Sil 20ac ht

For all measurements, a Shimadzu high performance liquid chromatography (HPLC) apparatus, including binary pumps, degasser, autosampler, column oven and control unit (LC-20AB, SIL-20AC HT, CTO-20AC, CBM-20A, Duisburg, Germany) was used. The HPLC was coupled to an API4000 triple quadrupole mass spectrometer (MS) from Sciex (Darmstadt, Germany). MS ion source parameters were set as follows: ionization voltage, 2.500 V; nebulizer gas, 50 psi; heating gas, 50 psi, curtain gas, 30 psi, temperature, 600 °C; collision gas level, 7. For data acquisition and processing, Analyst (Version 1.6.2, Sciex, Framingham, MA, USA) and MultiQuant software (Version 3.0.1, Sciex, Framingham, MA, USA) were used. Figures were drawn using OriginPro software (Version 2019, OriginLab, Northampton, MA, USA).

HPLC analysis was done on a Shimadzu LC-20AD (Kyoto, Japan) or a Merck Hitachi L-7100 (Darmstadt, Germany) system. Both were equipped with a autosampler (SIL-20AC HT ➔ Shimadzu, L-7250 ➔ Merck) and a refractive index detector (RID-20A ➔ Shimadzu, L-7490 ➔ Merck). A YMC-Pack Polyamine II/S-5 μm/12 nm column (250×4.6 mm) (YMC, Kyoto, Japan) (Holtkamp et al. 2009 (link)) with or without a guard column (20×4 mm) was used. Isocratic elution used acetonitrile/water (75/25, by volume). Twenty-microliter sample was injected. HPLC was operated at a flow rate of 1 mL min⁻¹ at 25 °C. The run time was 30 min. All components of the reaction (sucrose, 2-GG and the corresponding 1-O-regioisomer, fructose, and glucose) were separated, as shown in Figure S1, and quantified. The glycerol was also separated, but due to its presence in excess, it was not used for quantification. Reaction selectivity was calculated on a mole basis as [2-GG]/[Fructose] (see Equation 2 in Supplementary Information).

The HPLC-MSMS system was composed of a LC- Shimadzu prominence LC20AD (Shimadzu, Maryland), with an ultramino 3 um 150 x 2.1 mm 100A aminopropylsilaneRestek column (Restek, Bellafonte, PA) with a flow rate of 0.30 ml/min with degasser and auto-sampler SIL-20AC HT (Shimadzu, Maryland), and column oven CTO-20A (Shimadzu, MD) run at 40°C. Mobile phase A was HPLC grade water with 0.1% formic acid and phase B was acetonitrile with 0.1% formic acid. Elution was programmed to start at 70% phase B for 0.5 minutes, then fall to 5% B at 2 minutes, return to 70%B at 2.5 minutes and equilibrate for two minutes prior to the next sample injection. MS/MS-API 5500 (AB Sciex, US) with turbo-ion probe (ESI) operated at 600C. The transitions monitored described before are listed with the retention times in Table 1 [17 (link)].

The analysis was carried out on Shimadzu prominence HPLC separation module with configuration of LC-20AD binary pumps along with DGU-20A5 degasser unit, SPD-20A dual λ UV detector, SPD-M10Avp Photodiode array detector, SIL-20AC HT autosampler, and CTO-10AS vp column oven. System control, data acquisition, and processing were performed with LC Solutions chromatography software (Version 1.24 SP1). Standard substances were weighed on Sartorius CP 225D analytical balance. A glass vacuum-filtration apparatus (Alltech Associates) was employed for the filtration of buffer solution using 0.22 μm filter obtained from Pall Pvt. Ltd (Bangalore, India). Degassing of the mobile phase was performed by ultrasonication in Oscar Micro clean-103 Ultrasonic bath.
GraceSmart RP C₁₈ (250.0 × 4.6 mm, 5 μm) column was used as a stationary phase. The isocratic mobile phase used was acetonitrile and potassium phosphate buffer (25.0 mM; pH 3.5 ± 0.1, containing 0.2% triethylamine; pH adjusted using dilute orthophosphoric acid) in the ratio of 25 : 75% v/v. The mobile phase was pumped at a flow rate of 1.0 mL/min. The column was maintained at a temperature of 25°C and the eluent was monitored at 282.0 nm. All solutions were injected using acetonitrile: ultrapure water (1 : 1) as diluent. The injection volume was 20 μL with a total run time of 10.0 min.