*4.2. Sample Processing*

### 4.2.1. Creatine and GAA Analysis

Snap frozen placental tissues of su fficient weight (*n* = 19 control and *n* = 19 PE) were freeze dried overnight at −80 ◦C. Powdered samples were then weighed (3–4 mg) and extracted on ice using 0.5 M perchloric acid/1mM Ethylenediaminetetraacetic acid (EDTA) before being neutralized with 2.1 M potassium hydrogen carbonate as previously described [17]. Total creatine content (creatine + phosphocreatine) was measured using fluorometric assays [46]. GAA was measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) through a slight modification of the method of Tran et al. (2014) [47]. Briefly, 10 μL of unlabeled standard, placental tissue lysate or lysis solution (extraction blank) were deproteinized with 200 μL of methanol containing 2.5 μM 2,2-d2-GAA (Sigma, Rowville, Victoria, Australia deuterium labelled internal standard. Samples were vortexed for 20 min before centrifugation at 15,000 rpm for 5 min, followed by the transfer of 180 μL of the supernatant into 250 μL glass inserts. Samples were then dried in a speed vacuum prior to being derivatized (butylated) by the addition of 100 μL of 3 M butanol-hydrochloric acid and incubation at 60 ◦C for

30 min. After derivatization, samples were dried under speed vacuum and the residue re-suspended in 100 μL of methanol: water (1:1 *v*/*v*). The LC-MS/MS system comprised a vacuum degasser, binary pumps, column oven and a temperature-controlled autosampler (Shimadzu, Nexera ® UPLC, Rowville, Victoria, Australia) interfaced with a triple quadrupole mass spectrometer (Shimadzu, LC-MS-8040) with positive electrospray ionization and operated in multiple reaction monitoring (MRM) mode. The MRM transitions for the [M+H<sup>+</sup>] + butylated derivatives were: GAA 174.1 m/z–101.1 m/z, d2-GAA 176.1 m/z–103.1 m/z, the collision energy (−15 V) and quad bias voltages were optimized using standards. The LC column was a 2.1 mm × 100 mm, 1.8 μm C18 Zorbax Elipse plus (Agilent, Mulgrave, Victoria, Australia) maintained at 30 ◦C. The sample and standard injection volume was 5 μL. Samples were eluted with a binary mobile gradient at 0.4 mL/min (mobile phase A water 0.1% formic acid, mobile phase B acetonitrile 0.1% formic acid) with the following program: initial composition was 5% B followed by a linear increase to 50% B over 5 min. The column was then washed at 100% B for 2 min then re-equilibrated at 5% B for 3 min. Total run time was 11 min. GAA eluted at 2.6 min. Peak areas were determined using Lab Solutions Post-run Browser software (Shimadzu). The GAA concentration in the tissue lysate was calculated via linear regression of a serially diluted external (unlabeled) standard series using the isotope dilution technique, after which the concentration was adjusted to the total lysate volume, dilution factor and then normalized to tissue dry mass.
