Serum Folate Forms Are Stable during Repeated Analysis in the Presence of Ascorbic Acid and during Frozen Sample Storage

Abstract Background Serum folate forms, and particularly tetrahydrofolate, are sensitive to oxidation. Methods Using a repeated measures design, we investigated the stability of folate forms in convenience samples with added ascorbic acid (AA; 5 g/L) analyzed initially and after variable (approximately 1–33 weeks) storage time at −70 °C. We examined the recovery of tetrahydrofolate added at different spiking levels to serum with and without AA (5 g/L). We also assessed the long-term frozen storage stability of folate forms. Results Repeat analysis produced consistent results with the initial analysis; the mean relative change (95% CI; Lin's concordance correlation between initial and repeat result; sample size) was 0.08% (−0.24% to 0.39%; r c = 0.999; n = 301) for 5-methyltetrahydrofolate, 4.23% (2.44%–6.05%; r c = 0.984; n = 211) for pyrazino-s-triazine derivative of 4α-hydroxy-5-methyltetrahydrofolate (MeFox), −0.22% (−1.90% to 1.49%; r c = 0.986; n = 214) for folic acid, and 1.49% (−2.71% to 5.88%; r c = 0.889; n = 81) for tetrahydrofolate. Linear regression testing for a time trend indicated an estimated average percent change of less than ±5% for samples retested after 4 months: 5-methyltetrahydrofolate P trend = 0.0007, folic acid P trend < 0.0001, MeFox P trend = 0.38, and tetrahydrofolate P trend = 0.0256. The mean ± SD tetrahydrofolate spiking recovery was 96.7% ± 9.4% for serum with added AA, but <50% for serum without added AA. We observed ≤10% loss for most serum folate forms during 4 years of storage at −70 °C. Conclusions Serum containing added AA showed acceptable stability of folate forms during repeat analysis from the same vial within 4 months, complete spiking recovery of tetrahydrofolate during sample processing, and long-term frozen storage stability of folate forms.