Agilent 6410 triple quadrupole

The analysis of the endocannabinoids AEA and 2-AG in plasma was performed by a validated method previously described (Pastor et al. 2014 (link)). Briefly, aliquots of 0.5 mL of plasma were transferred to 12-mL glass tubes, spiked with Deuterated internal standards, diluted with 0.1 M ammonium acetate buffer (pH 4.0), and extracted with tert-butyl methyl ether. The dry organic extracts were reconstituted in 100 μL of a mixture water:acetonitrile (10:90, v/v) with 0.1% formic acid (v/v) and transferred to HPLC vials. Twenty microliters was injected into the LC/MS-MS system. An Agilent 6410 triple quadrupole (Agilent Technologies, Wilmington, DE, USA) equipped with a 1200 series binary pump, a column oven, and a cooled auto-sampler (4 °C) was used. Chromatographic separation was carried out with a ACQUITY UPLC C18-CSH column (3.1 × 100 mm, 1.8 μm particle size) (Waters, Yvelines Cedex, France) maintained at 40 °C with a mobile phase flow rate of 0.4 mL/min. The composition of the mobile phase was as follows: A: 0.1% (v/v) formic acid in water; B: 0.1% (v/v) formic acid in acetonitrile. Detection was done by selection reaction monitoring (SRM). Quantification was performed by isotope dilution. Deuterated internal standards were obtained from Cayman Chemical (Ann Arbor, MI, USA), and solvents were from Merck (Darmstadt, Germany).

[ABA], [PA] and [DPA] in xylem sap were measured using liquid chromatography/mass spectrometry (Agilent 6410 Triple Quadrupole LC-MS/MS with Agilent 1200 series HPLC, Agilent Technologies Inc., Santa Clara, USA) using a stable isotope dilution assay [30 (link)]. The dry samples of xylem sap were dissolved in 30 μL 10 % acetonitrile (v/v) containing 0.05 % acetic acid (v/v). This acetonitrile solution also contained the deuterated internal standards D3-7′,7′,7′-DPA, D3-7′,7′,7′-PA and D6-3′,5′,5′,7′,7′,7′-ABA, all at a concentration of 100 pg/μL. The column used was a Phenomenex C18(2) 75 mm × 4.5 mm × 5 μm and column temperature was set at 40 °C. The solvents used were nanopure water and acetonitrile, both added with 0.05 % acetic acid (v/v). Samples were eluted with a linear 15 min gradient starting at 10 % acetonitrile (v/v) and ending with 90 % acetonitrile (v/v). Compounds were identified by retention times (DPA = 7.25–7.75, PA = 9.0–9.5 and ABA = 10.5–11.0 min) and multiple reaction monitoring of mass-to-charge ratio (m/z) for parent and product ions of native (DPA = 281/284, PA = 279/282 and ABA = 263/269) and deuterated internal standards (DPA = 171/174, PA = 139/142 and ABA = 153/159) [30 (link)].

All assays were prepared as previously described Levin et al. (2017) (link). Briefly, assays contained 20 mM Tris-HCl pH 7.5, 100 mM KCl, 0.8 mM NADH, 3 µM P5CR, 0.2 mM Hyp, and 0.3 µM HypD. HypD was first activated under conditions described for EPR spectroscopic assays. All assays were carried out in triplicate and were initiated by adding Hyp into reaction mixtures, which were then incubated for 21 hr at 22°C. Upon removal from the anaerobic chamber, reactions were quenched with a 2 × volume of methanol and protein precipitates were removed by centrifugation (15,200 g, 10 min). Supernatants were further diluted 30-fold with water for Pro detection and 7.5-fold for Hyp detection by LC-MS/MS using previously published methods (Levin et al., 2017 (link)). Briefly, LC-MS/MS analysis of Pro and Hyp were performed on an Agilent 6410 Triple Quadrupole LC-MS instrument (Agilent Technologies) using a Luna SCX column (5 μm, 100 Å, 50 × 2.0 mm, Phenomenex). Precursor and product ions of m/z 116.1 and m/z 70.1 were monitored for proline detection whereas m/z 132.1 and m/z 86.1 were monitored for hydroxyproline. Amino acid standards were dissolved in water and diluted to a range of concentrations to generate standard curves used to quantify Pro and Hyp in samples. Source data can be found in Figure 5—source data 1.