Certification and long-term stability monitoring of low-content folic acid and 5-methyltetrahydrofolate in human plasma certified reference material by isotope dilution ultra-high performance liquid chromatography/tandem mass spectrometry

Abstract

A reliable measurement of blood folate levels is necessary in the clinical field to prevent serious diseases such as cognitive impairment and neural tube defects. Herein, the certification of the low-content folic acid (FA) and 5-methyltetrahydrofolate (5-Me-THF) in human plasma certified reference material (KRISS CRM 111-01-018) was performed. A human plasma pool obtained from the Korean Red Cross was used as a CRM candidate. The certification of the human plasma CRM was performed by isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry. Two-dimensional liquid chromatography was employed to confirm the validity of the analytical method for FA due to the susceptibility of FA to matrix effects because of its limited quantity. The CRM stability was evaluated at −20 °C for 2 months and at −70 °C for up to 12 months to determine the certified value of the CRM. The certified value of the CRM was (84.6 ± 4.3) ng kg⁻¹ and (5.80 ± 0.47) μg kg⁻¹ for FA and 5-Me-THF, respectively. The homogeneity of the CRM was 1.64% and 3.10% for FA and 5-Me-THF, respectively. Further long-term stability assessments were conducted, indicating that the CRM remains valid for at least 58 months at −70 °C for FA and 48 months for 5-Me-THF. Compared to other blood-based CRMs, this CRM has lower folate levels, making it helpful in establishing analytical methods for a broader range of folate levels.