Accq tag

To determine the concentration of 16 amino acids in the human milk samples, 10 mL of 6 M HCl (containing 0.1% phenol) was added to a hydrolysis tube that contained 1 mL of human milk. Following vacuum and nitrogen flush, repeated three times, the tube was sealed under a nitrogen blanket and the sample hydrolyzed by placing in an oven at 110°C for 24 h. The hydrolysate was then quantitatively transferred to a volumetric flask and made up to a volume of 50 mL using distilled water. A total of 15 µL of filtered hydrolysate solution was quantitatively pipetted into a derivatization tube, dried under vacuum, and then combined with alpha-amino butyric acid as an internal standard and analyzed using AccQ-Tag (Waters Corporation, Milford, MA) derivatization and high-performance liquid chromatography (HPLC) (25 , 26 ).

Phloem sap was extracted from plant treatments according to the methods of King and Zeevaart (1974 (link)) and Gould et al. (2007 (link)) on 11 November (Table 1). The terminal leaflet was excised from a compound leaf that was midway along the shoot axis, after which the excised leaflet was inserted immediately into a 1.5-ml solution of 20 mM ethylenediamine tetraacetic acid (EDTA; pH 7.0) for 2 h in the dark at 25°C. The leaflet samples were then transferred to 1.5-ml vials that contained deionized water for 12 h of sap collection in the dark. The phloem sap samples exuded in diH₂O for 12 h were used for chemical analysis. The petioles were removed from the vials, and the phloem sap fraction was frozen at −20°C until analysis. The amino acids in the phloem exudates were separated by reverse-phase HPLC (Jones et al., 1981 (link)) using a Perkin Elmer Series 200 LC system in conjunction with Waters AccQ•Tag™ derivatization chemistry, columns and fluorescence detection.

The amine metabolites were measured using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) employing an Accq-Tag derivatization strategy adapted from the protocol supplied by Waters. Sample preparation consisted of protein precipitation by the addition of methanol to 5 μL of urine spiked with internal standards. The centrifuged supernatant was then evaporated using a speedvac prior to reconstitution in borate buffer (pH 8.5) with AQC reagent. Chromatic separation was done on an Accq-Tag Ultra column (Waters Chromatography B.V., Etten – Leur, The Netherlands) using a UPLC Agilent Infinity II (1290 Multisampler, 1290 Multicolumn Thermostat and 1290 High Speed Pump; Agilent Technologies, Waldbronn, Germany) coupled to an AB SCIEX quadrupole-ion trap (QTRAP; AB Sciex, Massachusetts, USA). Analytes were detected in the positive ion mode and monitored in Multiple Reaction Monitoring (MRM) using nominal mass resolution. The amine method has been described in detail elsewhere (29 (link)). Metabolites are reported as ‘relative response ratios' (target area/area of internal standard) after quality control (QC) correction.

According to the manufacturer’s instructions, extracted samples were derivatised in AccQ•Tag™ (Waters, Milford, MA, United States). Briefly, ten10 μL of the extract was added to 70 μL of AccQ•Tag Ultra Borate buffer, and finally, 20 μL of the freshly prepared AccQ•Tag derivatisation solution was added, and the sample was immediately vortexed for 30 s. Samples were kept at room temperature for 30 min, followed by 10 min at 55°C. For each batch quality control sample and procedure, blanks were included. Calibration curves were prepared similarly by adding 10 µL of each point of the curve to 70 µL of AccQ•Tag Ultra Borate buffer and 20 μL of the freshly prepared AccQ•Tag s derivatisation solution.

Glucose, glutamine, and lactate quantification were performed using the Cedex Bio analyzer 7100 (Roche). Amino acids were determined by UPLC after derivatization using the AccQ-Tag method (Waters).