Qtrap 3200

The detailed procedures of analyzing hair cortisol contents (HCCs) were described elsewhere (Chen et al., 2019 (link)). Briefly, the 1-cm hair strands closest to the scalp were treated by a standard protocol: washing with methanol, cutting into pieces, incubation in methanol, centrifugation, solid-phase extraction, and drying with pure nitrogen gas. The dried residue was redissolved in 50-μl methanol for cortisol analysis that was done on a Qtrap 3200 liquid chromatography-tandem mass spectrometer (ABI, United States). Cortisol was ionized with an atmosphere pressure chemical ionization and identified in positive ion mode using multiple reactions monitoring mode. The assay method had good linearity in the range of 0.8–250.0 pg/mg, showing the square coefficient of correlation at more than 0.99. It also had good sensitivity, accuracy, and precision, showing limits of detection and quantitation at 0.3 and 0.8 pg/mg, intra-day and inter-day coefficients of variation less than 15%, and recovery ranging between 85 and 115% (Chen et al., 2019 (link)), which fit the requirements of hair cortisol measurement.

Proanthocyanidins were degraded in the presence of benzyl mercaptan, and then the degradation products were injected into an Agilent 1200 system (Agilent, Palo Alto, CA, USA) interfaced to a QTRAP 3200 (Applied Biosystems, Foster, USA) with a 250 mm× 4.6mm i.d. 5.0mm Hypersil ODS column (Elite, Dalian, China). The elution system was: 0–45 min, 12–80% B (linear gradient); 45–50 min, 80–12% B (linear gradient). The column temperature was 25°C and the flow rate was 1 mL/min.

The modified method described by Zhou et al. [9] was carried out to characterize Fp and obtain the mDP for each subfraction. Condensed tannins were thiolysis degraded by benzylmercaptan, and then the degradation products were analyzed on an Agilent 1200 system (Agilent, Palo Alto, CA, USA) interfaced to a QTRAP 3200 (Applied Biosystems, Foster, USA) with a 250 mm×4.6 mm i.d. 5.0 µm Hypersil ODS column (Elite, Dalian, China).