Acquity uplc tqd system

Amino acids were determined from 200 mg of sprouts combined with 1 mL of 80% (v/v) aqueous ethanol [24 (link)]. For accurate evaluation and correction of varied mass spectrometry reactions, norvaline was employed as an internal standard. The homogenate was centrifuged for 30 min at 14,000× g. The supernatant was added to fresh tubes and then dried. Next, chloroform (1 mL) was used for pellet resuspension followed by centrifugation (14,000× g for 30 min), then further extraction of the plant in water was performed and the extract was blended with the chloroform-suspended pellet. Then, the combined extract was centrifuged at 20,000× g for 10 min. Thereafter, the aqueous phase was filtered (Millipore microfilters, 0.2 μm pore size) for amino acid assessment. Amino acids were separated on a BEH amide 2.1 × 50 column (Waters Acquity UPLC-tqd system, Milford, MA, USA) [21 (link)]. A 10 μL sample was inserted and elution was carried out with a gradient of 0.1% formic acid (FA) in H₂O and 0.1% formic acid in acetonitrile. The sample was kept at 20 °C and at a 30 °C column temperature.

Ethanolic root and shoot extracts of S. nigrum plants were used for assaying free amino acids (FAAs) levels using a Waters Acquity UPLC-tqd system (Milford, MA, United States) equipped with a BEH amide 2.1 × 50 column according to (Sinha et al., 2013 (link)) with the minor modification previously described by (AbdElgawad et al., 2015 (link)).

In homogenized shoots, the amounts of chlorophyll a and b, as well as carotenoids, were measured in acetone [33 (link)]. The photorespiration-related essential enzymes including GO (Glycolate oxidase) and HPR (hydroxy pyruvate reductase) activities were assessed according to Feierabend and Beevers [34 (link)] and Schwitzguebel and Siegenthaler 1984, respectively. Moreover, the glycine/serine ratio known as an indicator of photorespiration) [35 (link)]. A Waters Acquity UPLC-TQD system (Waters, Milford, MA, USA) with a BEH amide 2.150 column was used to quantify glycine and serine.

The extraction of amino acids was performed using sprouts homogenized in 1 mL of 80% (v/v) aqueous ethanol, supplemented with norvaline, to control the loss of amino acids during extraction. Amino acid content was assessed using the Waters Acquity UPLC-tqd system (Waters Corporation, Milford, MA, USA) along with a 2.1 × 50 amide BEH column, as described by AbdElhawad et al. [35 (link)]. Nitrogen levels were assessed using a CN element analyzer (NC-2100; Carlo Erba Instruments, Milan, Italy).

Ethanolic extracts of rye seedlings were used to assay free amino acids (FAAs) levels using a Waters Acquity UPLC-tqd system (Milford, MA, USA) equipped with a BEH amide 2.1 × 50 column according to Sinha et al. (2013) (link) with the minor modification previously described by AbdElgawad et al. (2015) (link).