Agilent 1260 series instrument

Powdered sample (1.0 g) was extracted with water (50 mL) in a 100 mL conical flask. The mixture was sonicated for 30 min at the extraction temperature of 60 °C, and the supernatant was filtered under vacuum after cooling to room temperature. Subsequently, the solution was filtered through a 0.45 μm membrane before HPLC analysis. In order to obtain the linear range of quantification, a stock standard solution of L-theanine (6 mg/mL) was prepared with water and stored at 4 °C, and finally the standard curve of L-theanine in the range from 0.05–2 mg/mL was determined.
To determine the content of L-theanine, a RP-HPLC method was performed by using an Agilent 1260 series instrument (Palo Alto, CA, USA). A reverse-phase Agilent TP-C18 column (4.6 mm × 250 mm, 5 μm) was used. The column temperature was maintained at 30 °C. The injected volume was 10 μL and the flow rate was 1.0 mL/min. The mobile phases which were used for gradient HPLC were (A) 0.1% phosphoric aqueous acid and (B) methanol. The detailed gradient was given as follows: 2% B at 0–8 min, 2–95% B at 8–10 min, 95% B at 10–20 min, and 95–2% at 20–25 min. The DAD detection was performed at 210.4 nm. The determination of L-theanine in tea samples is calculated according to the chromatographic peak area of the L-theanine standard substance.

Blood samples were thawed at room temperature and then treated as follows. First, samples were centrifuged for 15 min at 8000 × g. The plasma was deprotonized, and supernatants (100 μL) were mixed with 300 μL methanol. The mixture was then centrifuged at 10000 × g for 12 min at 4°C. The supernatant was collected for UPLC-TQ-MS analysis, which was performed on an Agilent 6460 Triple Quadrupole Mass Spectrometer with an electrospray ionization (ESI) source, coupled with an UPLC Agilent 1260 series instrument.
The chromatographic separation was achieved with an Agilent Poroshell 120 EC-C18 column (2.1 mm × 100 mm, 2.7 μm). The mobile phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile containing 0.1% formic acid. The elution conditions were optimized as follows: 25–65% B (0–4 min), 65–85% B (4–6 min), 85–25% B (6–6.1 min), and 25% B (6.1–9 min). The flow rate was 0.25 mL/min. The column and autosampler were maintained at 30°C. The injection volume was 2 μL. Mass spectra were acquired in ESI mode using nitrogen gas at a temperature of 350°C, a nebulizer pressure of 15 psi, a flow rate of 12 L/min, and a capillary voltage of 3000 V. Total ion chromatograms were obtained from m/z 200 to m/z 1500 in MS/MS positive mode.

Saussurea costus (Falc.) Lipsch. root was obtained from the local market “El-Gelany” of Menoufia in January 2022. Dr Esraa Ammar from the Plant Ecology Department, Faculty of Science, Tanta University, kindly verified the plant identification. A voucher specimen (PG-A-SC-W-12) was kept at the Plant Ecology Department. Saussurea costus root powder (300 g) was extracted with 70% methanol in water three times. Next, the aqueous extract was evaporated under reduced pressure to yield SCRE residue (23.09 g).
HPLC analysis of the SCRE was performed according to Seliem et al. [41 (link)] with some modifications. The SCRE was analyzed using an Agilent 1260 series instrument and Eclipse C18 column (4.6 mm × 250 mm i.d., 5 µm). Separation was performed at a flow rate of 0.9 mL/min. The mobile phase consisted of water (reservoir A) and 0.5% trifluoroacetic acid in acetonitrile (reservoir B) at a concentration of 0.1%. The mobile phase was sequentially programmed with a linear gradient as follows: 0 min (82%A); 0–5 min (80%A); 5–8 min (60%A); 8–12 min (60%A); 12–15 min (82%A); 15–16 min (82%A); and 16–20 (82%A). A multi-wavelength UV detector was used for detection at 280 nm. The injection volume for each sample solution was 5 μL. The column temperature was retained at 40 °C.