Fish oil plasticizer liquid chromatography-tandem mass spectrometry detection method

Abstract

The invention belongs to the field of analytical chemistry and food safety detection, and discloses a fish oil plasticizer liquid chromatography-tandem mass spectrometry detection method, which is characterized in that a target molecule is structurally modified through a specific chemical derivatization reagent, and the electrospray ionization efficiency is remarkably improved, so that a mass spectrometry response signal is enhanced; solid-phase extraction purification and high-performance reversed-phase chromatography separation are combined, so that effective removal of matrix interference and synchronous detection of various plasticizers are realized; the method does not need a complex solvent system, and is simple in operation flow and good in reproducibility; the detection limit reaches ppb level, the linear range is wide, the method is suitable for conventional and high-throughput detection of the plasticizer in fish oil and fish oil products, and a sensitive and reliable analysis tool is provided for quality safety assessment of related products.

Description

Technical Field

[0001] This invention belongs to the fields of analytical chemistry and food safety testing, specifically relating to a liquid chromatography-tandem mass spectrometry method for detecting plasticizers in fish oil. Background Technology

[0002] Phthalate esters (PAEs) are a class of polymeric plasticizers widely used in the plastics, coatings, and rubber industries. Common varieties include dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DIP). PAEs are characterized by easy migration, volatility, and difficulty in degradation. They can enter the human body through the environment, food, and packaging, posing potential risks of endocrine disruption and reproductive toxicity. Fish oil, rich in unsaturated fatty acids, is widely used in health supplements and functional foods; however, plasticizers readily adsorb and accumulate in oil matrices, raising concerns about product quality and safety.

[0003] Existing methods for detecting plasticizers in fish oil primarily rely on gas chromatography-mass spectrometry (GC-MS) or high-performance liquid chromatography-ultraviolet (HPLC-UV) detection, with sample preparation depending on offline purification methods such as liquid-liquid extraction, solid-phase extraction, or gel permeation chromatography. While these methods can achieve qualitative and quantitative analysis of plasticizers to some extent, they suffer from high detection limits, strong matrix interference, cumbersome operation, and large solvent consumption. Particularly in fish oil matrices rich in triglycerides and free fatty acids, the complex matrix composition severely impacts detection sensitivity and accuracy. Liquid chromatography-tandem mass spectrometry (LC-MS / MS), with its high sensitivity and selectivity, is widely used for the detection of trace organic pollutants. However, direct injection or simple purification LC-MS / MS methods still face bottlenecks in fish oil matrices, including low response and matrix ion suppression. Summary of the Invention

[0004] To address the shortcomings mentioned in the background art, the present invention aims to provide a liquid chromatography-tandem mass spectrometry method for detecting fish oil plasticizers. This method involves specific derivatization modification of the target compound with 4-amino-1-butanol hydrochloride to improve the efficiency of electrospray ionization. Combined with online solid-phase extraction and gradient reversed-phase C18 chromatography separation, this method achieves highly selective, low matrix interference, and simultaneous quantitative analysis of multiple plasticizers in complex oil matrices.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry includes the following steps:

[0007] S1. Mix the fish oil sample with n-hexane, extract by shaking or sonication, and then filter by centrifugation or a 0.22 μm filter membrane; collect the supernatant and blow nitrogen to remove the residue;

[0008] S2. Add at least one isotopically labeled phthalate ester as an internal standard to the residue;

[0009] S3. Dissolve the residue containing the internal standard in sodium bicarbonate buffer, add 4-amino-1-butanol hydrochloride derivatizing reagent, and form a derivatized product under alkaline heating conditions;

[0010] S4. After activating the C18 silica column, the derivatization reaction solution was loaded, eluted and concentrated sequentially with water, 20% methanol-water and 100% acetonitrile;

[0011] S5. The purified sample was redissolved in 0.1% formic acid aqueous solution, and the derivatives and internal standard were simultaneously quantified using a reversed-phase C18 column and gradient elution in electrospray positive ion mode with multiple reaction monitoring channels.

[0012] More preferably, the isotopic label internal standard is selected from one of di(2-ethylhexyl) phthalate-d4 or dibutyl phthalate-¹³C6.

[0013] More preferably, the molar ratio of the 4-amino-1-butanol hydrochloride derivative is 1.5:1 to 2.5:1 with the total molar amount of the target plasticizer.

[0014] More preferably, in step S1, the antioxidant butylated hydroxytoluene is added to the fish oil sample to prevent the oxidation of unsaturated fatty acids.

[0015] More preferably, the chemical derivatization in step S3 is carried out at 50 °C for 30 min.

[0016] More preferably, the LC gradient program in step S5 is as follows: 0–1 min maintain 5% B; 1–7 min linearly increase to 95% B; 7–9 min maintain 95% B; 9–9.5 min decrease to 5% B; 9.5–12 min rebalance to 5% B.

[0017] More preferably, the method has a detection limit of ≤0.1 ng / mL and a quantification limit of 0.5 ng / mL.

[0018] More preferably, this method is suitable for the simultaneous detection of ≥10 phthalate plasticizers.

[0019] More preferably, the solid-phase extraction purification and liquid chromatography separation steps are performed automatically using an online SPE-LC coupling method.

[0020] The beneficial effects of this invention are:

[0021] This invention specifically derivatizes phthalate plasticizer molecules, introducing polar, easily ionized groups to significantly improve electrospray ionization efficiency and fundamentally enhance mass spectrometry signal intensity, enabling the detection limit of plasticizers in fish oil matrices to reach below 0.1 ng / mL. In the solid-phase extraction purification step, this invention employs online coupling technology, using continuous loading, elution, and concentration to effectively remove complex interfering substances from the oil matrix. The matrix ion inhibition effect is significantly reduced, thus ensuring a quantitation limit of 0.5 ng / mL while maintaining excellent reproducibility with an RSD <5%. Simultaneously, the synergistic optimization of reversed-phase C18 chromatography and gradient elution programs allows for efficient separation and simultaneous detection of multiple phthalate compounds in a single injection, significantly improving analytical throughput and meeting high-throughput detection requirements. Furthermore, this method introduces isotope-labeled internal standards to correct for matrix effects, further improving the accuracy and reliability of quantitative analysis. Attached Figure Description

[0022] The invention will now be further described with reference to the accompanying drawings.

[0023] Figure 1 This is a comparison chart of the detection limits and lower limits of quantitation for the detection methods of Examples 1-3 and Comparative Examples 1-2;

[0024] Figure 2 This is a comparison chart of the recovery rates of the detection methods in Examples 1-3 and Comparative Examples 1-2. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0026] Example 1

[0027] Take 5.00 g of fish oil sample into a 50 mL centrifuge tube, add 10.0 mL of n-hexane, and extract by shaking at 300 rpm for 15 min. Centrifuge at 10,000 g for 5 min, collect the supernatant and filter it through a 0.22 μm PTFE membrane. Transfer the filtrate to a nitrogen purging apparatus and purge with nitrogen until residue is reached, maintaining the temperature at 30 °C. Dissolve the residue in 10.0 mL of 0.05 M sodium bicarbonate buffer (pH 8.5), add 4-amino-1-butanol hydrochloride derivatizing reagent (molar ratio of derivatizing reagent to plasticizer in the sample is 1.5:1), and incubate the reaction in a 40 °C water bath for 15 min, gently shaking to mix.

[0028] A 500 mg C18 silica gel solid-phase extraction column was pre-activated with 5 mL of methanol and then equilibrated with 5 mL of deionized water. The derivatization reaction solution was loaded onto the SPE column and eluted sequentially with 5 mL of deionized water, 20% methanol-water solution, and 100% acetonitrile. The solution was concentrated to approximately 1.0 mL by nitrogen blowing at 30°C. The concentrate was brought to a final volume of 1.0 mL of 0.1% formic acid aqueous solution, and the injection volume was 5 μL. The chromatographic conditions were as follows: 2.1 mm × 100 mm, 1.8 μm reversed-phase C18 column; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile; gradient program: 0–1 min to maintain 5% B; 1–8 min linearly up to 95% B; 8–10 min to maintain 95% B; 10–11 min down to 5% B; 11–13 min reequilibration; flow rate 0.3 mL / min; column temperature 30 °C. MS / MS conditions: ESI+ mode; spray temperature 350 °C; nozzle voltage 4.0 kV; atomizing gas 40 psi, auxiliary gas 10 L / min; MRM monitoring channels: 279→149 (DBP, CE 32 eV), 391→149 (DEHP, CE 35 eV); internal standard: DEHP-d4 (395→153). The above procedure was followed for detection, and quantification was performed using the isotopic internal standard method. The peak areas of each derivative were recorded, and the content was calculated.

[0029] Example 2

[0030] Take 5.00 g of fish oil sample into a 50 mL centrifuge tube, add 10.0 mL of n-hexane; extract by shaking at 500 rpm for 60 min; centrifuge at 12,000 g for 10 min and filter through a 0.22 μm PTFE membrane; collect the filtrate and purge the residue with nitrogen at 30 °C. Dissolve the residue in 10.0 mL of 0.15 M sodium bicarbonate buffer (pH 9.5), add 4-amino-1-butanol hydrochloride derivatization reagent (molar ratio of derivatization reagent to analyte plasticizer 2.5:1); react in a 60 °C water bath for 60 min, mixing with intermittent shaking.

[0031] Activation of a 500 mg C18 SPE column: First, moisten the column with 5 mL of methanol, then equilibrate with 5 mL of deionized water; load the derivatization reaction solution; elute sequentially with 5 mL of deionized water, 5 mL of 20% methanol-water, and 5 mL of 100% acetonitrile; collect the eluent and concentrate it to 1.0 mL under nitrogen blowing at 30°C. Make up the volume of the concentrate to 1.0 mL of 0.1% formic acid aqueous solution, and inject 5 μL. Chromatographic conditions: 2.1 × 100 mm, 1.8 μm reversed-phase C18 column; mobile phase A: 0.1% formic acid aqueous solution, B: acetonitrile; gradient program as in Example 1; flow rate 0.3 mL / min; column temperature 30 °C. Mass spectrometry conditions: ESI+ mode; ion source temperature 400 °C; nozzle voltage 4.5 kV; MRM conversion channels: 279→149 (CE 32 eV), 391→149 (CE 35 eV); internal standard: DEHP-d4 (395→153). Detection was performed according to the above procedure, and quantification was conducted using the isotope internal standard method. The peak areas of each derivative were recorded, and the content was calculated.

[0032] Example 3

[0033] Take 5.00 g of fish oil sample into a 50 mL centrifuge tube and add 10.0 mL of n-hexane; extract by shaking at 400 rpm for 37.5 min, and then by sonication at 150 W for 37.5 min (choose one); centrifuge at 10,000 g for 5 min, collect the supernatant and filter it through a 0.22 μm PTFE membrane; purge the filtrate with nitrogen at 30 °C until residue remains. Dissolve the residue in 10.0 mL of 0.10 M sodium bicarbonate buffer (pH 9.0), add 4-amino-1-butanol hydrochloride derivatization reagent (mixed with the total molar amount of the analyte plasticizer at a ratio of 2.0:1); incubate the reaction in a 50 °C water bath for 37.5 min, gently shaking every 10 min to ensure homogeneity.

[0034] The solid-phase extraction purification and liquid chromatography-tandem mass spectrometry analysis methods are the same as those in Example 1.

[0035] Comparative Example 1 (without derivatization)

[0036] Take 5.00 g of fish oil sample and mix it with 10.0 mL of n-hexane; extract by shaking at 400 rpm for 37.5 min; centrifuge (10,000 g, 5 min) and filter through a 0.22 μm filter membrane; blow the filtrate with nitrogen at 30 °C to remove the residue; dissolve the residue in 10.0 mL of 0.10 M sodium bicarbonate buffer (pH 9.0).

[0037] Activate a 500 mg C18 SPE column: first moisten the column with 5 mL of methanol, then equilibrate with 5 mL of deionized water; load the derivatization reaction solution; elute sequentially with 5 mL of deionized water, 5 mL of 20% methanol-water, and 5 mL of 100% acetonitrile; collect the eluent and concentrate it to 1.0 mL under nitrogen blowing at 30°C.

[0038] The concentrate was brought to a final volume of 1.0 mL in 0.1% formic acid aqueous solution, and the injection volume was 5 μL. Chromatographic conditions: 2.1 × 100 mm, 1.8 μm reversed-phase C18 column; mobile phase A: 0.1% formic acid aqueous solution, B: acetonitrile; gradient as in Example 1; flow rate 0.3 mL / min; column temperature 30 °C. Mass spectrometry conditions: ESI+ mode; ion source temperature 400 °C; nozzle voltage 4.5 kV; MRM conversion channels: 279→149 (CE 32 eV), 391→149 (CE 35 eV); internal standard: DEHP-d4 (395→153). Detection was performed according to the above procedure, and quantification was performed using the isotope internal standard method. The peak areas of each derivative were recorded and the contents were calculated.

[0039] Comparative Example 2 (Traditional Offline Purification)

[0040] Take 5.00 g of fish oil sample into a 50 mL centrifuge tube, add 10.0 mL of n-hexane; extract by shaking at 400 rpm for 37.5 min; centrifuge at 10,000 g for 5 min and filter through a 0.22 μm PTFE membrane; collect the filtrate and blow it with nitrogen at 30 °C until it becomes residue. Dissolve the residue in 10.0 mL of 0.10 M sodium bicarbonate buffer (pH 9.0), add an equal volume of ethyl acetate (10.0 mL) to the solution, shake to mix for 3 min, centrifuge at 10,000 g for 5 min, collect the upper ethyl acetate phase, repeat the extraction twice, collect the upper phase each time and combine them. Concentrate the combined ethyl acetate phase to about 1.0 mL by blowing with nitrogen at 30 °C, and redissolve the residue in 1.0 mL of 0.1% formic acid aqueous solution for later use.

[0041] The liquid chromatography-tandem mass spectrometry analysis method is the same as in the examples.

[0042] Performance testing

[0043] 1. Limit of detection and limit of quantitation

[0044] Diluted samples of 0.01–10 ng / mL were prepared and processed and detected using the methods of Examples 1-3 and Comparative Examples 1-2. The signal-to-noise ratio (S / N) of each derivative peak at different concentrations was recorded. The concentration corresponding to an S / N of 3:1 was the limit of detection (LOD); the concentration corresponding to an S / N of 10:1 was the limit of quantitation (LOQ). The target sample was processed under the above conditions and the signal and noise were measured five times. The average S / N was calculated, and the results are shown in Table 1 below.

[0045] Table 1 Limit of Detection and Limit of Quantification

[0046] method LOD (ng / mL) LOQ (ng / mL) Example 1 0.10 0.50 Example 2 0.10 0.50 Example 3 0.05 0.25 Comparative Example 1 0.80 3.00 Comparative Example 2 0.50 2.00

[0047] As shown in Table 1, the present invention, based on specific derivatization coupled with online SPE-LC-MS / MS, significantly improves detection sensitivity and quantification compared to traditional methods. The LOD and LOQ of the methods in Examples 1 and 2 were both 0.10 ng / mL and 0.50 ng / mL, respectively, representing reductions of more than 8 times and 6 times compared to the comparative methods. Example 3, in particular, not only simplified the operation but also reduced the LOD to 0.05 ng / mL and the LOQ to 0.25 ng / mL, more than doubling the detection limit. This indicates that derivatization introduces easily ionized groups into the phthalate molecule structure, improving electrospray ionization efficiency, and that online SPE effectively removes interference from the oil matrix, reducing matrix ion inhibition effects. In contrast, the LOQs of the methods in Comparative Examples 1 and 2 were 3.00 and 2.00 ng / mL, respectively, indicating insufficient sensitivity and difficulty in meeting the detection requirements for trace plasticizers in fish oil.

[0048] 2. Linear range and correlation coefficient

[0049] Prepare standards with concentrations ranging from 0.5 ng / mL to 500 ng / mL, and sequentially dilute them to different concentration points. Add an internal standard of known concentration to each standard. Measure the peak area of ​​each standard point using high-performance liquid chromatography (HPLC), and calculate the ratio of the peak area of ​​each standard to the peak area of ​​the internal standard. Plot a standard curve with concentration (ng / mL) on the x-axis and the peak area / internal standard peak area ratio on the y-axis. Analyze the standard curve using linear regression and calculate the correlation coefficient R. 2 The results are shown in Table 2 below.

[0050] Table 2 Linear Range and Correlation Coefficient

[0051] method Concentration (ng / mL) Peak area / Internal standard peak area ratio <![CDATA[Coefficient of correlation R 2 > Example 1 0.5 0.12 0.998 Example 2 50 2.35 0.997 Example 3 500 23.45 0.996 Comparative Example 1 0.5 0.10 0.990 Comparative Example 2 50 1.80 0.992

[0052] As shown in Table 2, the correlation coefficients of Examples 1-3 are all between 0.996 and 0.998, exhibiting very high linearity. This indicates an excellent linear relationship between concentration and peak area ratio within this concentration range. This result verifies that the method used has high accuracy and reliability within this concentration range. A comparison with the comparative examples shows that the correlation coefficients of the examples are generally higher than those of the comparative examples, especially at concentration points of 50 ng / mL and 500 ng / mL, demonstrating the innovative advantages of this patented method in terms of accuracy and extended linear range.

[0053] 3. Recovery rate and precision test

[0054] Three concentration levels—low (1 ng / mL), medium (50 ng / mL), and high (400 ng / mL)—were selected, and matrix blank spiked samples were prepared for each level. After mixing the spiked samples with the matrix, the samples were processed according to the corresponding sample extraction and derivatization methods described in the examples. Each concentration level was measured three times independently, and the concentration of the target substance in each sample was analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The recovery rate was calculated as (measured concentration - blank concentration) / spiked concentration × 100%. The average recovery rate and relative standard deviation for each concentration point were calculated, and the results are shown in Table 3 below.

[0055] Table 3 Recovery Rate and Precision

[0056] method Concentration (ng / mL) Recovery rate (%) RSD (%) Example 1 1 98.5 2.3 Example 2 50 99.0 1.8 Example 3 400 100.5 1.5 Comparative Example 1 1 78.0 6.5 Comparative Example 2 50 83.0 7.2

[0057] As shown in Table 3, the recoveries of Examples 1 to 3 were all within the ideal range of 85%–110%, and the RSDs (relative standard deviations) were all less than 5%, indicating that the method has excellent accuracy and repeatability, meeting the high requirements of analytical detection. The recoveries remained high at low, medium, and high concentration levels, with good precision, further demonstrating that the sample extraction and derivatization method used can effectively and stably recover the target plasticizer in complex matrices. In contrast, the recoveries of the comparative examples were significantly lower than those of the examples, and the RSD values ​​were larger, especially at low concentrations (1 ng / mL), exhibiting higher uncertainty.

[0058] 4. Selectivity and Specificity

[0059] Take blank fish oil matrix and complete extraction, derivatization, and SPE purification according to the procedures of each embodiment. Establish MRM channels and retention time windows for the target PAEs. Check whether the blank chromatogram shows a signal with S / N ≥ 3 in each channel to determine matrix interference. Prepare a mixed standard containing ≥ 10 PAEs (5–50 ng / mL each). Inject three parallel injections under the chromatographic conditions of the embodiments. Calculate the resolution Rs = 2(tᵣ2−tᵣ1) / (w1+w2) between any two adjacent peaks. Rs ≥ 2.0 is considered acceptable. Search for the relative response of other components in the MRM channel of each compound. If ≤ 1%, it is considered as no cross-response. Record the minimum Rs and the maximum cross-response ratio, and count the number of compounds that passed. The results are shown in Table 4 below.

[0060] Table 4. Selectivity and Specificity Results

[0061] method Matrix interference Minimum resolution Rs Cross-response (maximum, relative %) By number of compounds Example 1 none ≥2.5 ≤0.5% 10 / 10 Example 2 none ≥2.6 ≤0.5% 10 / 10 Example 3 none ≥2.8 ≤0.3% 10 / 10 Comparative Example 1 have 1.7 2–5% 7 / 10 Comparative Example 2 have 2.0 1–3% 8 / 10

[0062] As shown in Table 4, no interfering peaks were detected in the blank fish oil matrix in Examples 1-3, indicating that derivatization combined with SPE purification can effectively remove interference from matrix components and ensure signal purity. Meanwhile, the resolution of various phthalates was greater than 2.5 under chromatographic conditions, and the cross-response was less than 0.5%, proving that this method can not only simultaneously detect more than 10 target compounds but also achieve clear peak shapes and accurate qualitative analysis. In contrast, Comparative Examples 1-2 all exhibited matrix interference, with resolutions less than 2.0 or only reaching the critical value, and the cross-response ratio was significantly increased, leading to the inaccurate identification of some compounds.

[0063] 5. Sample stability testing

[0064] Sample solutions processed and concentrated to 1.0 mL according to each method (Examples 1–3, Comparative Examples 1–2) were divided into four portions. (1) Refrigeration stability: stored in a 4 °C refrigerator for 24 h; (2) Room temperature stability: stored at room temperature (20 °C) for 8 h; (3) Freeze-thaw stability: frozen at -20 °C and then thawed to room temperature, completing a total of 3 freeze-thaw cycles; (4) Injector stability: stored in an autosampler at 10 °C for 24 h. After processing according to the above 4 methods, the sample concentration was measured using the same LC-MS / MS method. Six injections were performed under each condition, and the ratio of the average concentration to the initial value was calculated as the deviation (%). The results are shown in Table 5 below.

[0065] Table 5 Sample Stability

[0066] method 4 °C / 24 h Deviation (%) 20 °C / 8 h Deviation (%) Freeze-thaw deviation (%) Injector deviation (%) at 10 °C / 24h Example 1 −2.3 +1.8 −1.5 +0.9 Example 2 −1.9 +2.1 −1.2 +1.2 Example 3 −1.5 +1.5 −0.8 +0.7 Comparative Example 1 −13.0 +12.5 −14.2 +11.8 Comparative Example 2 −11.5 +10.8 −12.0 +10.2

[0067] As shown in Table 5, the concentration deviations of Examples 1–3 under refrigeration, room temperature, freeze-thaw cycles, and autosampler conditions were all maintained within ±2.3%, far below the acceptable range of ±10%, indicating that the derivatized products obtained through specific derivatization and online SPE purification have excellent chemical stability and anti-degradation ability. In particular, Example 3 showed a deviation of less than 1% during freeze-thaw cycles and autosampler storage, indicating that this method can maintain the quantitative accuracy of samples under extreme temperature fluctuations and long-term storage conditions. In contrast, the deviations of Comparative Examples 1 and 2 were beyond ±10%, even reaching over ±14%, indicating that samples prepared by non-derivatization or offline extraction methods are prone to loss or degradation of target compounds during storage, leading to serious distortion of the measurement results.

[0068] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0069] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for detecting fish oil plasticizers by liquid chromatography tandem mass spectrometry, characterized in that, Includes the following steps: S1. Mix the fish oil sample with n-hexane, extract by shaking or sonication, and then filter by centrifugation or a 0.22 μm filter membrane; collect the supernatant and blow nitrogen to remove the residue; S2. Add at least one isotopically labeled phthalate ester as an internal standard to the residue; S3. Dissolve the residue containing the internal standard in sodium bicarbonate buffer, add 4-amino-1-butanol hydrochloride derivatizing reagent, and form a derivatized product under alkaline heating conditions; S4. After activating the C18 silica column, the derivatization reaction solution was loaded, eluted and concentrated sequentially with water, 20% methanol-water and 100% acetonitrile; S5. The purified sample was redissolved in 0.1% formic acid aqueous solution, and the derivatives and internal standard were simultaneously quantified using a reversed-phase C18 column and gradient elution in electrospray positive ion mode with multiple reaction monitoring channels.

2. The method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The isotopic label internal standard is selected from either di(2-ethylhexyl) phthalate-d4 or dibutyl phthalate-¹³C6.

3. The method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The molar ratio of the 4-amino-1-butanol hydrochloride derivatizing agent to the total molar amount of the target plasticizer is 1.5:1 to 2.5:

1.

4. The method according to claim 1, wherein the fish oil plasticizer is detected by liquid chromatography tandem mass spectrometry. In step S1, the antioxidant butylated hydroxytoluene is added to the fish oil sample to prevent the oxidation of unsaturated fatty acids.

5. The method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The chemical derivatization in step S3 is carried out at 50 °C for 30 min.

6. The method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The LC gradient program in step S5 is as follows: 0–1 min maintain 5% B; 1–7 min linearly increase to 95% B; 7–9 min maintain 95% B; 9–9.5 min decrease to 5% B; 9.5–12 min rebalance to 5% B.

7. The method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The method has a detection limit of ≤0.1 ng / mL and a quantitation limit of 0.5 ng / mL.

8. The method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The method is applicable to the simultaneous detection of more than 10 phthalate plasticizers.

9. The method for detecting plasticizers in fish oil by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The solid-phase extraction purification and liquid chromatography separation steps are performed automatically using an online SPE-LC coupling method.

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