Fig. 1. Chemical structure for FA showing the two major ESI fragmentation pathways (A, B). Fragmentation at A results in the characteristic loss of the glutamic acid moiety from the intact molecule. The orientation of the α and γ carboxyl groups on the glutamic acid moiety are labeled. Fragmentation at B produces the characteristic folate degradation product, p-aminobenzoylglutamic acid (p-ABG, relative mass 266.3 g/mol). The relative mass of FA is 441.4 g/mol.

Fig. 2. A representative positive-ion mode CID spectrum (product ion spectrum) of a FA standard (200 ng/μL). The ions located at m/z 424 (a), m/z 313 (b) and at m/z 295 (c) can be tentatively identified as [M + H − H₂O]⁺, [M + H − glutamic acid]⁺ and [M + H − glutamic acid − H₂O]⁺, respectively. All profiles were collected using the LC/MS/MS conditions described in Section 3.

Fig. 3. A representative negative-ion mode CID spectrum (product ion spectrum) of a FA standard (200 ng/μL). The ions located at m/z 422 (a), m/z 396 (b), m/z 378 (c), m/z 311 (d) and at m/z 293 (e) can be tentatively identified as [M − H − H₂O]⁻, [M − H − CO₂]⁻, [M − H₂O − CO₂]⁻, [M − H − glutamic acid]⁻ and [M − H − glutamic acid − H₂O]⁻, respectively. All profiles were collected using the LC/MS/MS conditions described in Section 3.

Fig. 4. Typical positive-ion mode MRM chromatograms and ultraviolet absorbance chromatogram of FA extracted from SRM 3280. (A) MRM chromatogram of unlabeled FA. (B) MRM chromatogram of [¹³C₅]-FA. (C) UV absorbance chromatogram of tablet extract monitored at 282 nm. All profiles were collected using the LC/MS/MS conditions described in Section 3.

Fig. 5. Typical negative-ion mode MRM chromatograms and ultraviolet absorbance chromatogram of FA extracted from SRM 3280. (A) MRM chromatogram of unlabeled FA. (B) MRM chromatogram of [¹³C₅]-FA. (C) UV absorbance chromatogram of tablet extract monitored at 282 nm. All profiles were collected using the LC/MS/MS conditions described in Section 3.

LC/MS/MS instrument parameters for the quantitative determination of FA in multivitamin/multielement tablets^a

DP, declustering potential; CE, collision energy; CXP, collision exit potential; EP, entrance potential; CAD, collision activated dissociation.

Extraction solvents tested on SRM 3280^a

Method performance characteristics

Quantification of FA in NIST SRM 3280 using LC/MS/MS^a