# A Serum Metabolomic Signature for the Detection and Grading of Bladder Cancer
## 2 Materials and Methods
### Metabolome Analysis by GC-MS  
Serum metabolites were extracted, purified, and derivatized using the MetaboPrep GC kit (Theoreo srl, Montecorvino Pugliano, Italy) as reported in [PERSON] et al. [14; 15; 16; 17; 18], prior to GC-MS analysis. Briefly, 50 \(\upmu\)L of serum were pipetted into a microcentrifuge tube containing the extraction solution and the internal standard (2-isopropyl malic acid). The tubes were vortexed at 1250 rpm for 30 min; the solution was then centrifuged for 5 min at 16,000 rpm at 4 \({}^{\circ}\)C; 200 \(\upmu\)L of resultant supernatant was transferred to a separate microcentrifuge tube containing a purification solution and subsequently vortexed at 1250 rpm for 30 s and then centrifuged for 5 min at 16,000 rpm at 4 \({}^{\circ}\)C. The supernatant (175 \(\upmu\)L) was then transferred into a glass vial and frozen at \(-80\)\({}^{\circ}\)C prior to being freeze-dried overnight. Derivatization of the metabolites in the freeze-dried samples was conducted in two steps: first 50 \(\upmu\)L of methoxylamine hydrochloride in pyridine was added and vortexed at 1200 rpm for 90 min; secondly, 25 \(\upmu\)L of the derivatization solution containing N,O-Bis(trimethylsilyl)trifluoracetamide (BSTFA) and trimethylchlorosilane (TMCS) was added and the vials were vortexed again at 1200 rpm for another 90 min. The derivatized metabolites (75 \(\upmu\)L) were transferred to a 100 \(\upmu\)L vial insert to facilitate auto-sampler injection. Vials were centrifuged for 5 min at 16,000 rpm at 4 \({}^{\circ}\)C, before injection into GC-MS.  
Two \(\upmu\)L of derivatized samples was injected into the GCMS-2010 SE (Shimadzu Corp., Kyoto, Japan). Chromatographic separation was achieved with helium as carrier gas flowing through a 30 m \(\times\) 0.25 mm CP-Sil 8 CB fused silica capillary GC column with 1.00 \(\upmu\)m film thickness (Agilent, J&W, Santa Clara, CA, USA). The initial oven temperature of 100 \({}^{\circ}\)C was held for 1 min and subsequently raised at 6 \({}^{\circ}\)C/min to 320 \({}^{\circ}\)C and held for 2.33 min. The injector temperature was 280 \({}^{\circ}\)C and the MS transfer line was held at 290 \({}^{\circ}\)C. The gas flow was set to a constant linear speed of 39 cm/s, and the split flow was set to 1:5. The mass spectrometer operated with electron impact ionization (70 eV) in full scan mode with a range of 35-600 m/z and a scanning speed of 3333 amu/sec and a solvent cut time of 5 min. Samples were divided in batches, each one made up of 25 samples. Each individual batch was monitored using 4 controls: a solvent blank injection to monitor for carryover, an injection of a standard mix, an injection of a pooled sample solution, and a duplicate injection of a randomly selected sample in the batch. Two \(\upmu\)L of hexane was used for the instrument blank, while the analytical standards mix contains a solution of 15 analytes (organic acids, sugars, amino acids, steroids and fatty acids) derivatized in the same way as the unknowns. The pooled sample contained 2 \(\upmu\)L of each of 50 randomly selected derivatized unknowns while the duplicated injection was done using a randomly selected sample from the batch. Each analytical batch was validated only if four conditions were met: the solvent blank did not generate any chromatographic peaks; the peak areas of the 15 analytes in the analytical standard (normalized by the internal standard area) were within 10% of the expected value; the standard deviation of peak area (normalized to the internal standard) for the 100 highest intensity peaks of the repeated injection were \(\leq\)15% of the respective signals in the original injection; and, the pooled sample must cluster with all other pooled samples within 5% of the total area using a partial least square discriminant analysis (PLS-DA) model built using all the samples analyzed. Gas chromatography-mass spectrometry signals consistently found in at least 80% of the samples were considered.