Authentic reference compounds

Photometric assay (absorbance detection at 440 nm) was used for determination of the α‐keto‐acid product formed from d‐Met by Z‐DAAO. The assay is based on reaction of α‐keto‐acid with 2,4‐dinitrophenylhydrazine (1.0 mM in 2 M HCl; Bolivar et al., 2015 (link)). Samples were centrifuged shortly at 7,000 rpm (Phoenix Instrument CD‐1008 mini‐centrifuge) to remove the co‐immobilizate. The supernatant was directly incubated with 2,4‐dinitrophenylhydrazine.
Reaction samples were further analyzed with ion‐pairing HPLC. For this, they were mixed with the same volume of pure acetonitrile to stop the reaction. Solid material was centrifuged off for 30 min. d‐Met and α‐keto‐γ‐(methylthio)‐butyric acid were quantitated (UV absorbance at 210 nm) using authentic reference compounds (Sigma‐Aldrich) for calibration. A Shimadzu Prominence HPLC‐UV system (Shimadzu) equipped with a Chromolith® HighResolution RP‐18 column (100 × 4.6 mm²; Merck Chemicals) and a UV detector (210 nm) was used. Water containing tetrabutylammonium phosphate (6 mM) as an ion‐paring reagent was used for elution (40°C) at a flow rate of 2 ml/min.

The identification of the volatile oil components was performed by their Retention Indices (marked as RI in Table 2) and their mass spectra, by comparison with a NIST database mass spectral library [49 ], as well as with literature data [17 ,50 ].
Authentic reference compounds purchased from Sigma-Aldrich were also used. Retention indices were calculated using a n-alkane series (C₆–C₃₂) under the same GC conditions as for the samples. The relative amounts of individual components of the essential oil were expressed as percent peak area relative to total peak area from the GC/FID analyses of the whole extracts.