A method for discriminating endogenous and exogenous thiocyanate in milk samples based on stable carbon isotope ratio

Abstract

The application discloses a method for identifying endogenous and exogenous thiocyanate in a milk sample based on stable carbon isotope ratio, which is an analysis method for accurately tracing and distinguishing naturally occurring (endogenous) and added (exogenous) thiocyanate in a milk sample through stable carbon isotope ratio 13 C. The method comprises: performing isotope mass spectrometry on phase transfer catalytic derivatized milk samples to obtain mass spectrometry data; judging whether the thiocyanate in the milk sample is an endogenous risk substance or an exogenous risk substance based on the isotope ratio 13 C, and judging whether the thiocyanate in the milk sample is illegally added based on the risk substance type. The method can identify whether the thiocyanate in the milk sample is exogenously added, and has high accuracy. The method is a new identification method with high sensitivity and high accuracy based on stable carbon isotope analysis, and can clearly identify the behavior of illegally adding industrial thiocyanate in the milk sample.

Description

Technical Field

[0001] This invention relates to the fields of food safety and analytical chemistry, specifically to a method for identifying endogenous and exogenous thiocyanates in milk samples based on stable carbon isotope ratios. Background Technology

[0004] The current technical challenge lies in the fact that conventional concentration detection methods cannot effectively distinguish between trace amounts of naturally occurring endogenous thiocyanate in raw milk and dairy products and low levels of illegally added exogenous thiocyanate. Because the background values ​​of endogenous thiocyanate fluctuate significantly, their concentration range may overlap with the concentration range of illegally added low levels, making it impossible to reach a clear qualitative and tracing conclusion based solely on concentration. This poses a significant challenge to regulatory enforcement. Therefore, there is an urgent need for a novel identification and analysis technology that can transcend the limitations of concentration detection and clearly distinguish between endogenous and exogenous thiocyanate at their source. Summary of the Invention

[0005] This invention aims to solve the technical problem of the inability of existing technologies to effectively distinguish between endogenous and exogenous thiocyanates in milk samples (i.e., raw milk or dairy products to be tested). It provides a novel identification method based on stable carbon isotope analysis that is highly sensitive and accurate, so as to achieve clear identification and scientific confirmation of the illegal addition of industrial thiocyanates to milk samples.

[0006] To achieve the above objectives, this invention provides an analytical method for identifying the source of thiocyanate in milk samples. Its core lies in revealing and utilizing the stable carbon isotope ratio (δ¹⁸O) among thiocyanates from different pathways. 13 The significant difference in C) is based on Compound-Specific Isotope Analysis (CSIA) technology, which utilizes gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the stable carbon isotope ratio (δ¹²). 13 C), to detect naturally occurring (endogenous) and illegally added (exogenous) thiocyanates (containing SCN) in milk samples. - An analytical method for precise tracing and differentiation.

[0007] Based on research, the following technical solutions are proposed:

[0008] In a first aspect, the present invention provides a method for identifying endogenous and exogenous thiocyanates in milk samples based on stable carbon isotope ratios, comprising the following steps:

[0009] Step S1. Sample pretreatment:

[0010] Accurately measure the milk sample to be tested and divide it into the required number of portions. Add an organic solvent to each portion to remove more than 50 wt% of fat and protein from the milk sample. Then concentrate the solution to obtain an aqueous concentrate.

[0011] Step S2. Phase transfer catalytic derivatization:

[0012] Add appropriate amounts of phase transfer catalyst, alkaline solution and derivatization reagent sequentially to the aqueous concentrate obtained in step S1; then carry out the derivatization reaction; after the reaction is completed, remove the organic phase and filter it to obtain the filtrate to be tested, and store it frozen.

[0013] Step S3. Determine the stable carbon isotope ratio in the milk sample by GC-C-IRMS analysis.

[0014] The filtrate obtained in step S2 was analyzed using gas chromatography-combustion-isotope ratio mass spectrometry (GC-COMP-MS) to determine the stable carbon isotope ratio δ of the derivative. 13 C_PFB-SCN;

[0015] Step S4. Isotope Correction and Result Determination:

[0016] The derivatization reaction introduces carbon atoms from the derivatizing reagent into the thiocyanate molecules in the milk sample to be tested, thereby stabilizing the carbon isotope ratio δ of the measured derivative. 13 C_PFB-SCN correction was performed to calculate the stable carbon isotope ratio δ of the thiocyanate to be tested. 13 C_SCN; The correction calculation is based on the mass balance equation of Formula 1 below:

[0017]

[0018] Wherein, a, b, and c are the number of carbon atoms in the PFB-SCN derivative, the PFB derivatizing group, and the thiocyanate to be tested, respectively;

[0019] δ 13 C_PFB-SCN is the δ of the derivative PFB-SCN. 13 C measured value;

[0020] δ 13 C_SCN is the δ of the thiocyanate analyte obtained after correction. 13 C value, i.e., stable carbon isotope ratio δ 13 C_SCN;

[0021] δ 13 C_PFB is an effective δ-molecule derived from PFB groups. 13 C value is a correction constant;

[0022] The δ13 C_PFB is the effective isotope value of the derivatization reagent. Its value needs to be calculated by formula 1 after EA-IRMS and GC-C-IRMS analysis of pure thiocyanate standards.

[0023] If the stable carbon isotope ratio of the thiocyanate to be tested is δ 13 If the C_SCN is below 0‰, it can be determined that the milk sample contains illegally added industrial thiocyanate.

[0024] In this invention, "milk sample" should be interpreted broadly, and may include milk samples that have not been sterilized or processed (such as raw milk, fresh milk or milk powder), or milk samples that have been sterilized or processed. In short, in this invention, "milk sample" refers to raw milk or dairy products to be tested.

[0025] In this invention, "sample" and "sample" can be used interchangeably.

[0026] In this invention, the milk sample comprises at least one of cow's milk, goat's milk, mare's milk, and camel's milk. Preferably, the milk sample is a cow's milk sample.

[0027] In this invention, the milk sample is usually in liquid form, or it may be processed into a liquid form to facilitate detection using the method of this invention.

[0028] In this invention, "illegal addition" refers to the addition of "non-edible substances that are expressly prohibited by the state," and "illegal addition" refers to the use of legal food additives beyond the permitted scope or limit. "Illegal addition" or "illegal addition" should be understood in the broadest sense, that is, it includes any situation that violates the relevant regulations or laws of the state, industry, region, and relevant management or supervision units, that is, it includes "illegal addition."

[0029] In this invention, one of the main sources of endogenous sodium thiocyanate in milk samples is cruciferous animal feed.

[0030] In this invention, weak alkalinity is achieved at a pH of 7.1-8.5.

[0031] In this invention, the terms "industrial grade thiocyanate" and "industrial thiocyanate" are used interchangeably, and similarly, the terms "industrial grade sodium thiocyanate" and "industrial sodium thiocyanate" are also used interchangeably.

[0032] In this invention, "real milk sample" refers to a milk sample without exogenous thiocyanate added. This is the key to determining the correct correction constant for exogenous industrial thiocyanate. Similarly, for example, "real milk sample" refers to a milk sample without exogenous thiocyanate added.

[0033] In some preferred embodiments, to better extract thiocyanate from the milk sample to be tested, which is more conducive to subsequent detection and improves accuracy, step S1 includes: accurately measuring the milk sample to be tested and dividing it into the required number of portions; adding an organic solvent to each portion, wherein the ratio of the milk sample to the organic solvent in each portion is 0.5-1 g:1 mL; vortexing to precipitate and remove more than 50 wt% of fat and protein from the milk sample; then centrifuging at 1-6°C and combining the supernatants of each portion; and then concentrating to remove more than 90% of the organic solvent, thereby obtaining an aqueous concentrate.

[0034] In some preferred embodiments, in step S1, in order to better extract thiocyanate from the milk sample to be tested, which is more conducive to subsequent detection and improves accuracy, the organic solvent is an acetonitrile solution of 0.1-0.2% (v / v) formic acid.

[0035] In some preferred embodiments, in step S1, in order to further and better remove fat and protein from the milk sample, vortex oscillation is performed at an oscillation frequency of 2000-3000 rpm / min for 0.5-2 minutes.

[0036] In some preferred embodiments, in step S1, in order to further and better extract thiocyanate from the milk sample to be tested, which is more conducive to subsequent detection and improves accuracy, the combined extract is concentrated at room temperature using a gentle nitrogen flow to remove more than 95% of the organic solvent.

[0037] In some preferred embodiments, in order to further and better extract thiocyanate from the milk sample to be tested, which is more conducive to subsequent detection and improves accuracy, in step S1, the sample is centrifuged at 9000-10000 rpm / min for 10-20 minutes.

[0038] In some preferred embodiments, to further improve the extraction of thiocyanate from the milk sample to be tested, which is more conducive to subsequent detection and improves accuracy, in step S1, the milk sample to be tested is accurately measured and divided into the required number of portions. A 0.1-0.2% (v / v) formic acid acetonitrile solution is added to each portion, with the ratio of the milk sample to the formic acid acetonitrile solution in each portion being 0.5-1 g:1 mL. The mixture is vortexed at a frequency of 2000-3000 rpm / min for 0.5-2 minutes to completely precipitate the protein. Subsequently, the mixture is centrifuged at 9000-10000 rpm / min for 10-20 minutes at 1-6°C, and the supernatants from each portion are combined. At room temperature, the combined extract is concentrated using a gentle nitrogen flow to remove more than 95% of the organic solvent, thereby obtaining an aqueous concentrate for derivatization.

[0039] In some preferred embodiments, to facilitate the phase transfer derivatization reaction, the ionic thiocyanate in the aqueous phase is converted into a volatile derivative suitable for gas chromatography analysis, and to improve the accuracy of subsequent stable carbon isotope ratio determination, step S2 includes: sequentially adding 10%-20% (by volume of the aqueous phase concentrate) of a 0.05-0.3 mol / L aqueous solution of hexadecyltrimethylammonium bromide as a phase transfer catalyst, 10%-20% (by volume of the aqueous phase concentrate) of an alkaline solution with a concentration of 0.05-0.2 mol / L to the aqueous phase concentrate to ensure the reaction system is weakly alkaline, and 15%-30% (by volume of the aqueous phase concentrate) of a pentafluorobenzyl bromide derivatization reagent dissolved in ethyl acetate with a concentration of 3-6 mL / L; immediately after adding the materials, sealing the reaction vessel and vortexing at a frequency of 2000-3000 rpm / min for 1-3 minutes to form a homogeneous reaction mixture emulsion; placing the emulsion at 65-75°C... Incubate in a constant temperature water bath at °C for 20-40 minutes to ensure complete derivatization.

[0040] In some preferred embodiments, in step S2, after the reaction is complete, the mixture is centrifuged at 2-5°C at 9000-10000 rpm / min for 5-15 minutes to rapidly separate the phase.

[0041] In some preferred embodiments, in order to improve the accuracy of subsequent stable carbon isotope ratio determination, in step S2, the upper organic phase containing the target derivative pentafluorobenzyl thiocyanate is aspirated and filtered, the clarified filtrate is transferred to a container to obtain the test filtrate, and then frozen and stored at -15°C to -20°C.

[0042] In some preferred embodiments, to further facilitate the phase transfer derivatization reaction, the ionic thiocyanate in the aqueous phase is converted into a volatile derivative suitable for gas chromatography analysis, and to ensure the accuracy of subsequent stable carbon isotope ratio determination, step S2 includes: adding, sequentially, 10%-20% of the volume of the aqueous phase concentrate obtained in step S1, of a 0.05-0.3 mol / L aqueous solution of hexadecyltrimethylammonium bromide as a phase transfer catalyst; 10%-20% of the volume of the aqueous phase concentrate, of an alkaline solution with a concentration of 0.05-0.2 mol / L to ensure the reaction system is weakly alkaline; and 15%-30% of the volume of the aqueous phase concentrate, of a pentafluorobenzyl bromide derivatization reagent dissolved in ethyl acetate with a concentration of 3-6 mL / L; immediately after adding the materials, sealing the reaction vessel and vortexing at a frequency of 2000-3000 rpm / min for 1-3 minutes to form a homogeneous reaction mixture emulsion; placing the emulsion at 65-75°C... Incubate in a constant temperature water bath at °C for 20-40 minutes to ensure complete derivatization reaction; after the reaction, to quickly separate the phase, centrifuge the mixture at 9000-10000 rpm / min at 2-5°C for 5-15 minutes; aspirate the upper organic phase containing the target derivative pentafluorobenzyl thiocyanate and filter it, transfer the clear filtrate to a container to obtain the test filtrate, and freeze it at -15°C to -20°C for later detection.

[0043] In some preferred embodiments, to ensure the accuracy of the stable carbon isotope ratio determination, step S3 includes gas chromatography detection and isotope mass spectrometry detection, wherein: the gas chromatography detection includes: injecting the filtrate to be tested into the sample in splitless mode, with an injection volume of 1-2 µL, an injection time of 0.5-2 min, and an injection port temperature of 250-280 °C; the chromatographic column is a fused silica capillary column, the carrier gas is helium, and the flow rate is 1-3 mL / min; the temperature program is: initial temperature 95-105 °C held for 0.5-1.5 min, increasing to 110-130 °C at 2-5 °C / min and holding for 2-6 min, then increasing to 280-320 °C at 30-50 °C / min and holding for 1-3 min, to obtain the chromatographic effluent; and the isotope mass spectrometry detection includes: passing the chromatographic effluent through a combustion port, wherein the combustion temperature is 950-1100 °C. At °C, the combustion catalyst is a CuO / NiO catalyst, thereby quantitatively converting the chromatographic effluent into CO2. After drying with a Nafion membrane to remove water, the CO2 is introduced into an isotope ratio mass spectrometer through a continuous flow interface to determine the stable carbon isotope ratio δ of the product CO2. 13 C, and calibrated using the international standard reference material IAEA-600.

[0044] Preferably, the fused silica capillary column is DB-5 or HP-5.

[0045] In this invention, the step of determining the stable carbon isotope ratio of industrial thiocyanate by EA-IRMS analysis is not necessary for the subsequent step of determining the correction constant. However, if necessary, this step is added before step 4 to ensure the accuracy of the measurement results.

[0046] In some preferred embodiments, the step of determining the stable carbon isotope ratio of industrial thiocyanate by EA-IRMS analysis includes: determining the true δ¹⁸O of a pure sodium thiocyanate standard using EA-IRMS. 13 C value; 0.5 mg of the pure sodium thiocyanate standard was packaged and burned in an elemental analyzer at 950-980 °C to produce carbon dioxide gas. The product gas was purified by a reduction furnace at 620-680 °C and then sent to a mass spectrometer for analysis; all δ 13 C values ​​are reported in per mille (‰) relative to the VPDB standard and calibrated using the international standard reference material IAEA-600, with an analytical precision better than 0.2‰; wherein, IAEA-600 is caffeine, δ 13 C = -27.77‰.

[0047] Furthermore, the correction constant was determined by the following method: First, the true δ of the pure NaSCN standard was obtained by EA-IRMS analysis. 13 C value, i.e., δ 13 C_SCN; then, the pure NaSCN standard was derivatized and analyzed by GC-C-IRMS to obtain its corresponding δ 13 The C_PFB-SCN value; finally, δ is calculated by rearranging Formula 1. 13 C_PFB; then the δ 13 The value of C_PFB is used as a constant for the δ of all milk samples. 13 C_PFB-SCN is used for correction to calculate the final δ. 13 C_SCN value.

[0048] Secondly, the present invention provides the application of the above-described method in identifying endogenous and exogenous thiocyanates in milk samples.

[0049] The beneficial effects of the present invention include at least the following:

[0050] This invention provides a method for identifying endogenous and exogenous thiocyanates in milk samples based on stable carbon isotope ratios. This method confirms that thiocyanates from different sources (i.e., endogenous and exogenous) have distinctly different stable carbon isotope characteristics. Endogenous thiocyanates derived from animal (such as dairy cow) metabolism, whose carbon primarily comes from cruciferous plants in feed, exhibit significantly positive values, with an average stable carbon isotope ratio of approximately +12.5‰. Conversely, exogenous thiocyanates synthesized through the fossil fuel industry exhibit significantly negative values, with an average stable carbon isotope ratio of approximately -18.3‰. Since the distribution ranges of stable carbon isotope ratios for these two sources of thiocyanate do not overlap, this invention establishes a clear discrimination threshold: 0‰. In the analysis of unknown samples, if the calculated true stable carbon isotope ratio δ of the thiocyanate is... 13 If the C_SCN is below 0‰, it can be determined that the milk sample contains illegally added industrial thiocyanate.

[0051] Furthermore, the method of identifying endogenous and exogenous thiocyanate in milk samples based on stable carbon isotope ratios in this invention is the first to utilize stable carbon isotope ratios (δ¹⁸O⁻¹) to identify endogenous and exogenous thiocyanate in milk samples. 13 C) An analytical method for accurately tracing and distinguishing between naturally occurring (endogenous) and illegally added (exogenous) thiocyanates in milk samples. This invention, for the first time, combines sample pretreatment, phase transfer catalytic derivatization (PTC), GC-C-IRMS analysis to determine the stable carbon isotope ratio in milk samples, and isotope correction and result determination (especially combining PTC, GC-C-IRMS, and isotope correction and result determination). The derived raw milk or dairy products are then subjected to isotope mass spectrometry to obtain mass spectrometry data; based on the isotope ratio (δ¹⁸O⁻¹), the method... 13 The value of C) determines whether thiocyanate in the raw milk or dairy products is an endogenous or exogenous hazardous substance, and based on the hazardous substance, determines whether the milk has been illegally added. If the calculated stable carbon isotope ratio δ of thiocyanate is... 13 A C_SCN below 0‰ indicates the presence of illegally added industrial thiocyanate in the milk sample. This method can identify whether the thiocyanate in the milk sample is exogenously added, with high accuracy. This method is a novel, highly sensitive, and highly accurate identification method based on stable carbon isotope analysis, enabling clear identification and scientific confirmation of the illegal addition of industrial thiocyanate to milk samples. Attached Figure Description

[0052] Figure 1 This demonstrates the effect of different factors on signal strength or δ in embodiments of the present invention. 13 The effect of C, specifically... Figure 1Figure (A) shows the gas chromatography-mass spectrometry analysis of the PFB-SCN derivatives, with optimized total ion chromatogram (TIC) and ionization mass spectrum of the target peak; Figure 1 Figure (B) shows the gas chromatography-combustion-isotope ratio mass spectrometry analysis of PFB-SCN in dichloromethane; Figure 1 Figure (C) shows the gas chromatography-combustion-isotope ratio mass spectrometry analysis of PFB-SCN in ethyl acetate; Figure 1 Figures (D)-G show the optimization of sample pretreatment and derivatization conditions, where: Figure 1 The horizontal axis of graph (D) represents the enrichment factor (2.0 mg / L). Figure 1 In the graph (E), the horizontal axis represents the volume of the potassium hydroxide solution. Figure 1 In the graph (F), the horizontal axis represents the volume of sodium dodecyl sulfate solution. Figure 1 In the graph (G), the horizontal axis represents the reaction time.

[0053] Figure 2 Figure (A) shows the assessment of matrix effects and potential isotope fractionation under enriched and non-enriched conditions. Figure 2 Figure (B) shows the δ of exogenous thiocyanates from different sources. 13 Distribution of C_SCN values.

[0054] Figure 3 The intrinsic δ was observed on days 6 and 10. 13 A comparison of C_SCN values, where... Figure 3 Figure (A) shows the farm HYL. Figure 3 Figure (B) shows the farm at KM. Figure 3 Figure (C) in the diagram represents the farm MCY. Figure 3 Figure (D) in the diagram represents the QT farm. Figure 3 Figure (E) in the diagram represents farm CR. Figure 3 Figure (F) in the diagram represents farm XQ. Figure 3 Figure (G) in the figure shows the comparison results among the six farms. Figure 3 Figure (H) in the figure shows the comparison results of the merged datasets. ns indicates no significant difference (p>0.05). These results suggest that there is an endogenous δ difference between day 6 and day 10. 13 The C_SCN value did not show statistically significant changes over time.

[0055] Figure 4 The study showed endogenous delta-lactone levels in milk samples from five farms (HYL, KM, QT, CR, and XQ). 13 Distribution of C_SCN values ​​(n=12 for each sample).

[0056] Figure 5δ, indicating exogenous and endogenous sources of thiocyanate. 13 Comparison of C_SCN values. Figure 5 Figures (A)-(E) show a comparison between the exogenous group (n=28) and the individual farms: Figure 5 Figure (A) in the diagram corresponds to HYL. Figure 5 Figure (B) in the diagram corresponds to KM. Figure 5 The diagram (C) in the image corresponds to QT. Figure 5 Figure (D) in the diagram corresponds to CR and Figure 5 The diagram (E) in the figure corresponds to XQ. Figure 5 Figure (F) in the figure shows a comparison between the exogenous group and the endogenous summary data. Figure 5 Figure (G) in the figure shows a comprehensive comparison between the exogenous group and five individual farms. Detailed Implementation

[0057] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in the following description are merely illustrative examples of specific implementations of this invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0058] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In this invention, when a specific numerical value is mentioned, it means that the value can vary within ±5%. In this invention, unless otherwise stated, terms such as "multiple / a plurality of" mean two / a kind or more. The terms "containing," "comprising," or "including" can be open-ended, semi-closed, or closed.

[0059] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the invention. It should also be noted that if specific conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or the product instructions should be followed. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0060] Example 1

[0061] 1. Raw materials and instruments

[0062] 1.1 Standards, reagents and samples

[0063] Eight industrial-grade sodium thiocyanate (NaSCN, purity >99%) standards were purchased from Ampere Laboratory Technology Co., Ltd. (CNW, Shanghai, China), Tanmo Quality Testing Technology Co., Ltd. (TM, Beijing, China), Guangzhou Jiatu Technology Co., Ltd. (CA, Guangzhou, China), Henan Institute of Standard Materials (BW, Zhengzhou, China), Shanghai Yuyan Biotechnology Co., Ltd. (YY, Shanghai, China), Beijing Manhag Biotechnology Co., Ltd. (BE, Beijing), Sichuan Institute of Metrology (SIT, Chengdu, China), and Beijing PCL Technology Co., Ltd. (PCL, Beijing, China). The stable carbon isotope certification reference material IAEA-600 (caffeine) was purchased from the International Atomic Energy Agency (IAEA, Austria). The derivatization reagents pentafluorobenzyl bromide (PFBBr, 99%), hexadecyltrimethylammonium bromide (CTAB, ≥98%), and potassium hydroxide (KOH, 99%) were all purchased from Sigma-Aldrich (USA). HPLC-grade methanol, acetonitrile, formic acid, and ethyl acetate solvents were supplied by Thermo Scientific (China). High-purity gases (helium, oxygen, carbon dioxide, and compressed air) were purchased from Beijing.

[0064] 1.2 Instruments

[0065] The main analytical instruments used in this embodiment include a gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) system (Thermo Scientific, Germany), which consists of a TRACE 1310 gas chromatograph, a GC IsoLink II combustion interface, a ConFlo IV continuous flow interface, and a Delta V Advantage isotope ratio mass spectrometer. Simultaneously, an elemental analysis-isotope ratio mass spectrometry (EA-IRMS) system (Thermo Scientific, Germany) was used to analyze the standards. This system consists of an EA IsoLink elemental analyzer coupled with the aforementioned mass spectrometer.

[0066] 1.3. Collection of Real Milk Samples: Fresh milk samples (real milk samples) were collected from six commercial dairy farms. Considering potential isotopic variations due to seasonality, lactation stage, and feed type, the sampling activity lasted from 2024 to 2025. Participating dairy farms included: HYL (Ningxia, China), KM (Inner Mongolia, China), MCY (Ningxia, China), QT (Hebei, China), CR (Shanxi, China), and XQ (Gansu, China). These cows continuously received specific protein supplements: rapeseed meal intake of 1.5 kg per cow (HYL), 2.0 kg (KM and QT), and 2.5 kg per cow (MCY) per day; sesame meal intake of 2.0 kg per cow (CR); and flaxseed meal intake of 2.0 kg per cow (XQ). To eliminate residual effects from previous diets, all cows underwent a 14-day washout period before the start of the experiment and were initially fed a standardized basal feed. Subsequently, the cows continued to consume the designated experimental feed. On days 6 and 10 of the continuous feeding period, approximately 300 mL of morning milk samples were collected from each cow. Samples were immediately placed on ice after collection, transported to the laboratory within 24 hours, and stored at -20°C until further analysis. A total of 72 raw milk samples were collected.

[0067] 1.4 Preparation of milk samples simulating exogenous addition

[0068] Simulated exogenous additive samples (also known as adulterated samples) with a concentration of 10 mg / L were prepared by adding industrial-grade sodium thiocyanate standard solution to real milk samples, forming the exogenous group. Fifteen representative samples were selected from five compliant farms (excluding farm MCY), with three samples from each farm to represent different endogenous background levels. This concentration level was designed to raise the total thiocyanate concentration in milk to 14 mg / L, which is the effective threshold for activating the lactoperoxidase system to extend shelf life. After adding the standard, the samples were thoroughly mixed, placed in 50 mL sample tubes, and stored at -20°C for subsequent analytical procedures.

[0069] 2. Experimental conditions

[0070] Step S1. Sample pretreatment

[0071] Accurately measure 30 g of milk sample and divide it into three equal portions in 50 mL polypropylene centrifuge tubes. Add 20 mL of acetonitrile solution containing 0.1% (v / v) formic acid to each tube, with a milk sample to formic acid acetonitrile solution ratio of 1 g: 1 mL. Vortex at 3000 rpm / min for 0.5–2 minutes to completely precipitate the protein. Then, centrifuge at 9500 rpm / min for 15 minutes at 4 °C. Carefully combine the supernatants from the three tubes. At room temperature, concentrate the combined extract to approximately 15 mL using a gentle nitrogen flow to remove more than 95% of the organic solvent, thereby obtaining an aqueous concentrate for derivatization.

[0072] Step S2. Phase transfer catalytic derivatization:

[0073] To the 15 mL aqueous concentrate obtained in step S1, add 2.0 mL of a 0.1 mol / L hexadecyltrimethylammonium bromide (CTAB) aqueous solution as a phase transfer catalyst, 2.0 mL of a 0.1 mol / L potassium hydroxide (KOH) solution to ensure the reaction system is weakly alkaline, and 3.0 mL of a 4 mL / L pentafluorobenzyl bromide (PFBBr) derivatizing reagent dissolved in ethyl acetate. Immediately after adding the materials, seal the reaction vessel and vortex at 3000 rpm for 1 minute to form a homogeneous reaction mixture emulsion. Incubate the emulsion in a 70 °C water bath for 30 minutes to ensure complete derivatization. After the reaction, to quickly separate the phase, centrifuge the mixture at 9500 rpm / min for 10 minutes at 4 °C. Collect the upper organic phase containing the target derivative pentafluorobenzyl thiocyanate (PFB-SCN) and use 0.22... The solution was filtered through a µm polyvinylidene fluoride (PVDF) needle filter, and the clarified filtrate was transferred to an autosampler vial and stored frozen at -20°C for later testing.

[0074] The chemical structure of the PFB-SCN derivative was confirmed by GC-MS using a combination of an Agilent 7890A gas chromatograph and an Agilent 5975C mass spectrometer detector (Agilent Technologies, Santa Clara, USA). The chromatographic conditions, including the capillary column, injection parameters, and temperature program, were identical to those used in step S3 for the GC-C-IRMS mass spectrometry analysis. The mass spectrometer was operated in electrospray ionization mode at an ionization energy of 70 eV. The temperatures of the ion source and transfer line were maintained at 230°C and 250°C, respectively. Data were acquired in full scan mode, ranging from m / z 50 to 300, to obtain the mass spectrum of the derivative.

[0075] Step S3. Determine the isotope ratio by GC-C-IRMS analysis.

[0076] The δ-ray diffraction (δ) of PFB-SCN derivatives was determined using a Thermo Fisher Scientific TRACE 1310 gas chromatograph (connected to a Delta V Advantage isotope ratio mass spectrometer via a GC IsoLink II combustion interface and a ConFlo IV continuous flow interface). 13 C value (the instrument was provided by Thermo Fisher Scientific GmbH in Bremer, Germany).

[0077] The gas chromatography conditions included: injection of the analyte filtrate at a volume of 1 µL (splitless mode, 1.00 min), with an injection port temperature of 260 °C. The column was a DB-5 capillary column (30 m × 0.25 mm id, 0.25 µm film thickness), with helium as the carrier gas at a flow rate of 1.5 mL / min. The temperature program was as follows: initial temperature 100 °C, held for 1 min; increased at 5 °C / min to 120 °C, held for 4 min; then increased at 40 °C / min to 300 °C, held for 2 min.

[0078] Isotope mass spectrometry conditions: The chromatographic effluent was quantitatively converted into CO2 via a combustion interface (combustion furnace temperature 1000 °C, CuO / NiO catalyst). After drying with a Nafion membrane to remove water, the CO2 was introduced into an isotope ratio mass spectrometer through a continuous flow interface. The stable carbon isotope ratio (δ¹⁸O) of the product CO2 was determined systematically. 13 C), and calibrated using the international standard reference material IAEA-600.

[0079] δ 13 The C value is expressed in parts per thousand (‰) relative to VPDB. The measurement is calculated based on a carbon dioxide reference gas. This reference gas is used daily with the international standard substance IAEA-600 (caffeine; δ-Caffeine). 13 Calibration was performed using C = -27.77‰. All data acquisition and processing were performed using Thermo Fisher Scientific's Isodate software.

[0080] The stable carbon isotope ratio of industrial thiocyanate was determined by EA-IRMS analysis.

[0081] Undergenerated SCN - δ of standards and reagents 13 The C value was determined on a Thermo Fisher Scientific EA IsoLink elemental analyzer connected to a DELTA V Advantage isotope ratio mass spectrometer.

[0082] The true δ of pure sodium thiocyanate standard was determined using EA-IRMS. 13C value; Approximately 0.5 mg of the standard was packaged in a tin capsule and burned at 960 °C in an elemental analyzer to produce carbon dioxide as the product gas. The product gas was purified in a 650 °C reduction furnace and then analyzed by a mass spectrometer; all δ 13 C values ​​are reported as per per thousand (‰) relative to the VPDB standard, and the international standard reference material IAEA-600 (caffeine, δ) is used. 13 The analysis accuracy is better than 0.2‰ when calibrated by C = -27.77‰.

[0083]

[0084] Step S4. Isotope Correction and Result Determination:

[0085] δ obtained directly from GC-C-IRMS analysis 13 The C value represents the isotopic composition of the entire pentafluorobenzyl thiocyanate (PFB-SCN) derivative. Because the derivatization process introduces seven foreign carbon atoms into the pentafluorobenzyl (PFB) group, these carbon atoms affect the overall δ0. 13 The C value must therefore be corrected to obtain the target analyte (thiocyanate SCN). - The true δ 13 C value.

[0086] Therefore, the mass balance formula was used to correct all the measured data:

[0087]

[0088] in:

[0089] 8, 7, and 1 represent the number of carbon atoms in the PFB-SCN derivative, the PFB derivatizing agent group, and the thiocyanate to be tested, respectively.

[0090] δ 13 C_PFB-SCN is the δ of the derivative PFB-SCN. 13 C measurement value.

[0091] δ 13 C_SCN is the δ of the thiocyanate analyte obtained after correction. 13 C value.

[0092] δ 13 C_PFB is an effective δ-molecule derived from PFB groups. 13 C value.

[0093] δ 13 The value of C_PFB was determined empirically. First, the true δ of a pure NaSCN standard was determined by EA-IRMS. 13 C value (δ)13 C_SCN). Then, the standard was derivatized and analyzed by GC-C-IRMS to obtain its corresponding δ. 13 The C_PFB-SCN value. Finally, δ is calculated by rearranging Formula 1. 13 C_PFB. This value was subsequently used as a constant for δ across all milk samples. 13 C_PFB-SCN is corrected to calculate their final δ. 13 C_SCN value.

[0094] Statistical analysis

[0095] Data processing and statistical analysis were performed using GraphPad Prism software (version 9.0; GraphPad Software, San Diego, California, USA). For each sample, the final δ... 13 The C-value is the average of three repeated measures. Descriptive statistics are reported as mean ± standard deviation (SD) unless otherwise stated.

[0096] To establish an endogenous isotope baseline, the interquartile range (IQR) method was used to remove outliers and determine the central distribution range for each farm. One-way ANOVA was used to assess differences between farms. Paired t-tests (day 6 and day 10) were used to assess temporal variations in isotopic characteristics within the same sample. Independent samples t-tests were used to examine differences between the endogenous and exogenous groups, as well as matrix-related effects.

[0097] This embodiment successfully established endogenous and exogenous thiocyanate (containing thiocyanate ions SCN) - The characteristic stable carbon isotope range.

[0098] Experimental results show that SCN derived from industrial synthesis - It exhibits a consistent and significantly negative δ 13 The C value (average -18.3‰) reflects its fossil fuel origin. In contrast, the natural SCN in all real milk samples... - All showed significantly positive δ 13 The C value (average +12.5‰) is attributed to the fact that dairy cows are fed plants such as linaloides, sesame, and cruciferous plants (rapeseed usually refers to rapeseed).

[0099] Crucially, endogenous and exogenous SCN - δ 13 There is a significant difference exceeding 20‰ between the C-value ranges, with no overlap (p < 0.0001). This clear and robust difference makes it possible to establish a clear discrimination model: using δ 13C = 0‰ is the discrimination threshold; the corrected SCN in any milk sample - δ 13 If the C value is below this threshold, it can be clearly determined that industrial thiocyanate has been illegally added.

[0100] 3. Results and Discussion

[0101] 3.1. Method Development and Characteristic Analysis

[0102] 3.1.1. Identification and Chromatographic Analysis of PFB-SCN Derivatives

[0103] To verify the structure and chromatographic separation of the PFB-SCN derivative, thiocyanate standards were subjected to a PTC reaction, followed by analysis using gas chromatography-mass spectrometry (GC-MS). The total ion chromatogram (TIC) showed a single, dominant peak at a retention time of 5.079 min. The corresponding mass spectrum showed the presence of the molecular ion [M]+ at m / z 239, with a base peak at m / z 181 ([C7H2F5]). + ),like Figure 1 As shown in Figure (A), this is consistent with the expected PFB-SCN structure.

[0104] After structural confirmation, isotopic analysis was performed using GC-C-IRMS. Figure 1 As shown in Figure (C), the PFB-SCN derivative elutes as a sharp and symmetrical peak at 506.2 sec with a very small tail. The observed peak symmetry indicates stable ion beam generation and δ-wavelength peaks. 13 C measurements are reproducible. These results indicate that PTC derivatization will reduce SCN... - It is converted into a volatile derivative compatible with GC-C-IRMS analysis.

[0105] 3.1.2. Determination of isotope correction constants (δ) 13 C_PFB)

[0106] To determine the isotope correction constants, eight different batches of commercial SCN were analyzed. - The standards were analyzed. For each standard, its inherent δ was measured using EA-IRMS. 13 The C_SCN value was determined, and the corresponding derivative value (δ) was determined using GC-C-IRMS. 13 (C_PFB-SCN). All measurements were repeated three times to ensure statistical reliability, with a standard deviation of less than 0.3‰. The compared isotope data are summarized in Table 1. δ 13 The C_SCN values ​​range from -18.617‰ to -17.501‰ (average -18.093 ± 0.383‰). The corresponding δ13 The C_PFB-SCN values ​​range from -28.273‰ to -27.480‰ (mean -27.886 ± 0.281‰). Both datasets exhibit very low dispersion, with standard deviations below 0.4‰. The standard with the lowest intrinsic value (-18.617‰) also produced the lowest derivative value (-28.273‰), consistent with the linear relationship of isotopic signal propagation through derivation.

[0107]

[0108] By rearranging the isotopic mass balance equation, Formula 1, the δ of each standard was calculated. 13 C_PFB values. These eight independently calculated δ values. 13 The C_PFB values ​​exhibited extremely high consistency, ranging from -29.652‰ to -28.864‰, with an average of -29.284‰ and a standard deviation of 0.275‰. The δ values ​​between standards... 13 The limited dispersion of C_PFB values ​​indicates that the isotopic contributions of the derived groups are stable under the applied reaction conditions. Therefore, the average δ 13 The C_PFB value (-29.284‰) was used as a correction constant for subsequent analyses. All reported δ... 13 The C_SCN values ​​are all calculated using rearrangement formula 1 and represent the isotopic composition after mass balance correction.

[0109] 3.1.3. Optimization of Sample Pretreatment and Derivatization Conditions

[0110] To simulate the average endogenous background level of raw milk in China (approximately 2.11 mg / L), an SCN concentration of 2.0 mg / L was selected. - As the added concentration, the effects of pretreatment and PTC derivatization were evaluated by monitoring the peak area (m / z 44) of PFB-SCN in GC-C-IRMS analysis.

[0111] The ratio of the two-phase solvent has been optimized. Dichloromethane, commonly used in similar derivatization experiments, was initially chosen as the organic phase. However, significant interference phenomena were observed, such as... Figure 1 As shown in Figure (B), this problem arises because dichloromethane has a higher density than the aqueous phase. Therefore, after centrifugation, the organic phase containing the derivative precipitates to the bottom layer. Collecting the analyte requires the sampling needle to pass through the upper aqueous phase and the intermediate emulsion layer, thus increasing the risk of contamination.

[0112] To address these limitations, the inventors of this invention have developed an ethyl acetate-based solvent system. In this system, contamination associated with impregnating the inorganic layer is effectively reduced. For example... Figure 1As shown in Figure (C), the phase system was altered using ethyl acetate, causing the PFB-SCN derivative to concentrate in the upper organic layer. This configuration allows for clean collection of the analyte without interference with the underlying aqueous and emulsion phases. The resulting chromatograms showed significantly reduced matrix interference and a substantial increase in signal intensity. Therefore, ethyl acetate was selected as the optimal solvent system.

[0113] Accurate isotope ratio determination is limited by the instrument's linear dynamic range. Outside this range, the measurement results are more susceptible to background interference and instrument nonlinearity. Analytes below the linear threshold amplify ion beam fluctuations, thereby reducing δ¹⁸O₂ levels. 13 The accuracy of C is crucial. Given that the average endogenous thiocyanate content in raw milk from China is approximately 2.11 mg / L, direct analysis would result in signal intensity below the reliable operating range of an ionization mass spectrometer. Therefore, a pretreatment (pre-concentration) step is required to increase the analyte mass to a level suitable for high-precision isotope determination.

[0114] To determine the optimal enrichment factor, the weight of the initial milk sample was varied to achieve different theoretical concentration levels. The enrichment process utilized PTC extraction and derivatization to fix the final organic phase volume at 3.0 mL. Therefore, the initial sample weights of 12 g, 18 g, 24 g, and 30 g corresponded to theoretical enrichment folds of 4-fold, 6-fold, 8-fold, and 10-fold, respectively.

[0115] The effect of enrichment factor on signal intensity and isotope performance is as follows: Figure 1 As shown in Figure (D) in the diagram. In this biaxial plot, the box plot represents the measured δ. 13 The statistical distribution of C-values ​​is shown, with the bins defining the interquartile range (IQR, Q1-Q3), the center line representing the median, and the bands representing the entire data range. Overlaid line plots depict the corresponding average signal intensity. A significant positive correlation was observed between the enrichment factor and signal intensity and isotopic repeatability. Under 4-fold and 6-fold enrichment conditions, the signal intensity was below 200 mV, and δ... 13 The C value exhibits greater dispersion, manifested as a larger IQR and overall range. This behavior is consistent with the reduced ion statistics at low signal intensities and the effects of instrument nonlinearity. At 8-fold and 10-fold enrichment conditions, the signal intensity increases, and δ... 13 C variability decreased. Under 10-fold enrichment conditions (30 g sample), δ 13 The C value (-17.617±1.341‰) was consistent with the EA-IRMS reference value. Therefore, an initial sample weight of 30 g was selected for subsequent analysis.

[0116] After determining the enrichment factor, key parameters of the phase-transfer catalytic (PTC) reaction were systematically optimized to ensure quantitative conversion and minimize potential isotopic fractionation. The effect of basicity on derivatization efficiency was first investigated, such as… Figure 1 As shown in Figure (E). Due to SCN - The effect of SCN is limited to its anionic form as a nucleophile, thus requiring basic conditions. At lower base concentrations, the reaction yield decreases, likely due to the reduced yield of SCN. - The protonation of the analyte formed HSCN. The addition of 0.1 mol / L KOH increased the peak area to its maximum, indicating that the matrix acidity was effectively neutralized and the analyte was stabilized.

[0117] The dosage of phase transfer catalyst (CTAB) was then optimized, such as... Figure 1 As shown in Figure (F), increasing the amount of CTAB from 0.5 mL to 2.0 mL resulted in a significant increase in signal intensity, which is consistent with SCN. - The transfer from the aqueous phase to the organic phase enhances phase consistency. After exceeding 2.0 mL, the signal tends to stabilize, indicating that the catalyst concentration at the phase interface is sufficient. Therefore, 2.0 mL was selected as the optimal dosage.

[0118] Based on considerations of reaction stoichiometry and phase handling, the volume of the PFB-Br derivatization solution was set at 3.0 mL. This volume ensures adequate concentration relative to trace amounts of SCN. - An excess of molar concentration promotes complete reaction and provides sufficient organic phase for separation and purification. Extracting 30 g of milk sample into a fixed 3.0 mL organic phase corresponds to a theoretical enrichment factor of 10 times.

[0119] Finally, the reaction kinetics were evaluated at 70°C, such as... Figure 1 As shown in Figure (G), the reaction rapidly increased within the first 20 minutes and then stabilized after 30 minutes. Extending the reaction time beyond 30 minutes did not result in any significant change in signal intensity. Therefore, 30 minutes was chosen as the reaction time to ensure complete derivatization and minimize potential kinetic isotope effects.

[0120] 3.1.4. Verification of Method Accuracy: Assessment of Matrix Effects

[0121] Isotope fractionation during sample preparation is a major consideration in carbon isotope analysis (CSIA), as physical or chemical processes such as extraction and enrichment can lead to systematic changes in isotopic composition. To investigate the potential isotope fractionation caused by the milk matrix and to assess whether the pre-concentration process affects isotopic accuracy, a comparative study was conducted. Ultrapure water and blank milk samples were supplemented with 2.0 mg / L SCN. - Analysis was performed under optimized 10-fold enrichment conditions. This design enables the evaluation of δ... 13 The accuracy of C may be affected by isotopic fractionation caused by the matrix. Then, 20.0 mg / L of SCN was added to ultrapure water and blank milk samples. - The samples were analyzed without enrichment. These unenriched groups were compared with a 10-fold enriched control group to assess the isotopic fractionation that the enrichment step might introduce.

[0122] like Figure 2 As shown in Figure (A), δ for all four groups 13 The C-values ​​were comparable. Under 10-fold enrichment conditions, the average δ of the milk samples with added markers was... 13 The C-value was -16.374 ± 1.583‰ (n=6), while the corresponding value in the water matrix was -15.981 ± 2.652‰ (n=6). No significant isotopic differences were observed between the enriched samples and the direct analysis control group (p > 0.05). This result indicates that, under the test conditions, the pre-concentration step did not introduce detectable isotopic fractionation, and the milk matrix did not introduce a systematic isotopic bias. Therefore, the consistency between water and milk suggests that the matrix effect is minimal within the analytical precision range of this method.

[0123] 3.2. Isotopic Characterization of Exogenous and Endogenous Thiocyanate

[0124] 3.2.1. Isotopic Characterization of Exogenous Thiocyanate

[0125] To determine the isotopic characterization of exogenous sources, a series of industrial thiocyanate samples (including high-purity standards and commercially available reagents) were systematically analyzed. First, the delta content (δ¹⁸O) of eight thiocyanate standards was determined using gas chromatography-ion mass spectrometry (EA-IRMS). 13 C_SCN values ​​(Table 1). These values ​​range from -18.746‰ to -17.016‰, with an average of -18.093 ± 0.383‰, indicating a relatively narrow distribution.

[0126] To further assess potential differences between industrial sources, the analysis was expanded to include twenty batches of commercially available thiocyanate reagents from different suppliers. The δ0.05 values ​​of these reagents were analyzed. 13 The C_SCN value ranges from -18.416‰ to -13.341‰, with an overall average of -15.852 ± 1.621‰. Compared with high-purity standards, its isotope distribution is more extensive.

[0127] like Figure 2 As shown in Figure (B), a comparison of the two sets of data indicates that although the isotopic variability of industrial reagents is greater, their δ¹² values ​​are lower. 13 The C-values ​​remain within the overlapping negative range. Despite subtle differences between high-purity standards and commercial reagents, a comprehensive dataset of all industrial samples determined the exogenous δ... 13 The range of C_SCN is from -18.746‰ to -13.341‰.

[0128] The negative δ value exhibited by industrial thiocyanate 13 The C value is consistent with carbon sources derived from petrochemical feedstocks, which typically have relatively light carbon isotopic compositions. This isotopic pattern provides a practical basis for distinguishing industrial thiocyanates from endogenous sources.

[0129] 3.2.2. Endogenous δ 13 Characteristics and influencing factors of C_SCN value

[0130] Currently, the study on SCN in milk - Regulatory screening primarily relies on concentration thresholds. However, due to the influence of biological factors such as diet and season, endogenous SCN... - Concentrations can fluctuate significantly. This fluctuation can lead to overlap between natural background levels and industrial additives, thus limiting the reliability of concentration-based standards in source attribution. To address this limitation, stable carbon isotope ratios (δ¹²) were used. 13 C) Analysis to determine the range of endogenous isotopes. To ensure representativeness, a controlled feeding experiment was conducted at six farms, with milk samples collected on days 6 and 10 following the dietary intervention. This section assesses the effects of sampling time and feed composition on endogenous delta-lactone (δ¹²) levels. 13 The influence of the C marker. Through a systematic study of these variables, the endogenous δ was determined. 13 The natural range of C_SCN values.

[0131] 3.2.2.1. Sampling time on endogenous δ 13 The influence of C_SCN value

[0132] To assess potential time-varying patterns, endogenous δ 13C_SCN values ​​were compared. First, an analysis was conducted at the individual farm level to assess intragroup consistency, followed by a comprehensive population analysis to assess overall population trends.

[0133] Internal comparisons within the farm under six different dietary protocols showed that the isotopic distributions were fairly consistent between the two sampling times. Figure 3 As shown in Figures (A), (B), (C), (D), (E), and (F), the isotopic distributions at the two sampling times were highly similar across all surveyed farms (HYL, KM, MCY, QT, CR, and XQ). Independent samples t-tests confirmed that no statistically significant differences were observed between the two time points for all six farms (p > 0.05). Figure 3 Figure (G) in the figure provides a comprehensive overview of these results, highlighting the δ¹²⁺ within each farm despite isotopic differences between farms. 13 The C_SCN values ​​remained statistically consistent across the sampling interval. It is noteworthy that the δ of the farm MCY... 13 The C_SCN value is lower compared to the other five farms. This time stability is further confirmed by a comprehensive analysis of all six farms, as shown in the figure. Figure 3 (H) shows the total δ on days 6 and 10. 13 The C_SCN distributions were extremely similar, and no statistically significant differences were found (p>0.05).

[0134] 3.2.2.2. Effects of feed components on δ-carboxylates in vivo 13 The influence of C_SCN value

[0135] To investigate the potential causes of isotopic variations in the raw data, the five compliant farms were categorized based on their primary protein feed supplements: rapeseed meal (HYL, KM, QT), sesame meal (CR), and flaxseed cake (XQ). Statistical analysis revealed differences in δ¹⁸O values ​​among these dietary protocols. 13 The C_SCN values ​​showed significant differences (e.g. Figure 4 (As shown)

[0136] In the rapeseed meal group (HYL, KM, and QT), δ 13 Significant within-group differences were observed in C_SCN values ​​(p < 0.05). The rapeseed meal intake of farm HYLs was 1.5 kg per head, and their δ... 13The C_SCN values ​​ranged from 3.660‰ to 14.412‰, representing the lower limit of the distribution. In contrast, both farm KM and farm QT ​​supplemented each head with 2.0 kg of rapeseed meal, showing different central values. Farm KM had a wider distribution range, from 8.084‰ to 22.420‰, while farm QT's range was lower and more concentrated, from 3.228‰ to 16.116‰. These within-group differences indicate that despite similar feed types, the δc values ​​varied between farms. 13 The C_SCN values ​​varied, which may reflect differences in intake levels or local production conditions. In contrast, the sesame meal group (farm CR) had an intake of 2.0 kg per head, and its δ... 13 C_SCN values ​​are higher and have a wider distribution range, δ 13 The C_SCN values ​​range from 7.540‰ to 30.916‰, with most values ​​exceeding 15‰.

[0137] δ of flaxseed cake group (farm XQ) 13 The C_SCN values ​​were generally the highest, ranging from 19.380‰ to 35.868‰, significantly higher than those in the rapeseed meal group. One-way ANOVA confirmed significant differences among the dietary groups (p < 0.001).

[0138] The observed isotopic differences are consistent with variations in dietary composition and production conditions, factors that influence the conversion of carbon isotopes into thiocyanate during metabolism. The δ¹² values ​​of the flaxseed cake and sesame powder groups... 13 The C_SCN value was higher in the rapeseed meal group, indicating that these feed types are associated with relatively higher δ values ​​in milk. 13 These results indicate that feed type affects endogenous δ 13 Natural variations in C_SCN values ​​have a significant impact. Therefore, any discrimination threshold must take into account this variability caused by biological factors to avoid misclassification of true samples.

[0139] 3.2.2.3. Endogenous δ 13 Determination of the natural range of C_SCN value

[0140] The valid dataset contains milk samples from five compliant farms (HYL, KM, QT, CR, and XQ), representing authentic raw milk stored under standard conditions. To reflect endogenous delta... 13 To characterize the C_SCN values ​​and minimize the influence of extreme observations, the interquartile range (IQR) method was used to evaluate the data. Specifically, the values ​​between the first and third quartiles (Q1 - Q3) were used to describe the endogenous distribution of the center (e.g., Figure 4(As shown). This method excludes the lowest and highest 25% of observations, thereby reducing sensitivity to potential physiological or environmental anomalies while preserving inherent biological variability.

[0141] Of these five farms, δ 13 The C_SCN values ​​exhibit regional differences and partial overlap, forming a continuous distribution without obvious discontinuities (e.g., Figure 4 (As shown). δ of farm XQ 13 The C_SCN value is the highest, with a clustering range of 20.660‰ to 26.924‰, representing the upper limit of the observed distribution. In contrast, the farm HYL value is the lowest, with a minimum of 4.716‰ and a range of 14.412‰. The farm QT ​​distribution has the narrowest range (7.132‰ to 12.260‰), while the farm CR distribution has the widest range (9.516‰ to 26.732‰), spanning the middle value between the lower and higher values.

[0142] Based on a validation dataset built from 60 samples collected from five dairy farms, the PTC-GC-C-IRMS method, primarily employed in this invention, was used to analyze endogenous delta-index. 13 A systematic analysis of C_SCN values ​​was performed. Among all samples meeting the criteria, δ 13 C_SCN values ​​ranged from 4.716‰ to 26.924‰. Notably, all samples confirmed as endogenous showed positive δ0.05. 13 C_SCN value.

[0143] The lowest observed endogenous value (4.716‰) is clearly separated from the negative isotopic range of industrial thiocyanate, where values ​​consistently fall below -13‰. No overlap was observed between the endogenous and exogenous datasets in this study.

[0144] Therefore, within the current sampling framework, the endogenous δ in fresh milk 13 The C_SCN range was observed to be between 4.716‰ and 26.924‰. This range reflects natural biological variability related to feed composition and regional production conditions, and provides a data-supported reference range for distinguishing genuine milk from samples that may contain industrial thiocyanate.

[0145] 3.3. Establishment and Application of Discrimination Thresholds

[0146] 3.3.1. Comparison of Endogenous and Exogenous Fingerprints and Threshold Determination

[0147] To establish a data-driven discriminant model, a comprehensive analysis was performed on validated endogenous datasets (n=60) and exogenous datasets (n=28). Exogenous samples, including high-purity standards and industrial reagents, exhibited a limited negative distribution. The δ0 of these 28 industrial samples... 13 The C_SCN value ranges from -18.746‰ to -13.341‰. This depleted δ 13 Characteristic C is consistent with the characteristics of chemicals synthesized from petrochemical raw materials, which typically have relatively low... 13 C content.

[0148] In contrast, endogenous thiocyanate extracted from milk samples from five farms showed completely positive δ values. 13 The C_SCN values ​​exhibit distinct isotopic distribution characteristics. The endogenous data range from 4.716‰ (farm HYL) to 26.924‰ (farm XQ), forming two clearly separated isotopic populations, with no overlap observed in the current dataset.

[0149] Statistical analysis further confirmed the differences between the endogenous and exogenous groups. For example... Figure 5 As shown in Figures (A)-(E), independent samples t-tests were performed on the exogenous group and each individual farm (HYL, KM, QT, CR, and XQ), and the p-values ​​were all less than 0.0001, indicating high statistical significance. When the data from all five farms were aggregated to represent the overall natural variation in real milk, as shown in Figures (A)-(E), the results showed that the p-values ​​were all less than 0.0001, indicating high statistical significance. Figure 5 As shown in Figure (F), the difference from the exogenous group remained highly significant (p < 0.0001). Figure 5 The figure (G) in the figure summarizes the isotopic distance between the two groups in an intuitive way.

[0150] It is worth noting that the lowest endogenous δ 13 The C_SCN value (4.716‰, farm HYL) is 18.057‰ higher than the largest exogenous value (-13.341‰), indicating that there is a significant isotopic difference between endogenous thiocyanate and industrial thiocyanate in this study.

[0151] Based on this clear boundary, δ 13 C = 0‰ is set as the operational distinguishing threshold for identifying potential foreign additives. Zero is chosen as the threshold for two reasons. First, it provides a safety margin of 4.716‰ relative to the lowest observed endogenous value, thus reducing the risk of misclassifying genuine milk samples as foreign additives. Second, it is 13.341‰ higher than the maximum observed in the tested foreign samples, ensuring high sensitivity when detecting industrial thiocyanate in the evaluation dataset.

[0152] Therefore, if the integrity of the sample can be confirmed, when δ 13 A C_SCN value less than 0‰ may indicate the presence of exogenous thiocyanate. All validated endogenous samples in this invention showed a positive δ0.05. 13 The C_SCN value was negative for all tested industrial samples.

[0153] 3.3.2. Verification using simulated adulterated samples

[0154] To assess the robustness of the proposed identification method under real-world adulteration conditions, validation experiments were conducted using simulated adulterated samples. Fifteen real milk samples were selected from five compliant farms (HYL, KM, QT, CR, and XQ), while samples from farm MCY were excluded due to previous evidence of spoilage. This selection followed factor design principles to ensure coverage of four key categories: (1) high concentration and δ 13 High C_SCN content, (2) high concentration and δ 13 The C_SCN content is low, (3) the concentration is low and δ 13 High C_SCN content, (4) low concentration and δ 13 Low C_SCN content.

[0155] To improve representativeness, samples within the median quartile (Q1–Q3) of the endogenous distribution were selected. This selection also accounted for differences in dietary regimens by including samples from each farm group. By integrating diverse dietary contexts and sampling time points, this experiment assessed simulated adulteration under a wide range of biological and environmental conditions. These samples were spiked with 10 mg / L of YY industrial standard (δ¹²). 13 C_SCN = -18.104‰). This standard was chosen because it represents an intermediate isotope value from the industrial source being tested.

[0156] δ-thiocyanate in 15 representative milk samples 13 C-values ​​were determined in these samples, which were supplemented with 10 mg / L of exogenous thiocyanate. The isotopic results of the spiked samples are listed in Table 2. As expected, the addition of exogenous thiocyanate resulted in δ-values. 13 The C_SCN values ​​showed a significant negative shift. The measured values ​​for the spiked mixtures ranged from -18.804‰ to -2.284‰. The δ values ​​for all simulated adulterated samples... 13 The C_SCN values ​​were all below the set operating threshold of 0‰. Even the samples from farms XQ and CR (whose endogenous δ before spiking) were below this threshold. 13The C_SCN value was the highest, and it also entered the negative region after addition. This behavior conforms to the isotopic mass balance principle: the mass introduced from exogenous thiocyanate... 13 C-deficient carbon causes the overall isotopic composition of the mixture to shift towards the negative characteristics of the exogenous source. These results demonstrate that the proposed method can distinguish between exogenous additives and natural biological variation under controlled simulation conditions.

[0157]

[0158] 3.3.3. Application of Commercial Milk Samples

[0159] To evaluate the applicability of the PTC-GC-C-IRMS method in routine monitoring, 30 commercial liquid milk samples were randomly collected from the market and analyzed. Detailed isotopic data are summarized in Table 3.

[0160] δ 13 The C_SCN values ​​have a wide distribution, reflecting the diversity of milk sources in the retail market. Quartile analysis shows that most values ​​are concentrated between 5.527‰ and 14.905‰ (the central Q1-Q3 interval), which significantly overlaps with the endogenous reference dataset established in this invention.

[0161]

[0162] It is noteworthy that the lowest observed value (2.764‰) is still positive, although it exceeds the middle quartile range, it is still significantly positive. δ for all analyzed samples 13 The C_SCN values ​​were all above the 0‰ operating threshold and none approached the negative isotope range of industrial thiocyanate.

[0163] Within the scope of this investigation, the isotopic characteristics of commercial milk samples from endogenous sources were consistent. These findings indicate that the proposed method is applicable to distinguishing genuine milk samples from those containing industrial thiocyanate under actual market conditions.

[0164] in conclusion:

[0165] This invention successfully developed and validated a novel method including PTC-GC-C-IRMS for determining trace levels of SCN in milk. - The carbon isotope ratio was used for the first time to detect trace amounts of thiocyanate (containing thiocyanate ions SCN) in milk samples (such as cow's milk). - This method enables precise source identification of analytes. By integrating an innovative derivatization strategy, it simultaneously extracts, concentrates, and converts analytes into volatile derivatives, overcoming the challenge of compound-specific isotope analysis of non-volatile ions in complex food matrices.

[0166] To the inventor's knowledge, this is the first reported instance of SCN in milk. - Isotopic characterization analysis. This invention mainly combines phase-transfer catalytic derivatization with isotopic mass balance correction and isotopic ratio mass spectrometry. This method can analyze the SCN in complex milk matrices. - Reliable analysis was performed. Stable analytical performance and satisfactory accuracy were achieved.

[0167] A clear isotopic segregation was observed between endogenous and industrial thiocyanate sources. Endogenous delta-hydroxyl content was found in validated fresh milk samples. 13 The C_SCN value ranges from 4.716‰ to 26.924‰, while the value of industrial thiocyanate is consistently negative (-18.746‰ to -13.341‰). Based on this, an operational identification threshold of 0‰ is proposed. Validation through simulated adulteration experiments and commercial milk samples demonstrates the applicability of this method in distinguishing between exogenous additives and natural biological variations.

[0168] In summary, this invention establishes an isotopic identification framework for thiocyanates contained in milk and other substances, and provides a data-based analytical method for authenticity assessment.

[0169] The core discovery of this invention is the interplay between endogenous and exogenous SCN. - There are fundamental isotopic differences between them, proving the endogenous SCN. - It consistently exhibits a positive δ 13 C characteristics, while all tested industrial-grade SCN - The standard samples all have significantly negative δ. 13 C-characteristics. δ from these two sources 13 The C range is completely separated, thus allowing for the establishment of a clear discrimination threshold.

[0170] Therefore, the method of this invention enables regulatory agencies to clearly identify exogenous SCN additives in milk samples. - This approach provides a scientifically robust tool. The principles of this method have great potential for application in the source analysis of other challenging analytes in other food matrices, contributing a novel strategy to the fields of food forensic science and authenticity verification.

[0171] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," 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, the 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.

[0172] Although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple variations can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple variations and combinations should also be considered as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. A method for identifying endogenous and exogenous thiocyanate in milk samples based on stable carbon isotope ratios, characterized in that, Includes the following steps: Step S1. Sample pretreatment: Accurately measure the milk sample to be tested and divide it into the required number of portions. Add an organic solvent to each portion to remove more than 50 wt% of fat and protein from the milk sample. Then concentrate the sample to obtain an aqueous concentrate. Step S2. Phase transfer catalytic derivatization: Add appropriate amounts of phase transfer catalyst, alkaline solution and derivatization reagent sequentially to the aqueous concentrate obtained in step S1; Then, a derivatization reaction was carried out; after the reaction was completed, the organic phase was aspirated and filtered to obtain the filtrate to be tested, which was then frozen and stored. Step S3. Determine the stable carbon isotope ratio in the milk sample by GC-C-IRMS analysis. The filtrate obtained in step S2 was analyzed using gas chromatography-combustion-isotope ratio mass spectrometry (GC-COMP-MS) to determine the stable carbon isotope ratio δ of the derivative. 13 C_PFB-SCN; Step S4. Isotope Correction and Result Determination: The derivatization reaction introduces carbon atoms from the derivatizing reagent into the thiocyanate molecules in the milk sample to be tested, thereby stabilizing the carbon isotope ratio δ of the measured derivative. 13 C_PFB-SCN correction was performed to calculate the stable carbon isotope ratio δ of the thiocyanate to be tested. 13 C_SCN; The correction calculation is based on the following formula 1: Wherein, a, b, and c are the number of carbon atoms in the PFB-SCN derivative, the PFB derivatizing group, and the thiocyanate to be tested, respectively; δ 13 C_PFB-SCN is the δ of the derivative PFB-SCN. 13 C measured value; δ 13 C_SCN is the δ of the thiocyanate analyte obtained after correction. 13 C value, i.e., stable carbon isotope ratio δ 13 C_SCN; δ 13 C_PFB is an effective δ-molecule derived from PFB groups. 13 C value is a correction constant; The δ 13 C_PFB is the effective isotope value of the derivatization reagent. Its value needs to be calculated by formula 1 after EA-IRMS and GC-C-IRMS analysis of pure industrial thiocyanate standards. If the stable carbon isotope ratio of the thiocyanate to be tested is δ 13 If the C_SCN is below 0‰, it can be determined that the milk sample contains illegally added industrial thiocyanate.

2. The method according to claim 1, characterized in that, Step S1 includes: Accurately measure the milk sample to be tested and divide it into the required number of portions. Add an organic solvent to each portion, with the ratio of milk sample to organic solvent in each portion being 0.5-1 g:1 mL. Vortex the sample to precipitate and remove more than 50 wt% of fat and protein from the milk sample. Then, centrifuge at 1-6°C and combine the supernatants from each portion. Then concentrate the sample to remove more than 90% of the organic solvent, thereby obtaining an aqueous concentrate.

3. The method according to claim 2, characterized in that, Step S1 includes: The organic solvent is an acetonitrile solution of 0.1-0.2% (v / v) formic acid; and / or In step S1, vortex oscillation is performed at an oscillation frequency of 2000-3000 rpm / min for 0.5-2 minutes; and / or In step S1, centrifuge at 9000-10000 rpm / min for 10-20 minutes.

4. The method according to claim 1, characterized in that, Step S2 includes: Add, sequentially, 10%-20% (by volume of the aqueous concentrate) of a 0.05-0.3 mol / L hexadecyltrimethylammonium bromide aqueous solution as a phase transfer catalyst, 10%-20% (by volume of the aqueous concentrate) of an alkaline solution with a concentration of 0.05-0.2 mol / L to the aqueous concentrate to ensure a weakly alkaline reaction system, and 15%-30% (by volume of the aqueous concentrate) of a 3-6 mL / L pentafluorobenzyl bromide derivatizing reagent dissolved in ethyl acetate. Immediately after addition, seal the reaction vessel and vortex at a frequency of 2000-3000 rpm / min for 1-3 minutes to form a homogeneous reaction mixture emulsion. Incubate the emulsion in a constant temperature water bath at 65-75 °C for 20-40 minutes to ensure complete derivatization; and / or After the reaction is complete, to rapidly separate the phase, the mixture is centrifuged at 9000-10000 rpm / min for 5-15 minutes at 2-5°C; and / or The upper organic phase containing the target derivative pentafluorobenzyl thiocyanate was aspirated and filtered. The clarified filtrate was transferred to a container to obtain the test filtrate, which was then frozen and stored at -15°C to -20°C.

5. The method according to claim 4, characterized in that, Step S2 includes: To the aqueous concentrate obtained in step S1, add 10%-20% (by volume of the aqueous concentrate) of a 0.05-0.3 mol / L aqueous solution of hexadecyltrimethylammonium bromide as a phase transfer catalyst, 10%-20% (by volume of the aqueous concentrate) of an alkaline solution with a concentration of 0.05-0.2 mol / L to ensure the reaction system is weakly alkaline, and 15%-30% (by volume of the aqueous concentrate) of a 3-6 mL / L pentafluorobenzyl bromide derivatizing reagent dissolved in ethyl acetate. Immediately after addition, seal the reaction vessel and vortex at a frequency of 2000-3000 rpm / min for 1-3 minutes to form a homogeneous reaction mixture emulsion. Incubate the emulsion in a constant temperature water bath at 65-75 °C for 20-40 minutes to ensure complete derivatization. After the reaction, to rapidly separate the phases, incubate the mixture at 2-5 °C with a flow rate of 9000-10000 rpm. Centrifuge at rpm / min for 5-15 minutes; aspirate the upper organic phase containing the target derivative pentafluorobenzyl thiocyanate and filter it. Transfer the clear filtrate to a container to obtain the test filtrate and store it frozen at -15°C to -20°C for later detection.

6. The method according to claim 1, characterized in that, Step S3 includes gas chromatography detection and isotope mass spectrometry detection, wherein: The gas chromatography detection includes: injecting the filtrate to be tested into the sample in splitless mode, with an injection volume of 1-2 µL and an injection port temperature of 250-280 °C; using a fused silica capillary column and helium as the carrier gas at a flow rate of 1-3 mL / min; and a temperature program of: initial temperature 95-105 °C held for 0.5-1.5 min, increasing to 110-130 °C at 2-5 °C / min and holding for 2-6 min, then increasing to 280-320 °C at 30-50 °C / min and holding for 1-3 min, to obtain the chromatographic eluent; and The isotope mass spectrometry detection includes: passing the chromatographic effluent through a combustion interface at a temperature of 950-1100 °C using a CuO / NiO catalyst to quantitatively convert the chromatographic effluent into CO2. After drying with a Nafion membrane to remove water, the CO2 is introduced into an isotope ratio mass spectrometer through a continuous flow interface to determine the stable carbon isotope ratio δ of the product CO2. 13 C, and calibrated using the international standard reference material IAEA-600.

7. The method according to claim 1, characterized in that, Prior to step S4, the stable carbon isotope ratio of industrial thiocyanate is determined by EA-IRMS analysis.

8. The method according to claim 7, characterized in that, The determination of the stable carbon isotope ratio of industrial thiocyanate by EA-IRMS analysis includes the following steps: determining the true δ¹⁸O of pure sodium thiocyanate standard using EA-IRMS. 13 C value; 0.5 mg of the pure sodium thiocyanate standard was packaged and burned in an elemental analyzer at 950-980 °C to produce a product gas containing carbon dioxide. The product gas was purified in a reduction furnace at 620-680 °C and then analyzed by a mass spectrometer; all δ 13 C values ​​are reported in per mille (‰) relative to the VPDB standard and calibrated using the international standard reference material IAEA-600, with an analytical precision better than 0.2‰.

9. The method according to claim 1, characterized in that, The correction constant was determined by the following method: First, the true δ of the pure NaSCN standard was obtained by EA-IRMS analysis. 13 C value, i.e., δ 13 C_SCN; then, the pure NaSCN standard was derivatized and analyzed by GC-C-IRMS to obtain its corresponding δ 13 The C_PFB-SCN value; finally, δ is calculated by rearranging Formula 1. 13 C_PFB; then the δ 13 The value of C_PFB is used as a constant for the δ of all milk samples. 13 C_PFB-SCN is used for correction to calculate the final δ. 13 C_SCN value.

10. The application of the method according to any one of claims 1-9 in identifying endogenous and exogenous thiocyanates in milk samples.

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