Prominence model

The identification of flavonoids and other phenolic compounds was conducted in the liquid chromatography (LC) (Prominence model, Shimadzu, Japan) linked to a mass spectrometer ion trap (Esquire HCT model, Bruker Daltonics, Germany) and electrospray ionization interface (ESI). The separation conditions were the same as those used for the HPLC-DAD, described in section High-Performance Liquid Chromatography With Diode Array Detector (HPLC-DAD). After passage through the DAD, the flow was changed to 0.2 ml/min to the passage in the mass spectrometer. The ESI was maintained in positive mode. The mass detector was programmed to perform full scan between m/z 100–1000. The ionization energy for the positive mode was 3500 V. The identity of the compounds was evaluated by comparing the mass spectrum obtained with the commercial standards and, or literature data (Lee and Mitchell, 2011 (link)). To confirm the identity, the HPLC retention time of commercial flavonoid standards (quercetin 3-O-glucoside, quercetin aglycone, and cyanidin 3-O-glucoside) was used for comparison.

Aflatoxin M₁ levels in bokina and milk were analyzed using high-performance liquid chromatography (HPLC) techniques as described with slight modification. Samples were analyzed using reverse-phase HPLC (Shimadzu Prominence model; Kyoto, Japan) consisting of a binary solvent delivery system (LC-20AB), a degasser (DGU-20A3), an auto-sampler (SIL-20ACHT), a column temperature controller (CTO - 10AS VP) and a fluorescence detector (RF-10AXL).5 (link) The latter was set at a wavelength of excitation 360 nm and emission 440 nm. The mobile phase consisted of 45% methanol and 55% water and the flow rate was 1 ml/min. A C18 column (Tskgel ODS, diameter 5 µm, length x width, 150 mm ×4.6 mm) was used. The column temperature was maintained at 40°C and the injection volume was 20 µl.

MBP-11901 (Figure 1) was prepared at 99.64% purity as previously reported [10 (link)] with further modifications for pilot-scale production. HPLC analysis was performed using a Prominence model (Shimadzu Corporation, Kyoto, Japan) equipped with a photodiode array detector (SPD-M20A) to confirm the purity of MBP-11901 (Figure S1). Test sample solution was prepared in water at a concentration of 200 ppm, and a SunFire C18 column (100 Å, 5 µm, 4.6 mm × 250 mm) was used for analysis. Mobile phases consisted of 0.1% formic acid-buffered water (A) and acetonitrile (B), with the gradient mode at a flow rate of 1 mL/min. Injection volume was 10 µL and the wavelength was fixed at 310 nm for UV detection. Data were acquired based on the following gradient system: T_min/B%—T₀/10, T_1.5/10, T_4.5/60, T_10.5/80, T₁₅/80, T_28.5/10, T₃₀/10.