A method for detecting six p-phenylenediamine compounds in milk

Abstract

This invention belongs to the field of organic matter detection technology and relates to a method for detecting six p-phenylenediamine compounds in milk. The method includes: mixing saturated saline solution, glutathione solution, extraction solvent, milk, and internal standard; sequentially subjecting the mixture to vortexing, sonication, and centrifugation; collecting the upper organic phase; adding the extraction solvent again to the lower aqueous phase; repeating the extraction and combining the organic phases; concentrating the mixture under nitrogen and redissolving it in acetonitrile; filtering to obtain the sample; and finally, using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) for detection, calculating the content based on peak area and a standard curve. This invention employs liquid-liquid extraction pretreatment combined with isotope internal standard method and optimized chromatographic and mass spectrometric conditions. It is simple, efficient, low-cost, has good repeatability, high sensitivity, and a recovery rate that meets detection requirements. It can simultaneously achieve qualitative and quantitative detection of six p-phenylenediamine compounds in milk.

Description

Technical Field

[0001] This invention belongs to the field of organic matter detection technology, and relates to a method for detecting six p-phenylenediamine compounds in milk. Background Technology

[0002] p-Phenylenediamine compounds, as antioxidants, are widely used in rubber products to prevent rubber oxidation, especially in tires. The increasing use of antioxidants in tire-related products has significantly increased their release into ecosystems, including the atmosphere, stormwater runoff, and soil sediments. Related studies have shown that p-Phenylenediamine compounds, such as N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, seriously threaten the survival of aquatic organisms. Some mouse toxicology experiments have also demonstrated their effects on liver and reproductive functions in these organisms.

[0003] Currently, monitoring technology mainly focuses on environmental and human samples, lacking monitoring techniques for food samples like milk. Furthermore, it primarily monitors N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine and N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone. Therefore, in order to simultaneously detect multiple p-phenylenediamine compounds and expand the scope of detection, a simple and convenient method for detecting multiple p-phenylenediamine compounds in food samples is urgently needed. Summary of the Invention

[0004] To address the above problems, this invention provides a method for detecting six p-phenylenediamine compounds in milk, specifically comprising the following steps:

[0005] A method for detecting six p-phenylenediamine compounds in milk includes the following steps:

[0006] Step 1: Mix saturated saline, glutathione solution, extraction solvent, milk and internal standard, vortex at 4500 rpm for 3-8 min, sonicate at 100 kHz for 15 min, and centrifuge at 4500 rpm for 5 min to separate the oil phase and aqueous phase, and collect the upper organic phase.

[0007] Preferably, the volume ratio of the saturated saline solution, glutathione solution, extraction solvent and milk is (0.7-1):0.6:5:1, and the ratio of milk to internal standard is (300-800) μL:(3-8) ng.

[0008] Preferably, the concentration of the glutathione solution is 0.5-1 mM, and most preferably 1 mM.

[0009] Preferably, the extraction solvent comprises acetonitrile, n-hexane, and dichloromethane in a volume ratio of 2:1:1.

[0010] Preferably, the internal standard is N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediaminequinone-d5(6PPD-Q-d5).

[0011] Step 2: Add extraction solvent to the lower aqueous phase, using the same amount as in Step 1. Perform vortexing, sonication, and centrifugation sequentially according to the method in Step 1, collect the organic phase, repeat 1-3 times, combine all organic phases, dry with nitrogen, dissolve the obtained solid with acetonitrile, filter to obtain the sample to be tested, and detect it by ultra-high performance liquid chromatography-tandem mass spectrometry. Calculate the content of the six p-phenylenediamine compounds based on the measured peak area and standard curve.

[0012] Preferably, the volume ratio of acetonitrile to milk is 1:1.

[0013] Preferably, filtration is performed using a 0.22μm filter membrane.

[0014] Preferably, the chromatographic conditions are as follows: using a C18 column; injection volume of 3-5 μL; flow rate of 0.3-0.5 mL / min; using 0.1% formic acid aqueous solution as mobile phase A and methanol as mobile phase B; the volume percentage of mobile phase B varies as follows: 0-1.5 min, mobile phase B is 10%; 1.5-4 min, mobile phase B is 10-95%; 4-7 min, mobile phase B is 95-100%; 7-8 min, mobile phase B is 10-100%; 8-8.1 min, mobile phase B is 10%.

[0015] Most preferably, the C18 chromatographic column has a column temperature of 38-40℃, a length of 90-100mm, an inner diameter of 1.8-2.1mm, and a packing particle diameter of 4-5μm.

[0016] Preferably, the mass spectrometry conditions are: drying gas temperature 250°C, drying gas flow rate 5 L / min, nebulizing gas pressure 35 psi, sheath gas temperature 350°C, sheath gas flow rate 11 L / min, capillary voltage 3500 V, and nozzle voltage 500 V.

[0017] The present invention has the following advantages:

[0018] The method of this invention employs liquid-liquid extraction as a sample pretreatment method that is simple, efficient, low-cost, and highly reproducible. It is combined with isotope labeling and ultra-high performance liquid chromatography-tandem mass spectrometry to achieve qualitative and quantitative detection and separation of six p-phenylenediamine compounds in different samples. The spiked recovery rate is 60-120%. It also has the advantages of high sensitivity, small amount of isotope internal standard, and low solvent loss, meeting the requirements of actual detection and having greater simplicity and a wider detection limit. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 This is a graph showing the elution performance in the first stage.

[0021] Figure 2 This is a graph showing the elution performance in the second stage.

[0022] Figure 3 This is a graph showing the elution performance in the third stage. Detailed Implementation

[0023] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] The names, corresponding abbreviations, and CAS numbers of the six p-phenylenediamine compounds are as follows:

[0026] N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD, CAS:793-24-8);

[0027] N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-Q, CAS:2754428-18-5);

[0028] N-Isopropyl-N'-Phenylene-p-phenylenediamine (IPPD, CAS: 101-72-4);

[0029] N,N'-Diphenyl-1,4-phenylenediamine (DPPD, CAS: 74-31-7);

[0030] N-Cyclohexyl-N'-phenyl-1,4-phenylenediamine (CPPD, CAS: 101-87-1);

[0031] N,N'-Bis(1,4-Dimethylpentyl)p-phenylenediamine (DNPD.CAS:93-46-9).

[0032] The names and corresponding abbreviations of isotopically labeled internal standards are as follows:

[0033] N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediaminequinone-d5(6PPD-Q-d5).

[0034] Preparation of standard solutions:

[0035] A mixed standard stock solution containing six p-phenylenediamine compounds was prepared using acetonitrile: the concentration of the standard for each p-phenylenediamine compound was 1.00 µg / mL. Subsequently, the final concentrations of the standards for each p-phenylenediamine compound were serially diluted with acetonitrile to 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL to obtain mixed standard working solutions of the corresponding dilution concentrations.

[0036] Detection method:

[0037] Step 1: Take 500 μL of milk sample containing the contents of the six p-phenylenediamine compounds to be tested, add 5 ng of each isotopically labeled internal standard, and then mix with 300 μL of 1 mmol / L glutathione solution, 400 μL of saturated saline, and 2.5 mL of extraction reagent (acetonitrile, n-hexane, and dichloromethane in a volume ratio of 2:1:1). Vortex for 5 min, sonicate at 100 kHz for 15 min, and centrifuge at 25℃ and 4500 rpm for 5 min to separate the organic reagent and aqueous phase. Collect the upper organic phase. Add 2.5 mL of extraction reagent to the remaining lower aqueous phase and extract using the same method, repeating the extraction of the lower aqueous phase a total of 3 times. Combine the upper organic phases from the three extractions, dry them with nitrogen, and finally redissolve them in 500 μL of acetonitrile. Filter through a 0.22 μm filter membrane to obtain the sample to be tested, ready for use.

[0038] Step 2, the detection conditions for ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry are as follows: the liquid chromatography instrument is an Agilent 1290 Infinity II ultra-high performance liquid chromatography system, the mass spectrometry instrument is an Agilent G6470B electrospray triple quadrupole mass spectrometer, and the chromatographic column is a Waters XBridge® C18 high performance liquid chromatography column with a length of 100 mm, an inner diameter of 2.1 mm, and a packing particle diameter of 5.00 μm.

[0039] Gradient elution was performed using a mobile phase A containing 0.1% formic acid (volume concentration) and a mobile phase B containing methanol. The column temperature was 40℃, the injection volume was 4 μL, and the flow rate was 0.4 mL / min. The gradient elution program is shown in Table 1.

[0040] Data were collected in positive ion mode. Tandem mass spectrometry was performed using an AJS electrospray ionization source with the following parameters: drying gas temperature 300℃, drying gas flow rate 5 L / min, nebulizing gas pressure 35 psi, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, capillary voltage 3500 V, and nozzle voltage 500 V. Specific multiple reaction mass spectrometry parameters are shown in Table 2.

[0041] Standard curves were plotted based on the peak areas and concentrations of six p-phenylenediamine compounds in their standard working solutions. The x-axis of the standard curves represented relative concentration, and the y-axis represented relative response values. Each p-phenylenediamine compound was quantified using standard curves at more than ten concentration levels. As shown in Table 3, the six p-phenylenediamine compounds exhibited good linearity and a good correlation coefficient (R²) in the concentration range of 0.01–50 ng / mL. 2 All values ​​are greater than 0.99, and can be used for subsequent calculations of the contents of the six p-phenylenediamine compounds.

[0042] A standard curve was established based on the peak area and concentration of the standard solutions of the six p-phenylenediamine compounds. Then, the content of the six p-phenylenediamine compounds in the milk sample to be tested was calculated based on the peak area and the standard curve.

[0043] Table 1 Gradient elution program

[0044] Time (min) Mobile phase A (v / v%) Mobile phase b (v / v%) Flow rate (mL / min) 0.00 90 10 0.40 1.50 90 10 0.40 4.00 5 95 0.40 7.00 0 100 0.40 8.00 90 10 0.40 8.10 90 10 0.40

[0045] Table 2 Specific parameters of multiple reaction mass spectrometry

[0046]

[0047] Table 3. Linearity results of the detection methods for six p-phenylenediamine compounds.

[0048]

[0049] Experimental Example 1

[0050] Following the steps in Example 1, internal standards and six p-phenylenediamine compounds were added to 500 μL of milk to verify their recovery rates. The spiking concentrations of the six p-phenylenediamine compounds were set at 1 ng / mL, 5 ng / mL, and 10 ng / mL, respectively, with each concentration performed in triplicate. A blank control without spiking was also included. At each concentration gradient, the spiking amount of each p-phenylenediamine compound was the same; that is, the same spiking amount was added for each p-phenylenediamine compound. The spiking recovery results were measured, and the results are shown in Table 4.

[0051] Table 4. Limits of Quantitation and Spiked Recovery Results for Six p-Phenylenediamine Compounds

[0052]

[0053] As can be seen from Table 4, in the blank control, except for IPPD having a background value, the background values of the other p-phenylenediamine antioxidants and quinone derivatives were lower than the limit of quantification (<LOQ). The recoveries at three concentrations were between 60.2 - 96.3%, and the relative deviation was less than 10%, meeting the recognized recovery standard (60 - 120%), and the simultaneous extraction and determination of 6 p-phenylenediamine compounds could be accurately achieved.

[0054] Test Example 2

[0055] Collect 60 milk samples from various places in China, and use the method of Example 1 to detect the contents of 6 p-phenylenediamine compounds in the milk samples. The results are shown in Table 5. Due to instrument limitation in detection and the fact that not all 6 p-phenylenediamine compounds may be contained in the milk samples, in Example 1 of the present invention, 6 p-phenylenediamine compounds were actually detected in the milk samples. Among them, the detection rate of 6PPD was the highest, at 86.67%, followed by IPPD, with a corresponding detection rate of 80.00%. This indicates that the method of Example 1 of the present invention can detect the presence and content of p-phenylenediamine compounds in milk samples.

[0056] Table 5 Detection situations of 6 p-phenylenediamine compounds in 60 milk samples

[0057] IPPD CPPD DPPD DNPD 6PPD 6PPD-Q LOQ 0.01 0.01 0.02 0.05 0.02 0.05 Detection rate (%) 80.00% 5.00% 35.00% 6.67% 86.67% 40.00% Minimum value <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Median 0.01 <LOQ <LOQ <LOQ 0.23 <LOQ Maximum value 4.98 0.02 0.22 0.15 3.90 0.09

[0058] Test Example 3

[0059] In order to explore the influence of the extraction reagent on the results, a standard working solution of 10 ng / mL was used, and the detection was carried out using the method of Example 1, with the difference that the mixed solvent was replaced by acetonitrile, dichloromethane, n-hexane, ethyl acetate or methyl tert-butyl ether. The results are shown in Table 6.

[0060] Table 6 Comparison results of the recoveries of 6 bisphenol compounds extracted by the mixed reagent and the single reagent

[0061]

[0062] When using nonpolar, weakly polar reagents (pure hexane, pure dichloromethane, and pure methyl tert-butyl ether) for extraction, their poor polarity means they do not dissolve well in water when mixed with milk. Instead, they interact with large molecules such as fats and proteins in the milk, potentially causing these molecules to aggregate and form a gel-like structure. However, p-phenylenediamine compounds have good solubility in hexane and dichloromethane. Therefore, a mixed reagent in a certain proportion was used to extract p-phenylenediamine compounds from milk. As shown in Table 6, the mixed reagent (acetonitrile, hexane, and dichloromethane in a volume ratio of 2:1:1) combines the good extraction effects of hexane and dichloromethane with the high polarity of acetonitrile, ensuring mutual solubility without forming a gel structure. This successfully extracted six p-phenylenediamine compounds, improving extraction efficiency and covering a wider range of compounds.

[0063] Therefore, within the recovery range of 60-120%, the mixed reagent (acetonitrile, n-hexane, and dichloromethane in a volume ratio of 2:1:1) was identified as the optimal extraction reagent, with wider applicability and higher extraction efficiency.

[0064] Test Example 4

[0065] To investigate the effect of the number of extractions on the results, a standard working solution of 10 ng / mL was used, and the method of Example 1 was employed for detection, except that the number of extractions was changed to 2 or 4 times.

[0066] Table 7. Comparison of recoveries of six p-phenylenediamine compounds extracted with different extraction cycles.

[0067]

[0068] As shown in Table 7, when using 2.50 mL of a mixed reagent (acetonitrile, n-hexane, and dichloromethane in a volume ratio of 2:1:1) for two extractions, the recoveries of 6PPD, DPPD, and DNPD were all below 60%. When extracting four times, the recoveries were not significantly different from those extracted three times. Furthermore, three extractions ensured good recoveries while also being economical and feasible. Therefore, considering factors such as recovery rate, time consumption, and cost, three extractions were the optimal number of extractions.

[0069] Experimental Example 5

[0070] Given the complex matrix of dairy products and the trace amounts and strong lipophilicity of target compounds (PPDs and 6PPD-Q), this experimental example aims to optimize the gradient elution procedure to address matrix inhibition, shorten the detection cycle, and improve detection sensitivity, thereby confirming the optimal liquid chromatography analysis conditions.

[0071] Phase 1: Initial Exploration Gradient (15-minute gradient, specific gradient settings are shown in Table 8). Typical 90% aqueous phase initial conditions (10% B phase) were used, with a relatively long elution cycle (15 minutes) designed to investigate the basic retention behavior and elution performance of the target compound in the complex matrix of dairy products. Results are shown in Table 9 and [Table data missing]. Figure 1 .

[0072] The second stage involved optimizing the gradient (8.1 min gradient, specific gradient settings are shown in Table 10). While maintaining a 90% aqueous initial phase, the efficiency of high-throughput detection and instrument response were improved by compressing the equilibrium and reducing the gradient rise time (to 8.1 min). The impact of increased elution rate on the target peak shape was also observed. Results are shown in Table 11. Figure 2 .

[0073] Phase 3: Performance Breakthrough Gradient (specific gradient settings are consistent with Example 1). Based on the experimental results of the previous phase, while ensuring that the responses of the six PPDs remain basically unchanged, the chromatographic conditions were improved by slowing down the rise rate of the organic phase in the middle and later stages, widening the retention time distribution of the target analytes, improving the interpeak separation and overall peak width, thereby obtaining chromatographic conditions more suitable for qualitative and quantitative analysis. The results are shown in Table 12 and... Figure 3 .

[0074] Table 8 Gradient Settings for the First Stage

[0075] Time (min) Water (v / v%) Methanol (v / v%) Flow rate (mL / min) 0.00 90 10 0.40 1.50 90 10 0.40 4.00 0 100 0.40 11.00 0 100 0.40 11.10 90 10 0.40 15.00 90 10 0.40

[0076] Table 9 Elution performance in the first stage

[0077] substance Retention time (min) IPPD 3.80 CPPD 4.07 DPPD 4.74 DNPD 5.07 6PPD 4.14 6PPD-Q 4.81

[0078] Table 10 Gradient Settings for the Second Stage

[0079] Time (min) Water (v / v%) Methanol (v / v%) Flow rate (mL / min) 0.00 90 10 0.40 1.50 90 10 0.40 3.00 5 95 0.40 4.00 0 100 0.40 7.00 0 100 0.40 8.00 90 10 0.40

[0080] Table 11 Elution performance in the second stage

[0081] substance Retention time (min) IPPD 3.45 CPPD 3.65 DPPD 4.16 DNPD 4.58 6PPD 3.70 6PPD-Q 4.19

[0082] Table 12 Elution performance in the third stage

[0083] substance Retention time (min) IPPD 3.85 CPPD 4.15 DPPD 4.94 DNPD 5.33 6PPD 4.22 6PPD-Q 4.97

[0084] From Table 8-12 and Figure 1-3It can be seen that the response intensity and material separation of the first-stage gradient are inferior to those of the optimal gradient, so this gradient is abandoned. The material response of the second-stage gradient is similar to that of the optimal gradient, but a comparison of the overall peak width and the aforementioned retention time table reveals that (3.45-4.58 vs 3.85-5.33), the material separation under this gradient is not as good as that of the first gradient, so this gradient is also abandoned.

[0085] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting six para-phenylenediamine compounds in milk, characterized in that, It comprises the following steps: Step one, mix saturated brine, glutathione solution, extraction solvent, milk and internal standard, and sequentially perform vortex, ultrasonic and centrifugal treatment, collect the upper organic phase; Step two, add extraction solvent to the lower aqueous phase, the amount is the same as step one, sequentially perform vortex, ultrasonic and centrifugal treatment according to the method of step one, collect the organic phase, repeat 1-3 times, combine all the organic phases, blow dry with nitrogen, dissolve the obtained solid with acetonitrile, filter to obtain the sample to be tested, and detect by ultra-high performance liquid chromatography tandem mass spectrometry, calculate the content of the six p-phenylenediamine compounds according to the measured peak area and standard curve; The volume ratio of the saturated brine, glutathione solution, extraction solvent and milk is (0.7-1):0.6:5:1, and the ratio of the milk and internal standard is (300-800) μL:(3-8) ng; The volume ratio of the acetonitrile and milk is 1:

1.

2. The method of detecting six para-phenylenediamine compounds in milk according to claim 1, characterized in that, The concentration of the glutathione solution in step one is 0.5-1 mM.

3. The method of claim 1, wherein the six para-phenylenediamine compounds are detected in milk. The extraction solvent in step one comprises acetonitrile, n-hexane and dichloromethane, and the volume ratio is 2:1:

1.

4. The method of claim 1, wherein the six para-phenylenediamine compounds are detected in milk. The internal standard in step one is 6PPD-Q-d5.

5. The method of detecting six para-phenylenediamine compounds in milk according to claim 1, wherein, In step two, filter with a 0.22 μm filter membrane.

6. The method of detecting six para-phenylenediamine compounds in milk according to claim 1, wherein, In step two, the chromatographic conditions are as follows: use a C18 chromatographic column, the sample amount is 3-5 μL; the flow rate is 0.3-0.5 mL / min; use 0.1% formic acid aqueous solution as mobile phase A and methanol as mobile phase B, and the volume percentage of mobile phase B changes as follows: 0-1.5 min, mobile phase B is 10%; 1.5-4 min, mobile phase B is 10-95%; 4-7 min, mobile phase B is 95-100%; 7-8 min, mobile phase B is 10-100%; 8-8.1 min, mobile phase B is 10%.

7. The method of detecting six para-phenylenediamine compounds in milk according to claim 6, characterized in that, The C18 chromatographic column has a column temperature of 38-40℃, a length of 90-100 mm, an inner diameter of 1.8-2.1 mm, and a filler particle diameter of 4-5 μm.

8. The method of detecting six para-phenylenediamine compounds in milk according to claim 1, wherein, In step two, the mass spectrometry conditions are as follows: dry gas temperature 250℃, dry gas flow rate 5 L / min, atomization gas pressure 35 psi, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, capillary voltage 3500 V, and nozzle voltage 500 V.

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