4.3.2. Urine Normalization by Specific Gravity

Specific gravity (SG) measurements were made with a portable digital refractometer (Atago UG-α, Tokyo, Japan). The refractometer had a urinary SG range from 1.000 to 1.060 with a resolution of 0.001. Urine samples were thawed at room temperature in an ultrasonic bath for 10 min and then centrifuged (4000 rpm). An aliquot of urine (100 μL) was placed upon the lens of the refractometer previously calibrated with LC–MS-grade water to measure SG values. Samples were then split into two aliquots. Urinary metabolite levels were normalized by SG-diluting each aliquot with water or ACN:H2O in variable amounts for RP and HILIC analysis, respectively. Dilutions were performed to bring all samples to the same specific gravity value.

#### 4.3.3. Urine Samples Preparation

All samples were further diluted by 3-fold with ISWS-A for RP analysis or ISWS-B for HILIC analysis. Samples were vortexed and centrifuged (13,000 g for 10 min), and the supernatant (350 μL) was transferred to a 96-well plate and randomized for LC–MS analyses.
