A method for detecting phenolic antioxidants by dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry

Abstract

The present application relates to a kind of dan sulfonyl chloride pre-column derivatization-high performance liquid chromatography-mass spectrometry detection phenolic antioxidant method.Dan sulfonyl chloride pre-column derivatization-high performance liquid chromatography-mass spectrometry detection phenolic antioxidant method, comprising the following steps: using dan sulfonyl chloride to the sample to be measured is carried out pre-column derivatization processing, acid neutralization treatment to terminate derivatization reaction, obtain derivatization sample;Using high performance liquid chromatography-mass spectrometry to determine derivatization sample, to realize the ultra trace detection of phenolic antioxidant in the sample to be measured.The present application solves the problem that existing determination method cannot meet the ultra trace detection requirement of phenolic antioxidant, also solves the problem that existing determination method is difficult to realize the ultra trace detection of multiple phenolic antioxidant.

Description

Technical Field

[0001] This invention relates to the field of phenolic antioxidant detection technology, specifically to a method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Phenolic antioxidants are a class of synthetic chemical additives containing one or more phenolic structures, typically ranging from 0.05% to 1%. These antioxidants are widely used in food contact materials and products, plastics, coatings, rubber, inks, adhesives, and paper products. Their main function is to inhibit the rate of thermal and photo-oxidative reactions during production or use, slowing down degradation and yellowing aging processes, thereby extending service life. According to statistics, synthetic phenolic antioxidants have the largest usage and widest application among synthetic antioxidants. For example, di-tert-butyl-p-methylphenol (BHT) is one of the earliest and most widely used synthetic phenolic antioxidants in the world. However, recent studies have shown that phenolic antioxidants leave residues in various media, such as indoor dust, various foods, drinking water, human urine, and even in the bodies of infants and young children. A regional study found that indoor dust contained phenolic antioxidants ranging from 1560 ng / g dry weight to 20300 ng / g dry weight, with BHT being the most abundant (median: 1880 ng / g dry weight), and the levels were 2 to 5 times higher than in 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, and its thermal decomposition produces irritating fumes; animal studies have also shown potential carcinogenicity. Butylhydroxyanisole (BHA) releases pungent, irritating fumes upon thermal decomposition; long-term exposure can adversely affect organs such as the liver, spleen, and lungs, and may also be carcinogenic. 2,6-Di-tert-butylphenol (2,6-TDBP) also exhibits some biotoxicity, is harmful to some aquatic organisms, and is irritating; its vapor can irritate the eyes, skin, and respiratory system. Ingestion or skin absorption can cause headaches, dizziness, nausea, and may even lead to coma or death. Given these potential hazards of phenolic antioxidants, foreign companies have begun to boycott the use of BHT in recent years. Furthermore, relevant domestic and international regulations and standards impose strict usage requirements or specific migration limits (SML) on several common phenolic antioxidants. For example, EU regulations stipulate that the standard minimum concentration (SML) of BHT in food contact plastic materials and articles is 3 mg / kg, and that of BHA is 30 mg / kg. Chinese standard GB 9685—2016 also specifies SMLs of 3 mg / kg and 30 mg / kg for BHT and BHA additives in food contact materials and articles, respectively. Therefore, establishing rapid, accurate, and sensitive methods for determining phenolic antioxidants is of paramount importance for the 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 (GC-MS / MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). The applicable scope of these five methods is specified in the standard "Determination of Nine Antioxidants in Food" (GB 5009.32-2016). Specifically, liquid chromatography is suitable for the determination of PG, THBP, TBHQ, NDGA, BHA, BHT, Ionox-100, OG, and DG in food; liquid chromatography-tandem mass spectrometry (LC-MS / MS) is suitable for the determination of THBP, PG, OG, NDGA, and DG in food; gas chromatography-mass spectrometry (GC-MS) is suitable for the determination of BHA, BHT, TBHQ, and Ionox-100 in food; gas chromatography is suitable for the determination of BHA, BHT, and TBHQ in food; and colorimetry is suitable for the determination of PG in oils and fats. Therefore, each detection method has a different applicable scope. Traditional liquid chromatography (LC) and gas chromatography (GC) require high purification levels for complex matrix samples. Inadequate purification can lead to interfering peaks co-eluting with the target analyte, affecting the integrated quantification results. Furthermore, the instrument detection limits of traditional LC and GC are at most sub-ppm, which is insufficient for ultra-trace detection (ppt to ppb) of phenolic antioxidants. Currently, some researchers have attempted to use LC-MS / MS to detect ultra-trace amounts of phenolic antioxidants such as BHT and AO246 in the environment. However, LC-MS / MS detection of phenolic antioxidants presents two main problems: first, phenolic antioxidants such as BHT and AO246 do not readily form charged ions in mass spectrometry, resulting in low electrospray ionization efficiency and affecting the measurement results; second, there are instrument contamination issues caused by BHT, TBHQ, and BHA. Therefore, it is necessary to develop an analytical method capable of simultaneously detecting ultra-trace amounts of phenolic antioxidants such as BHT, BHA, TBHQ, 4-tOP, and AO246. Summary of the Invention

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

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0008] S1. Dansyl chloride was used to perform pre-column derivatization of the test sample, followed by acid neutralization to terminate the derivatization reaction, resulting in a derivatized sample.

[0009] S2. High performance liquid chromatography-tandem mass spectrometry is used to determine the derivatized samples in order to achieve ultra-trace detection of phenolic antioxidants in the test samples.

[0010] Based on the aforementioned techniques, pre-column derivatization of phenolic antioxidants using dansyl chloride significantly enhances the fluorescence intensity and mass spectrometry response of the target compounds, thereby improving detection sensitivity and meeting the requirements for ultra-trace detection. Simultaneously, the specificity of the derivatization reaction enables highly selective separation and detection of multiple phenolic antioxidants using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), solving the problem of existing methods' difficulty in simultaneously detecting multiple phenolic antioxidants and achieving ultra-trace detection of various phenolic antioxidants.

[0011] The derivatization reaction principle of dansyl chloride is as follows: phenolic antioxidants possess one or more phenolic hydroxyl functional groups. Dansyl chloride readily undergoes chlorination with the phenolic hydroxyl group under alkaline conditions (as shown in the attached figure). Figure 1 ), forming a stable derivative. This derivative readily fragments in a liquid MS ESI+ source to form [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, dansyl chloride derivatization can also improve the reversed-phase chromatographic retention behavior and sample stability of phenolic antioxidants. Therefore, experimental studies have shown that phenolic antioxidants derivatized with dansyl chloride are very suitable for detection by LC-ESI-MS / MS.

[0012] The detection principle of HPLC-MS / MS is as follows: When phenolic antioxidant components, with the mobile phase as the carrier, pass through the stationary phase of the C18 column, their residence times in the stationary phase vary due to the different strengths of their interaction with the stationary phase. As a result, they elute from the column sequentially and enter the triple quadrupole tandem mass spectrometer. In electrospray ionization (ESI) mode, they are bombarded by electrons to form charged particles. These particles are then separated in the mass analyzer according to their mass-to-charge ratio (m / z) using an electric field and detected.

[0013] Preferably, the pre-column derivatization treatment of the test sample using dansyl chloride includes: concentrating the treated test sample to near dryness by nitrogen blowing, adding acetonitrile, vortexing, adding dansyl chloride and sodium carbonate buffer solution, vortexing, and heating in the dark.

[0014] Nitrogen blowing concentration to near-dryness refers to evaporating the solvent using a nitrogen stream until the solvent is almost completely removed from the sample, leaving no free-flowing solvent within the sample itself. The purpose of this process is to reduce the amount of solvent, thereby increasing the concentration of the target compound in the sample to facilitate subsequent analysis or detection.

[0015] Preferably, the volume ratio of acetonitrile, dansyl chloride solution and sodium carbonate buffer is 10 μL:50 μL:100 μL.

[0016] 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 solution is 0.1 M.

[0017] 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. Preparation of Na2CO3 buffer solution: Weigh 8.00 g of NaHCO3 powder and 0.51 g of Na2CO3 powder, dissolve them in a beaker containing 800 mL of deionized water, stir, adjust the pH to 8.77 with NaOH or HCl (if pH > 9.0, add 1 M HCl to adjust to 9.0; if pH < 9.0, add 1 M NaOH), and add deionized water to a volume of 1000 mL.

[0018] Preferably, the vortex treatment time is 30 s.

[0019] Preferably, the light-shielding heating treatment is a light-shielding water bath heating treatment, with the water bath heating temperature being 28~32 ℃ and the time being 8~12 min.

[0020] Because the sulfonyl chloride group and naphthalene ring structure in the dansyl chloride molecule make it highly sensitive to light (especially ultraviolet light), it is prone to decomposition under light irradiation. Therefore, operating in the dark effectively ensures the stability of the reagent and guarantees the normal progress of the reaction.

[0021] By selecting an appropriate temperature for water bath heating treatment, the derivatization reaction rate was significantly accelerated, the time required to reach reaction equilibrium was shortened, and the thermal stability of dansyl chloride was balanced, thus avoiding high-temperature decomposition.

[0022] Preferably, the water bath heating temperature is 30 ℃ and the time is 10 min.

[0023] Preferably, the acid neutralization treatment includes: adding an aqueous methanol solution to the sample to be tested after the light-shielded heating treatment, vortexing, adjusting the pH value to below 7 to terminate the derivatization reaction, centrifuging and membrane filtration to obtain the derivatized sample.

[0024] By adding an aqueous methanol solution to the sample before adjusting the pH value, the solvent composition of the sample is made consistent with the composition of the liquid chromatography mobile phase, ensuring the full dissolution of the target antioxidant and avoiding the solvent effect.

[0025] Preferably, the volume ratio of methanol to water in the methanol-water solution is 4:1.

[0026] Preferably, the amount of the methanol aqueous solution used is 840 μL.

[0027] Preferably, formic acid is used to adjust the pH value to below 7.

[0028] Preferably, the filter membrane used in the membrane process is a polypropylene (PP) filter membrane.

[0029] Preferably, the chromatographic conditions for the determination of derivatized samples by the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) are as follows: 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: hold 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, for a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 5~10 μL.

[0030] Preferably, the mass spectrometry conditions for determining derivatized samples using the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) are as follows: spray ionization source (ESI+), multiple reaction monitoring mode (MRM), capillary voltage: 2.0 kV, ion source temperature: 110~130 ℃, desolvation gas temperature: 300-400 ℃, 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 nebulizing gas: 7 bar.

[0031] Preferably, the sample to be tested is selected from dust, food, human blood, or urine.

[0032] 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).

[0033] Preferably, the detection limit of the method for phenolic antioxidants in the sample to be tested is 0.2~2.5 μg / L.

[0034] 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.

[0035] Preferably, the detection limit of the method for tert-butylhydroquinone (TBHQ) in the sample to be tested is 0.2 μg / L.

[0036] Preferably, the detection limit of the method for butylated hydroxyanisole (BHA) in the sample to be tested is 0.5 μg / L.

[0037] Preferably, the detection limit of the method for 4-tert-butylphenol (4-tOP) in the sample to be tested is 1.2 μg / L.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The beneficial effects of this invention are:

[0042] The present invention provides a method for the detection of phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry. By employing dansyl chloride for pre-column derivatization of phenolic antioxidants, the fluorescence intensity and mass spectrometric response of the target compounds are significantly enhanced, thereby improving detection sensitivity and meeting the requirements for ultra-trace detection. Simultaneously, 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 system, solving the problem of existing methods' difficulty in simultaneously detecting multiple phenolic antioxidants. This method achieves ultra-trace detection of multiple phenolic antioxidants and has significant application value in the field of phenolic antioxidant detection technology. Attached Figure Description

[0043] Figure 1 Total ion current (TIC) plot for seven phenolic antioxidants;

[0044] Figure 2 This is a comparison of the mass spectrometry response signals of phenolic antioxidants before and after the dansyl chloride derivatization reaction. Detailed Implementation

[0045] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0046] Where specific techniques or conditions are not specified in the detailed embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0047] Example 1

[0048] A method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0049] S1. Pre-column derivatization: 1 mL of standard working solution (containing 7 target phenolic antioxidants BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300 and AO246, with a concentration of 50 ng / mL for each target phenolic antioxidant, and acetonitrile as the solvent in the standard working solution) was used as the test sample. The test sample was concentrated to near dryness by nitrogen blowing, 10 μL of acetonitrile was added, and after vortexing for 30 s, 50 μL of 5 mg / mL dansyl chloride solution and 100 μL of 0.1 M Na2CO3 buffer solution were added. After vortexing for another 30 s, the sample was heated in a water bath at 30 ℃ for 10 min in the dark.

[0050] The preparation of dansyl chloride solution involves dissolving dansyl chloride in acetonitrile or acetone to obtain a dansyl chloride solution with a concentration of 5 mg / mL. The pH of the Na₂CO₃ buffer solution is 8.77. To prepare the Na₂CO₃ buffer solution, weigh 8.00 g of NaHCO₃ powder and 0.51 g of Na₂CO₃ powder, dissolve them in a beaker containing 800 mL of deionized water, stir, and adjust the pH to 8.77 with NaOH or HCl (if pH > 9.0, add 1 M HCl to adjust to 9.0; if pH < 9.0, add 1 M NaOH). Add deionized water to a volume of 1000 mL.

[0051] 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 was completed by heating in a water bath in the dark, vortex mix, and then use formic acid to adjust the pH value of the sample to below 7 to terminate the derivatization reaction. Then, centrifuge the sample to be tested at 4℃ and 5000 r / min for 5 min. The sample is then filtered through a polypropylene (PP) filter membrane to obtain the derivatized sample.

[0052] S3, HPLC-MS / MS detection: The derivatized sample obtained in S2 was determined using high performance liquid chromatography-tandem mass spectrometry;

[0053] The chromatographic conditions were as follows: 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 was maintained for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, with a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 5~10 μL.

[0054] The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring mode (MRM), capillary voltage: 2.0 kV, ion source temperature: 110~130 ℃, desolvation gas temperature: 300~400 ℃, 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;

[0055] The detection conditions for the target phenolic antioxidants are shown in Table 1. The total ion current (TIC) chromatograms of the seven target phenolic antioxidants are shown below. Figure 1 As shown.

[0056] Table 1 shows the retention times of the compounds and the parameters of multiple reaction monitoring (MRM) in mass spectrometry.

[0057]

[0058] Example 2

[0059] A method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0060] S1. Pre-column derivatization: 1 mL of standard working solution (containing 7 target phenolic antioxidants BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300 and AO246, with a concentration of 50 ng / mL for each target phenolic antioxidant, and acetonitrile as the solvent in the standard working solution) was used as the test sample. The test sample was concentrated to near dryness by nitrogen blowing, 10 μL of acetonitrile was added, and after vortexing for 30 s, 50 μL of 5 mg / mL dansyl chloride solution and 100 μL of 0.1 M Na2CO3 buffer solution were added. After vortexing for another 30 s, the sample was heated in a water bath at 30 ℃ for 10 min in the dark.

[0061] The preparation of dansyl chloride solution involves dissolving dansyl chloride in acetonitrile or acetone to obtain a dansyl chloride solution with a concentration of 5 mg / mL. The pH of the Na₂CO₃ buffer solution is 8.77. The preparation of the Na₂CO₃ buffer solution involves weighing 8.00 g of NaHCO₃ powder and 0.51 g of Na₂CO₃ powder, dissolving them in a beaker containing 800 mL of deionized water, stirring, and adjusting the pH to 8.77 with NaOH or HCl (if pH > 9.0, add 1 M HCl to adjust to 9.0; if pH < 9.0, add 1 M NaOH). Deionized water is then added to a volume of 1000 mL.

[0062] 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 was completed by heating in a water bath in the dark, vortex mix, and then use formic acid to adjust the pH value of the sample to below 7 to terminate the derivatization reaction. Then, centrifuge the sample to be tested at 4℃ and 5000 r / min for 5 min. The sample is then filtered through a polypropylene (PP) filter membrane to obtain the derivatized sample.

[0063] S3, HPLC-MS / MS detection: The derivatized sample obtained in S2 was determined using high performance liquid chromatography-tandem mass spectrometry;

[0064] The chromatographic conditions were as follows: 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 was maintained for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, with a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 10 μL.

[0065] The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring mode (MRM), capillary voltage: 2.0 kV, ion source temperature: 120 ℃, desolvation gas temperature: 350 ℃, 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.

[0066] Comparative Example 1

[0067] A conventional method for detecting phenolic antioxidants using high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0068] S1. HPLC-MS / MS detection: 1 mL of standard working solution (containing 7 target phenolic antioxidants BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300 and AO246, with a concentration of 50 ng / mL for each target phenolic antioxidant, and acetonitrile as the solvent in the standard working solution) was used as the test sample. The test sample was analyzed by high performance liquid chromatography-tandem mass spectrometry.

[0069] The chromatographic conditions were as follows: 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 ℃; injection volume: 5 μL.

[0070] The mass spectrometry conditions were as follows: electrospray ionization source (ESI-), multiple reaction monitoring mode (MRM), capillary voltage: 3.0 kV, ion source temperature: 150 ℃, desolvation gas temperature: 300 ℃, 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.

[0071] The mass spectrometry response signals of phenolic antioxidants before and after the dansyl chloride derivatization reaction are compared, for example... Figure 2 As shown, Figure 2 Before derivatization, the mass spectrometry response signal corresponds to that of the test sample in Example 1 that was not derivatized by dansyl chloride before column derivatization, which was directly detected by HPLC-MS / MS. After derivatization, the mass spectrometry response signal corresponds to that of the test sample in Example 2 that was derivatized by dansyl chloride before column derivatization, which was detected by HPLC-MS / MS.

[0072] from Figure 2 Comparative analysis shows that, compared with the existing LC-MS / MS method in Comparative Example 1, the method of pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry for detecting phenolic antioxidants 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 spectrometric signal intensity generated by the dansyl chloride derivative is one to two orders of magnitude greater than that of the underrivatized phenolic antioxidant standard, resulting in stronger instrument response and higher sensitivity; 3) Since this method detects derivatives rather than the target phenolic antioxidant itself, it also has the ability to resist instrument contamination and mobile phase contamination.

[0073] Furthermore, the method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry of the present invention has a detection time of 10 min, which is significantly faster than the existing GC-MS method with a detection time of 20-30 min, making it more suitable for large-scale sample analysis. Compared with existing GC or LC methods, this method, using tandem mass spectrometry as the detector, can detect only the parent and daughter ions of the target phenolic antioxidants in complex matrix samples using a tandem quadrupole, without interference from chromatographic co-eluting components. Therefore, this method has higher accuracy in both qualitative and quantitative analysis. Moreover, this method has a lower detection limit (Table 2), making it suitable for ultra-trace analysis (ppb level).

[0074] Instrumental detection limit analysis of target phenolic antioxidants

[0075] The instrument detection limit for the target phenolic antioxidant is calculated as follows:

[0076] The detection limit of the instrument is defined as the concentration with a signal-to-noise ratio (S / N) of 3:1, and its calculation formula is shown in Equation (I):

[0077] D=3N×Q / I (Ⅰ)

[0078] In formula (I), Q represents the instrument concentration (μg / L), N represents noise, I represents the signal response value, and D represents the instrument detection limit of the target phenolic antioxidant (μg / L). I / N is the signal-to-noise ratio (S / N) for the corresponding instrument concentration. The workstation automatically analyzes the chromatogram to obtain the S / N. The specific steps are as follows: the standard solution of the target phenolic antioxidant with a known concentration is serially diluted until the instrument S / N is between 3 and 10 (read directly by the instrument). The instrument detection limit (D) of the target phenolic antioxidant is then calculated using the above formula, and the results are shown in Table 2.

[0079] Table 2 shows the instrument detection limits for seven target phenolic antioxidants determined using the pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry method with dansyl chloride.

[0080]

[0081] Example 3

[0082] To determine the optimal pH, water bath temperature, and reaction time for the dansyl chloride derivatization reaction, a full factorial experimental design was employed in this embodiment, with a 3-factor, 3-level, center-point replication, and two parallel experiments for each group. The experimental steps are as follows:

[0083] S1. Take 10 μL of 100 mg / L standard working solution (the standard working solution contains seven target phenolic antioxidants: BHT, TBHQ, BHA, 4-tOP, 2,6-TDBP, AO300, and AO246, with a concentration of 100 mg / L for each target phenolic antioxidant, and acetonitrile as the solvent) as the stock solution. Place the stock solution in a 2 mL centrifuge tube, and add 50 μL of 5 mg / mL dansyl chloride solution and 100 μL of 0.1 M Na2CO3 buffer (pH values ​​of the buffers are 8.77, 9.4, and 9.9, respectively) sequentially to reconstitute the sample. After adding the reagents, vortex for 30 seconds to ensure the reaction system is fully homogenized. Subsequently, place the centrifuge tube in a water bath and perform water bath heating reactions at three temperatures of 30, 40, and 50 °C for 10, 20, and 30 minutes, respectively, under light-protected conditions. After the reaction was completed, 840 μL of methanol-water (volume ratio 4:1) solution was added and vortexed to prepare a mixed standard solution with a concentration of 100 ng / L.

[0084] The preparation of dansyl chloride solution involves 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: For a 0.1 M pH 8.77 Na₂CO₃ buffer solution: Weigh 7.56 g of NaHCO₃ powder and 1.06 g of Na₂CO₃ powder, dissolve them in a beaker containing 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 adjust to 8.77; if pH < 8.77, add 1 M NaOH). Add deionized water to a volume of 1000 mL. For a 0.1 M pH 9.4 Na₂CO₃ buffer solution: Weigh 5.88 g of NaHCO₃ powder and 3.18 g of Na₂CO₃ powder, dissolve them in a beaker containing 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 adjust to 9.4; if pH < 9.4, add 1 M NaOH to adjust to 9.4). Add NaOH to deionized water to a volume of 1000 mL; Prepare a 0.1 M pH 9.9 Na2CO3 buffer solution: Weigh 3.36 g of NaHCO3 powder and 6.63 g of Na2CO3 powder, dissolve them in a beaker containing 800 mL of deionized water, stir, adjust the pH to 9.9 with NaOH or HCl, and add deionized water to a volume of 1000 mL;

[0085] 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 value to below 7, and then centrifuge at 4 ℃ and 5000 rpm for 5 minutes. Take 800 μL of the supernatant and filter it through a membrane filter.

[0086] S3. The supernatant after membrane filtration was analyzed by LC-MS / MS.

[0087] The chromatographic conditions were as follows: 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 was maintained for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, with a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 5~10 μL.

[0088] The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring mode (MRM), capillary voltage: 2.0 kV, ion source temperature: 110~130 ℃, desolvation gas temperature: 300~400 ℃, 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.

[0089] Through full factorial design experiments, the optimal derivatization conditions for each compound were obtained (the experimental conditions at which the peak area is the largest are the optimal derivatization conditions), as shown in Table 3.

[0090] Table 3 shows the optimal derivatization conditions for the target compound.

[0091]

[0092] As shown in Table 3, the optimal derivatization conditions for the seven target phenolic antioxidants are a buffer solution pH of 8.77, a water bath reaction temperature of 30 °C, and a water bath reaction time of 10 min. Therefore, these parameters are used as the actual optimal derivatization conditions in practical testing.

[0093] Example 4

[0094] A method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0095] S1. Sample Pretreatment: 500 mg of each of the five unknown indoor dust samples collected in Guangzhou was weighed into five 15 mL glass test tubes. 3 mL of n-hexane-dichloromethane (1:3, v / v) solution was added to each tube, and the mixture was vortexed twice, 1 min each time. Then, each tube was placed in a water bath for ultrasonic extraction for 30 min. After each ultrasonic extraction, the supernatant was collected in a glass test tube and centrifuged at 4500 r / min for 6 min. The supernatant was collected after centrifugation. The extraction was repeated three times, and the supernatants were combined to obtain five dust samples for testing.

[0096] S2, Pre-column derivatization: The five dust samples obtained in S1 were subjected to the following operations:

[0097] The solution was concentrated to near dryness by nitrogen blowing, 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 solution were added. After vortexing for another 30 s, the solution was heated in a water bath at 30 ℃ for 10 min in the dark.

[0098] The preparation of dansyl chloride solution involves dissolving dansyl chloride in acetonitrile or acetone to obtain a dansyl chloride solution with a concentration of 5 mg / mL. The pH of the Na₂CO₃ buffer solution is 8.77. To prepare the Na₂CO₃ buffer solution, weigh 8.00 g of NaHCO₃ powder and 0.51 g of Na₂CO₃ powder, dissolve them in a beaker containing 800 mL of deionized water, stir, and adjust the pH to 8.77 with NaOH or HCl (if pH > 9.0, add 1 M HCl to adjust to 9.0; if pH < 9.0, add 1 M NaOH). Add deionized water to a volume of 1000 mL.

[0099] 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 S2 has been completed by heating in a water bath in the dark. Vortex mix well, 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 ℃ and 5000 r / min for 5 min. The sample is then filtered through a polypropylene (PP) membrane to obtain the derivatized sample.

[0100] S4, HPLC-MS / MS detection: The derivatized sample obtained in S3 was determined using high performance liquid chromatography-tandem mass spectrometry;

[0101] The chromatographic conditions were as follows: 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 was maintained for 5 min, then linearly increased to 100% B over 3 min, and then linearly decreased to 45% B over 2 min, with a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 10 μL.

[0102] The mass spectrometry conditions were as follows: electrospray ionization source (ESI+), multiple reaction monitoring mode (MRM), capillary voltage: 2.0 kV, ion source temperature: 120 ℃, desolvation gas temperature: 350 ℃, 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.

[0103] The measurement results are shown in Table 4.

[0104] Table 4 shows the detection results (ng / g dry weight) of seven phenolic antioxidants in five indoor dust samples from Guangzhou.

[0105]

[0106] As shown in Table 4, all seven phenolic antioxidants were detected to varying degrees; the detection concentration ranged from 4.8 ng TBHQ / ng dry weight to 3450 ng BHT / g dry weight. Among them, BHT had the highest content. Phenolic antioxidants were detected in all five indoor dust samples, with a detection rate of 100%. The detection rate = number of detected samples / total number of samples * 100%.

[0107] In summary, the novel method and procedure for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry of the present invention includes dansyl chloride derivatization, formic acid neutralization, and analytical detection steps. (1) Compared with existing LC-MS / MS methods, this 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 existing GC-MS methods, the detection time of the method used in this invention is 10 min, which greatly improves the analysis speed and is more suitable for large-scale sample detection. (3) Compared with existing GC or LC methods, this method has the advantages of high accuracy, small sample volume, lower detection limit, and suitability for ultra-trace analysis.

[0108] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection 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, Includes the following steps: S1. The sample to be tested was derivatized using dansyl chloride before column derivatization, and acid neutralization was performed to terminate the derivatization reaction, thus obtaining the derivatized sample. S2. High performance liquid chromatography-triple quadrupole tandem mass spectrometry was used to determine the derivatized samples in order to achieve ultra-trace detection of phenolic antioxidants in the test samples; The pre-column derivatization of the test sample using dansyl chloride includes: concentrating the test sample to near dryness by nitrogen blowing, adding acetonitrile, vortexing, adding dansyl chloride solution and sodium carbonate buffer, vortexing, and heating in the dark. The acid neutralization treatment includes: adding an aqueous methanol solution to the sample to be tested after the light-shielded heating treatment, vortexing, adjusting the pH value to below 7 to terminate the derivatization reaction, centrifuging and passing through a membrane to obtain the derivatized sample. The phenolic antioxidants include 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. The detection limit of the method for phenolic antioxidants in the test sample is 0.2~2.5 μg / L; The chromatographic conditions for the determination of derivatized samples using the high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (HPLC-MS / MS) were as follows: column: C18 column, 50 mm × 2.1 mm, 1.7 μm; mobile phase: methanol: 0.2 mM ammonium acetate aqueous solution; gradient elution: 45% methanol was maintained for 5 min, then linearly increased to 100% methanol over 3 min, and then linearly decreased to 45% methanol over 2 min, with a total run time of 10 min; flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 5~10 μL. The mass spectrometry conditions for the determination of derivatized samples using the high performance liquid chromatography-triple quadrupole tandem mass spectrometry system are as follows: spray ion source, multiple reaction monitoring mode, capillary voltage: 2.0 kV, ion source temperature: 110~130 ℃, desolvation gas temperature: 300-400 ℃, 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.

2. The method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The volume ratio of 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 solution is 0.1 M. And / or, the vortex processing time is 30s.

3. The method for detecting phenolic antioxidants using dansyl chloride pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The light-shielding heating treatment is a light-shielding water bath heating treatment, with the water bath heating temperature being 28~32℃ and the time being 8~12 min.

4. The method for detecting phenolic antioxidants by pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry of dansyl chloride according to claim 1, 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 used is 840 μL; And / or, adjust the pH value to below 7 using formic acid; And / or, the filter membrane material used in the membrane is a polypropylene filter membrane.

5. The method for detecting phenolic antioxidants by pre-column derivatization-high performance liquid chromatography-tandem mass spectrometry of dansyl chloride according to claim 1, characterized in that, The samples to be tested are selected from dust, food, human blood, or urine.

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