Identification, Organic Synthesis, and Sensitive Analysis of a cis-Homosalate-Specific Exposure Biomarker

Abstract

Homosalate (HMS) is an organic UV filter used in sunscreens and personal care products. Despite its widespread use and detection in environmental matrices, little is known regarding its exposure in humans. HMS is used as a mixture of cis-and transisomers, and we recently revealed major differences in human toxicokinetics, indicating the need to consider these isomers separately in exposure and risk assessments. In the course of these previous investigations of human HMS toxicokinetics, we identified two trans-HMS-specific and one cis-HMS-specific biomarker candidates. However, the latter lacks sensitivity due to only low amounts excreted in urine, prompting the search for another cis-HMS-specific biomarker. Our toxicokinetic investigations revealed a total of five isomers of HMS carboxylic acid metabolites (HMS-CA). Of these, only one was specifically formed from cis-HMS (HMS-CA 5), but its full identity in terms of constitution and configuration had, so far, not been elucidated. Here, we describe the synthesis of three HMS-CA isomers, of which the isomer (1R,3S,5S)/ (1S,3R,5R)-3-((2-hydroxybenzoyl)oxy)-1,5-dimethylcyclohexane-1-carboxylic acid turned out to be HMS-CA 5. Taken together with two previously synthesized HMS-CA isomers, we were able to identify the constitution and configuration of all five HMS-CA isomers observed in human metabolism. We integrated the newly identified cis-HMS-specific metabolite HMS-CA 5 into our previously published human biomonitoring LC-MS/MS method. Intra-and interday precisions had coefficients of variation below 2% and 5%, respectively, and the mean relative recovery was 96%. The limit of quantification in urine was 0.02 μg L −1 , enabling the quantification of HMS-CA 5 in urine samples for at least 96 h after sunscreen application. The extended method thus enables the sensitive and separate monitoring of cis-and trans-HMS in future human biomonitoring studies for exposure and risk assessment.

Toxicokinetics of homosalate in humans after dermal application: applicability of oral-route data for exposure assessment by human biomonitoring

“…5 , and numerically, as the ratio between the relative dose recoveries of the metabolite in question and the sum of all metabolites of the respective parent HMS isomer: Table 3 ). In the case of HMS-CA 5 , the F ue s and relative dose recoveries were obtained by quantitative analysis of pooled urine samples using a revalidated method (Ebert et al 2023 ) as described in Sect. " Confirmation of HMS-CA 5 relative dose recovery ".…”

“…Data are shown as mean values with ranges in parentheses. Semi-quantitatively analyzed metabolites are given in italics a ratio between the relative dose recoveries of the metabolite in question and the sum of all metabolites of the respective parent HMS isomer (including unchanged HMS after deglucuronidation), the dermal/oral ratio indicates the differences between both uptake routes b urinary excretion fractions (F ue ) reported in Ebert et al ( 2022 ) c data for HMS-CA isomers 2 and 3 are less reliable due to overlap with t HMS-CA tailing, low concentrations, and a minor matrix peak co-eluting with HMS-CA 3 d investigated in pool urine samples using HMS-CA 5 calibration function and revalidated method described in Ebert et al ( 2023 )…”

“…HMS metabolite analytical standards and aryl-deuterated internal standards ( t HMS-CA, t HMS-CA- d 4 , c HMS-CA, c HMS-CA- d 4 , HMS-CA 5 , 3OH- t HMS, and 3OH- c HMS) were obtained by custom synthesis in cooperation with the Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany. See Ebert et al ( 2021 ) and ( 2023 ) for further reagents, including a detailed description of the syntheses of the metabolite standards. See Fig.…”

“…An analytical standard for the c HMS-specific metabolite HMS-CA 5 (Fig. 1 ) could only be obtained after the analysis and evaluation of the dermal study were already concluded (see Ebert et al ( 2023 ) for the standard synthesis as well as the description and revalidation results of the analytical method). To enable as accurate a comparison as possible of HMS-CA 5 elimination between the oral and dermal pathways and provide the most accurate F ue s for future application in HMS exposure assessments, the revalidated method was used to analyze pooled urine samples of both the dermal (96 h) and the previously described oral (48 h) study (Ebert et al 2022 ), and the respective dose recoveries were calculated for each volunteer after dermal and oral HMS exposure.…”

“…To enable as accurate a comparison as possible of HMS-CA 5 elimination between the oral and dermal pathways and provide the most accurate F ue s for future application in HMS exposure assessments, the revalidated method was used to analyze pooled urine samples of both the dermal (96 h) and the previously described oral (48 h) study (Ebert et al 2022 ), and the respective dose recoveries were calculated for each volunteer after dermal and oral HMS exposure. The comparability of the two methods was shown by also analyzing the metabolism samples of one volunteer using the method described in Ebert et al ( 2023 ) and comparing the two excretion curves (see Fig. S2).…”