Determination of Ethyl Maltol Content in Soybean Oil by Wayeal LCMS-TQ9200 Liquid Chromatography-Tandem Mass Spectrometry System

Ethyl maltol is widely used in the food industry as a highly efficient and broad‑spectrum flavor enhancer and modifier to intensify and harmonize sweet, fruity, and creamy notes in food products. According to China’s National Food Safety Standard for the Use of Food Additives (GB 2760‑2024), ethyl maltol is permitted to be used at appropriate levels according to production needs in specific food categories.

However, since ethyl maltol can effectively mask the oxidative off-flavors in oils and enhance their taste, there have been repeated incidents in recent years involving its unauthorized addition to edible vegetable oils, including soybean oil. Therefore, establishing an accurate, sensitive, and efficient analytical method for monitoring the illegal addition of ethyl maltol in soybean oil and other edible oils holds significant practical importance for ensuring food safety, safeguarding consumer rights, and maintaining market order.

Soybean oil has a complex matrix composition, primarily consisting of triglycerides, free fatty acids, phospholipids, and natural tocopherols. These coexisting components can cause significant matrix interference during the analysis, severely affecting the detection of the target analyte ethyl maltol. Liquid chromatography - tandem mass spectrometry combines the excellent separation capability of liquid chromatography with the high selectivity and high sensitivity of tandem mass spectrometry. This technique has become a powerful tool for the analysis and identification of trace illegal additives in food products.

Keywords: LCMS, Soybean oil, Ethyl maltol.

1. Instruments and Reagents

1.1 Configuration List of LCMS

Table 1 Instrument Configuration List

1.2 Reagents and Standards

Table 2 Table of Reagents and Standards

1.3 Experimental Materials and Auxiliary Equipment

Ultrasonic cleaner

Vortex mixer

High-speed centrifuge

2. Experiment Method

2.1 Liquid Chromatography Conditions

Column: C18, 1.7 µm, 2.1×50 mm

Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in methanol

Flow rate: 0.3 mL/min

Column temperature: 40 ℃

Injection volume: 2µL

2.2 Pretreatment Method

Accurately weigh 10g of the sample (accurate to 0.01g) and transfer it into a 50mL polypropylene centrifuge tube. Precisely add 10 mL of methanol using a pipette, vortex for 2 minutes, and centrifuge at 9,000rpm/min for 10 minutes at 4°C. Transfer the supernatant to a 20mL graduated stoppered test tube. Repeat the extraction of the remaining oil layer with another 10mL of methanol, combine the supernatants, and dilute to 20mL with methanol. Filter the solution through a microporous membrane (0.22µm, organic phase) and use it for liquid chromatography-tandem mass spectrometry analysis.

2.3 Solution Preparation

2.3.1 Ethyl maltol standard solution: 100µg/mL

2.3.2 Ethyl Maltol Standard Working Solutions: Dilute the ethyl maltol standard stock solution stepwise with methanol to prepare standard solutions at concentrations of 1.25µg/mL, 2.5µg/mL, 5µg/mL, 25µg/mL, and 50µg/mL. Accurately weigh 10g (accurate to 0.01 g) of a negative sample corresponding to the test sample, add 200µL of each standard solution separately, and process the mixtures simultaneously with the test samples through extraction. Then prepared the working solutions with final concentrations of 12.5ng/mL, 25ng/mL, 50ng/mL, 250ng/mL, and 500ng/mL. Prepare these solutions immediately before use.

2.3.3 0.1% Formic Acid Aqueous Solution: Take 1mL of formic acid and dilute to 1000mL with water. Filter through a membrane (0.22µm, aqueous phase) before use.

2.3.4 0.1% Formic Acid in Methanol Solution: Take 1mL of formic acid and dilute to 1000mL with methanol. Filter through a membrane (0.22µm, organic phase) before use.

3. Experiment Result

3.1 Standard Chromatogram

The determination of ethyl maltol content was completed within 7 minutes, with good peak shape and compound response, meeting the requirements of experimental analysis.

Fig 1 Ethyl Maltol Chromatogram (250ng/mL)

3.2 Linear Range

The standard curve was prepared through serial dilution of the ethyl maltol standard solution. The linear range was 12.5–500ng/mL, with deviations between the linear detection results and known concentrations less than the maximum allowable deviation. All R² values exceeded 0.9999, indicating excellent linearity.

Table 3 Linearity Range Table of Compounds

Fig 2 Linearity Results of Compounds

3.3 LOD and LOQ

In this method, the signal-to-noise ratio (S/N) of ethyl maltol at 12.5ng/mL is significantly greater than 10. When S/N = 10 is defined as the theoretical limit of quantitation (LOQ), and S/N = 3 is defined as the theoretical limit of detection (LOD). The calculated limit of detection (LOD) is 1.07ng/mL, and the limit of quantitation (LOQ) is 3.56ng/mL.

Table 4 LOQ and LOQ of Compounds

Compound	Signal-to-Noise Ratio (S/N)	Limit of Detection (ng/mL)	Limit of Quantitation (ng/mL)
Ethyl Maltol	35.079	1.07	3.56

Fig 3 Chromatogram of Compounds LOQ

3.4 Precision

Take blank solvent and negative samples to the ethyl maltol standard solution to prepared a final concentration of 25ng/mL, and injected consecutively for 7 replicates to evaluate precision. The results are shown in the table below. The RSD of ethyl maltol was within 3%, meeting the standard precision requirement of ≤15%.

Table 5 Compounds Precision

Compound	Sample	RSD
Ethyl Maltol	Spiked Methanol	2.407%
	Spiked Soybean Oil	2.915%

Fig 4 Precision Chromatogram of Methanol Spiked Sample

Fig 5 Precision Chromatogram of Soybean Oil Spiked Sample

3.5 Recovery Rate

A 100µg/mL ethyl maltol standard solution was diluted with soybean oil to 25ng/mL. After sample pretreatment, extraction, and filtration, seven consecutive injections were performed to compare recovery rates. The results are shown in the table below. The recovery rate of ethyl maltol was 102.53%, meeting the acceptable recovery range of 80%–120%.

Table 6 Compounds Recovery Rate

Compound	Spiked Concentration (ng/mL)	Recovery (%)
Ethyl Maltol	25	102.53%

3.6 Blank Residue

After injecting the highest point (500ng/mL) of the standard curve, a blank reagent was injected. No blank carryover was detected after calculation.

Fig 6 Blank Residue Chromatogram

3.7 Sample Test

After pretreatment, a sample of a certain brand of soybean oil was injected and no target compound was detected.

Fig 7 Sample Test Chromatogram

4. Conclusion

This method employs the Wayeal’s LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system for the determination of ethyl maltol in soybean oil. The data show that the chromatographic peaks of the method show good peak shape with no tailing, and the sensitivity meets the requirements. The linear correlation coefficients are all greater than 0.9999, the precision is within 3%, and the spike recovery rates range between 100% and 105%. No system carryover was observed for high-concentration samples. These results demonstrate that the method, equipped with the Wayeal’s LC-MS/MS system, fulfills the routine qualitative and quantitative detection requirements for the target analyte in samples.

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