J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 21, March 2023, pages 28-34

LC-MS analysis of urolithin-related metabolites in human plasma reveals glucuronide conjugates as the primary species after 4-weeks of pecan consumption

Figures

Figure 1. LC-MS MRM chromatograms of (a) EA, (b) Uro-B-G, (c) Uro-A-G, (d) Uro-C, (e) Uro-A, (f) Uro-B, and (g) Uro-C-G (tentatively identified) in plasma.

Figure 2. Linearity for EA, Uro-A, Uro-B, Uro-C, Uro-A-G, and Uro-B-G.

Tables

**Table 1.** Optimal MS/MS parameters for EA, Uro-A, Uro-B, Uro-C, Uro-A-G, Uro-B-G and Uro-C-G
Analytes	Retention time (min)	Polarity	Precursor ion (m/z)	Product ion (m/z)	Collision energy (V)	Fragmentor (V)	Cell accelerator voltage (V)
EA	3.0	Negative	301.0	283.6	36	176	4
Uro-A	3.5	Negative	227.0	197.7	36	134	4
Uro-B	4.0	Negative	211.0	166.7	28	134	4
Uro-C	3.2	Negative	243.0	186.7	32	156	4
Uro-A-G	2.9	Negative	403.0	226.6	30	120	4
Uro-B-G	3.2	Negative	387.0	210.6	30	100	4
Uro-C-G	2.8	Negative	419.0	242.6	30	120	4

**Table 2.** Comparison of limit of quantification (LOQ) for EA, Uro-A, Uro-B, Uro-C, Uro-A-G, and Uro-B-G in plasma
Analytes	The present study	Reference paper¹
Analytes	LOQ (ng/mL)	LOQ (ng/mL)
¹Quantification results of urolithin-related metabolites (García-Villalba et al., 2016).
EA	<1.0	–
Uro-A	<0.1	3.9
Uro-B	3.0	13.2
Uro-C	0.5	7.3
Uro-A-G	<1.0	20.9
Uro-B-G	<0.5	16.7

**Table 3.** Evaluation of batch reliability using pooled QC samples (n = 7)
	Precision (RSD, %)
	EA	Uro-A	Uro-B	Uro-C	Uro-A-G	Uro-B-G	Uro-C-G
QCs	–	–	–	12.9	5.3	4.6	11.4

**Table 4.** Concentrations of EA, Uro-A, Uro-B, Uro-C, Uro-A-G, Uro-B-G, and Uro-C-G in plasma samples of human study after 0 week and 4 weeks of 68g/day intake of pecans
Sample group	Daily pecan consumption	Concentration (mean ± SE, ng/mL)
Sample group	Daily pecan consumption	EA	Uro-A^***	Uro-B	Uro-C^***	Uro-A-G^***	Uro-B-G^***	Uro-C-G^***
ND not detected. Values with different letters (a, b) mean a statistically significant difference (p < 0.05) between groups. ^***indicates p < 0.001.
Control (n = 25)	0 week	0.05 ± 0.03	ND^b	ND	ND^b	0.81 ± 0.39^b	ND^b	0.01 ± 0.01^b
Control (n = 25)	4 weeks	0.19 ± 0.10	ND^b	ND	ND^b	4.69 ± 2.38^b	ND^b	0.02 ± 0.01^b
Pecan (n = 20)	0 week	0.08 ± 0.06	0.00 ± 0.00^b	ND	0.01 ± 0.00^b	2.57 ± 1.35^b	0.27 ± 0.19^b	ND^b
Pecan (n = 20)	4 weeks	0.23 ± 0.07	0.10 ± 0.03^a	ND	0.46 ± 0.14^a	106.06 ± 16.50^a	15.90 ± 6.53^a	0.31 ± 0.09^a