MSACL 2024 Abstract

1. Problem
Method comparison of 25-hydroxyvitamin D3 showed significant number of samples with >20% positive bias while compared to reference method during validation of new instrumentation in the laboratory while 25-hydroxyvitamin D2 was within acceptable limits.

2. Method Information
200 uL serum sample prepared by acetonitrile protein precipitation.
TSQ Altis with Transcend TLX-LC; APCI
Turbo column: C18 XPL 0.5x50 mm
Turbo Mobile Phases: A- 0.1% Formic acid/ Water, B- 0.1% Formic acid/ Methanol; C- 40/40/20 ACN/IPA/Acetone
Analytical column: C18 Accucore 2.6 um 50 x 3mm
Analytical Mobile Phases: A- 0.1% Formic acid/Water, B- 0.1% Formic acid/Methanol

3. Troubleshooting Steps
Analysis of NIST standards, particularly the NIST standard with >20% epimer, showed the method was unable to separate 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3, thus showing >20% positive bias in the patients samples. We developed a new LC gradient using fluorophenyl 2.7um 100x3 mm column that was able to obtain baseline resolution of 25-hydroxyvitamin D3 from 3-epi-25-Hydroxyvitamin D3. Further verified accuracy using CAP PT samples.

4. Outcome
The new flurophenyl method was able to obtain baseline resolution of 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3 while maintaining the extra online extraction provided by the turbo column on the TLX Transcend HPLC. While this method is longer, performance of the new method resolved the positive bias in 25-hydroxyvitamin D3 as compared to the reference method. Additionally, the number of samples with detectable 3-epi-25-Hydroxyvitamin D3 corresponded with the number of samples with >20% bias seen during previous validation studies. While 3-epi-25-Hydroxyvitamin D3 was not quantitated, Tracefinder, data analysis software, allows for an estimation of the amount present, which was in agreement with NIST standards with known 3-epi-25-hydroxyvitamin D3 values. Separation of 25-hydroxyvitamin D2 from 3-epi-25-hydroxyvitamin D2 was also achieved. No significant amount of 3-epi-25-hydroxyvitamin D2 was seen in patient samples.