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Simultaneous Quantitation of 2-Hydroxy-4-Methoxybenzophenone, a Sunscreen Ingredient, and its Metabolites in Harlan Sprague Dawley Rat Plasma Following Perinatal Dietary Exposure

Mutlu E, Pierfelice J, McIntyre BS, Cunny HC, Kissling GE, Burback B, Waidyanatha S.
Journal of Analytical Toxicology (2017) DOI: https://doi.org/10.1093/jat/bkx070 PMID: 28977387


Publication


Abstract

2-Hydroxy-4-methoxybenzophenone (HMB) is a common ingredient in sunscreens and other personal care products and thus significant potential exists for human exposure. HMB was nominated to the National Toxicology Program (NTP) for testing due to its high exposure through consumer products and inadequate toxicological data at the time, which also included increasing concern for the potential effects of HMB on reproduction and development. HMB is metabolized to numerous metabolites in vivo and in vitro including 2,4-dihydroxybenzophenone (DHB), 2,3,4-trihydroxybenzophenone (THB) and 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB) as well as their corresponding glucuronide and/or sulfate conjugates. In this study, we have developed and validated a liquid chromatography-tandem mass spectrometry method to quantitate free (unconjugated) HMB and DHB, and total (combined conjugated and unconjugated) HMB, DHB, THB and 2,5-DHMB. The method was successfully applied to quantitate these analytes in plasma from postnatal day 28 and 56 male and female Harlan Sprague Dawley rat pups following perinatal dietary exposure to 0 (control), 3,000, 10,000 and 30,000 ppm HMB beginning on gestational Day 6. All determined analyte concentrations increased with increasing dose and were significantly higher than the controls at both timepoints. All the total analytes were quantified in all plasma samples and total concentrations were considerably higher than free, suggesting extensive conjugation. Mean concentrations of total HMB and DHB were higher (~100-300-fold) than the free HMB and DHB concentrations, and total concentrations in plasma were approximately HMB≈DHB > 2,5-DHMB»THB. Free and total analyte plasma concentrations were not sex-dependent and in general, both free and total analytes were detected in the control samples. Comparison of our rat data, using the internal dose, with human data available in the literature suggests that the rat doses used in our studies were within 4-fold of the human dose.

Figures


Figure 1. HMB and its free (DHB) and total metabolites (HMB, DHB, THB, and 2,5-DHMB).

2-Hydroxy-4-methoxybenzophenone (HMB) and its free (DHB) and total metabolites (HMB, DHB, 2,3,4-trihydroxybenzophenone (THB) and 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB)).

Figure 2. Selected ion chromatograms for HMB, DHB, THB, 2,5-DHMB from a male PND56 plasma.

Selected ion chromatograms for HMB, DHB, THB, 2,5-DHMB from a male PND56 plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed.

Figure 3. Free HMB (A) and DHB (B) concentrations in PND28 and PND56 male and female rat plasma.

Free HMB (A) and DHB (B) concentrations in PND28 and PND56 male and female rat plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed.

Figure 4. Total HMB (A), DHB (B), THB (C), and 2,5-DHMB (D) concentrations.

Total HMB (A), DHB (B), THB (C), and 2,5-DHMB (D) concentrations in PND28 and PND56 male and female rat plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed.

Tables


Table 1. Method validation and stability data for free and total analyses.

Table 2. Free HMB and DHB concentrations in PND28 and PND56 male and female rats.

Free HMB and DHB concentrations in PND28 and PND56 male and female rats (n = 4–5) following perinatal exposure to HMB via feed.

Table 3. Total HMB and metabolite concentrations in PND28 and PND56 male and female rats.

Total HMB and metabolite concentrations in PND28 and PND56 male and female rats (n = 4–5) following perinatal exposure to HMB via feed.

Table 4. Comparison of animal data to human data based on free HMB plasma levels and external dose.