Data on the optimisation of a solid phase extraction method for fractionating estrogen metabolites from small urine volumes

Certain estrogen metabolites have been implicated in the pathophysiology of breast cancer. Moreover, the estrogen metabolite profiles of healthy women and those with (a high risk of) breast cancer differ significantly. The development of an analytical method to determine the relative levels of all the estrogen biotransformation products has been described in van der Berg et al. [1]. An improvement on previously developed methods was the ability to also detect molecules such as sulphate and glucuronide conjugates as well as progesterone, estradiol precursors, and metabolites from the 16-hydroxylation metabolic pathway of estrogens simultaneously with all other estrogen metabolites. The data presented here describe the optimisation of a solid phase extraction method with different fractionation steps for LC-MS/MS analysis of 27 estrogen-related metabolites from small urine volumes. Conditions that were optimised include the elution and washing solvent concentration, the urine, loading, washing, and elution volumes, as well as pH. All raw data used to construct the bar graphs presented in this article are included in the supplementary data file. The data indicated that fractionation was necessary in order to elute estrogen metabolites with different chemical properties at different eluate compositions. Only one of the fractions (containing the less water-soluble metabolites) underwent derivatisation before LC-MS/MS analysis.

the fractions (containing the less water-soluble metabolites) underwent derivatisation before LC-MS/MS analysis.
© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Data description
The data presented here were obtained during the development of an LC-ESI-MS/MS method for the detection and quantification of various urinary estrogen metabolites [1]. A number of conditions for solid phase extraction (SPE) were tested by determining the response of estrogen-related metabolites on the LC-MS/MS after each adjustment. Before LC-MS/MS analysis, SPE eluates were dried and dansyl derivatised (if applicable). The integrated chromatographic spectral results are available as raw data tables in the supplementary file accompanying this article. This data was plotted as response or log transformed response values over different variables. The composition of the estrogen metabolite mixture that was used for these assays is given in Table 1 in the Materials and Methods section. Due to the large variation in the water solubility of estrogen metabolites and the fact that the sulphate and glucuronide conjugates (most water soluble) do not dansyl derivatise, we considered the possibility of analysing the derivatising and non-derivatising metabolites separately (i.e. by fractionating the sample).
Specifications Table   Subject Biochemistry Specific subject area Metabolite extraction for estrogen biotransformation analysis Type of data In order to determine the optimal conditions to fractionate the samples before derivatisation, we first tested different washing and elution solvent concentrations. The plotted bar graphs of the response of each metabolite at different (5e40%) organic solvent concentrations in the washing solution are presented in Fig. 1 a-aa. The raw data used to compile the graphs shown in Fig. 1 are available in Table 1 of the supplementary data file. Fig. 2 shows the response after a second 40% methanol wash, while Fig. 3 shows a comparison of the responses after washing with different methanol compositions (40e55%). The raw data of Figs. 2 and 3 are available in Supplementary Table 2. In addition, the urine and SPE loading, washing, and elution volumes, as well as the pH, were optimised. The log transformed data presented in Fig. 4 (raw data in Supplementary Table 3) show the metabolite responses when 1 ml, 2 ml or 5 ml urine was used. Fig. 5 shows the effect of different SPE loading volumes on the metabolite responses while the raw data before log transformation is available in Supplementary Table 4. The log transformed responses of the conjugated (Fig. 6a) and the unconjugated (Fig. 6b) metabolites after washing with either 3 ml or 6 ml was also determined. The graphs were plotted separately because the metabolites in Fig. 6a underwent only a single 5% washing step, whereas the metabolites in Fig. 6b underwent multiple washing steps (5%, and 45%). Furthermore, metabolite responses were compared using different elution volumes (3 ml or 6 ml; Fig. 7). The raw data supporting Figs. 6 and 7 is given in Supplementary Table 5. Finally, in Fig. 8 the effect of the pH of the buffer or washing and elution steps on the response values can be seen. Fig. 8 represents the log values of the raw data found in Supplementary Table 4. Another urine volume comparison was done ( Fig. 9) to confirm that, after all other parameters were optimised, the most optimal urine volume was chosen (raw data in Supplementary Table 6). Washing was done with 5%, 10%, 15%, 20%, 30%, 35%, and 40% (v/v) methanol (MeOH) and the washed-out fractions were analysed. This was followed by an elution step with methanol and acetone, followed by the analysis of the eluate. Blue bars represent the response of metabolites washed from the SPE column, while the red bars represent the response of the metabolites that eluted with methanol and acetone at the end of the sample preparation method.  Relative responses of each of the final eluate metabolites on the mass spectrometer after washing only with 5% MeOH or first with 5% and then 40% (v/v) MeOH. Blue bars represent the response of the eluate after a single 5% MeOH in water wash, followed by elution with methanol and acetone (i.e. no fractionation, conjugates co-eluted with other metabolites). Orange bars represent the response of the SPE eluates after first washing with 5% MeOH, eluting conjugates with 20% MeOH, washing a second time with 40% MeOH, and finally eluting the remaining metabolites with methanol and acetone.

Working solution preparations
The stock solutions of all the estrogens (Table 1) were prepared at concentrations of 0.1 mg/ml in methanol, and further diluted to 50 ng/ml working stock solutions in MeOH. The standards were then stored at À80 C until used for method development. For spiked urine solutions, the same concentration was spiked in a single urine sample to be used for all analysis.

LC-MS/MS analysis
Liquid chromatography mass spectrometry was performed using an Agilent 1290 Infinity series LC system (Agilent Technologies, Santa Clara, CA, USA) consisting of a micro vacuum degasser (G1330B); binary pump (G4220A); preparative autosampler HiP-ALS (G4226A); and thermostatted column compartment (G1216C). The LC system was coupled to the Agilent 6460 triple quadrupole mass spectrometer (G6460A), with a jet stream electrospray ionisation (ESI) source. The Agilent Technologies, Zorbax Eclipse plus rapid resolution high definition (RRHD) C8-E chromatographic column (959758e906), were used for liquid chromatographic separations. The LC-MS/MS methods as described in Van der Berg et al. (2020) was followed for sample analysis [1]. These methods consisted of two different runs, one for the more water soluble and one for the less water soluble metabolites.

Solid phase extraction method outline
The general solid phase extraction (SPE) sample preparation method was followed. The adsorbent in the SPE cartridges (Strata C18-E) was conditioned and equilibrated with two cartridge volumes of each, acetone, methanol (MeOH), and water at a flow rate of 3 ml/min. The adsorbent bed was not allowed to run dry before the next step, which included sample loading. Before loading, the samples (standard or matrix) were centrifuged and only the supernatant was diluted with water to a total volume of twice the SPE column capacity, and, unless indicated otherwise, the pH was adjusted to 7. The sample was then loaded onto the adsorbent. The adsorbent was washed with single or multiple water or higher organic composition solvents and the adsorbent was allowed to dry for 10 min under high vacuum. The last step of the SPE included the elution of the metabolites of interest into a polypropylene tube at approximately 1 ml/min with at least two column volumes of methanol or acetone. Any of the collected washing or eluate fractions containing solvent were dried under a gentle stream of nitrogen gas and those that contained water were freeze-dried. If both were present, the organic solvent was evaporated first, followed by freeze-drying the remaining aqueous solution. The less water-soluble fractions were dansyl derivatised and the more water-soluble, conjugated fraction was resuspended in a 50:50 water:MeOH mixture before both fractions were filtered through 0.2 mm Nylon Spin-X filters for LC, after which the filtrate was transferred to inserts in LC-MS analysis vails.

Solid phase extraction method optimisation
2.4.1. Washing and elution solvent SPE optimisation was done using either a 50 ng/ml standard mixture prepared in methanol or a urine sample spiked with the same concentration (50 ng/ml) of estrogen metabolite mixture. To test the possible advantage of fractionation of the sample, standard estrogen metabolite mixtures were loaded onto SPE cartridges, which were then washed with 5%, 10%, 15%, 20%, 25%, 30%, 35% and 40% (v/ v) organic solvent (MeOH) in the aqueous washing step to give an indication of which metabolites will elute at which stage. Both the wash and elution steps were collected, dried, derivatised (if applicable), and analysed using the LC-MS/MS method described in van der Berg et al. [1]. For confirmation if fractionating the samples in different elution steps (one for the more and a second for the less watersoluble metabolites) would increase responses, the following procedure was followed. The estrogen standard mixture spiked in urine was loaded on SPE columns, after which the first column was washed with 5% MeOH and eluted, and the second was washed with 5% MeOH followed by a first elution with 20% MeOH and a 40% MeOH second wash. To optimise the organic composition of the second washing step, standard estrogen metabolite mixtures were again loaded to the SPE columns and, first, washed with 5% (v/v) MeOH in water, followed by the first elution with 20% (v/v) methanol, and then a second wash with 40%, 45%, 50% or 55% (v/v) MeOH in water. Again, all fractions were collected and the response of the last eluting metabolites (unconjugated and dansylated metabolites) was plotted for comparison.
2.4.2. Urine, loading, washing, and elution volumes Following optimisation of the SPE washing and elution solvent composition, different urine, loading, washing and elution volumes were tested. For this, a urine sample spiked with 50 ng/ml standard mixture was loaded to SPE columns, washed with 5% (v/v) MeOH, then 20% (v/v) MeOH (elution 1), then 45% (v/v) MeOH, and metabolites finally eluted with methanol and acetone. The urine volumes that were tested included 1 ml, 2 ml and 5 ml. For the loading volume analysis, 1 ml urine was diluted to a final volume of 6 ml, 9 ml or 12 ml, while washing and elution volumes of either 3 ml or 6 ml water and methanol (premixed) were tested for all the washing and elution steps. The SPE eluates were dried and derivatised (if applicable) after sample clean-up and analysed in order to compare metabolite responses.

pH
The use of a buffering agent, such as 10 mM ammonium formate, and different pH levels during different steps in the SPE procedure was also investigated. We compared (1) no pH buffering (in washing and elution steps), with (2) pH buffering at pH 7 with 10mM ammonium formate, and (3) conditioning with a more acetic solvent and subsequent elution with an alkaline solvent [2,3].

Confirmation of optimal urine volume
Finally, the optimal urine volume for SPE was confirmed by applying the optimised conditions for all other SPE parameters. For this, the spiked urine samples were diluted to a 6 ml loading volume either by adding 5 ml of water to 1 ml of the urine sample, or by adding 3 ml of water to 3 ml of the urine sample. The SPE columns were conditioned with 6 ml acetone, 6 ml methanol and 6 ml distilled water. The prepared urine sample supernatant was then loaded onto the SPE columns. Each column was washed with 6 ml 5% (v/v) MeOH in water followed by a 6 ml 20% (v/v) MeOH in water, from which the sulphate and glucuronide conjugates were collected. The SPE cartridge was then washed again with a 45% (v/v) MeOH in water solution and dried under vacuum. The sample was next eluted with 2 ml MeOH and 4 ml acetone. The SPE elutes were then evaporated to dryness, resuspended or derivatised, analysed by LC-MS/MS, and the response of each metabolite was log transformed and plotted for comparison of the different urine volumes.