Data on UPLC/MS method validation for the biodegradation of pharmaceuticals and intermediates by a fungal consortium and on T47DK-Bluc reporter gene assay to assess the reduction of their estrogenic activity

In term of pharmaceutical and their intermediate compounds analysis, UPLC/MS method is a valuable equipment to achieve better confirmation on their biodegradation by fungi. The T47D-KBluc reporter gene assay is an appropriate tool to investigate to removal of estrogenic and antiestrogenic activities of pharmaceuticals and their metabolites from a synthetic wastewater. A consortium of isolated South African indigenous fungi Aspergillus niger, Mucor circinelloides, Trichoderma longibrachiatum, Trametes polyzona and Rhizopus microspores was found to perform a removal of pharmaceuticals and their metabolites and to reduce their estrogenic activity below the limit of detection in a sequencing batch reactor. Here are presented data regarding the phenolic compounds list and the method validation for UPLC/MS analysis used for selected pharmaceutical compounds namely carbamazepine, diclofenac, ibuprofen and their metabolites, as well as the T47D-KBluc bioassay using as positive control, the agonist E2 for estrogenic activity and the antagonist ICI 182,780 for antiestrogenic activity. For better understanding of the data presented in this paper, please see the research paper “Removal of pharmaceutical’ estrogenic activity of sequencing batch reactor effluents assessed in the T47DK-Bluc reporter gene assay” [1].

paper, please see the research paper "Removal of pharmaceutical' estrogenic activity of sequencing batch reactor effluents assessed in the T47DK-Bluc reporter gene assay" [1]. © 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Data
In the present report, authors present data on the method development and validation to confirm the removal of pharmaceutical compounds (PhCs) and their metabolites, especially the reduction of the metabolite' estrogenic activity in a sequencing batch reactor (SBR) [1], as it has been reported that intermediate products might be more toxic than the initial parent compounds [2]. The UPLC-MS Specifications Table   Subject

Value of the data
The data highlight characteristics of positive controls 17-b estradiol (E2) and ICI 182,780 used as positive control in the T47D-KBluc bioassay and their scientific names according to the International Union of Pure and Applied Chemistry (UIPAC). The data show a list of the potential estrogenic active phenolic compounds displaying endocrine-disrupting properties in the environment, because of their complex structures and masses normally ranging from 200 to 1000 Da. The data provide details in the development and validation of the UPLC/MS method used for the analysis of pharmaceutical compounds and their transformation fragment ions referred as metabolites, for better understanding of the results in the research paper. A protocol is provided with details for the preparation of media, general cell culture procedure of T47D-KBluc breast cancer cells, to be used for further insights. The data display the T47D-KBluc method validation in terms of the calibration curves of E2 and ICI 182,780 using Graphpad Prism Software (version 4).

UPLC/MS method development and validation
The Ultra-Performance Liquid Chromatography tandem Mass Spectrometry (UPLC/MS) method was used for the present data. Pharmaceuticals separation and their detection were carried out using a Waters® Synapt G2 high definition mass spectrometry system (Waters Inc., Milford, Massachusetts, USA). The system consisted of a Waters Acquity UPLC® system hyphenated to a quadrupole-time-offlight (QToF) instrument operating with MassLynx™ (version 4.1) software (Waters Inc., Milford, Massachusetts, USA) for data acquisition and processing. The internal control was used to compensate for instrumental drift, ensuring good mass accuracy, throughout the duration of the runs. The instrument was calibrated using sodium formate clusters and Intellistart functionality (mass range 112.936 -1 132.688 Da). Resolution of 20,000 at m/z 200 [full width at half maximum (FWHM)] and mass error within 5 mDa were obtained. The source conditions were as follows: the capillary voltage for ESI was 2.6 kV for positive mode ionisation. The source temperature was set at 120 C, the sampling cone voltage at 25 V, extraction cone voltage at 4.0 V and cone gas (nitrogen) flow at 10.0 L/Hr. The desolvation temperature was set at 350 C with a gas (nitrogen) flow of 600.0 L/Hr. Quantitative dataindependent acquisition (DIA) was done using two simultaneous acquisition functions with low and high collision energy (MS E approach) with a QTOF instrument. Fragmentation was performed using high energy collision induced dissociation (CID). The fragmentation energy was set at 2 V and 3 V for the trap and collision energy, respectively. The ramping was set from 3 to 4 V and 20e40 V for the trap and transfer collision energy, respectively. Mass spectral scans were collected every 0.3 seconds. The raw data was collected in the form of a continuous profile. Mass to charge ratios (m/z) between 50 and 1 200 Da were recorded. Mobile phase and other parameters including the column used, flow rate and injection conditions are provided within the paper.

Method development and validation
A control blank matrix extract with increments of known analyte concentrations of selected drugs (CBZ, DCF and IBP) was used for the method development. The blank matrix (ddH 2 O) was spiked with a mixture of pharmaceutical standard solutions (CBZ, DCF and IBP) at the concentration of 0.01, 1 and 5 mg/L. According to their monoisotopic mass, all targeted drugs were quantified in positive ion mode (ESIþ) while their detection in low energy was performed in a reaction monitoring mode of respectively m/z 237.10 (CBZ), m/z 296.02 (DCF) and m/z 229.12 (IBP for IBP þ Na). The following Table 1 showed the method development parameters set to perform the UPLC/MS.
Considering the endogenous compounds from the aerated batch flasks (including fungal enzymes and other compounds), the ionisation of the targeted analytes could be increased or suppressed. The  effect of the fungal medium constituents as a matrix effect was evaluated by comparing the slope of the selected pharmaceutical calibration curves in spiked medium extracts and in spiked pure solvent [7]. The matrix effect using SPE HLB column extracted was evaluated and the values less than 20% were considered acceptable [8]. The procedure described by Kasonga et al. (2019) was used. The following Equation (1) was used to determine the matrix effect (ME) in the working conditions.

ME ¼
Slope of fungal medium À matched calibration curve Slope of the s tan dard calibration curve x100 (1) The method was thereafter validated in terms of required parameters including: linearity of the calibration curve, specificity and selectivity, sensitivity, precision and accuracy, and recovery [9].

Linearity of calibration curve
Calibration curves (Fig. 1) were carried out by plotting the peak area ratios (or signal response) of each analyte versus the theoretical eight (8) point concentrations of the spiked drug in methanol-water (1:1, v/v). The mixture CBZ, DCF and IBP at individual concentrations of 0.001, 0.01, 0.1, 0.25, 0.5, 1, 1.5 and 2 mg/L were prepared in triplicate of each concentration from filtered fungal batch media. The filtered medium without selected pharmaceuticals was used as a blank. Prior to the injection in UPLC/ MS, the SPE was performed for all blank and analyte media. The linearity of the calibration curves was assessed throughout the recorded r 2 values and equations given by the instrument. The recorded values were found to be in the acceptable range >0.9 as suggested in literature [3,10].

Sensitivity
The sensitivity of the method was evaluated in terms of the limit of detection (LoD) and the limit of quantification (LoQ) for each selected pharmaceutical and was assessed during the determination of the linear range of the calibration standards as those concentrations giving a signal to noise ratio ( LoQ ¼ lowest concentration of standard instrumental S=N for lowest concentration of standard x 10 (3)

Accuracy and precision
In respect of assessing the reproducibility and measuring the closeness among the replicates, daily precision and accuracy, CBZ, DCF and IBP (mixed working standard solution) at low (0.01 mg/L and 0.1 mg/mL), middle (0.75 mg/L and 1 mg/L), and high (2 mg/L) concentrations were spiked in triplicates (n ¼ 3) in accordance with the method proposed by previous investigators [4,11]. The daily precision (intra-day/inter-day assay), one spike per day over three days, was then assessed by determining the method accuracy (expressed as % recovery) and precision [expressed as repeatability in terms of % relative standard deviation (RSD)]. The criteria for the tolerability were that the precision level should not exceed 15%e20% and the averaged assessment of the accuracy should be within ±15e20% (Wang et al., 2011). The % Recovery and % RSD of the method were determined using Equations (4) and (5) respectively. % Recovery ¼ drug experimentally determined concentration true spike concentration x 100 (4) % RSD ¼ standard deviation mean x 100 (5)

Specificity and selectivity
These parameters were evaluated in terms of chromatographic interferences from endogenous compounds which were eliminated by comparing the chromatograms of the blank medium and the analyte spiked medium (CBZ, DCF and IBP, Table 2) as described by previous investigators [10,11]. The analyte samples at the concentrations of 0.001, 1 and 5 mg/L were used for the data.

. Bioassay media preparation
Media preparation and the assay procedure were performed according to the standard assay procedures [5,12,13]. Media components were prepared in double distilled water (ddH 2 O) from a Milli-Q synthesis ultrapure water system (Merck, Germany), equipped with an EDS-Pak Polisher (an activated carbon-based filter) to produce EDC-free water. Briefly explained, the RPMI medium (medium formulation developed at Roswell Park Memorial Institute) was prepared by dissolving in 1L ddH 2

T47D-KBluc general cell culture procedures
The T47D-KBluc cells used for the bioassay were purchased from the American Type Culture Collection (ATCC, USA) and aseptic procedures were performed in a Type II bio-hazardous safety cabinet. Cell stock cultures were stored at À80 C for 6e12 months and were thawed in a water bath at 37 C. Cells were grown and maintain in RPMI medium (medium formulation developed at Roswell Park Memorial Institute, Sigma Aldrich, USA) with 10% foetal bovine serum, characterized, (FBS, Hyclone Laboratories, USA) and antibiotic/antimycotic solution using 25 cm 2 or 75 cm 2 tissue culture flasks and were incubated at 37 C in a humidified 5%CO 2 water-jacketed incubator (Nuaire™, USA). The cells were trypsinized and subcultured when confluent, at 3 to 4-day intervals. To trypsinize, the cells were rinsed 2x using 5 mL of Hank's balanced salt solution (HBSS, Gibco Life Technologies corporation, UK). The HBSS was then removed and 5 mL of trypsin was added to each flask. The flasks were incubated again for 3 min in the same conditions (37 C and 5% CO 2 ). The excess trypsin was discarded from the tissue culture flasks and the cells detached from the surface by gently tapping the flask against the hand. Cells were thereafter suspended in 10 mL of maintenance RPMI medium and divided 1/3 into subcultures.

Ethics approval
The human cells used in these data were not harvested by us. We used an established and modified cell line from the ATCC® CRL-2865™.