An analytical method to investigate the estrogen metabolism in women taking combined oral contraceptives

Abstract

The human body is exposed to numerous (endogenous and exogenous) toxins daily. The body has a protective mechanism to help detoxify certain toxins, through biotransformation processes. However, when exposure exceeds the capacities of the biotransformation systems, it may result in pathophysiological conditions. The additional intake of estrogens are a good example thereof, where exposure (through intake) might contribute to a disturbance in the balance between the different phases of liver detoxification and allow toxic metabolites to be circulated in the blood (rather than quickly excreted in the urine). Evidence suggests that the estrogen metabolism and an imbalance thereof could be associated with an increased risk of breast cancer development. The intake of estrogens through combined oral contraceptives is one of these exogenous sources of estrogens. A large part of the South African population might be unknowingly affected by the additional intake of estrogens, as more than half of married or in-union females use this type of contraceptive method. Determining the effect of exogenous estrogens, on the normal physiological detoxification capacities, requires certain markers to be monitored. A link between the urinary estrogens and relative cancer risk has been made in previous studies, and evidence has shown that the evaluation of the estrogen-related metabolites in the urine, might aid in breast cancer risk evaluation. This study focused on the need for a non-invasive method for the quantification of the estrogen metabolites excreted in the urine. These metabolite concentrations and their ratios of excretion, resulting from either normal estrogen metabolism or an additional estrogen intake, could be evaluated as possible markers for breast cancer development risks. Previous methods for the quantification of some of these metabolites have been developed but limitations still exist and in-house implementation always requires adaptation of previously developed methods due to variation in factors such as the availability of infrastructure, analytical standards and the sensitivity of the available instrumentation. This study, therefore, aimed to develop a highly sensitive and selective urine-based liquid chromatography—electrospray tandem mass spectrometry method to investigate the estrogen metabolism in women. Outcomes of this study included the development of analytical methods to measure the metabolic products of estrogen metabolism in the urine. The first part of the study comprised of the development of a sensitive, specific, accurate and precise liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify the levels of estrogens and related metabolites in the urine. A non-derivatised method was developed, however, during validation, the limitations of the capacity of the method became known. This was then followed by the development of a derivatised method that enabled the quantification of 27 estrogen and related metabolites. This was achieved after a single sample clean-up with solid phase extraction, derivatisation through dansylation, and two different (polar and non-polar) analyses on the LC-MS/MS instrument. The metabolites that could be accurately quantified with the developed methods included parent estrogens (E1, E2, E3), their hydroxylated forms (2OH, 4OH, 16OH), metabolites of the 16α-hydroxyestrogen pathway, sulphate and glucuronide conjugated forms, methylated forms (2 & 4-methoxy), a precursor (testosterone and androstenedione) and related steroid hormones (progesterone). The developed method was then validated and allows quantification in the high pictogram and low nanogram per millilitres range. The developed method is now available for laboratory use and for use in a research setting to investigate estrogen metabolism and the effect that exogenous sources of estrogens, for examples combined oral contraseptives (COCs), might have on metabolic profiles. Furthermore, in a more clinical setting, the method could be beneficial for monitoring the relative estrogen metabolite-related cancer risk in females over time. The study was concluded by testing the utility of the developed method on actual clinical samples, merely to confirm detection and quantification of each of the metabolites in urine samples and to compare the concentrations obtained through urine analysis to those of published reference ranges.