A method for detecting 22 kinds of organophosphate esters in fish meat by LC-MS / MS

Abstract

The application provides a kind of LC-MS / MS detection method for 22 kinds of organophosphate in fish meat, and relates to the technical field of food safety detection.The detection method of the application includes steps such as sample extraction, sample purification and concentration, LC-MS / MS determination, etc.Through optimizing the acetonitrile extraction of fish meat sample, salting-out, HLB-P Glass SPE flow-through purification, vacuum parallel concentration, 40% acetonitrile water reconstitution, delay column system background control and C18 analysis column chromatographic separation conditions, the simultaneous, accurate and sensitive detection of 16 kinds of organophosphate triesters and 6 kinds of organophosphate diesters in fish matrix is realized, which has the characteristics of simple operation, low matrix interference, controllable system blank, less target loss, good separation effect and reliable quantitative result, and is suitable for monitoring the residual level of organophosphate in fish samples, and has good application prospect in food safety detection.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to an LC-MS / MS method for the detection of 22 organophosphates in fish meat. Background Technology

[0002] Organophosphate esters (OPEs) are a class of synthetically produced phosphoric acid derivatives used in transportation, electronic and electrical equipment, furniture, and building materials due to their flame-retardant and plasticizing properties. Organophosphate triesters are typically present in products or raw materials through mechanical addition and are easily released into the environment during production, use, and disposal. Organophosphate diesters can originate from production and emissions, as well as be generated from the conversion of organophosphate triesters. Fish, as a common animal-derived food in the diet, has organophosphate residue levels that are relevant to food safety risk monitoring; therefore, it is necessary to establish organophosphate detection methods suitable for fish matrices.

[0003] The detection of organophosphates in animal-derived foods typically employs liquid chromatography-tandem mass spectrometry (LC-MS / MS), with sample processing generally including organic solvent extraction, purification, isotope internal standard correction, and multiple reaction monitoring (MRM). However, fish samples contain abundant proteins, lipids, and other co-extracts, resulting in a complex matrix composition. This matrix interference and solvent effects can easily occur in the detection of trace organophosphates, affecting peak shape, response, and quantitative accuracy. Furthermore, the mobile phase, post-mixer flow path, and tubing materials in the LC system may introduce background contaminants such as TEP and TCiPP, which are similar to or identical to the analytes. These contaminants can easily superimpose on the target analyte signal at low concentrations, thus affecting the detection limit and quantitative reliability.

[0004] Furthermore, the organophosphates to be detected in fish meat include both organophosphate triesters and organophosphate diesters, with significant differences in polarity, retention behavior, and mass spectrometric response among different targets. Isomers such as TiBP and TnBP also present chromatographic separation challenges. Existing methods have limitations in the number of detectable targets, particularly in organophosphate diester coverage. Some methods lack optimization for sample concentration, reconstitution, and solid-phase extraction column activation conditions specifically for fish meat matrix and trace organophosphate detection, leading to issues such as target analyte loss, increased blank contamination, or deteriorated peak shape. Therefore, a method for detecting organophosphates in fish meat samples is needed that can simultaneously control system background, reduce matrix interference, minimize target analyte loss, achieve multi-component separation, and simultaneously detect organophosphate triesters and organophosphate diesters. Summary of the Invention

[0005] In view of this, the present invention provides an LC-MS / MS method for the detection of 22 organophosphates in fish meat. This invention optimizes acetonitrile extraction, salting out, HLB-P Glass SPE flow-through purification, vacuum parallel concentration, redissolution with 40% acetonitrile in water, background control of the delayed column system, and chromatographic separation conditions of the C18 analytical column. This achieves simultaneous, accurate, and sensitive detection of 16 organophosphate triesters and 6 organophosphate diesters in the fish meat matrix. It features simple operation, low matrix interference, controllable system blank, minimal target analyte loss, good separation effect, and reliable quantitative results. It is suitable for monitoring organophosphate residue levels in fish meat samples and has promising applications in food safety testing.

[0006] The present invention provides an LC-MS / MS method for the detection of 22 organophosphates in fish meat samples, comprising the following steps: S1. Sample extraction: Take a well-mixed fish meat sample, add pure water and isotope mixed internal standard in sequence, mix well, add acetonitrile, vortex and sonicate to extract, add NaCl for salting out, vortex and centrifuge, and take the supernatant. S2. Sample purification and concentration: The HLB-P Glass SPE column was activated with acetonitrile. The supernatant obtained in step S1 was passed through the column and the eluent was collected. The HLB-P Glass SPE column was then eluted with acetonitrile. The eluents were combined and concentrated under vacuum to 0.4 mL. The volume was then adjusted to 1 mL with pure water. The mixture was then centrifuged to obtain the test solution. S3, LC-MS / MS determination: The test solution was determined using a liquid chromatography system including a delay column and an analytical column and a tandem mass spectrometry system. The delay column was located between the mixer and the injector. The delay column was a C18 column with a higher carbon content than the analytical column. The analytical column was a C18 column. The 22 organophosphates were determined using an isotope internal standard working curve and multiple reaction monitoring.

[0007] Preferably, the 22 organophosphates include 16 organophosphate triesters and 6 organophosphate diesters; the 16 organophosphate triesters are TMP, TEP, TiPP, TnPP, TiBP, TnBP, TCEP, TPhP, TCiPP, EHDPP, TMPP, TBOEP, TDCiPP, TEHP, V6, and TDBPP; the 6 organophosphate diesters are DnBP, DPhP, BCiPP, DoCP, BBOEP, and BDCiPP.

[0008] Preferably, in step S1, the fish meat sample is 1.0 g, weighed to an accuracy of 0.001 g; 5 mL of pure water, 10 ng of isotope mixed internal standard, 10 mL of acetonitrile, and 2.5 g of NaCl are added; the vortexing is performed by vortexing for 2 min after adding acetonitrile and vortexing for 2 min after adding NaCl; the ultrasonic extraction is performed by ultrasonic extraction at 25 kHz for 10 min; and the centrifugation is performed by centrifugation at 3000 r / min and 4℃ for 10 min.

[0009] Preferably, in step S2, the HLB-P Glass SPE column is of 300 mg and 6 mL specification; it is activated with 5 mL of acetonitrile, and 5 mL of the supernatant obtained in step S1 is passed through the column and eluted with 5 mL of acetonitrile.

[0010] Preferably, in step S2, the HLB-P Glass SPE column adopts a flow-through purification mode, and no pressure is applied during sample loading; the sample passes through the HLB-P Glass SPE column by its own gravity; the activation solution is discarded, and 5 mL of sample and 5 mL of eluent are collected, and the HLB-P Glass SPE column does not need to be dried after activation.

[0011] Preferably, the stock solution of the isotope mixed internal standard is a commercially available standard with a concentration of 10 μg / mL for each monomer, diluted to 100 ng / mL before use; when using, take 100 μL of the 100 ng / mL isotope mixed internal standard intermediate solution as 10 ng of isotope mixed internal standard; the target analyte mixed standard is a commercially available standard with a concentration of 100 μg / mL for each monomer, diluted to 100 ng / mL before use.

[0012] Preferably, the analytical column is a Shim-pack GISS-HP C18 metal-free column with a particle size of 3 μm, a size of 2.1×150 mm, and a carbon content of 9%; the delay column is a Shim-pack Scepter C18-120 with a particle size of 3 μm, a size of 2.1×50 mm, and a carbon content of 19.5%.

[0013] Preferably, in step S3, the liquid chromatography conditions include: flow rate 0.4 mL / min, column temperature 40℃, injection volume 5 μL; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile; the gradient elution program is: A 85% and B 15% at 0.0 min; A 85% and B 15% at 0.5 min; A 30% and B 70% at 16.0 min; A 2% and B 98% at 18.0 min; A 2% and B 98% at 20.0 min; A 85% and B 15% at 20.1 min; and A 85% and B 15% at 25.0 min.

[0014] Preferably, in step S3, the tandem mass spectrometry conditions include: positive and negative ion scanning of the electrospray ion source, multiple reaction monitoring; the interface voltage is 4 kV in both positive and negative ion modes, the interface temperature is 150℃, the desolvation temperature is 261℃, the DL temperature is 250℃, the heating block temperature is 400℃, the nebulizing gas flow rate is 3 L / min, the heating gas flow rate is 10 L / min, and the drying gas flow rate is 10 L / min.

[0015] Preferably, the preparation of the working curve includes: taking 7 glass centrifuge tubes, adding 5 mL of pure water and 10 ng of isotope mixed internal standard to each tube, then adding 0.5~50 ng of OPEs mixed standard solution to each tube, and processing according to steps S1 and S2. Since blank fish meat matrix is ​​difficult to obtain and is easily contaminated by organophosphates in the environment during storage, this invention uses a pure water spiking system to prepare the working curve; the isotope internal standard is used to correct for target analyte loss and matrix effect during sample pretreatment. The working curve prepared using the pure water spiking system can meet the quantitative requirements for spiked recovery of fish meat samples.

[0016] Preferably, the content X of each analyte in the sample is calculated according to the following formula: X = m1 / m × 1000 / 1000, where X is the content of each analyte in the sample, in μg / kg; m1 is the content of each analyte in the sample solution obtained from the working curve, in ng; and m is the sample weight, in g.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention addresses the challenges of fish meat samples containing numerous proteins, lipids, and other co-extractants, and having complex matrix compositions. It employs a sample pretreatment method combining acetonitrile extraction, NaCl salting out, and HLB-P Glass SPE column flow-through purification. This effectively reduces the impact of co-extractants in the fish meat matrix on the peak shape, mass spectrometry response, and quantitative accuracy of the target analytes, thereby improving the stability and reliability of trace organophosphate detection in the fish meat matrix.

[0018] This invention employs a glass HLB-P SPE column and activates the SPE column with acetonitrile, which can reduce the background contamination of organophosphates that may be caused by the solid phase extraction column itself. At the same time, it adopts a flow-through purification mode, where the SPE column is activated with acetonitrile, the activation solution is discarded, and the sample is directly loaded and eluted without drying. The sample and eluent are collected, which reduces the risk of target adsorption loss and contamination introduction in the sample processing steps.

[0019] This invention incorporates a delay column in a liquid chromatography system, positioned between the mixer and the injector. This allows for the temporal separation of background contaminants such as TEP and TCiPP, which may dissolve from the mobile phase, the pre-column flow path, and tubing materials, from the target analytes in the sample. This reduces the interference of system background on the detection of low-concentration samples, improving the method's detection limit control and quantitative reliability. Furthermore, this invention utilizes a C18 delay column with a carbon content of 19.5%. Compared to C18 short columns with carbon contents of 9% and 13%, the 19.5% carbon content delay column exhibits stronger retention of system-derived TCiPP, resulting in more significant temporal separation between the system background peak and the target peak, thus improving system background control and the quantitative reliability of low-concentration samples.

[0020] This invention addresses the significant differences in polarity, retention behavior, and mass spectrometry response between organophosphates (OPPs) and organophosphates (EPDs). It establishes an LC-MS / MS detection method that simultaneously covers 16 OPPs and 6 EPDs, expanding the detection range of organophosphate contaminants in fish samples and solving the problem of limited detection targets in existing methods, especially insufficient coverage of EPDs.

[0021] This invention enables the effective chromatographic separation of 22 target analytes by selecting a suitable C18 analytical column and gradient elution conditions, and improves the separation effect of isomers such as TiBP and TnBP, while reducing the impact of isomer co-elution on qualitative and quantitative results.

[0022] This invention employs a vacuum parallel concentration method, controlling the concentration endpoint at 0.4 mL, and then bringing the volume to 1 mL with pure water to form a 40% acetonitrile-water reconstitution system. This avoids the loss of target analytes and the introduction of exogenous contamination caused by complete evaporation or nitrogen blowing concentration. At the same time, it takes into account the absolute recovery rate and chromatographic peak shape of the target analytes, reduces the solvent effect caused by high proportions of acetonitrile, improves the peak shape of early-eluting target analytes such as TMP, TEP, and BCIPP, and enhances the sensitivity and repeatability of the method.

[0023] This invention employs isotope internal standard working curves and multiple reaction monitoring for quantitative analysis, which can compensate for the loss of target substances and matrix effects during sample pretreatment, improve the quantitative accuracy of organophosphates with different polarities and response characteristics, and is suitable for food safety risk monitoring of trace organophosphates in fish meat. Attached Figure Description

[0024] The present invention will be further described below with reference to the accompanying drawings.

[0025] Figure 1 A comparison of the background contamination removal effect of HLB-P SPE columns after activation with different volumes of acetonitrile; Figure 2A comparative graph showing the response loss of some target substances under different concentration endpoint conditions; Figure 3 The chromatograms show the separation of 22 organophosphate target compounds, among which Figure 3 (a) is the chromatogram of the high-sensitivity target analyte. Figure 3 (b) is the chromatogram of the low-sensitivity target analyte; Figure 4 To compare the chromatograms of TEP and TCIPP before and after setting the delay column; Figure 5 A schematic diagram showing the location of the delay column; Figure 6 A comparison of the separation effects of TiBP and TnBP under different analytical column conditions; Figure 7 The graph shows a comparison of the background response of TCiPP after 10 mL of acetonitrile was concentrated by nitrogen blowing and by vacuum parallel concentration, respectively. Figure 8 This is a comparison of the separation effect between the target peak of TCiPP and the system background peak under C18 delayed column conditions with different carbon contents. Figure 8 (a) is a delayed column with a carbon content of 9%. Figure 8 (b) is a delayed column with a carbon content of 13%. Figure 8 (c) is a delayed column with a carbon content of 19.5%; Figure 9 The chromatograms of TMP, TCEP and TEP in different acetonitrile ratio resolution systems are shown. Among them, Figures (a) to (g) are the chromatograms obtained by resolution with acetonitrile-water solution with volume fractions of 10%, 20%, 30%, 40%, 50% and 75% acetonitrile and 100% acetonitrile, respectively. Figure 10 The chromatograms of TiPP, TCiPP and TnPP in different acetonitrile ratio resolution systems are shown. Among them, Figures (a) to (g) are the chromatograms obtained by resolution with acetonitrile-water solution with volume fractions of 10%, 20%, 30%, 40%, 50% and 75% acetonitrile and 100% acetonitrile, respectively. Figure 11 The chromatograms of V6, TiBP and TDCiPP in different acetonitrile ratio resolution systems are shown. Figures (a) to (g) are chromatograms obtained by resolution with acetonitrile-water solution with volume fractions of 10%, 20%, 30%, 40%, 50% and 75% acetonitrile and 100% acetonitrile, respectively. Figure 12The chromatograms of TnBP, TDBPP and TPHP in different acetonitrile ratio resolution systems are shown in Figures (a) to (g), which are obtained by resolution with acetonitrile-water solution with volume fractions of 10%, 20%, 30%, 40%, 50% and 75% and 100% acetonitrile, respectively. Figure 13 The chromatograms of TBOEP, EHDPP and TMPP in different acetonitrile ratio resolution systems are shown. Among them, Figures (a) to (g) are the chromatograms obtained by resolution with acetonitrile-water solution with volume fractions of 10%, 20%, 30%, 40%, 50% and 75% acetonitrile and 100% acetonitrile, respectively. Figure 14 The chromatograms of TEHP, DnBP and DpHP in different acetonitrile ratio resolution systems are shown. Among them, Figures (a) to (g) are the chromatograms obtained by resolution with acetonitrile-water solution with volume fractions of 10%, 20%, 30%, 40%, 50% and 75% acetonitrile and 100% acetonitrile, respectively. Figure 15 The chromatograms of DoCP, BBOEP, BCIPP and BDCiPP in different acetonitrile ratio resolution systems are shown. Among them, Figures (a) to (g) are the chromatograms obtained by resolution with acetonitrile-water solution with volume fractions of 10%, 20%, 30%, 40%, 50% and 75% acetonitrile and 100% acetonitrile, respectively. Figure 16 The graph shows a comparison of the absolute recoveries of 22 organophosphate target compounds in different acetonitrile resolution systems, where the volume fractions of acetonitrile in the acetonitrile-water resolution systems are 10%, 20%, 30%, 40%, and 50%, respectively. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Detection method of 22 OPEs in fish meat samples 1. Reagents and consumables: NaCl was of analytical grade, calcined at 550℃ for 5 h and then stored in a glass container; formic acid was of LC-MS grade; acetonitrile was of LC-MS grade; HLB-P Glass SPE column was 300 mg, 6 mL specification; 30 mL glass centrifuge tubes were used.

[0028] 2. Standards: A mixed standard of 22 organophosphate esters including TMP, TEP, TiPP, TnPP, TiBP, TnBP, TCEP, TPhP, TCiPP, EHDPP, TMPP, TBOEP, TDCiPP, TEHP, V6, TDBPP, DnBP, DPhP, BCIPP, DoCP, BBOEP, and BDCiPP. Eighteen organophosphate deuterated internal standard mixtures were used, including TMP-D9, TEP-D15, TnPP-D21, TnBP-D27, TPhP-D15, TCiPP-D18, TDCiPP-D15, TEHP-D51, TCEP-D12, TBOEP-D27, V6-D16, TDBPP-D15, DnBP-D18, DPhP-D10, BCIPP-D12, DoCP-D14, BBOEP-D8, and BDCiPP-D10. The stock solution of the isotope mixture was 10 μg / mL, diluted to 100 ng / mL before use. 100 μL of the 100 ng / mL isotope mixture intermediate solution was taken, i.e., 10 ng of the isotope mixture internal standard was added. The concentration of the target analyte stock solution was 100 μg / mL, which was diluted to 100 ng / mL before use.

[0029] 3. Sample Extraction: Weigh 1.0 g of the well-mixed fish meat sample, accurate to 0.001 g, and place it in a 30 mL glass centrifuge tube. Add 5 mL of pure water and 10 ng of isotope mixed internal standard, and mix well. Add 10 mL of acetonitrile, vortex for 2 min, and sonicate at 25 kHz for 10 min. Add 2.5 g of NaCl, vortex for 2 min, and centrifuge at 3000 r / min and 4℃ for 10 min. Collect the supernatant for purification.

[0030] 4. Sample purification and concentration: The HLB-P Glass SPE column was activated with 5 mL of acetonitrile, and the activation solution was discarded. 5 mL of the supernatant was directly loaded onto the column without pressure, allowing it to pass through the SPE column by gravity. The eluent was collected in a parallel concentration bottle. The SPE column was then eluted with 5 mL of acetonitrile, and the eluents were combined, collecting 5 mL of sample and 5 mL of eluent. The eluent was concentrated to 0.4 mL using a high-throughput vacuum parallel concentrator, and then diluted to 1 mL with pure water. After thorough mixing, the mixture was transferred to a sample bottle and centrifuged at 3000 r / min for 10 min for LC-MS / MS analysis. This example uses a pure water spiking system to prepare the working curve. Blank fish meat matrix is ​​difficult to obtain and is susceptible to contamination by environmental organophosphates during storage. Using a pure water spiking system reduces the risk of contamination from the blank matrix itself, and after isotope internal standard correction, it meets the spike recovery requirements for fish meat samples.

[0031] 5. Preparation of the working curve: Take seven 30 mL glass centrifuge tubes, add 5 mL of pure water and 10 ng of a mixed isotope internal standard to each, then add 0.5–50 ng of OPE mixed standard solution sequentially, and process them according to the sample extraction, purification, and concentration steps described above. This method uses a working curve; the contribution of the system blank has already been fitted into the intercept of the calibration curve and does not need to be deducted separately when calculating the sample content. If the blank value of the entire procedure is still higher than the detection limit, system contamination investigation and quality control explanation should be performed. The working curve, samples, and blanks should be pretreated simultaneously in the same batch and using the same batch of reagents and consumables.

[0032] 6. Chromatographic conditions: The analytical column was a Shim-pack GISS-HP C18 metal-free column (3 μm, 2.1 × 150 mm) with a carbon content of 9%; the delay column was a Shim-pack Scepter C18-120, 3 μm, 2.1 × 50 mm, with a carbon content of 19.5%; the delay column was installed between the mixer and the injector; the flow rate was 0.4 mL / min, the column temperature was 40℃, and the injection volume was 5 μL; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, and the gradient elution program is shown in Table 1.

[0033] Table 1 Gradient elution program for liquid chromatography

[0034] 7. Mass spectrometry conditions: Ion source and scanning mode were electrospray ionization source for positive and negative ion scanning (ESI+, ESI-); detection mode was MRM; interface voltage was 4 kV in both positive and negative ion modes, interface temperature was 150℃, desolvation temperature was 261℃, DL temperature was 250℃, heating block temperature was 400℃, nebulizing gas flow rate was 3 L / min, heating gas flow rate was 10 L / min, and drying gas flow rate was 10 L / min. Target analyte MRM parameters are shown in Table 2, and internal standard MRM parameters are shown in Table 3.

[0035] Table 2. Multiple reaction monitoring parameters for 22 organophosphate target compounds

[0036] Note: * indicates quantitative ions.

[0037] Table 3. Multiple reaction monitoring parameters for 18 isotopic internal standards

[0038] Under the above chromatographic and mass spectrometric conditions, the chromatographic separation results of the 22 organophosphate target compounds are as follows: Figure 3 As shown, where, Figure 3 (a) is the chromatogram of the highly sensitive target analyte. Figure 3(b) is the chromatogram of the low-sensitivity target analyte.

[0039] 8. Calculation: The content X of each analyte in the sample is calculated according to the formula X = m1 / m × 1000 / 1000. In the formula, X is the content of each analyte in the sample, in μg / kg; m1 is the content of each analyte in the sample solution obtained from the working curve, in ng; and m is the sample weight, in g.

[0040] 9. The names and CAS numbers of the target objects are shown in Table 4.

[0041] Table 4. Names, abbreviations, and CAS numbers of 22 organophosphate targets.

[0042] Example 2: Screening of SPE column activation reagent dosage The difference from Example 1 is that: the HLB-P Glass SPE column was activated with 2 mL, 5 mL, and 10 mL of acetonitrile, respectively, and then eluted with 5 mL of acetonitrile. The eluent was collected, concentrated to 1 mL, and analyzed. The results showed that 2 mL of acetonitrile could not remove the column contamination itself, while 5 mL and 10 mL of acetonitrile could effectively control the background contamination of the SPE column. Common background contaminants in the finished SPE column include TCIPP, TEP, TPhP, TEHP, and TCEP, which can be effectively eluted with 5 mL of acetonitrile.

[0043] The effect of SPE column background removal is shown in [the image]. Figure 1 .

[0044] Example 3: Validation of chromatographic separation, delay column setup, and system background control Separation tests were performed on different C18 columns, and the results are shown in [Figure number missing]. Figure 6 Therefore, it can be seen that the Shim-pack GISS-HPC18 metal-free column (3 μm, 2.1×150 mm) can effectively separate isomers such as TiBP and TnBP; for cluster peaks such as TCiPP and BCIPP, changing the column cannot effectively improve the peak shape and resolution, but the cluster peaks can be integrated simultaneously without affecting the quantitative analysis.

[0045] Further investigation was conducted into the system's baseline control effect before and after setting the delay column, and the results are as follows: Figure 4 As shown. By Figure 4 As can be seen, after setting the delay column, the system background peaks of TEP and TChiPP are separated from the target peaks of the sample in terms of retention time, thereby reducing the interference of system background on the detection of target analytes.

[0046] To verify the effect of delay column type and carbon content on the system's background separation performance, three different types of C18 short columns were selected as delay columns for comparison. Other methods and parameter settings were the same as in Example 1. The column types are as follows:

[0047] The chromatographic separation results are shown below. Figure 8 .

[0048] All three conditions were tested using a 10 ng / mL TCiPP standard solution.

[0049] Figure 8 (a) Under the delayed column condition with a carbon content of 9%, the TCiPP target peak and the system background peak could not be effectively separated. Figure 8 (b) Under the delayed column conditions with a carbon content of 13%, the two peaks can be initially separated, but the peak bottoms are close together and are prone to merging when the target concentration is high. Figure 8 (c) shows the best separation between the two peaks under the delayed column condition with a carbon content of 19.5%, with a retention time difference of approximately 0.3 min between the two peaks. Under other conditions, the two peaks are adjacent or merged, with no gap. Due to the irregular shape of the system background delayed peak, it is not suitable to use the conventional peak width to calculate the resolution. Therefore, the evaluation is based on the distance between the two peaks and the actual chromatographic separation.

[0050] Further analysis was conducted on the placement of the delay columns.

[0051] If the delay column is set after the injector and before the analytical column: When the delay column is positioned between the injector and the analytical column, both the TCiPP contamination from the system tubing carried by the mobile phase and the TCiPP components from the sample injected by the injector will flow through the delay column together. Since the TCiPP from the tubing and the TCiPP in the sample are the same substance, their retention behavior is identical under the same delay column chromatographic conditions, and there is no retention time difference between them. Even if the analytical column is subsequently connected, the TCiPP dissolved from the system tubing and the TCiPP in the sample will still co-elute, making it impossible to chromatographically separate the interfering substance from the sample analyte. This fails to solve the technical problem of TCiPP background contamination in the LC-MS system interfering with sample determination, and thus cannot achieve the purpose of this invention. Therefore, this position is not suitable.

[0052] If the delay column is placed before the mixer / before the pump: First, from the perspective of mass transport pathway analysis: Before the HPLC pump, each mobile phase has its own independent solvent line. Even if a delay column is installed in each solvent line before the pump, it can only trap TCiPP contamination brought in from the solvent bottle. TCiPP will dissolve inside the pump body, in the post-pump tubing, in the mixer, and at all connectors. This contamination is downstream of the delay column before the pump, completely bypassing it and entering the injector directly. This most significant system tubing interference cannot be delayed and separated by the delay column, and the background interference from the tubing cannot be eliminated.

[0053] Second, the feasibility at the instrument hardware level is poor: multi-channel gradient liquid chromatography has multiple independent mobile phase lines before the pump, requiring each mobile phase to be equipped with a delay column, which results in high hardware modification costs and significant disassembly and reassembly. Multiple delay columns introduce additional dead volume, which will cause gradient delay and pressure fluctuations, affecting the accuracy of liquid chromatography gradients. At the same time, connecting a chromatographic column before the pump will change the pump inlet pressure conditions, which can easily introduce air bubbles, causing unstable pump suction, affecting the stability of the liquid chromatography-mass spectrometry system, and even causing hardware damage, making it impractical for actual application.

[0054] This invention installs a delay column between the liquid phase mixer and the injector: TCiPP dissolved from the pump, mixer, and upstream tubing flows through the delay column with the mobile phase, where it is retained chromatographically and thus delayed; while the sample is injected through the injector and bypasses the delay column, directly entering the subsequent analysis system. This creates a retention time difference between the TCiPP from the instrument tubing and the TCiPP in the sample, achieving chromatographic separation and effectively eliminating background interference. This location requires only a single delay column, without modifying the pump or injector hardware, allowing for controllable dead volume, preserving the sample's composition, and stably achieving the detection objectives of this invention.

[0055] The location of the delay column in this invention is as follows: Figure 5 As shown, the delay column is positioned between the liquid phase mixer and the injector, so that the mobile phase flows through the delay column before entering the injector, while the sample injected by the injector does not pass through the delay column.

[0056] In summary, placing the delay column between the injector and the analytical column, or before the mixer / pump, cannot achieve the purpose of this invention, either from the perspective of chromatographic separation mechanism or practical instrument application. Therefore, this invention selects the area between the mixer and the injector as the optimal and only feasible installation location.

[0057] Example 4: Optimization of Concentration Endpoint, Concentration Method, and Reconstitution System (1) The difference from Example 1 is that nitrogen blowing concentration and vacuum parallel concentration are compared. Blank experiments show that the nitrogen blowing process introduces some analytes, especially TCiPP in the blank sample, which is clearly detected after the nitrogen blowing step, indicating that there are interfering substances such as TCiPP in the purge gas, nitrogen blowing pipeline and ambient air.

[0058] Background contamination was mainly concentrated in TCiPP. Under vacuum parallel concentration conditions, the average background peak area of ​​TCiPP was approximately 240,000, with an RSD of approximately 12%, which was below the detection limit. Under nitrogen blowing concentration conditions, the blank background peak area of ​​TCiPP was approximately 3,750,000, with an RSD of approximately 24%. The estimated amount of TCiPP impurities introduced by a single nitrogen blowing process was approximately 10 ng. These results further demonstrate that vacuum parallel concentration can significantly reduce exogenous TCiPP contamination introduced during the concentration process. (See attached figures.) Figure 7 .

[0059] (2) The difference from Example 1 is that a comparison was made between two concentration endpoints: complete evaporation and incomplete evaporation. The results showed that after evaporation, the target substances such as TMP, TEP, and TiPP in the sample were significantly lost, and the other analytes also showed varying degrees of loss. The response loss of some target substances under different concentration endpoint conditions is as follows: Figure 2 As shown, the specific data results are as follows:

[0060] Note: A recovery rate exceeding 100% is normal fluctuation for the instrument and does not necessarily indicate better recovery under evaporation conditions.

[0061] (3) The difference from Example 1 is that seven redissolution systems—10% acetonitrile water, 20% acetonitrile water, 30% acetonitrile water, 40% acetonitrile water, 50% acetonitrile water, 75% acetonitrile water, and 100% acetonitrile—were used for redissolution and then transferred to the vial. See the detailed data below. Figures 9-15 .

[0062] From the perspective of chromatographic peak shape, an ideal peak shape should be symmetrical and sharp, and the solvent effect is one of the most significant factors affecting peak shape. The influence of the solvent effect on peak shape gradually decreases with retention time. Figures 9-15 Chromatographic peaks of 22 analytes were analyzed in acetonitrile reconstitution systems at seven ratios: 10%, 20%, 30%, 40%, 50%, 75%, and 100%. Peak shapes revealed that substances eluting earlier exhibited significant peak bifurcation and tailing in reconstitution systems with acetonitrile concentrations of 50% or higher. This was most pronounced in substances like BChiPP, TMP, and TEP, which eluted earlier. For instance, TMP showed significant peak broadening in a 50% acetonitrile reconstitution system; TEP showed peak bifurcation in the same system; and BChiPP showed significantly reduced cluster peak resolution in a 50% acetonitrile reconstitution system, affecting the integration.

[0063] On the other hand, by Figure 16 As can be seen, the absolute recoveries of the 40% reconstitution systems were all above 70%, while the absolute recoveries of the 30% and lower reconstitution systems were as low as below 40%. This will affect the detection limit of the analyte, but since internal standard correction was used, it does not affect the detection value.

[0064] The absolute recoveries of 16 OPEs in the 40% acetonitrile-water reconstitution system were no different from those in the acetonitrile-water reconstitution system with a ratio of 50% or higher. The only differences were in TMP (73% absolute recovery), TEP (75%), TiPP (73%), TnPP (77%), TiBP (83%), and TEHP (83%). However, the chromatographic peak responses of the 40% acetonitrile-water reconstitution system were all higher, which could improve the peak shape of the compounds, lower the detection limit, and reduce the solvent effect. Isotope internal standard correction could compensate for the loss of recovery.

[0065] Example 5 Methodological Validation Linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and precision were validated according to the method in Example 1. The 22 analytes showed good linearity, with correlation coefficients (r) ranging from 0.991 to 0.9999. The LOD was evaluated using the signal-to-noise ratio (SNR) method in GB 5009.295-2023, with an S / N ratio of 3:1 confirmed as the LOD and a LOQ ratio three times the LOD confirmed as the LOQ. The linear range, LOD, and LOQ are shown in Table 5.

[0066] Table 5. Linear range, limit of detection, and limit of quantitation for 22 organophosphate esters.

[0067] 1 g of fish meat sample was spiked with 1 ng, 5 ng, and 20 ng to determine the recovery rate and precision. The recoveries of 22 organophosphates ranged from 57% to 127%, with relative standard deviations ranging from 0.1% to 8.9%. Specific results are shown in Table 6.

[0068] Table 6. Spiking recoveries and precision of 22 organophosphate targets in fish meat samples

[0069] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An LC-MS / MS method for the detection of 22 organophosphates in fish meat samples, characterized in that, Includes the following steps: S1. Sample extraction: Take a well-mixed fish meat sample, add pure water and isotope mixed internal standard in sequence, mix well, add acetonitrile, vortex and sonicate to extract, add NaCl for salting out, vortex and centrifuge, and take the supernatant. S2. Sample purification and concentration: The HLB-P Glass SPE column was activated with acetonitrile. The supernatant obtained in step S1 was passed through the column and the eluent was collected. The HLB-P Glass SPE column was then eluted with acetonitrile. The eluents were combined and concentrated under vacuum to 0.4 mL. The volume was then adjusted to 1 mL with pure water. The mixture was then centrifuged to obtain the test solution. S3, LC-MS / MS determination: The test solution was determined using a liquid chromatography system including a delay column and an analytical column and a tandem mass spectrometry system; The delay column is located between the mixer and the injector. The delay column is a C18 column with a higher carbon content than the analytical column. The analytical column is a C18 column. The 22 organophosphates are determined using isotope internal standard working curves and multiple reaction monitoring.

2. The detection method according to claim 1, characterized in that, The 22 organophosphates include 16 organophosphate triesters and 6 organophosphate diesters; the 16 organophosphate triesters are TMP, TEP, TiPP, TnPP, TiBP, TnBP, TCEP, TPhP, TCiPP, EHDPP, TMPP, TBOEP, TDCiPP, TEHP, V6, and TDBPP; the 6 organophosphate diesters are DnBP, DPhP, BCiPP, DoCP, BBOEP, and BDCiPP.

3. The detection method according to claim 1, characterized in that, In step S1, the ultrasonic extraction is performed at 25 kHz for 10 min; the centrifugation is performed at 3000 r / min and 4℃ for 10 min.

4. The detection method according to claim 1, characterized in that, The HLB-P Glass SPE column is a 300 mg, 6 mL specification; it is activated with 5 mL of acetonitrile, and 5 mL of the supernatant obtained in step S1 is passed through the column and eluted with 5 mL of acetonitrile.

5. The detection method according to claim 1, characterized in that, In step S2, the HLB-P Glass SPE column adopts a flow-through purification mode, and no pressure is applied during sample loading. The sample passes through the HLB-P Glass SPE column by its own gravity. The activation solution is discarded, and 5 mL of sample and 5 mL of eluent are collected. The HLB-P Glass SPE column does not need to be dried after activation.

6. The detection method according to claim 1, characterized in that, The analytical column has a carbon content of 9%, and the delayed column has a carbon content of 19.5%.

7. The detection method according to claim 1, characterized in that, In step S3, the liquid chromatography conditions include: flow rate 0.4 mL / min, column temperature 40℃, injection volume 5 μL; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile; the gradient elution program is as follows: at 0.0 min, A is 85% and B is 15%; at 0.5 min, A is 85% and B is 15%; at 16.0 min, A is 30% and B is 70%; at 18.0 min, A is 2% and B is 98%; at 20.0 min, A is 2% and B is 98%; at 20.1 min, A is 85% and B is 15%; at 25.0 min, A is 85% and B is 15%.

8. The detection method according to claim 1, characterized in that, In step S3, the tandem mass spectrometry conditions include: positive and negative ion scanning from the electrospray ion source, and multiple reaction monitoring; the interface voltage is 4 kV in both positive and negative ion modes, the interface temperature is 150℃, the desolvation temperature is 261℃, the DL temperature is 250℃, the heating block temperature is 400℃, the nebulizing gas flow rate is 3 L / min, the heating gas flow rate is 10 L / min, and the drying gas flow rate is 10 L / min.

9. The detection method according to claim 1, characterized in that, The content X of each analyte in the sample is calculated according to the following formula: X = m1 / m × 1000 / 1000, where X is the content of each analyte in the sample, in μg / kg; m1 is the content of each analyte in the sample solution obtained from the working curve, in ng; and m is the sample weight, in g.

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