Method for Determining 4 Antioxidants in Steamed Flour Products Using the Anyeep TQ9120 Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry System

Yeast products are derived from yeast cultures using sugars and grains as raw materials, supplemented with necessary nutrients. Through biotechnology and fermentation processes, these yield nutrient-rich yeast or yeast-derived products widely used in food, health supplements, biopharmaceuticals, brewing, biochemical engineering, petroleum, and agriculture/livestock sectors. To enhance product stability, antioxidants and preservatives are added during production. Due to 2, 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and 2,6-di-tert-butyl-4-methylphenol (BHT) offering low cost and effective antioxidant properties, they are the most widely used antioxidants in China. However, all pose potential health risks; therefore, their use is strictly limited under China's Hygienic Standards for Food Additives. Consequently, monitoring and testing antioxidant levels is a key responsibility of quality supervision authorities. GB2760-2014 also specifies maximum usage limits. This method follows GB 5009.32-2016 and employs external standard quantification.

Instruments and Reagents

AnyeepTQ9120 Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry System

Acetonitrile (MS grade, Thermo Fisher, USA)

Methanol (mass spectrometry grade, Thermo Fisher Scientific, USA)

n-Hexane (mass spectrometry grade, Thermo Fisher Scientific, USA)

Formic acid (mass spectrometry grade, Anpel Shanghai)

C18 SPE Cartridge (Shanghai ANPEL)

Liquid Chromatography Conditions

Mobile Phase A: 0.1% Formic Acid in Water

Mobile Phase B: Methanol

Column: ACQUITY UPLC® BEH C18 1.7 μm, 2.1×100 mm

Column Temperature: 40 ℃

Flow rate: 0.3 mL/min

Injection Volume: 10 μL

Mass Spectrometry Conditions

Ionization Source: Electrospray Ionization (ESI-)

Source Temperature: 550°C

Spray voltage: 5000V

Air Curtain Air: 45 psi

Mist: 60 psi

Auxiliary Air: 60 psi

Crash Air: 12

Scan Mode: MRM

Sample Processing

Sample ProcessingWeigh 2 g of the sample (to the nearest 0.01 g) into a 50 mL capped centrifuge tube. Add 10 mL water and 5 mL n-hexane saturated with acetonitrile, then vortex and sonicate for 3 min. Add 10 mL acetonitrile saturated with n-hexane, vortex thoroughly at 2500 r/min for 3 min, add 2 g NaCl, vortex for 3 min, and sonicate for 5 min. Centrifuge at 10000 r/min for 5 min. Transfer 5 mL of the acetonitrile layer to a 15 mL capped centrifuge tube, evaporate to dryness under nitrogen stream in a water bath at 40°C, and reconstitute with mobile phase [V(0.1% formic acid-water):V(methanol) = 1:1] (1 mL). Add 100 mg C18 SPE sorbent, mix thoroughly, and centrifuge at 8000 r/min for 10 min. Filter the supernatant through a 0.22 μm nylon filter membrane and analyze by liquid chromatography-tandem mass spectrometry.

Standard Curve PreparationAccurately pipette an appropriate volume of the mixed standard working solution, add acetonitrile to prepare a series of mixed standard solutions with BHA and Ionox concentrations of 0.5 μg/L, 1 μg/L, 2 μg/L, 5 μg/L, 10 μg/L, 20 μg/L, 50 μg/L, 100 μg/L, 200 μg/L, and 500 μg/L; and BHT and TBHQ concentrations of 10 μg/L, 20 μg/L, 50 μg/L, 100 μg/L, 200 μg/L, 500 μg/L, 1000 μg/L, 2000 μg/L, and 5000 μg/L. These solutions are intended for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Summary

This study developed a UHPLC-MS/MS method using the Anyeep TQ9120 system for analyzing 4 antioxidants. Results demonstrate that the method offers excellent sensitivity, stability, and linearity, making it suitable for quantifying antioxidant residues in steamed flour products.