*3.4. UPLC–MS Analysis*

#### 3.4.1. Identification of Flavonoid Glycosides by UPLC-TOF/MS

The Shimadzu UPLC ((Shimadzu, Kyoto, Japan) system consists of an online degasser (DGU-20A5R), an auto-sampler (SIL-30AC), two pumps (LC-30AD), and a column oven (CTO-30aHE). Chromatographic separation was performed on a Waters BEHC18 analytical column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA) at 40 ◦C. The mobile phase consisted of 0.2% formic acid and acetonitrile. The linear gradient elution with a constant flow rate of 0.2 mL/min was 10%~10%~40%~95%~10% acetonitrile at 0~1~10~13~15 min. The sample solution and mixed working solutions of 5 μL were injected into the UPLC system by the auto-sampler.

TOF/MS measurements in negative ion mode were performed on a 4600 Q-TOF mass spectrometer (AB Sciex, Concord, CA, USA) equipped with an electrospray ionization (ESI) source with the following parameters: Ion source gas 1 (GS1) at 50 psi, ion source gas 1 (GS1) (N2) at 50 psi, curtain gas at 35 psi, temperature at 500 ◦C, and ionspray voltage floating at −4500 V. The mass range was set to *m*/*z* 100–800. The system was operated under Analyst 1.6 and Peak 2.0 (AB Sciex, Concord, CA, USA) and used an APCI negative calibration solution to calibrate the instrument's mass accuracy in real-time.

#### 3.4.2. Determination of Flavonoid Glycosides by UPLC-QqQ/MS

Chromatographic separation was the same as that used in UPLC-TOF/MS analysis described above. QqQ/MS measurements in negative ion mode were accomplished by a triple quadrupole mass spectrometer equipped with an ESI source (Thermo Fisher Scientific, San Jose, CA, USA). The determination of the target analytes was performed in a multi-reaction monitoring mode. The MS parameters were as follows: Vaporizer temperature and capillary temperature both 350 ◦C, aux gas pressure of 10 Arb, sheath gas pressure of 40 Arb, ion sweep gas pressure of 2 Arb, discharge current of 4.0 μA, and spray voltage of −2000 V. Data collection and processing were conducted with Thermo Xcalibur Workstation (Version 2.2, Thermo).

#### *3.5. Analytical Figures of Merit*

Method validation was performed according to the above UPLC–QqQ/MS conditions. After it was determined by the mixed working solutions I–V, the calibration curves of five analytes were obtained as shown in Table 3 by taking the concentration of each authentic standard as the abscissa (x) and the corresponding peak area as the ordinate (y), respectively. The limit of detection (LOD) and the limit of quantification (LOQ) were measured by a gradual dilution process of the standard stock solutions until the signal-to-noise ratio of 3:1 and 10:1, respectively. The precision was evaluated by standard working solution III, which was tested within one day to determine the intra-day precision and was tested within 3 days to determine the inter-day precision. The repeatability was evaluated by analyzing six independent portions of sample S4 with parallel running. The recovery was carried out by spiking an amount of about 1:1 of authentic standards to six independent portions of sample S4 with parallel running. The validation results are summarized in Table 3, which show that the present developed UPLC–QqQ/MS method meets the requirements of quantitative analysis and was appropriate for the determination of five flavonoid glycosides in lemonade. The analytical figures of merit were compared with those of several other quantitative methods reported for flavonoid glycosides in lemon as shown in Table 4.


**Table 3.** The results of method validation.

HPLC/UV [34] narirutin 2–50 mg/L 1.25 mg/L 2.5 mg/L 83%

hesperidin 2–50 mg/L 1.0 mg/L 2.5 mg/L 74%

HPLC/UV [35] eriocitrin 1.01–50.50 μg/mL 0.02 μg/mL 0.065 μg/mL 103.10%

UPLC/UV [36] eriocitrin 0.5–130 mg/L 6 μg/kg - 90.50%

narirutin 0.05–300 mg/L 5 μg/kg - 87.40% hesperidin 0.05–500 mg/L 8 μg/kg - 92.70% rutin 0.05–310 mg/L 5 μg/kg - 88.40% diosmin 0.01–200 mg/L 8 μg/kg - 100.80%

narirutin 0.505–10.10 μg/mL 0.024 μg/mL 0.18 μg/mL 99.14% hesperidin 5.00–100.00 μg/mL 0.04 μg/mL 0.132 μg/mL 99% rutin 0.101–10.100 μg/mL 0.079 μg/mL 0.263 μg/mL 98.37%
