Bstfa

A total of 10 µl of pyridine with 2.5 mg/ml hexachlorobenzene (internal standard), along with 20 µl of N, O-Bitrifluroacetamide (BSTFA) (Thermoscientific, Waltham, MA, USA) were added to 30 µl of the diluted hemolymph solution (previously lyophilized) inside a 1.5 ml microcentrifuge tube, sealed with parafilm to minimize evaporation. This was then heated to 70° C for 3 hr to allow for the derivatization reaction to go to completion. The mixture was then transferred to a 2 ml amber microvial with a spring loaded 100 µl glass insert.

Prior to the injection on the GC–MS equipment, approximately 2 mg of the samples were: (1) hydrolysed: (a) using acid conditions heating at 110 °C in 6 M HCl for 24 h; (b) using moderately basic conditions and heating at 140 °C in a phosphate buffer solution (0.01 M, pH 8) for three days; (c) using basic conditions and heating at 110 °C in 5 N NH₄OH for 24 h. Then, the samples were freeze-dried. (2) Each hydrolysed sample in 100 μL of BSTFA with 1% TMCS (BSTFA = N,O-bis(trimethylsilyl)trifluoroacetamide, TMCS = trimethylchlorosilane, obtained from Thermo Scientific) was heated at 80 °C for 3 h to obtain the respective trimethylsilyl derivatives. The derivatized samples were analysed by GC–MS using the same GC oven programme described in^{28 (link)}, method a, or described in^{29 (link)}, method b. For the sample hydrolysed using the phosphate solution, the solvent delay was 20 min.

Dried samples were washed three times with methanol and derivatized first with 20 μl methoxyamine hydrochloride (Merck, 89803) solution in pyridine (Merck, 270970) (20 mg ml⁻¹) for 16 h and then with 20 μl BSTFA + 1% TMCS silylation reagent (Thermo Fisher Scientific, TS-38831) for 30 min. Metabolite analysis was performed by GC–MS using an Agilent 7890B-7000C GC-triple-quadrupole MS. Splitless injection (injection temperature 250 °C) onto a 30 m + 10 m × 0.25 mm DB-5MS + DG column (Agilent J&W) was used, using helium as the carrier gas, in electron ionization mode. The initial oven temperature was 70 °C (2 min), followed by temperature gradients to 295 °C at 12.5 °C min⁻¹, then to 320 °C at 25 °C min⁻¹ (held for 3 min). Data analysis was performed using our in-house-developed software MANIC (v.3.0), based on the software package GAVIN^{74 (link)}. Label incorporation was calculated by subtracting the natural abundance of stable isotopes from the observed amounts. Metabolites were identified and quantified in comparison to authentic standards and scyllo-Inositol as an internal standard (Sigma, I8132).

For each of the compounds considered here (Fig. 1), aqueous solutions of known concentrations were prepared using Milli-Q water at an initial concentration of 1000 ppm. In some cases, it was necessary to adjust the pH to achieve complete dissolution of the standards. All these stock solutions were in turn diluted 1:10. From these samples, 100 µL was frozen and freeze-dried and then derivatized with BSTFA with 1% TMCS (BSTFA = N,O-bis(trimethylsilyl)trifluoroacetamide, TMCS = trimethylchlorosilane, obtained from Thermo Scientific) to obtain their corresponding TMS derivatives, as explained below, and used for analysis by GC–MS, as indicated in the following section.
All the pyrimidines and triazines shown in Fig. 1 are available commercially and were supplied by Sigma–Aldrich, Merck, Fluka, or Panreac (for further details, see Supplementary Information).
In addition, barbituric acid (8), orotic acid (10), melamine (11) and cyanuric acid (14) were quantified by multiple-point external standardization method. Calibrate lines for each analyte were calculated using at least six standard solutions with concentration from 10 to 150 ppm. The standard solutions were injected × 3 in the GC–MS equipment to sure the reproducibility of the measurements.