Table of Contents

Component Description

Measurement of folic acid and 5-methyltetrahydrofolate (5-methylTHF) in a subset of stored serum samples from NHANES 2001-2002 to allow comparison of CDC liquid chromatography tandem mass spectrometry (LC-MS/MS) method to data generated previously by Tufts University HPLC method with electrochemical detection.

Eligible Sample

Selected participants 60 years and older who provided informed consent for further testing of stored specimen. See “Analytic Notes” session for details on selection criteria.

Description of Laboratory Methodology

Measurement of folic acid and 5-methyltetrahydrofolate.

An isotope-dilution tandem mass spectrometry method coupled to liquid chromatography (LC-MS/MS) was used to measure folate forms in serum. To prepare samples for analysis, serum (275 µL) was mixed with ammonium formate buffer and amended with internal standard mixture that contained 13C5-labeled folate forms. Sample clean-up was performed using a 100-mg phenyl solid phase extraction (SPE) 96-well plate (Bond Elut 96; Agilent Technologies) and an automated 8-probe SPE system (Gilson 215; Gilson Inc.). Samples were eluted from the SPE plate with an organic elution buffer containing ascorbic acid and analyzed overnight by LC-MS/MS in positive ion mode using electrospray ionization on an AB Sciex 5500 triple-quadrupole MS system (Applied Biosystems) coupled to a HP1200C LC system (Agilent Technologies). Chromatographic separation was achieved using a Luna C-8 (2) analytical column (Phenomenex) with an isocratic mobile phase and a total run time of 7 min. Quantitation was performed by peak area ratio (analyte to internal standard) and based on a 6-point aqueous calibration curve that was carried through all sample preparation steps. The calibration range was 0–100 nmol/L for 5-methylTHF, and 0–50 nmol/L for folic acid. Values higher than the highest calibrator were diluted with 0.1% ascorbic acid and repeated for confirmation.

Limits of detection (LOD)
 Analyte Name  Variable Name  LOD (nmol/L)
 Serum 5-methyltetrahydrofolate  SSSF1  0.31
 Serum folic acid  SSSF2  0.09


Laboratory Quality Assurance and Monitoring

The laboratory and method were certified according to the Clinical Laboratory Improvement Amendments (1988) guidelines.

In-house prepared serum quality control pools at three levels were analyzed in every run in duplicate and evaluated for validity against pre-established means and control limits by use of a multi-rule quality control program.

Performance of QC pools during the study period (Oct–Dec 2013)
QC Pool   Analyte Mean (nmol/L)  SD (nmol/L)  CV (%) 
 LS11430d_LC  SSSF1  10  18.6  0.54  2.9%
 MS11431d_LC  SSSF1  10  34.2  0.81  2.4%
 HS11432d_LC  SSSF1  10  49.7  1.54  3.1%
 LS11430d_LC  SSSF2  12  0.66  0.10  14.7%
 MS11431d_LC  SSSF2  12  5.46  0.44  8.0%
 HS11432d_LC  SSSF2  12  10.2  0.62  6.1%

The method achieved satisfactory performance using the National Institute of Standards and Technology standard reference material 1950, matching the certified value [uncertainty] for 5-methylTHF (26.7 vs. 26.9 [0.70] nmol/L) and the reference value for folic acid (4.03 vs. 3.42 [1.02] nmol/L).

Data Processing and Editing

Data were received after all the laboratory testing was complete. The data were not edited for extreme values.

Data Access: Data will only be available through the Research Data Center.

Analytic Notes

Samples were selected from 650 surplus serum samples from participants 60 years and older from NHANES 2001-2002 who provided informed consent for further testing of stored specimen and where there was sufficient specimen volume for the proposed analyses. Samples were selected based on their folic acid concentration (as measured in a previous surplus specimen project by Tufts University) to ensure a folic acid distribution throughout the reported concentration range.

The current project did not use pristine (never thawed) samples, because the laboratory showed that multiple freeze/thawing cycles do not cause a noticeable loss of folate forms (1). In recent in-house studies, the laboratory also saw that multiple serum QC pools stored for up to 10 years at -70°C did not appear to lose folic acid, that three serum QC pools stored for up to 1 year at -20°C did not lose folic acid, and that three serum QC pools stored refrigerated for 1 year lost on average only 6.6% folic acid (unpublished data). While a storage-related increase in serum folic acid may be rationalized as a result of tetrahydrofolate (THF) oxidation, this is unlikely if samples are stored at -70°C. In three serum QC pools stored for up to 7 years at -70°C the laboratory did not observe a loss of trace levels of THF (≤1 nmol/L). Furthermore, THF concentrations are generally very low in serum, often below the LOD. Lastly, the serum samples used by the Tufts method in the previous project were also surplus specimens that had been through two or more freeze/thawing cycles and would therefore be expected to show similar changes in folate forms, if any.

Correction Needed for Serum Folic Acid Results for 2001-2002 NHANES Surplus Sera Project

For NHANES samples collected in the 2001-2002 cycle, the CDC Nutritional Biomarkers Laboratory measured serum 5-methyl-tetrahydrofolate and folic acid by isotope-dilution high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to compare their results to Tufts University HPLC method. These data were first published to the Research Data Center in July 2014. In 2015, the CDC laboratory discovered a calibration bias in the folic acid determination that resulted in overestimation of folic acid concentrations by about 25% as a result of solubility issues with the calibrator (see Analytic Notes for “Folate Forms – Serum” in NHANES 2011–2012). The laboratory corrected the assay and conducted a crossover study to assess whether folic acid data from various NHANES survey cycles needed to be adjusted. Based on the crossover study, the CDC folic acid results in this dataset have been corrected for the calibration bias using the following equation: New folic acid = 0.7586 * Original folic acid – 0.00016.

Comparability of Serum 5-methylTHF and Folic Acid Concentrations Measured by CDC versus NHANES 1999-2002 Concentrations Measured by Tufts

Serum 5-methylTHF and folic acid concentrations were measured by Tufts University using HPLC with electrochemical detection in American seniors (≥60 y) in NHANES 1999–2002 as part of a surplus specimen project to assess the relationship to anemia, macrocytosis and cognitive test performance (Bailey, et al. 2010; Morris, et al. 2010). To assess method differences between Tufts and CDC, a method comparison study was performed in 2013 on 311 surplus serum specimens from American seniors (≥60 y) in NHANES 2001–2002. Results for serum 5-methylTHF compared well between the two methods: mean (95% CI, nmol/L): 47.7 (44.5–50.9) [CDC] and 48.6 (45.8–51.4) [Tufts]; median (95% CI, nmol/L): 42.9 (38.2–45.4) [CDC] and 45.0 (41.3–48.0) [Tufts]; Pearson r: 0.89; no bias based on Deming regression (using log-transformed data) and Bland Altman relative difference analysis.

As mentioned above, the CDC folic acid results reported in this dataset have been corrected for the calibration bias. The CDC method detected folic acid in all samples (LOD = 0.09 nmol/L), while the detection rate of the Tufts method (LOD = 0.18 nmol/L) was ~35% in the full sample set (n = 1330; See the 2001-2002 Surplus Sera Folate (SSFOL_B) data). Overall, the correlation was low in the surplus sample set (Pearson r: 0.64, n = 311). In the subset of samples that had detectable folic acid results by the Tufts method (n = 163) and that did not include extreme outliers (n = 156; difference between the two results was <20 nmol/L), the CDC folic acid results were on average comparable to the Tufts results: mean (95% CI, nmol/L): 3.71 (2.40–5.02) [CDC] and 3.11 (2.19–4.02) [Tufts]; median (95% CI, nmol/L): 1.10 (0.99–1.37) [CDC] and 1.20 (0.88–1.62) [Tufts]; Pearson r: 0.92; Deming slope using log-transformed data (95%CI): 0.89 (0.76 to 1.02); Deming intercept using log-transformed data (95% CI): 0.06 (0.00 to 0.12); Bland Altman relative difference (95% CI): 10.3% (-1.9% to 22.5%).

Samples with sufficient sample volume (n = 17) were repeated by the CDC method; the repeat results confirmed the original results and were on average (SD) 5% (7%) higher for folic acid and 2% (5%) higher for 5-methylTHF.

The findings from this method comparison study suggest that the NHANES 1999–2002 Tufts surplus results can be compared for 5-methylTHF to the NHANES 2007–2008 CDC results. However, the comparability for folic acid is limited due to the different detection rates for these two methods.  

The current study is a method comparison project and there was no intent to establish population estimates, therefore, no special sample weights are used with these analyses or prepared for this dataset.


Codebook and Frequencies

SEQN - Respondent sequence number

Variable Name:
SAS Label:
Respondent sequence number
English Text:
Respondent sequence number.
Both males and females 60 YEARS - 150 YEARS

SSSF1 - 5-Methyltetrahydrofolic acid (nmol/L)

Variable Name:
SAS Label:
5-Methyltetrahydrofolic acid (nmol/L)
English Text:
5-Methyltetrahydrofolic acid (nmol/L)
Both males and females 60 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
6.8 to 130.3 Range of Values 252 252
. Missing 45 297

SSSF2 - Folic Acid (nmol/L)

Variable Name:
SAS Label:
Folic Acid (nmol/L)
English Text:
Folic Acid (nmol/L)
Both males and females 60 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.18 to 66.71 Range of Values 297 297
. Missing 0 297