua Akin (Presenter) UC San Diego Health Presenter Bio: Joshua Akin is a Clinical Laboratory Scientist (CLS) Specialist in the Clinical Mass Spectrometry Laboratory, Center for Advanced Laboratory Medicine at UC San Diego Health. He received his Bachelor of Science degree in Microbiology at California State University, Chico and a Master's Degree in Clinical Research from UC San Diego. His research interests include clinical mass spectrometry, ICP-MS analysis, and clinical trial design.

Authors: Josh Akin (1), Heather Tone (1), Dawn Francisco (1), Judy Stone (2), Audie Cabero (3), Robert Fitzgerald (1)
(1) UC San Diego Health, San Diego, CA (2) UC San Francisco Health, San Francisco, CA (3) Waters Corporation, Milford, MA

Overview/Problem:

Our vitamin D assay presented occasionally with unacceptably low 25-OH-D2 QC values over the course of an 18 month period. A thorough investigation was conducted, ranging from new reagents to major system cleanings and replacement parts. QC values would improve for weeks at a time, but then drop out of range without any clear indication of why it became unacceptable. After several months of exhaustive troubleshooting of the assay, we determined that our solvent mobile phase was of question, specifically certain lots of Fisher Optima™ grade MeOH. Further testing suggested that there was significant lot-to-lot variance in D2 signal observed, with certain lots of Fisher Optima™ MeOH being unacceptable for production in the clinical lab.

In order to further characterize this phenomenon, and to determine whether other sources MeOH exist that would be suitable for our use, we tested 4 different brands of LC-MS grade MeOH for use as a mobile phase in our vitamin D assay. We found that vitamin D area counts varied >50% between the different MeOH manufacturers, and more importantly, QC acceptability was determined not only to be dependent upon the lot and/or manufacturer of methanol used, but the country of origin in which the MeOH was produced.

Method information

• MS: Waters Xevo TQS triple quadruple mass spectrometer, operating in positive ESI mode

• LC: Waters Acquity Classic system with a Waters X-select HSS T3 2.5um 2.1x 75mm column

• Loop-mode injector in partial mode

• Solvent mobile phase: 2 mM ammonium acetate, 0.1% formic acid in MeOH

• Aqueous mobile phase: 2mM ammonium acetate, 0.1% formic acid in H2O

• Sample prep: crash precipitation and dilution

o 300 uL internal standard in MeOH

o 300 uL sample

o 300 uL Acetonitrile

Troubleshooting Steps:

• Problem observed on all instruments utilized for Vitamin D testing

• The QC problem was only with 25-OH-D2. QC for 25-OH-D3 was always acceptable

• The six other methods (drug confirmations) running on the same instruments were unaffected

• Chromatography acceptable: no leaks or irregular pressure traces

• 6-month PM performed regularly by vendor

• Several iterations of instrument cleaning, repair and consumable replacement including: new guard/column, new reagent bottles/sinkers, new needle/sample syringe, system flush, new ion guide, new quadrupole

• Updated software, verified acquisition parameters and tune settings were correct

• Acquired spectral data for possible ion suppression and/or contamination

Outcome:

A lot-screening protocol was instituted in our lab. With the aide of Fisher Scientific, we are able to sequester a specific lot, test it for acceptability, and then proceed to purchase from that lot when needed.

A specific cause of MeOH unacceptability was not fully determined. Spectra acquired from unacceptable lots show high levels of common phalates/plasticizers. Additional investigation would be needed to further characterize these contaminants and their effect vitamin D analysis.

We also validated two alternative MeOH manufacturers for production use, in the instance that we could not find a suitable lot from the primary supplier. The two primary origins of MeOH are from the US and Venezuela. The MeOH tested from US origin was all acceptable, while unacceptable MeOH was only found from Venezuelan origin.

Pinpointing the nature of this issue would not have been feasible without the use of a dedicated reagent tracking system that was implemented in our lab 4 years prior. Using this data, we were able to surmise a historical trend that correlated with our suspicion of a questionable MeOH lot.