One-time detection of 13 hormones in milk powder using Waters UPLC-Xevo TQ system

Wang Bing, Yuan Hancheng, Wang Shaozhen, Xia Xia Waters Technology (Shanghai) Co., Ltd.

In July 2010, a news report about “dairy premature babies” caused widespread concern in society. This is another incident related to dairy food safety incidents after the “Sanlu Milk Powder” incident in 2008. When the incident occurred, the Ministry of Agriculture immediately issued a test method to detect the hormones in the relevant milk powder. The test results showed that the hormone content in the dairy product was within the national limit. In this incident, people have been widely discussed about whether the levels of estrogen and progesterone in infant formula are exceeded. At the same time, higher requirements are put forward for related indicators of hormone substance detection. Some experts have pointed out that there is no specific national standard test method for hormone detection in milk powder in China, and the method of hormone detection in animal food is adopted. In response to this problem, Waters has introduced the UPLC ® -Xevo TM TQ mass spectrometry system for rapid detection and quantification of estrogen and progesterone substances in milk powder.

The principle of the rapid detection of neutral powder in milk powder by Waters UPLC-Xevo TQ system

UPLC (Ultra High Performance Liquid Chromatography) ultra-efficient separation capability is first used to achieve effective separation of the tested milk powder, and the Xevo TQ's IntelliStartTM function is used to quickly and easily establish mass spectrometry, PICS (sub-ion confirmation scan) function assisted qualitative, and Applying Quanpedia (Method Library) to automatically import and export MRM and liquid phase methods, and finally using UPLC/Xevo TQ's positive and negative ionization mode fast switching function to achieve one injection and simultaneously detect multiple hormones (7 estrogens, 6 progestogens) significantly shorten the detection cycle and increase the detection throughput.

Analysis conditions and steps

This test is applicable to the analysis of exogenous hormones. If the endogenous substances are analyzed, the test samples can be enzymatically hydrolyzed, and the treatment methods after enzymatic hydrolysis are the same.

Sample preparation

• Accurately weigh 1g milk powder sample in 8ml water, mix well, and sonicate for 5min
• Add 1 ml of glacial acetic acid to the mixture, acidify and add 16 ml of acetonitrile to further precipitate the water-soluble protein. Centrifuge at 7500 rpm for 10 min, and take the supernatant under a nitrogen stream until the acetonitrile content is <5%.
• Activate the balanced Oasis HLB (200mg/6cc) column with 6ml of methanol and 6ml of water respectively. • Load and control the flow rate at 3-4ml/min.
• Cleaning: Wash with 6ml acid wash (5% methanol, 2% glacial acetic acid), 6ml alkali wash (5% methanol, 2% ammonia), 6ml methanol solution (10% methanol) • Oasis HLB after washing The column is drained for 10 minutes.
• Ethyl acetate/methanol (9/1 v/v) activated amino column (Sept-pak NH2 500mg/6cc), activated equilibrium and in series with Oasis HLB column • Elution: 6ml ethyl acetate / methanol (9 /1 v/v ), 6ml 5% ammonia in methanol solution elute two columns in series, collect the eluent • blow the eluate to 0.2ml under nitrogen flow
• Add 0.4 ml of acetonitrile and 0.4 ml of aqueous solution in sequence, and vortex them to a final volume of 1 ml. The ratio of acetonitrile to water is 1:1.
• Centrifuge at 7000 rpm for 10 min or pass through the membrane and collect the supernatant for analysis by UPLC/MS/MS

LC condition

LC System: ACQUITY UPLC ® System Columns: ACQUITY UPLC HSS T3 , 2.1 × 50mm , 1.8μm
Column temperature: 40 °C
Sample chamber temperature: 10 °C
Flow rate: 0.6mL/min
Mobile phase A: acetonitrile mobile phase B: water

gradient:

Weak needle washing solution: 10% acetonitrile aqueous solution strong washing solution: 90% acetonitrile water soluble total running time: 6min
Injection volume: 5μL, not filled with loop (needle overflow)

MS condition

MS System: Xevo TQ MS
Ionization mode: ESI+/ESI
Spray voltage: 3.2kv ( ESI+ ) /2.8kv ( ESI- )
Desolvent gas: nitrogen, 1000L / Hr, 450 °C
Cone gas: nitrogen, 50L / Hr
Source temperature: 150 °C
Acquisition mode: Multiple Reaction Monitoring (MRM) Other mass spectrometry parameters:

Data collection and processing

The data processing system of this test is Waters® MassLynxTM 4.1 SCN729. The mass spectrometry method uses the IntelliStart function of this software. It does not require experienced mass spectrometry operators to develop and optimize the method. With TargetLynx® software, only a few parameters can be set. Get the full report. The IntelliStart function simultaneously develops positive and negative ion mass spectrometry parameters and simultaneously scans both positive and negative ions, as shown in Figure 1 and Figure 2.

figure 1

figure 2

Figure 2 Extracted ion chromatograms of 13 hormones

Figure 3 Extracted ion chromatograms of eight hormonal substances in milk powder matrix

Results and discussion

Figures 2 and 3 show the extracted ion chromatograms of 13 hormonal substances and the extracted ion chromatograms of 8 hormonal substances in the milk powder matrix.

By analyzing the 13 hormonal substances using UPLC/Xevo TQ MS, the analysis results can be obtained in 6 minutes, and satisfactory results can be obtained in terms of sensitivity and resolution. The efficient separation of the Waters ACQUITY UPLC System allows the mass spectrometer to have sufficient residence time for each substance during multi-substance acquisition, increasing sensitivity and increasing qualitative reliability. Xevo TQ MS's fast positive and negative ion switching allows for one injection and simultaneous detection of multiple hormones (7 estrogens, 6 progestins), significantly reducing detection cycles and increasing throughput.

The Xevo TQ system uses the IntelliStart feature of MassLynx 4.1 to automatically, easily and quickly automate the optimization of sample mass spectrometry conditions. The mass spectrometry method was developed using the mass spectrometry parameters automatically optimized by the IntelliStart function, and the test results were satisfactory. At the same time, because the system does not require experienced mass spectrometry operators to develop and optimize methods, it saves a lot of manpower and material resources.

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