Folic acid purity determination by q1H-NMR

The chemical purity (mass fraction) of the neat folic acid calibration standard was evaluated using a q¹H-NMR primary measurement procedure calibrated via internal standard (23–25). Three samples containing accurately weighed quantities of folic acid material (4.68 mg to 5.03 mg) and dimethyl sulfone internal standard (2.70 mg to 4.52 mg) were prepared in D₂O phosphate buffer solution (pH ≈7.2). Purity of the dimethyl sulfone internal standard was previously determined by q¹H-NMR comparison to the NIST PS1 Primary Standard for quantitative NMR (Benzoic Acid) (26) and is treated as 1.001 g/g, u=0.001. The purity of dimethyl sulfone was calculated using an average value for relative molar mass of dimethyl sulfone (based on standard atomic weights). Treatment in the purity calculations, whereby the limit of 1 g/g is not observed, is appropriate and does not contribute to bias when the dimethyl sulfone is used as a calibrant for qNMR measurement of the folic acid. ¹H-NMR measurement of folic acid samples was conducted using a Bruker AVANCE II spectrometer operating at 600.14 MHz with a double-resonance inverse broadband probe (BBI) optimized for ¹H observation. Experiments were performed using a 90° single-pulse sequence with the following parameters: 12 019 Hz (20.028 ppm) spectral width; 3 706 Hz (6.175 ppm) transmitter frequency offset; 4 dummy scans; signal averaging of 128 scans; 60 s relaxation delay; and 5.453 s data acquisition time. Data files were apodized using an exponentially decaying window function for 0.3 Hz line broadening and processed with Bruker TopSpin Version 3.2 software. Confirmation of folic acid chemical structure and determination of purity were assessed through properties of Fourier transformed ¹H spectra and the respective integral ratios for folic acid and internal standard signals. The folic acid purity results are metrologically traceable to the International System of Units (SI) unit for mass, expressed as mass fraction of folic acid in the calibration standard, through linkage of the known purity value of the dimethyl sulfone internal standard to that of the NIST PS1 Primary Standard for quantitative NMR (Benzoic Acid).