Reverse-Phase HPLC Analysis of Venoms

Reverse-phase high-performance liquid chromatography (RP-HPLC, or LC for short) was carried out using a Shimadzu HPLC system managed by Shimadzu LabSolution software (Shimadzu, s-Hertogenbosch, The Netherlands). Four types of samples were analyzed in the order specified in Table 1 of the SI: 50 μL water injections acting as blanks, 50 μL of each of the mentioned venoms spiked with d9Caffeine (2.5 µM; used as a standard), a mixture of standards (Phenylalanine 2.5 μM, Caffeine 250 nM, d9-Caffeine 250 nM, Nortriptyline 250 nM and Metoprolol 250 nM) and 50 μL of Naja siamensis venom spiked with the said mixture to test the repeatability of the method and the capability of those standards to normalize the intensities of the features found in Naja siamensis. All samples were injected using a SIL-30AC autosampler, and the column used was a Waters Xbridge Peptide BEH300 C18 analytical column (100 × 4.6 mm, 3.5 µm particle size and 300Å pore size with a flow rate of 0.5 mL/min). Mobile phase A consisted of 97.9% H₂O, 2% ACN, 0.1% FA, and mobile phase B consisted of 97.9% ACN, 2% H₂O, 0.1% FA. The gradient program was set as follows: linear increase to 50% B in 30 min, followed by linear increase to 90% B in 4 min, isocratic elution for 5 min at 90% B, down to 5% B in 1 min and then equilibration for 10 min.