Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism and elimination of approximately 25% of clinically used drugs, including antidepressants and antipsychotics, and its activity varies considerably on a population basis, primarily because of genetic variation. CYP2D6 phenotype can be assessed in vivo following administration of an exogenous probe compound, such as dextromethorphan or debrisoquine, but use of a biomarker that does not require administration of an exogenous compound (i.e., drug) has considerable appeal for assessing CYP2D6 activity in vulnerable populations, such as children. The goal of this study was to isolate, purify, and identify an “endogenous” urinary biomarker (M1; m/z 444.3102) of CYP2D6 activity reported previously. Several chromatographic separation techniques (reverse phase HPLC, cation exchange, and analytical reverse phase UPLC) were used to isolate and purify 96 μg of M1 from 40 l of urine. Subsequently, 1D and 2D NMR, and functional group modification reactions were used to elucidate its structure. Analysis of mass spectrometry and NMR data revealed M1 to have similar spectroscopic features to the nitrogen-containing steroidal alkaloid, solanidine. 2D NMR characterization by HMBC, COSY, TOCSY, and HSQC-TOCSY proved to be invaluable in the structural elucidation of M1; derivatization of M1 revealed the presence of two carboxylic acid moieties. M1 was determined to be a steroidal alkaloid with a solanidine backbone that had undergone C-C bond scission to yield 3,4-seco-solanidine-3,4-dioic acid (SSDA). SSDA may have value as a dietary biomarker of CYP2D6 activity in populations where potato consumption is common.

SIGNIFICANCE STATEMENT Endogenous or dietary biomarkers of processes involved in drug disposition and response may allow improved individualization of drug treatment, especially in vulnerable populations, such as children. Given that several CYP2D6 substrates are commonly used in pediatrics and the ubiquitous nature of potato consumption in Western diets, SSDA has considerable appeal as a noninvasive biomarker of CYP2D6 activity to guide treatment with CYP2D6 substrates in children and adults.

Funding support for this article was provided by the National Institutes of Health (NIH) and National Science Foundation (NSF) (R01HD058556, S10RR024664, P30GM110761, NSF #1625923)

The longitudinal CYP2D6 phenotyping study in which SSDA was discovered and originally described as an “endogenous” biomarker of CYP2D6 activity was funded by [Grant R01-HD058556], Exogenous and Endogenous Biomarkers of CYP2D6 Variability in Pediatrics (Leeder and Lin, co-PIs). Isolation, purification, and identification of M1 as SSDA was supported by internal funding, Children’s Mercy Kansas City (van Haandel, PI). Support for the 500 MHz NMR was provided by National Institutes of Health Shared Instrumentation [Grant S10-RR024664] and NSF Major Research Instrumentation Grant # 1625923. The 800 MHz instrument is supported by an Institutional Development Award (IDeA) from the NIH National Institute of General Medical Sciences [Grant P30-GM1110761].

↵1 Current affiliation: Department of Bioanalytical Chemistry, Charles River Laboratories, Mattawan, Michigan.

↵2 Current affiliation: Analytical Research and Development, NCE Project Team, Abbvie, North Chicago, Illinois.

Part of this work was presented as follows: (2017) Metabolomics in Translational Research Summit. Vanderbilt University, October 30, 2017

(2019) Found in Translation: Adaptive CMPK Strategies for Emerging Technologies. 22nd Annual Land O’ Lakes Drug Metabolism and Applied Pharmacokinetics Conference, September 9–12, 2019.

dx.doi.org/10.1124/dmd.122.000957.

↵This article has supplemental material available at dmd.aspetjournals.org.