Method for detecting phenolic antioxidant through dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to a method for detecting a phenolic antioxidant through dansyl chloride precolumn derivatization-high performance liquid chromatography-tandem mass spectrometry. The invention relates to a method for detecting a phenolic antioxidant by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: carrying out pre-column derivatization treatment and acid neutralization treatment on a sample to be detected by dansyl chloride to terminate derivatization reaction to obtain a derivatized sample; and determining the derivatization sample by adopting a high performance liquid chromatography-tandem mass spectrometer so as to realize the ultra-trace detection of the phenolic antioxidant in the sample to be detected. The method solves the problem that the existing determination method cannot meet the ultra-trace detection requirement of the phenolic antioxidants, and also solves the problem that the existing determination method is difficult to realize the ultra-trace detection of various phenolic antioxidants.

Description

Technical Field

[0001] The present invention relates to the technical field of phenolic antioxidant detection, and in particular to a method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Phenolic antioxidants are a class of synthetic chemical additives containing one or more phenolic structures, typically present in concentrations of approximately 0.05% to 1%. These antioxidants are widely used in food contact materials and products, plastics, coatings, rubber, inks, adhesives, and paper products. Their primary function is to inhibit the rate of thermal and photooxidation during product production and use, slowing degradation, yellowing, and aging, thereby extending product life. According to relevant statistics, synthetic phenolic antioxidants are the most widely used and widely used of all synthetic antioxidants. For example, butylated tert-methylphenol (BHT) is one of the oldest and most widely used synthetic phenolic antioxidants worldwide. However, recent studies have shown that phenolic antioxidants can remain in a variety of media, including indoor dust, various foods, drinking water, human urine, and even in infants and young children. In a regional study, the content of phenolic antioxidants in indoor dust was found to be as high as 1560 ng / g dry weight to 20300 ng / g dry weight, among which BHT content was the highest (median value: 1880 ng / g dry weight), which was 2-5 times that of its rural areas.

[0003] Compared to natural antioxidants, synthetic phenolic antioxidants are more toxic. Toxicological studies have shown that BHT is corrosive to the skin and cornea, produces irritating fumes upon thermal decomposition, and animal studies have shown it to be potentially carcinogenic. Butylated hydroxyanisole (BHA) releases acrid fumes upon thermal decomposition. Long-term exposure can adversely affect organs such as the liver, spleen, and lungs, and may be carcinogenic. 2,6-Di-tert-butylphenol (2,6-TDBP) also exhibits certain biotoxicity and is harmful to some aquatic organisms. It is an irritant, and its vapor can irritate the eyes, skin, and respiratory system. Ingestion or absorption through the skin can cause symptoms such as headaches, dizziness, nausea, and even coma or death. In light of these potential hazards of phenolic antioxidants, international companies have recently begun to resist the use of BHT. Furthermore, relevant domestic and international regulations and standards have strict usage requirements or specific migration limits (SMLs) for several common phenolic antioxidants. For example, European Union (EU) regulations stipulate that the SML for BHT in food contact plastic materials and articles is 3 mg / kg, and the SML for BHA is 30 mg / kg. my country's standard GB 9685-2016 also stipulates that the SMLs for BHT and BHA in additives for food contact materials and articles are 3 mg / kg and 30 mg / kg, respectively. Therefore, establishing a rapid, accurate, and sensitive method for the determination of phenolic antioxidants is extremely important for safety risk assessment of phenolic antioxidants.

[0004] Currently, there are five main methods for detecting phenolic antioxidants: colorimetry, gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-tandem mass spectrometry. The "Determination of Nine Antioxidants in Foods" (GB 5009.32-2016) specifies the applicable scopes of these five methods. Liquid chromatography is applicable to the determination of PG, THBP, TBHQ, NDGA, BHA, BHT, Ionox-100, OG, and DG in foods; liquid chromatography-tandem mass spectrometry (LC-MS / MS) is applicable to the determination of THBP, PG, OG, NDGA, and DG in foods; gas chromatography-mass spectrometry (GC-MS) is applicable to the determination of BHA, BHT, TBHQ, and Ionox-100 in foods; gas chromatography is applicable to the determination of BHA, BHT, and TBHQ in foods; and colorimetry is applicable to the determination of PG in oils and fats. Therefore, each detection method has a different scope of application. Traditional liquid chromatography and gas chromatography require high cleanup requirements for complex matrix samples. Inadequate cleanup can produce interfering peaks that coelute with the target analytes, affecting integrated quantitative results. Furthermore, the instrumental detection limits of traditional liquid chromatography and gas chromatography are typically sub-ppm, which cannot meet the ultratrace detection requirements (ppt to ppb) for phenolic antioxidants. Currently, researchers have attempted to use LC-MS / MS to detect ultratrace levels of phenolic antioxidants such as BHT and AO246 in the environment. However, the detection of phenolic antioxidants using LC-MS / MS is subject to two major challenges: first, BHT and AO246 do not readily form charged ions in the mass spectrometer, resulting in low electrospray ionization efficiency, which can affect the results; second, instrument contamination caused by BHT, TBHQ, and BHA is a concern. Therefore, it is necessary to develop an analytical method capable of simultaneously detecting phenolic antioxidants such as BHT, BHA, TBHQ, 4-tOP, and AO246 at ultratrace levels. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry, so as to solve the problem that existing determination methods cannot meet the requirements for ultratrace detection of phenolic antioxidants, and solve the problem that existing determination methods are difficult to achieve ultratrace detection of multiple phenolic antioxidants.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry comprises the following steps: S1. Performing a pre-column derivatization treatment on the sample to be tested with dansyl chloride, and performing an acid neutralization treatment to terminate the derivatization reaction to obtain a derivatized sample; S2. Derivatized samples were determined using high performance liquid chromatography-tandem mass spectrometry to achieve ultra-trace detection of phenolic antioxidants in the sample.

[0007] The above-mentioned technical approach, through pre-column derivatization of phenolic antioxidants with dansyl chloride, significantly enhances the fluorescence intensity and mass spectrometric response of the target compounds, thereby improving detection sensitivity and meeting the requirements of ultratrace detection. Furthermore, the specificity of the derivatization reaction enables highly selective separation and detection of multiple phenolic antioxidants on a high-performance liquid chromatography-tandem mass spectrometer, overcoming the difficulty of existing assay methods in simultaneously detecting multiple phenolic antioxidants and enabling ultratrace detection of multiple phenolic antioxidants.

[0008] The principle of dansyl chloride derivatization reaction is that phenolic antioxidants have one or more phenolic hydroxyl functional groups. Dansyl chloride is very easy to react with phenolic hydroxyl groups under alkaline conditions (such as the attached Figure 1 ), to generate a stable derivative. This derivative is easily fragmented in the liquid mass ESI+ source to generate [C 12 H 12 N+H] + (m / z = 171), thereby improving the electrospray ionization efficiency and detection sensitivity of phenolic antioxidants, resulting in better quantitative results. Furthermore, derivatization with dansyl chloride can improve the reversed-phase chromatographic retention and sample stability of phenolic antioxidants. Therefore, experimental studies have shown that phenolic antioxidants derivatized with dansyl chloride are highly suitable for detection by LC-ESI-MS / MS.

[0009] The detection principle of HPLC-MS / MS is as follows: when the phenolic antioxidant components with the mobile phase as the carrier pass through the C18 chromatographic column stationary phase, due to the different strengths of the interaction with the stationary phase, they have different retention times in the stationary phase, and thus flow out of the chromatographic column one after another and enter the triple quadrupole tandem mass spectrometer. After electron bombardment in the electrospray ionization (ESI) mode, charged particles are formed, which are separated according to the mass-to-charge ratio (m / z) by the electric field in the mass analyzer and then detected.

[0010] Preferably, the pre-column derivatization treatment of the sample to be tested with dansyl chloride comprises: concentrating the treated sample to be tested to near dryness by nitrogen blowing, adding acetonitrile, vortexing, adding dansyl chloride and sodium carbonate buffer, vortexing, and heating in the dark.

[0011] Concentration to near dryness with nitrogen evaporation involves evaporating the solvent from the sample using a nitrogen stream until the solvent is almost completely removed and there is no free-flowing solvent in the sample. The purpose of this process is to reduce the amount of solvent, thereby increasing the concentration of the target compound in the sample for subsequent analysis or detection.

[0012] Preferably, the volume ratio of the acetonitrile, dansyl chloride solution and sodium carbonate buffer is 10 μL:50 μL:100 μL.

[0013] Preferably, the concentration of dansyl chloride in the dansyl chloride solution is 5 mg / mL, and the concentration of sodium carbonate in the sodium carbonate buffer is 0.1 M.

[0014] To prepare the dansyl chloride solution, dissolve dansyl chloride in acetonitrile or acetone to a concentration of 5 mg / mL. To prepare the Na2CO3 buffer, weigh 8.00 g of NaHCO3 powder and 0.51 g of Na2CO3 powder and dissolve them in 800 mL of deionized water in a beaker. Stir and adjust the pH to 8.77 with NaOH or HCl (if the pH is > 9.0, add 1 M HCl to 9.0; if the pH is < 9.0, add 1 M NaOH). Add deionized water to bring the volume to 1000 mL.

[0015] Preferably, the vortex treatment time is 30 s.

[0016] Preferably, the light-proof heating treatment is a light-proof water bath heating treatment, the water bath heating temperature is 28-32° C., and the time is 8-12 min.

[0017] Because the sulfonyl chloride group and naphthalene ring structure in the dansyl chloride molecule make it very sensitive to light (especially ultraviolet light), it easily decomposes under light. Therefore, the light-proof operation is adopted to effectively ensure the stability of the reagent and ensure the normal progress of the reaction.

[0018] By selecting a suitable temperature for water bath heating treatment, not only the derivatization reaction rate is significantly accelerated and the time required to reach reaction equilibrium is shortened, but also the thermal stability of dansyl chloride is balanced and high-temperature decomposition is avoided.

[0019] Preferably, the water bath heating temperature is 30° C. and the heating time is 10 min.

[0020] Preferably, the acid neutralization treatment comprises: adding methanol aqueous solution to the sample to be tested after the light-proof heat treatment, vortexing, adjusting the pH value to below 7 to terminate the derivatization reaction, and centrifuging through a membrane to obtain a derivatized sample.

[0021] By adding methanol aqueous solution to the sample to be tested before adjusting the pH value, the solvent composition of the sample to be tested is made consistent with the liquid chromatography mobile phase composition, thereby ensuring the full dissolution of the target antioxidant and avoiding the occurrence of solvent effect.

[0022] Preferably, the volume ratio of methanol to water in the methanol aqueous solution is 4:1.

[0023] Preferably, the amount of the methanol aqueous solution is 840 μL.

[0024] Preferably, formic acid is used to adjust the pH value to below 7.

[0025] Preferably, the filter membrane used for the membrane is made of polypropylene (PP) filter membrane.

[0026] Preferably, the chromatographic conditions for the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) for the determination of the derivatized sample are as follows: chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; mobile phase: methanol: 0.2 mM ammonium acetate aqueous solution (B:A = 45:55); gradient elution: maintaining at 45% B for 5 min, then linearly increasing to 100% B within 3 min, and then linearly decreasing to 45% B over 2 min, with a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 ° C; injection volume: 5~10 μL.

[0027] Preferably, the mass spectrometry conditions for the high performance liquid chromatography-tandem mass spectrometry for the derivatized sample are: spray ion source (ESI+), multiple reaction monitoring mode (MRM), capillary voltage: 2.0 kV, ion source temperature: 110~130 ° C, desolvation temperature: 300-400 ° C, desolvation gas flow rate: 800~900 L / h, cone backflush gas flow rate: 50~150 L / h, collision gas flow rate: 0.12 mL / min, nebulizer gas: 7 bar.

[0028] Preferably, the sample to be tested is selected from dust, food, human blood or urine.

[0029] Preferably, the phenolic antioxidant includes at least one of 2,6-di-tert-butyl-p-cresol (BHT), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), 4-tert-butylphenol (4-tOP), 2,6-di-tert-butylphenol (2,6-TDBP), 4,4'-thiobis(6-tert-butyl-m-cresol) (AO300) and 2,4,6-tri-tert-butylphenol (AO246).

[0030] Preferably, the detection limit of the method for phenolic antioxidants in the sample to be tested is 0.2~2.5 μg / L.

[0031] Preferably, the detection limit of the method for 2,6-di-tert-butyl-p-cresol (BHT) in the sample to be tested is 2.2 μg / L.

[0032] Preferably, the detection limit of the method for tert-butylhydroquinone (TBHQ) in the sample to be tested is 0.2 μg / L.

[0033] Preferably, the detection limit of the method for butylated hydroxyanisole (BHA) in the sample to be tested is 0.5 μg / L.

[0034] Preferably, the detection limit of the method for 4-tert-butylphenol (4-tOP) in the sample to be tested is 1.2 μg / L.

[0035] Preferably, the detection limit of the method for 2,6-di-tert-butylphenol (2,6-TDBP) in the sample to be tested is 1.4 μg / L.

[0036] Preferably, the detection limit of the method for 4,4'-thiobis(6-tert-butyl-m-cresol) (AO300) in the sample to be tested is 1.4 μg / L.

[0037] Preferably, the detection limit of the method for 2,4,6-tri-tert-butylphenol (AO246) in the sample to be tested is 2.5 μg / L.

[0038] Beneficial effects of the present invention: The present invention discloses a method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry. By using dansyl chloride to perform pre-column derivatization treatment on the phenolic antioxidants, the fluorescence intensity and mass spectrometric response of the target compound are significantly enhanced, thereby improving detection sensitivity and meeting the requirements of ultra-trace detection. Furthermore, the specificity of the derivatization reaction enables highly selective separation and detection of multiple phenolic antioxidants on a high performance liquid chromatography-tandem mass spectrometry instrument, resolving the difficulty of simultaneous detection of multiple phenolic antioxidants in existing assay methods. This method enables ultra-trace detection of multiple phenolic antioxidants and has application value in the field of phenolic antioxidant detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Total ion current (TIC) diagram of seven phenolic antioxidants; Figure 2 This is a comparison of the mass spectrometric response signals of phenolic antioxidants before and after dansyl chloride derivatization reaction. DETAILED DESCRIPTION

[0040] The following will describe the embodiments of the present invention with reference to preferred embodiments. Those skilled in the art will readily appreciate the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0041] If no specific techniques or conditions are specified in the specific examples, the techniques or conditions described in the literature in this field or the product instructions were used. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional products.

[0042] Example 1 A method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry comprises the following steps: S1. Pre-column derivatization: Use 1 mL of standard working solution (the standard working solution contains 7 target phenolic antioxidants BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300 and AO246, each with a concentration of 50 ng / mL, and the solvent in the standard working solution is acetonitrile) as the sample to be tested. Concentrate the sample to be tested to near dryness by nitrogen purge, add 10 μL of acetonitrile, vortex for 30 s, then add 50 μL of 5 mg / mL dansyl chloride solution and 100 μL of 0.1 M Na2CO3 buffer, vortex for another 30 s, and heat in a water bath at 30°C for 10 min in the dark. Preparation of dansyl chloride solution: Dissolve dansyl chloride in acetonitrile or acetone to prepare a dansyl chloride solution with a concentration of 5 mg / mL. The pH value of Na2CO3 buffer is 8.77. Preparation of Na2CO3 buffer: Weigh 8.00 g of NaHCO3 powder and 0.51 g of Na2CO3 powder, dissolve them in a beaker filled with 800 mL of deionized water, stir, and adjust the pH to 8.77 with NaOH or HCl (if the pH is > 9.0, add 1 M HCl to adjust to 9.0; if the pH is < 9.0, add 1 M NaOH). Add deionized water to bring the volume to 1000 mL. S2. Acid neutralization: Add 840 μL of methanol-water (volume ratio 4:1) solution to the sample to be tested after the reaction in S1 is completed by heating in a dark water bath, vortex mix, and then adjust the pH value of the sample to below 7 with formic acid to terminate the derivatization reaction. Then, centrifuge the sample at 4°C and 5000 r / min for 5 min, and filter the sample with a polypropylene (PP) membrane to obtain a derivatized sample. S3. HPLC-MS / MS detection: The derivatized sample obtained in S2 was measured using high performance liquid chromatography-tandem mass spectrometry; The chromatographic conditions were as follows: chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; mobile phase: methanol: 0.2 mM ammonium acetate aqueous solution (B:A = 45:55); gradient elution: 45% B for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, for a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 5–10 μL; The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring (MRM) mode, capillary voltage: 2.0 kV, ion source temperature: 110-130 °C, desolvation temperature: 300-400 °C, desolvation gas flow rate: 800-900 L / h, cone backflush gas flow rate: 50-150 L / h, collision gas flow rate: 0.12 mL / min, nebulizer gas: 7 bar; The detection conditions of target phenolic antioxidants are shown in Table 1. The total ion current (TIC) of the seven target phenolic antioxidants is shown in Figure 1 shown.

[0043] Table 1 shows the compound retention times and mass spectrometry multiple reaction monitoring (MRM) parameters Example 2 A method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry comprises the following steps: S1. Pre-column derivatization: Use 1 mL of standard working solution (the standard working solution contains 7 target phenolic antioxidants BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300 and AO246, each with a concentration of 50 ng / mL, and the solvent in the standard working solution is acetonitrile) as the sample to be tested. Concentrate the sample to be tested to near dryness by nitrogen purge, add 10 μL of acetonitrile, vortex for 30 s, then add 50 μL of 5 mg / mL dansyl chloride solution and 100 μL of 0.1 M Na2CO3 buffer, vortex for another 30 s, and heat in a water bath at 30°C for 10 min in the dark. Preparation of dansyl chloride solution: Dissolve dansyl chloride in acetonitrile or acetone to prepare a dansyl chloride solution with a concentration of 5 mg / mL. The pH value of Na2CO3 buffer is 8.77. Preparation of Na2CO3 buffer: Weigh 8.00 g of NaHCO3 powder and 0.51 g of Na2CO3 powder, dissolve them in a beaker filled with 800 mL of deionized water, stir, and adjust the pH to 8.77 with NaOH or HCl (if the pH is > 9.0, add 1 M HCl to adjust to 9.0; if the pH is < 9.0, add 1 M NaOH), and add deionized water to a volume of 1000 mL. S2. Acid neutralization: Add 840 μL of methanol-water (volume ratio 4:1) solution to the sample to be tested after the reaction in S1 is completed by heating in a dark water bath, vortex mix, and then adjust the pH value of the sample to below 7 with formic acid to terminate the derivatization reaction. Then, centrifuge the sample at 4°C and 5000 r / min for 5 min, and filter the sample with a polypropylene (PP) membrane to obtain a derivatized sample. S3. HPLC-MS / MS detection: The derivatized sample obtained in S2 was measured using high performance liquid chromatography-tandem mass spectrometry; The chromatographic conditions were as follows: chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; mobile phase: methanol: 0.2 mM ammonium acetate aqueous solution (B:A = 45:55); gradient elution: 45% B for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, for a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 10 μL; The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring (MRM) mode, capillary voltage: 2.0 kV, ion source temperature: 120 °C, desolvation temperature: 350 °C, desolvation gas flow rate: 850 L / h, cone backflush gas flow rate: 100 L / h, collision gas flow rate: 0.12 mL / min, and nebulizer gas: 7 bar.

[0044] Comparative Example 1 A method for detecting phenolic antioxidants by high performance liquid chromatography-tandem mass spectrometry comprises the following steps: S1. HPLC-MS / MS detection: 1 mL of standard working solution (the standard working solution contains seven target phenolic antioxidants: BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300, and AO246, each with a concentration of 50 ng / mL, and the solvent in the standard working solution is acetonitrile) is used as the test sample and the sample is measured by high performance liquid chromatography-tandem mass spectrometry; The chromatographic conditions were as follows: chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; mobile phase: methanol: 0.2 mM ammonium acetate aqueous solution (B:A = 25:75); gradient elution: 0–0.5 min: 25% B, 0.5–7.5 min: 25%–100% B, 7.5–8.5 min: 100% B, 8.5–9.0 min: 100% B–25% B, 9.0–10 min: 25% B, total run time: 10 min; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 5 μL; The mass spectrometry conditions were as follows: electrospray ionization source (ESI-), multiple reaction monitoring (MRM) mode, capillary voltage: 3.0 kV, ion source temperature: 150 °C, desolvation temperature: 300 °C, desolvation gas flow rate: 800 L / h, cone backflush gas flow rate: 150 L / h, collision gas flow rate: 0.17 mL / min, and nebulizer gas: 7 bar.

[0045] Comparison of mass spectrometric response signals of phenolic antioxidants before and after dansyl chloride derivatization Figure 2 As shown, Figure 2 The mass spectrometric response signal before derivatization corresponds to the mass spectrometric response signal of the sample to be tested that was not subjected to pre-column derivatization treatment with dansyl chloride in Control Example 1 and was directly subjected to HPLC-MS / MS detection. The mass spectrometric response signal after derivatization corresponds to the mass spectrometric response signal of the sample to be tested that was subjected to pre-column derivatization treatment with dansyl chloride in Example 2 and was subjected to HPLC-MS / MS detection.

[0046] from Figure 2From the comparative analysis, it can be seen that, compared with the existing LC-MS / MS method in Control Example 1, the method of detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry of the present invention has at least the following advantages: 1) it can simultaneously detect seven phenolic antioxidants, including BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300, and AO246; 2) the mass spectrometry signal intensity generated by the dansyl chloride derivative is one to two orders of magnitude greater than the signal intensity of the underivatized phenolic antioxidant standard, resulting in a stronger instrument response and higher sensitivity; 3) since this method detects the derivative rather than the target phenolic antioxidant itself, it is also resistant to instrument contamination and mobile phase contamination.

[0047] Furthermore, the present method for detecting phenolic antioxidants using pre-column derivatization with dansyl chloride followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) has a detection time of 10 minutes, significantly improving analysis speed compared to existing GC-MS methods, which typically have a detection time of 20-30 minutes. This makes it more suitable for large-scale sample testing. Compared to existing GC or LC methods, the present method for detecting phenolic antioxidants using pre-column derivatization with dansyl chloride followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) utilizes a tandem mass spectrometer as a detector. This method can detect only the parent and daughter ions of the target phenolic antioxidant in complex matrix samples via a tandem quadrupole, without interference from chromatographic co-eluting components. Consequently, this method offers higher accuracy in both qualitative and quantitative analysis. Furthermore, this method exhibits a lower limit of detection (Table 2), making it suitable for ultratrace analysis (ppb level).

[0048] Instrumental detection limit analysis of target phenolic antioxidants The instrument detection limits of the target phenolic antioxidants were calculated as follows: The instrument detection limit is defined as the concentration at which the signal-to-noise ratio (S / N) is 3:1, and its calculation formula is shown in formula (I): D=3N×Q / I (Ⅰ) In formula (I), Q represents the instrument concentration (μg / L), N represents the noise level, I represents the signal response value, and D represents the instrument detection limit (IDL) of the target phenolic antioxidant (μg / L). I / N represents the signal-to-noise ratio (S / N) at the corresponding instrument concentration. The workstation automatically analyzes the chromatogram to obtain the S / N ratio. Specifically, a standard working solution of a known concentration of the target phenolic antioxidant was serially diluted until the instrument S / N ratio (S / N) was between 3 and 10 (read directly by the instrument). The IDL (IDL) of the target phenolic antioxidant was then calculated using the above formula. The results are shown in Table 2.

[0049] Table 2 shows the instrument detection limits of seven target phenolic antioxidants determined by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry. Example 3 To determine the optimal pH, water bath temperature, and reaction time for the dansyl chloride derivatization reaction, a full factorial experimental design was used in this example, with three factors and three levels, and three replicates per experiment. The experimental steps were as follows: S1. Prepare 10 μL of a 100 mg / L standard working solution (containing seven target phenolic antioxidants, BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300, and AO246, at a concentration of 100 mg / L for each, using acetonitrile) as the stock solution. Place the stock solution in a 2 mL centrifuge tube and add 50 μL of a 5 mg / mL dansyl chloride solution and 100 μL of 0.1 M Na2CO3 buffer (pH 8.77, 9.4, and 9.9, respectively) to reconstitute the sample. After adding the reagents, vortex mix for 30 seconds to ensure thorough homogenization of the reaction system. Subsequently, place the centrifuge tube in a water bath and heat the reaction in the dark at 30, 40, and 50°C for 10, 20, and 30 minutes, respectively. After the reaction, 840 μL of methanol-water (volume ratio 4:1) solution was added and vortexed to mix to prepare a mixed standard solution with a concentration of 100 ng / L; The dansyl chloride solution is prepared by dissolving dansyl chloride in acetonitrile or acetone to prepare a dansyl chloride solution with a concentration of 5 mg / mL. Preparation of Na2CO3 buffer solution: Preparation of 0.1 M Na2CO3 buffer solution with pH 8.77: Weigh 7.56 g of NaHCO3 powder and 1.06 g of Na2CO3 powder, dissolve them in a beaker filled with 800 mL of deionized water, stir, and adjust the pH to 8.77 with NaOH or HCl (if pH>8.77, add 1 M HCl to 8.77; if pH<8.77, add 1 M NaOH). Add deionized water to a volume of 1000 mL; Preparation of 0.1 M Na2CO3 buffer solution with pH 9.4: Weigh 5.88 g of NaHCO3 powder and 3.18 g of Na2CO3 powder, dissolve them in a beaker filled with 800 mL of deionized water, stir, and adjust the pH to 9.4 with NaOH or HCl (if pH>9.4, add 1 M HCl to 9.4; if pH<9.4, add 1 M 3.36 g of NaHCO3 powder and 6.63 g of Na2CO3 powder were weighed and dissolved in a beaker containing 800 mL of deionized water. The mixture was stirred and the pH was adjusted to 9.9 with NaOH or HCl. The volume was then filled with deionized water to 1000 mL. S2. To terminate the reaction and adjust the pH of the mixed standard solution, add an appropriate amount of formic acid to the mixed standard solution to adjust the pH to below 7. Centrifuge at 4°C and 5000 rpm for 5 minutes. Take 800 μL of the supernatant and filter it through a membrane. S3. Analyze the supernatant after membrane filtration using LC-MS / MS; The chromatographic conditions were as follows: chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; mobile phase: methanol: 0.2 mM ammonium acetate aqueous solution (B:A = 45:55); gradient elution: 45% B for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, for a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 5–10 μL; The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring (MRM) mode, capillary voltage: 2.0 kV, ion source temperature: 110-130 °C, desolvation temperature: 300-400 °C, desolvation gas flow rate: 800-900 L / h, cone backflush gas flow rate: 50-150 L / h, collision gas flow rate: 0.12 mL / min, and nebulizer gas: 7 bar.

[0050] Through full factorial design experimental analysis, the optimal derivatization conditions for each compound were obtained (the experimental conditions with the largest peak area were the optimal derivatization conditions), as shown in Table 3.

[0051] Table 3 shows the optimal derivatization conditions for the target compounds. As shown in Table 3, the optimal derivatization conditions for the seven target phenolic antioxidants were a buffer pH of 8.77, a water bath reaction temperature of 30 °C, and a water bath reaction time of 10 min. Therefore, these parameters were used as the optimal derivatization conditions in the actual test.

[0052] Example 4 A method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry comprises the following steps: S1. Sample pretreatment: Weigh 500 mg of each of five unknown indoor dust samples collected in Guangzhou into five 15-mL glass test tubes. Add 3 mL of n-hexane-dichloromethane (1:3, v / v) solution to each of the five test tubes and vortex twice for 1 minute each. Then, ultrasonically extract each of the five test tubes in a water bath for 30 minutes. After each ultrasonic extraction, collect the supernatant in a glass test tube and centrifuge at 4500 rpm for 6 minutes. After centrifugation, collect the supernatant. Repeat the extraction three times and combine the supernatants to obtain the five dust samples to be tested. S2, pre-column derivatization: Perform the following operations on the five dust samples obtained in S1: The solution was concentrated to near dryness by nitrogen purge, and 10 μL of acetonitrile was added. After vortexing for 30 s, 50 μL of 5 mg / mL dansyl chloride and 100 μL of 0.1 M Na2CO3 buffer were added. After vortexing for another 30 s, the solution was heated in a water bath at 30 °C for 10 min in the dark. Preparation of dansyl chloride solution: Dissolve dansyl chloride in acetonitrile or acetone to prepare a dansyl chloride solution with a concentration of 5 mg / mL. The pH value of Na2CO3 buffer is 8.77. Preparation of Na2CO3 buffer: Weigh 8.00 g of NaHCO3 powder and 0.51 g of Na2CO3 powder, dissolve them in a beaker filled with 800 mL of deionized water, stir, and adjust the pH to 8.77 with NaOH or HCl (if the pH is > 9.0, add 1 M HCl to adjust to 9.0; if the pH is < 9.0, add 1 M NaOH). Add deionized water to bring the volume to 1000 mL. S3, acid neutralization: add 840 μL of methanol-water (volume ratio 4:1) solution to the dust sample to be tested after the reaction in a dark water bath in S2, vortex mix, and then adjust the pH value of the sample to below 7 with formic acid to terminate the derivatization reaction. Then, centrifuge the sample at 4°C and 5000 r / min for 5 min, and filter the sample with a polypropylene (PP) membrane to obtain a derivatized sample. S4. HPLC-MS / MS detection: The derivatized sample obtained in S3 was measured using high performance liquid chromatography-tandem mass spectrometry; The chromatographic conditions were as follows: chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; mobile phase: methanol: 0.2 mM ammonium acetate aqueous solution (B:A = 45:55); gradient elution: 45% B for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, for a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 10 μL; The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring (MRM) mode, capillary voltage: 2.0 kV, ion source temperature: 120 °C, desolvation temperature: 350 °C, desolvation gas flow rate: 850 L / h, cone backflush gas flow rate: 100 L / h, collision gas flow rate: 0.12 mL / min, and nebulizer gas: 7 bar.

[0053] The measurement results are shown in Table 4.

[0054] Table 4 shows the test results of seven phenolic antioxidants in five indoor dust samples in Guangzhou (ng / g dry weight) As shown in Table 4, all seven phenolic antioxidants were detected to varying degrees; the detection concentration range was 4.8 ngTBHQ / ng dry weight to 3450 ng BHT / g dry weight, among which BHT had the highest content. Phenolic antioxidants were detected in all five indoor dust samples, with a detection rate of 100%. Detection rate = number of detected samples / total number of samples * 100%.

[0055] In summary, the present invention provides a novel method and process for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry, including dansyl chloride derivatization, formic acid neutralization, and analytical detection steps. (1) Compared with the existing LC-MS / MS method, the present method can simultaneously detect seven phenolic antioxidants, including BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300, and AO246, and has a stronger instrument response and higher sensitivity. (2) Compared with the existing GC-MS method, the method adopted by the present invention has a detection time of 10 minutes, which greatly improves the analysis speed and is more suitable for large-scale sample detection. (3) Compared with the existing GC or LC methods, the present method has the advantages of high accuracy, low sample consumption, lower detection limit, and suitability for ultra-trace analysis.

[0056] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or modification made by those skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. A method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: S1. Performing a pre-column derivatization treatment on the sample to be tested with dansyl chloride, and performing an acid neutralization treatment to terminate the derivatization reaction to obtain a derivatized sample; S2. Derivatized samples were determined using high performance liquid chromatography-tandem mass spectrometry to achieve ultra-trace detection of phenolic antioxidants in the sample.

2. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The method of using dansyl chloride to perform pre-column derivatization treatment on the sample to be tested comprises: concentrating the sample to be tested to near dryness by nitrogen blowing, adding acetonitrile, vortexing, adding dansyl chloride solution and sodium carbonate buffer, vortexing, and heating in the dark.

3. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that: The volume ratio of the acetonitrile, dansyl chloride solution and sodium carbonate buffer is 10 μL:50 μL:100 μL; The concentration of dansyl chloride in the dansyl chloride solution is 5 mg / mL, and the concentration of sodium carbonate in the sodium carbonate buffer is 0.1 M; And / or, the vortex treatment time is 30 s.

4. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that: The light-proof heating treatment is a light-proof water bath heating treatment, the water bath heating temperature is 28-32° C., and the time is 8-12 min.

5. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that: The acid neutralization treatment includes: adding methanol aqueous solution to the sample to be tested after the light-proof heating treatment, vortexing, adjusting the pH value to below 7 to terminate the derivatization reaction, and centrifuging through a membrane to obtain a derivatized sample.

6. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 5, characterized in that: The volume ratio of methanol to water in the methanol-water solution is 4:1; and / or, the amount of the methanol aqueous solution is 840 μL; and / or, adjusting the pH value to below 7 using formic acid; And / or, the filter membrane used for the membrane filtration is made of polypropylene filter membrane.

7. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The chromatographic conditions for the determination of the derivatized samples by the high performance liquid chromatography-tandem mass spectrometry were as follows: chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; Mobile phase: Methanol: 0.2 mM ammonium acetate aqueous solution; gradient elution: maintain at 45% B for 5 min, then linearly increase to 100% B over 3 min, and then linearly decrease to 45% B over 2 min, total run time 10 min; flow rate: 0.3 mL / min; Column temperature: 40°C; injection volume: 5–10 μL.

8. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions for the derivatized sample determination using the high performance liquid chromatography-tandem mass spectrometry instrument are as follows: spray ion source (ESI+), multiple reaction monitoring mode (MRM), capillary voltage: 2.0 kV, ion source temperature: 110-130°C, desolvation gas temperature: 300-400°C, desolvation gas flow rate: 800-900 L / h, cone backflush gas flow rate: 50-150 L / h, collision gas flow rate: 0.12 mL / min, and nebulizer gas: 7 bar.

9. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The sample to be tested is selected from dust, food, human blood or urine; And / or, the phenolic antioxidant includes at least one of 2,6-di-tert-butyl-p-cresol, tert-butylhydroquinone, butylated hydroxyanisole, 4-tert-butylphenol, 2,6-di-tert-butylphenol, 4,4'-thiobis(6-tert-butyl-m-cresol) and 2,4,6-tri-tert-butylphenol.

10. The method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The detection limit of the method for phenolic antioxidants in the test samples is 0.2~2.5 μg / L.

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