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

Participants ≥60 y from NHANES 2001-2002.

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.

Analyte codes and limits of detection (LOD)
 Analyte name  Analyte code  LOD (nmol/L)
 Serum 5-methyltetrahydrofolate  SF1  0.31
 Serum folic acid  SF2  0.14


Data Processing and Editing

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


Laboratory Quality Assurance and Monitoring

The laboratory and method were certified according to the Clinical Laboratory Improvement Amendment (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) 


 LS11430d_LC  SF1  10  18.6  0.54  2.9%
 MS11431d_LC  SF1  10  34.2  0.81  2.4%
 HS11432d_LC  SF1  10  49.7  1.54  3.1%
 LS11430d_LC  SF2  12  0.66  0.10  14.7%
 MS11431d_LC  SF2  12  5.46  0.44  8.0%
 HS11432d_LC  SF2  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 Access: Data will only be available through the Research Data Center.

Analytic Notes

Samples were selected from 650 surplus serum samples from participants ≥60 y 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 y at -70°C did not appear to lose folic acid, that three serum QC pools stored for up to 1 y at -20°C did not lose folic acid, and that three serum QC pools stored refrigerated for 1 y 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 y 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.

Because this was a method comparison project and there was no intent to establish population estimates, there are no weights.

The format of the subject person data file for the two analytes is as follows:

Column A: Respondent sequence number (SEQN)
Column B: Analyte code (SF1=5-methyltetrahydrofolate; SF2=folic acid)
Column C: Serum concentration (nmol/L)



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 263 263
. Missing 48 311

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
0.26 to 87.96 Range of Values 311 311
. Missing 0 311