Interference due to lipemia by was investigated during validation of an LC-MS/MS assay for Vitamins A&E. A pool of grossly lipemic serum was serially diluted with serum calibrators, and it appeared to have minimal effect on the results. However, after the assay went live, a subset of lipemic clinical specimens was found to cause ion suppression for Vitamin E and its internal standard, Vitamin E (phenyl-5,7-dimethyl-d6). This had not been observed in any prior specimens extracted for correlation during validation (n=364).
• Serum combined with internal standard in isopropanol and extracted by supported liquid extraction with hexane, reconstituted in methanol
• 0.5µL injection
• Acquity I-Class UPLC, column manager at 45 °C
• Acquity BEH C18 UPLC column, 2.1x50 mm, 1.7 µm
• Waters TQ-XS, TQ-S micro
• Mobile Phase A: 0.1% formic acid, 2 mM ammonium acetate in water
• Mobile Phase B: 0.1% formic acid, 2 mM ammonium acetate in methanol
• 3.4 min method, linear gradient 90-100% mobile phase B at 0.5 mL/min
• Quantitative MRM acquisition
Why did only certain lipemic samples cause ion suppression for Vitamin E? Chart reviews revealed patients displaying this interference pattern were on total parenteral nutrition (TPN), which typically contains lipid emulsions in its mix of macro- and micronutrients. The problem was investigated in three parts. First, the effects on quantitation were investigated by serially diluting affected specimens with a standard of known value, and the expected and measured concentrations were compared. Second, we assessed the use of variable/reduced injection volumes versus dilutions to reduce the amount of interfering lipid entering the LC-MS/MS. Third, specimens were clarified by centrifugation, and then extracted and analyzed by LC-MS/MS to examine changes in signal intensity.
Lipemic specimens from patients who were given TPN caused ion suppression for our Vitamin E LC-MS/MS assay. These samples demonstrated a negative bias of up to 15% when compared to the expected results, and the severity of this bias correlated with the degree of turbidity in the specimen as measured by serum indices on the Ortho Clinical Diagnostics Vitros 4600. Both dilution and smaller injection volumes improved the accuracy of the result. Additionally, centrifugation was able to remove the lipemic layer in these specimens with adequate Vitamin E recovery. Our current practice is to reinject any specimens with <25% of the IS intensity of the highest standard at 0.1 µL rather than the standard 0.5 µL. The benefit of this procedure is that it does not require re-extraction of a diluted sample, allowing for faster turnaround time of the result.