Turbo ion spray source

All measurements were performed using a 4000 quadrupole linear-ion trap (QTrap) mass spectrometer equipped with a Turbo Spray ion source (AB SCIEX, Darmstadt, Germany): The QTrap was connected to a Agilent Cap HPLC comprising of 1200 Capillary Pump, 1200 Micro Wellplate Sampler and 1200 Thermostat Module (Agilent, Böblingen, Germany). Data analysis was carried out using Analyst 1.5 software (AB SCIEX, Darmstadt, Germany). For determination of phosphatidylcholine, phospatidylcholine-plasmalogen, lyso-phosphatidylcholine, sphingomyelin and ceramide levels 20μl sample was injected into sample loop with the following running solvent gradient (0.0–2.4min, 30μl; 2.4–3.0min, 200μl; 3.0min, 30μl). Mass spectrometry settings and composition of running solvents used is specified below.

The mass spectrometry measurements were performed with the same instrument previously described^{38 (link)}. Briefly, the liquid chromatography system was an UltiMate 3000 LC Systems (Dionex, Sunnyvale, CA, USA), with autosampler, binary pump, and column oven (Thermo Fisher Scientific, USA). The tandem mass spectrometer was an AB Sciex 3200 QTRAP LC–MS/MS instrument with electrospray ionization (ESI) TurboIonSpray source (AB Sciex Framingham, MA, USA). Instruments were managed with manufacturers' software and according to manufacturers' instructions. The analytical data were processed by Analyst software (version 1.6.2).

For the MS/MS analysis, a previously validated method published by our lab was used [49 (link)]. Briefly, a 4000 QTRAP^® triple quadrupole mass spectrometer with a Turbo Ion Spray source (AB Sciex, Redwood City, CA, USA) was used in the positive mode. The MRM transitions for the [M + H]+ ion of AC1LSPZG was (m/z 457.10 → 349.00) and that of Griseofulvin (IS) was (m/z 353.27 → 285.10). Representative LC chromatogram for tandem mass spectrometry is shown in Appendix (Figure A4).

Samples were analysed for the five main D₄-DEHP metabolites: D₄-lablled mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono-[2-(carboxymethyl), hexyl]phthalate (2cx-MMHP) [33 (link)]. Extract (1 μL) was injected onto LC-MS/MS. A C18 column (2.1 mm i.d. × 50 mm; Grace Genesis Lighting; Sunrise, FL, USA) was used before the injector to reduce the interference of contaminants during the mobile phase. A C18 column (1.5 μm, 2.0 mm i.d. × 30 mm VisionHT; Grace) was used, and the mobile phases were water and acetonitrile with 0.1% formic acid. The samples were analysed on a Shimadzu UFLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a QTRAP5500 (triple quadrupole linear ion trap mass spectrometer) equipped with a TurboIon Spray source (AB Sciex, Framingham, MA, USA). The samples were analysed in duplicates. The determined LOD for all analysed metabolites were 0.5 ng/mL (D₄-MEP), 0.9 ng/mL (D₄-MEHP), 0.1 ng/mL (D₄-5OH-MEHP), 0.2 ng/mL (D₄-5oxo-MEHP), 0.1 ng/mL (D₄-5cx-MEPP), and 0.1 ng/mL (D₄-2cx-MMHP). For more details, see Supplementary Information (Table S6).

Isolated sample fractions were analysed by LC–MS/MS using a PerkinElmer Series 200 high-performance liquid chromatography (HPLC) system coupled to an API5000 triple-quadrupole tandem mass spectrometer fitted with a TurboIonSpray™ source (ABSciex, Concord, ON, Canada). Compounds of interest were separated on a reversed phase C18 column (Xterra® MS C18 column, 100 × 2.1 mm, 3.5 μm, Waters, Milford, MA, USA) protected by a VanGuard® cartridge of the same material (Xterra® MS C18; 5 × 2.1 mm, 3.5 μm, Waters, Milford, MA, USA). The column oven was set at 40°C, and the injection volume was 50 μl. Optimized instrumental conditions are shown in Table 1. With the exception of DHEAs and triclosan, which were analysed in negative electrospray ionization (ESI) mode, steroid hormones and PPCPs were detected in positive ESI mode with multiple reaction monitoring of the two most abundant product ions for each analyte. For each MRM transition, the dwell time was 40 ms and the entrance potential (EP) was 10 volts.

The mass spectrometer was operated with an electrospray ionization (ESI) source in positive ion mode using Turbo Ion Spray source (AB Sciex, Foster City, CA, USA). The singly protonated ion of TA and sulphadimethoxine (SDM, IS) were observed at m/z 435.2 and m/z 311.0 in ESI positive ion mode. Fragmentation spectra of TA were recorded, and abundant daughter ions were selected for the analysis. Multiple reaction monitoring (MRM) mode was used for the quantification of TA using SDM as an internal standard. Quantification of TA was performed using precursor-to-product ion transition m/z 435.2/397.2 (quantifier) and m/z 435.2/415.2 (qualifier). The mass transition used for the internal standard (SDM) was m/z 311.0/156.0.