Method for determining origin of pagrus major by using ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The present invention relates to an anserin quantitative analysis method using ultra-high performance liquid chromatography-tandem mass spectrometry. It has been identified that, compared to a conventional method, the quantitative analysis method of the present invention is accurate and can significantly shorten analysis time In addition, through receiver operating characteristic (ROC) analysis, it has been identified that anserin can be used as a biomarker for determining the origin of Pagrus major, and, on the basis that anserin content is higher than 227 mg / 100 g, Pagrus major with higher levels and lower levels can be determined as Japanese and Korean, respectively.

Description

A method for determining the origin of red sea bream using ultra-high-performance liquid chromatography-tandem mass spectrometry.

[0001] The present invention relates to a method for quantitative analysis of anserine in red sea bream using an ultra-high performance liquid chromatography-tandem mass spectrometry and a method for determining the origin of red sea bream using anserine.

[0002] Red sea bream (Pagrus major) is a migratory fish native to East Asia, including Korea, Japan, and China. Its optimal water temperature is 18–25°C. Rich in essential amino acids, this species is highly valued as a high-value protein source and is widely consumed in Korea and Japan.

[0003] Meanwhile, South Korea implements a country-of-origin labeling system for fishery products. This system aims to promote fair trade, guarantee consumers' right to know, and protect producers and consumers by ensuring appropriate and reasonable country-of-origin labeling and distribution history management for fishery products and processed products. Furthermore, a distribution history system for imported fishery products is implemented to address concerns about market disruption and social safety, such as falsely labeling the country of origin as domestic after importation. This system applies to 21 products, including sea bream. However, with distribution history systems applied to only 5% of all imported fishery products, it is difficult to expect any real impact.

[0004] According to the Ministry of Oceans and Fisheries' inspection and crackdown on country of origin labeling over the past five years, 818 companies were caught in 2018, 916 in 2019, 543 in 2020, 783 in 2021, and 519 in 2022 for failing to label the country of origin, violating labeling methods, or falsely labeling. As of August 2023, the number of companies in violation reached 565, and the number of companies violating the country of origin labeling system is believed to have increased after the discharge of contaminated water from the Fukushima nuclear power plant into the ocean in August. Furthermore, as of the end of August 2023, 57 companies were caught falsely labeling Japanese seafood as domestic, and among them, red sea bream was the species with the most false country of origin, with 27 cases. Therefore, there is an urgent need for a method to accurately determine the country of origin of Korean and Japanese red sea bream.

[0005] DNA profiling, metabolomic profiling, fatty acid profiling, elemental analysis, and stable isotope analysis have been utilized to determine the origin of seafood. While DNA analysis is a successful method for identifying seafood species, it has limitations in distinguishing between geographically close regions, such as Korea and Japan, within the same species. Another key technology for determining the geographical origin of seafood is metabolomic profiling, which involves a comprehensive analysis of metabolites, the end products of cellular regulatory processes within an organism. This analytical approach allows for the investigation of responses to genetic and environmental changes and provides insight into unique metabolic signatures associated with specific geographical regions. In the case of red sea bream, untargeted metabolomic analysis suggested anserine as a powerful discriminant marker for distinguishing Korean and Japanese red sea bream.

[0006] Anserine, along with carnosine, is abundant in meat and certain fish. The two substances, both methylated forms of carnosine, share similar chemical properties. Differences in the content or ratio of the two substances have been used to identify meat types. According to Article 8. General Test Methods, Section 5.2.2 Physicochemical Test Methods of the Food Code, a method for identifying meat species such as beef, pork, horse, chicken, duck, and turkey using the carnosine / anserine ratio is described. However, this method is limited by the cumbersome derivatization process and the time-consuming analysis time of over an hour. Therefore, a more advanced analytical method for the quantitative analysis of anserine and carnosine is needed.

[0007] Therefore, the present inventors developed a new quantitative analysis method using ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to confirm whether the anserine content, carnosine content, and anserine / carnosine ratio in red sea bream can be applied to determine the origin of red sea bream. It was confirmed that the quantitative analysis time can be significantly shortened through the new quantitative analysis method compared to the existing analysis method, and anserine and carnosine can be separated, detected, and quantitatively analyzed. In addition, the present invention was completed by confirming that the anserine detected by the quantitative analysis method can be used as a biomarker for determining the origin of red sea bream.

[0008] The purpose of the present invention is to provide a biomarker composition for determining the origin of red sea bream (Pagrus major) containing anserine as an active ingredient.

[0009] In addition, the present invention aims to provide a kit for determining the origin of red sea bream, which includes a preparation for measuring the concentration of anserine in red sea bream.

[0010] In addition, the present invention aims to provide a method for determining the origin of red sea bream, including the steps of 1) measuring the concentration of anserine in red sea bream; and 2) comparing the concentration of anserine measured in step 1) with a reference value to determine the origin.

[0011] In addition, the present invention aims to provide a method for quantitative analysis of anserine, a biomarker, comprising the steps of: 1) preparing a standard solution including anserine and carnosine as standards, and preparing an internal standard solution including an internal standard; 2) pretreating a red sea bream sample; 3) obtaining a chromatogram of the pretreated red sea bream sample using an ultra-high performance liquid chromatography-tandem mass spectrometer; and 4) measuring the content of anserine through the peak and area of ​​the obtained chromatogram.

[0012] To achieve the above purpose, a biomarker composition for determining the origin of red sea bream (Pagrus major) containing anserine as an active ingredient is provided.

[0013] In addition, the present invention provides a kit for determining the origin of red sea bream, including a preparation for measuring the concentration of anserine in red sea bream.

[0014] In addition, the present invention provides a method for determining the origin of red sea bream, including: 1) a step of measuring the concentration of anserine in red sea bream; and 2) a step of determining the origin by comparing the concentration of anserine measured in step 1) with a reference value.

[0015] In addition, the present invention provides a method for quantitative analysis of anserine, a biomarker, comprising the steps of: 1) preparing a standard solution including anserine and carnosine as standards, and preparing an internal standard solution including an internal standard; 2) pretreating a red sea bream sample; 3) obtaining a chromatogram of the pretreated red sea bream sample using an ultra-high performance liquid chromatography-tandem mass spectrometer; and 4) measuring the content of anserine through the peak and area of ​​the obtained chromatogram.

[0016] The present invention relates to a biomarker composition for determining the origin of red sea bream (Pagrus major) containing anserine as an active ingredient, and has developed a quantitative analysis method that is accurate and rapid compared to existing methods, and can determine the origin of domestic and Japanese red sea breams using the anserine standard suggested in the present invention, and can be usefully used to crack down on false labeling of the origin of aquatic products that occurs for economic gain.

[0017] Figure 1 is a diagram showing the sampling site of a red sea bream sample.

[0018] Figure 2 is a diagram showing the chromatographic separation of six standard substances under the conditions of HILIC column and pH 5 in positive ion mode.

[0019] Figure 3 is a diagram showing the chromatographic separation of six standard substances under the conditions of an amide column and pH 5 in positive ion mode.

[0020] Figure 4 is a diagram showing the chromatographic separation of six standard substances under pH 8 conditions on a HILIC column in positive ion mode.

[0021] Figure 5 is a diagram showing the chromatographic separation of six standard substances under the conditions of an amide column and pH 8 in positive ion mode.

[0022] Figure 6 is a diagram showing the ionic intensity according to pH under HILIC column, 75% acetonitrile, positive ion mode conditions.

[0023] Figure 7 is a diagram showing the ionic intensity under HILIC column, 75% acetonitrile, and high pH conditions.

[0024] Figure 8 is a diagram showing the retention time of six standard substances according to pH and organic solvent under 70% acetonitrile conditions.

[0025] Figure 9 is a diagram showing the retention time of six standard substances according to pH and organic solvent under 75% acetonitrile conditions.

[0026] Figure 10 is a diagram showing the retention time of six standard substances according to pH and organic solvent under 80% acetonitrile conditions.

[0027] Figure 11 is a diagram showing a chromatogram under the final analysis conditions (HILIC column, 75% acetonitrile pH 5).

[0028] Figure 12 is a diagram showing the results of ROC curve analysis on the anserine content of red sea bream samples harvested in spring.

[0029] Figure 13 is a diagram showing the results of ROC curve analysis on the anserine content of red sea bream samples collected in summer.

[0030] Figure 14 is a diagram showing the results of ROC curve analysis on the anserine content of red sea bream samples collected in the fall.

[0031] Figure 15 is a diagram showing the results of ROC curve analysis on the anserine content of red sea bream samples collected in winter.

[0032] Figure 16 is a diagram showing the results of ROC curve analysis on the carnosine content of red sea bream samples harvested in spring.

[0033] Figure 17 is a diagram showing the results of ROC curve analysis on the carnosine content of red sea bream samples collected in summer.

[0034] Figure 18 is a diagram showing the results of ROC curve analysis on the carnosine content of red sea bream samples collected in the fall.

[0035] Figure 19 is a diagram showing the results of ROC curve analysis on the carnosine content of red sea bream samples collected in winter.

[0036] Figure 20 is a diagram showing the results of ROC curve analysis on the anserine / carnosine ratio of red sea bream samples harvested in spring.

[0037] Figure 21 shows the results of ROC curve analysis for anserine / carnosine in red sea bream samples collected in summer.

[0038] Figure 22 is a diagram showing the results of ROC curve analysis for anserine / carnosine in red sea bream samples collected in the fall.

[0039] Figure 23 shows the results of ROC curve analysis for anserine / carnosine in red sea bream samples collected in winter.

[0040] Figure 24 is a diagram showing the results of ROC curve analysis for the anserine content of all red sea bream samples.

[0041] Figure 25 is a diagram showing the results of ROC curve analysis for the carnosine content of all red sea bream samples.

[0042] Figure 26 is a diagram showing the results of ROC curve analysis for the anserine / carnosine ratio of all red sea bream samples.

[0043] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments of the present invention. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0044] In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0045] The present invention provides a biomarker composition for determining the origin of red sea bream (Pagrus major) containing anserine as an active ingredient.

[0046] In one embodiment of the present invention, the origin may be domestic or Japanese, but is not limited thereto.

[0047] In one embodiment of the present invention, the concentration of anserine may be lower in domestic red sea bream than in Japanese red sea bream, but is not limited thereto.

[0048]

[0049] In addition, the present invention provides a kit for determining the origin of red sea bream, including a preparation for measuring the concentration of anserine in red sea bream.

[0050]

[0051] In addition, the present invention provides a method for determining the origin of red sea bream, including: 1) a step of measuring the concentration of anserine in red sea bream; and 2) a step of determining the origin by comparing the concentration of anserine measured in step 1) with a reference value.

[0052] In one embodiment of the present invention, the step of measuring the concentration of anserine may use an ultra-high performance liquid chromatography-tandem mass spectrometer, but is not limited thereto.

[0053] In one embodiment of the present invention, the step of determining the country of origin may be characterized in that when the measured concentration of anserine exceeds 227 mg / 100 g, it is determined to be of Japanese origin, and when it is 227 mg / 100 g or less, it is determined to be of Korean origin, but is not limited thereto.

[0054] In one embodiment of the present invention, when the red sea bream is collected from March to May, the step of determining the country of origin may be characterized in that when the measured concentration of anserine exceeds 173 mg / 100 g, it is determined to be from Japan, and when it is 173 mg / 100 g or less, it is determined to be from Korea, but is not limited thereto.

[0055] In one embodiment of the present invention, when the red sea bream is collected from June to August, the step of determining the country of origin may be characterized in that when the measured concentration of anserine exceeds 235 mg / 100 g, it is determined to be from Japan, and when it is 235 mg / 100 g or less, it is determined to be from Korea, but is not limited thereto.

[0056] In one embodiment of the present invention, when the red sea bream is collected from September to November, the step of determining the country of origin may be characterized in that when the measured concentration of anserine exceeds 160 mg / 100 g, it is determined to be from Japan, and when it is 160 mg / 100 g or less, it is determined to be from Korea, but is not limited thereto.

[0057] In one embodiment of the present invention, when the red sea bream is collected in December to February, the step of determining the country of origin may be characterized in that when the measured concentration of anserine exceeds 144 mg / 100 g, it is determined to be from Japan, and when it is 144 mg / 100 g or less, it is determined to be from Korea, but is not limited thereto.

[0058] In addition, the present invention provides a method for quantitative analysis of anserine, a biomarker, comprising the steps of: 1) preparing a standard solution including anserine and carnosine as standards, and preparing an internal standard solution including an internal standard; 2) pretreating a red sea bream sample; 3) obtaining a chromatogram of the pretreated red sea bream sample using an ultra-high performance liquid chromatography-tandem mass spectrometer; and 4) measuring the content of anserine through the peak and area of ​​the obtained chromatogram.

[0059] In one embodiment of the present invention, the internal standard may be, but is not limited to, phenylephrine hydrochloride.

[0060] In one embodiment of the present invention, the ultra-high performance liquid chromatography-tandem mass spectrometer may use a BEH HILIC column, use a 50% acetonitrile and 10 mM ammonium acetate mixture solution as mobile phase A, and use a 75% acetonitrile and 10 mM ammonium acetate mixture solution as mobile phase B, the pH of mobile phases A and B is 5 in the aqueous phase, the flow rate of the mobile phase is 0.2 mL / min, the injection volume is 2 μL, the temperature of the column oven is 35°C, the ion mode is positive mode, the spray voltage is 3500 V, the temperature of the ion transfer tube is 350°C, and the temperature of the vaporizer is 275°C, but is not limited thereto.

[0061] In one embodiment of the present invention, the gradient elution of the mobile phase A and the mobile phase B may be characterized by being performed under the following mobile phase concentration gradient conditions, but is not limited thereto.

[0062] - Start ~ 10 minutes - Mobile phase A: 0%, Mobile phase B: 100%

[0063] - 10.1 to 15 minutes: Mobile phase A: 100%, Mobile phase B: 0%

[0064] - 15.1 min ~ 25 min: Mobile phase A: 0%, Mobile phase B: 100%

[0065] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0066]

[0067] <Preparation Example 1> Preparing a red sea bream sample

[0068] A total of 180 red sea breams (90 each from Korea and Japan) were purchased from Busan's Jagalchi Fish Market. The 180 red sea breams were purchased in spring (March to May, n=60), summer (June to August, n=60), fall (September to November, n=30), and winter (December to February, n=30). Table 1 presents information about the purchased red sea bream samples.

[0069] SeasonYearNumberKoreanJapaneseTotal length(cm)Weight(kg)Total length(cm)Weight(kg)Spring20211041.7 ± 5.11.1 ± 0.444.3 ± 1.81.4 ± 0.220221042.5 ± 2.11.3 ± 0.257.4 ± 1.83.0 ± 0.220231044.8 ± 2.11.3 ± 0.251.6 ± 1.22.1 ± 0.1Summer20211045.3 ± 2.21.5 ± 0.245.3 ± 1.41.5 ± 0.120221044.5 ± 1.81.3 ± 0.247.0 ± 1.21.8 ± 0.220231043.9 ± 3.31.5 ± 0.350.3 ± 1.62.0 ± 0.2Fall20211544.4 ± 1.81.3 ± 0.147.9 ± 1.71.7 ± 0.2Winter20211540.0 ± 10.01.2 ± 0.247.0 ± 4.21.5 ± 0.4

[0070]

[0071] All red sea breams were immediately euthanized by decapitation, and tissue samples were collected from the dorsal region, specifically the central region between the bones and the dorsal spine. Additionally, muscle tissue from the posterior abdomen was collected. The sampling sites are shown in Figure 1.

[0072] The obtained samples were cut into 1 cm cubes and stored frozen at -80°C. After frozen storage, they were freeze-dried to maintain the moisture content of the samples below 3%. After the pretreatment was completed, the samples were crushed, vacuum-packed, and stored at -80°C.

[0073]

[0074] <Preparation Example 2> Preparation of compounds and reagents

[0075] Anserine (CAS: 584-85-0, MedChemExpress, USA, HY-113354) and carnosine (CAS: 305-84-0, MedChemExpress, USA, HY-W013494) were purchased from MedChemExpress. In addition, phenylephrine hydrochloride (CAS: 61-76-7, Sigma-aldrich, USA, P6126), histidylleucine (His-Leu), 4-imidazoleacrylic acid (4-IAA), and L-norvaline (Nor) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the above four compounds were used as candidates for internal standards.

[0076] Standard solutions were prepared by adjusting the concentration of anserine and carnosine to 1 mg / mL each, and the prepared standard solutions were aliquoted, stored frozen, and then used for analysis.

[0077] Phenylephrine hydrochloride, histidylleucine, 4-imidazoleacrylic acid, and L-norvaline were adjusted to a concentration of 1 mg / mL, and aliquoted like the above standard solution, stored frozen, and then used for analysis.

[0078]

[0079] <Preparation Example 3> Preprocessing of red sea bream samples

[0080] 0.5 g of lyophilized sample was placed in a 15 ml conical tube, and 50 μl of internal standard at a concentration of 1 mg / mL was added. Then, 7 mL of 10% (w / v) trichloroacetic acid (TCA) was added to the conical tube to extract anserine and carnosine. After addition, vortexing was performed and extraction was performed by sonication for 5 minutes. The extract was maintained at a refrigerated temperature for more than 20 minutes and centrifuged at 10,000 × g and 4°C for 15 minutes. The supernatant of the centrifuged solution was obtained, and the above process was repeated once more and mixed with the existing supernatant. Thereafter, 10% TCA was added and the volume was adjusted to 25 mL.

[0081] Afterwards, it was diluted 20-fold using 75% acetonitrile. After filtering using a 0.2 μm PTFE (polytetrafluoroethylene) filter (Whatman, Maidstone, England), the sample solution was placed in a vial for HPLC analysis, and the solution was prepared.

[0082] The standard solutions (anserine, carnosine) diluted by concentration were also filtered using the above filter and placed in a vial for HPLC analysis to prepare for HPLC analysis.

[0083]

[0084] <Example 1> UHPLC-MS / MS analysis method

[0085] 1-1. Optimization of Separation Conditions for Anserine and Carnosine

[0086] To clearly separate anserine and carnosine and to determine how to detect anserine in a short time, a Thermo Fisher TSQ Endura triple-stage quadrupole mass spectrometer (Thermo Fisher Scientific, Middlesex, MA, USA) coupled to an Ultimate 3000 LC System was used.

[0087] Analyses were performed using an Acquity UPLC BEH amide column (2.1 × 100 mm i.d., particle size 1.7 μm; Waters) and an Acquity UPLC BEH HILIC column (2.1 × 100 mm i.d., particle size 1.7 μm; Waters). In addition, to determine the effect of mobile phase pH on the separation of anserine, carnosine, and candidate internal standards, as well as the stability of the column, analyses were performed at pHs of 3, 4, 5, 6, 8, and 9.

[0088] Additionally, the mobile phase composition was adjusted considering the pH range of buffer solutions. For the low pH mobile phase, the pH was adjusted to 3 and 4 by adding formic acid to 10 mM ammonium formate, and to 5 and 6 by adding acetic acid to 10 mM ammonium acetate. The high pH mobile phases (pH 8 and pH 9) consisted of 10 mM ammonium bicarbonate, and the pH was adjusted using ammonium hydroxide solution. All aqueous mobile phases were adjusted to the appropriate pH. Chromatographic separations were performed using 70%, 75%, and 80% acetronitrile.

[0089]

[0090] 1-2. Selection of internal standard material

[0091] Internal standards are used to improve accuracy and precision by compensating for analytical variations that occur during extraction, and therefore, internal standards that exhibit optimal efficiency were selected. Table 2 shows the chemical properties, such as log P and pKa, of phenylephrine hydrochloride (PEH), histidylleucine, 4-imidazoleacrylic acid, and L-norvaline (Nor) prepared in Preparation Example 2. The suitability of internal standards was evaluated based on chemical similarity, stability under analytical conditions, compatibility with the sample matrix, and presence in the sample.

[0092] Compound Molecular weight (g / mol) Log PpKa Ion mode SRM transition Collision energy (V) Precursor ion (m / z) Product ion (m / z) Anserine 24 0.26-5.0 6 3.42, 6.36, 9.12 Positive 24 1.110 9.123 17 0.116 Negative 23 9.116 8.113 16 6.114 Carnosine 22 6.23-4.8 13.36, 6.55, 9.13 Positive 227.111 0.12 12 10.110 Negative 225.115 4.113 110.119 Phenylehrin hydrochloride (PEH) 203.67-0.08 9.07, 9.67Positive168.1150.11091.121Negative―――――Histidyl-leucine(His-Leu)268.31-3.253.61, 6.60, 7.74Positive269.2110.12383.140Negative267.2130.117223.1124-imidazoleacrylic acid(4-IAA)138.12-0.984.68, 6.15Positive139.11211493.123Negative137.093.11266.022L-Norvaline (L-Nor)117.15-2.492.71, 9.53Positive118.172.11030.020Negative―――――

[0093]

[0094] 1-3. Verification of analysis method

[0095] The analytical method was verified for linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision according to the ISO / IEC guidelines. Linearity was confirmed by performing seven replicate measurements against a calibration curve of anserine and carnosine in the concentration range of 0.005 to 100 μg / mL. The LOD and LOQ were calculated using the standard deviation of the responses obtained from the seven replicate experiments at the lowest concentration and the slope of the calibration curve. Accuracy and precision were determined by performing nine replicate measurements with samples spiked with different concentrations (low, medium, and high) of anserine and carnosine. Precision was expressed as the percentage relative standard deviation (RSD, %).

[0096]

[0097] 1-4. Data Analysis

[0098] To assess statistical significance, Student's t-tests were performed using the SPSS program (IBM Corp., Armonk, NY, USA). The suitability of anserine, carnosine, and the anserine / carnosine ratio as candidate biomarkers was assessed through receiver operating characteristic (ROC) curve analysis. This analysis was performed using MetaboAnalyst 5.0 (https: / www.metaboanalyst.ca), and the discriminatory performance between domestic and Japanese red sea bream was determined by area under the curve (AUC).

[0099]

[0100] <Example 2> UHPLC-MS / MS analysis results

[0101] 2-1. Optimization of Separation Conditions for Anserine and Carnosine

[0102] The chromatographic separation conditions were optimized according to the above Example 1-1. The evaluation for optimizing the separation conditions was repeated five times, and the analysis was performed in the positive ion mode. Figures 2 to 5 show the chromatographic separation according to the column and pH in the positive ion mode. Figures 2 and 4 show the chromatographic separation according to the use of the BEH HILIC column, and Figures 3 and 5 show the chromatographic separation according to the use of the BEH amide column. According to the results using the HILIC column, the retention time of anserine was measured to be longer than that of carnosine at both low and high pH. On the other hand, when the BEH amide column was used, the retention time of carnosine was measured to be longer than that of anserine at high pH conditions, but anserine and carnosine were simultaneously detected and not separated at low pH conditions.

[0103] Additionally, the retention times of all candidate internal standards were measured to be smaller than those of anserine and carnosine.

[0104] Based on the above results, a BEH HILIC column capable of clearly separating anserine and carnosine under both low and high pH conditions was used for UHPLC-MS / MS analysis.

[0105]

[0106] To determine the optimal separation conditions for the HILIC column, chromatographic separation was performed by adjusting the ratio of organic and aqueous solvents and pH. Figures 6 and 7 show the ionization efficiency according to the pH of the mobile phase. In the positive ion mode, the intensities of anserine and carnosine varied depending on the pH of the ammonium solution. The highest intensities were observed at pH 8 and 9 containing 10 mM ammonium nitrate, followed by pH 5 and 6 containing 10 mM ammonium acetate, and then pH 3 and 4 containing 10 mM ammonium formate.

[0107] Additionally, in positive ion mode, all candidate internal standards except PEH showed increased intensities with increasing pH levels. However, in negative ion mode, PEH and Nor were not detected, while the other candidate internal standards were detected but showed higher intensities in positive ion mode.

[0108] The above results show that most compounds exhibited increased strength at higher pH in the positive ion mode. However, the precision measured at high pH was lower than at low pH. Furthermore, considering the pKa values, anserine and carnosine exist in a single dissociated form at pH 5. Considering these factors, pH 5 was confirmed to be the most suitable for analysis, and subsequent analyses were performed with the mobile phase pH adjusted to 5.

[0109]

[0110] The organic solvent ratio was evaluated under conditions of 70%, 75%, and 80% acetonitrile. The results of the analysis are shown in Figures 8 to 10. As the acetonitrile ratio increased, anserine and carnosine were clearly separated. However, the retention time was found to be longer, which increased the analysis time. Therefore, considering peak stability, separation of anserine and carnosine, and pKa comprehensively, it was confirmed that using 75% acetonitrile at pH 5 as the organic solvent was the most suitable. The final determined analysis method is shown in Table 3 below.

[0111] ColumnACQUITY UPLC BEH HILIC Column (130Å, 1.7 μm, 2.1 mm acid)(*pH is based on water)TimeMobile phase AMobile phase B0.0000.0100.010.0000.0100.010.100100.00.015.000100.00.015.1000.0100.025.0000.0100.0Flow rate0.2 mL / minInjection Volume2 μLColumn Oven35℃Ion modePositiveSprayVoltage3,500 VIon transfer tube Temp350℃Vaporizer Temp275℃

[0112]

[0113] 2-2. Selection of internal standard material

[0114] Internal standards were selected from the compounds of Examples 1-2 above based on chemical similarity, stability under analytical conditions, compatibility with the sample matrix, and absence in the sample. Because 4-IAA and Nor were present in the red sea bream sample, these two compounds were excluded from the internal standard candidates. Furthermore, His-Leu exhibited a broad peak, resulting in poor resolution, and was therefore excluded from the candidate material. PEH exhibited a sharp peak shape and was not present in the red sea bream sample, so PEH was selected as the internal standard.

[0115]

[0116] 2-3. Verification of analysis method

[0117] The accuracy, precision, and sensitivity of the UHPLC-MS / MS method were validated according to the conditions selected in Examples 2-1 and 2-2. The validation results are shown in Table 4 below.

[0118] Compound Detection Limit (mg / 100g) (n=7) Quantitation Limit (mg / 100g) (n=7) Linear Range (μg / mL) Regression Equation (y=area, x=μg / mL) R² Spiking Level (mg / g) Recovery (%) (n=9) RSD (%) (n=9) Anserine 0.177 0.538 0.005-100 y = 540,140x - 687,087 0.99 59 Low 0.60 10 2.26.7 Middle 2.00 10 0.12.8 High 6.00 10 0.73.2 Carnosine 0.012 0.036 0.005-100 y = 740,560x - 575,4880.9987Low0.0199.39.7Middle0.03102.55.2High0.10101.11.9

[0119]

[0120] The limit of detection (LOD) and limit of quantification (LOQ) were determined using the standard deviation and the slope of the regression equation. The LOD and LOQ of anserine were 0.177 mg / 100 g and 0.538 mg / 100 g, respectively, and those of carnosine were 0.012 mg / 100 g and 0.036 mg / 100 g, respectively. The standard solution concentrations of anserine and carnosine ranged from 0.005 to 100 μg / mL, and the R² value was 0.9959 for anserine and 0.9987 for carnosine.

[0121] Additionally, recovery experiments were conducted to evaluate accuracy and precision. Accuracy and precision were measured by adding three levels of standard mixtures (low, medium, and high) to homogenized red sea bream. The experiments were repeated three times to determine daily variations. A recovery rate within the range of 80 to 120% was considered acceptable. The recovery rates at low, medium, and high addition levels ranged from 99.3 to 102.5%. Precision (RSD) was less than 9.7% for all three addition levels.

[0122]

[0123] 2-4. Quantitative analysis results

[0124] Quantitative analysis was performed on 90 domestic and 90 Japanese red sea bream samples each using the analytical methods according to the conditions of Examples 2-1 and 2-2. To determine seasonal content changes, quantitative analysis was performed on red sea bream samples collected in spring (n=30), summer (n=30), fall (n=15), and winter (n=15).

[0125] The results of UHPLC-MS / MS analysis are shown in Fig. 11. The retention time of phenylephrine hydrochloride was 2 minutes 31 seconds, that of carnosine was 6 minutes 31 seconds, and that of anserine was 8 minutes 8 seconds. Through the above results, it was confirmed that anserine and carnosine in red sea bream could be detected separately under the conditions of Example 1-1. In addition, unlike the existing analysis method that took more than 1 hour, the retention time of anserine was significantly shortened to 8 minutes 8 seconds, confirming that anserine detection was possible quickly. In addition, the contents of anserine and carnosine were confirmed based on the peak areas of Fig. 11. The contents of anserine and carnosine in red sea bream samples are shown in Tables 5 and 6 below.

[0126] Anserine content (mg / 100g) Spring Summer Fall Winter Korean Japanese Korean Japanese Korean Japanese Korean Japanese 116.9 ± 2.5 3 5 4.0 ± 40.7 1 9 1.2 ± 3.0 2 8 0.6 ± 12.8 4 4.6 4 3.0 ± 40.3 1 9.3 ± 2.0 3 3.2 ± 27.2 2 5.2 ± 3.1 4 0 1.4 ± 57.3 1 0 8.6 ± 2.7 2 7 1.0 ± 23.2 5 9.3 ± 8.9 5 4 6.6 ± 51.0 1 9.6 ± 2.3 2 8 2.0 ± 45.2 3 7.6 ± 2.5 2 9 2.3 ± 11.5 2 1 9.9 ± 6.3 2 3 5.7 ± 15.038.7 ± 1.8435.9 ± 6.119.5 ± 1.6174.4 ± 2.8418.4 ± 2.4367.4 ± 11.1230.4 ± 6.4295.7 ± 18.865.3 ± 2.3340.1 ± 15.217.1 ± 1.5221.5 ± 10.1520.4 ± 2.7392.8 ± 18.3221.0 ± 10.8307.5 ± 10.645.9 ± 6.2288.1 ± 7.016.7 ± 2.5237.5 ± 6.4663.2 ± 9.0364.7 ± 17.7201.5 ± 8.8285.2 ± 10.373.7 ± 2.9246.3 ± 20.318.2 ± 2.2228.2 ± 8.877.0 ± 2.0284.3 ± 14.3111.3 ± 7.1286.7 ± 16.974.0 ± 3.7247.2 ± 8.218.4 ± 2.5261.6 ± 19.2852.1 ± 4.8330.1 ± 27.8223.3 ± 9.2241.9 ± 11.472.1 ± 8.2271.9 ± 27.214.2 ± 2.6171.1 ± 19.398.9 ± 1.1332.2 ± 28.1233.5 ± 9.9295.2 ± 7.062.0 ± 7.2281.3 ± 26.316.7 ± 1.5244.7 ± 27.21021.5 ± 2.9219.3 ± 24.6225.4 ± 12.5301.6 ± 18.172.1 ± 8.5290.7 ± 27.017.5 ± 1.6250.7 ± 21.31178.5 ± 8.6258.7 ± 31.367.2 ± 6.3547.1 ± 41.429.9 ± 1.8421.2 ± 18.2115.9 ± 1.7417.7 ± 26.81264.4 ± 3.6215.2 ± 30.8107.6 ± 12.6481.7 ± 40.557.3 ± 0.4460.9 ± 25.115.3 ± 1.6336.0 ± 15.41351.1 ± 1.6238.8 ± 12.0104.6 ± 1.2557.9 ± 9.971.1 ± 1.4472.1 ± 17.439.0 ± 1.5421.8 ± 20.51461.3 ± 5.7255.6 ± 17.361.0 ± 2.7435.4 ± 10.848.7 ± 1.0259.6 ± 13.929.1 ± 1.6414.2 ± 15.51563.1 ± 2.1177.8 ± 13.289.4 ± 7.8613.7 ± 8.140.9 ± 2.4466.7 ± 27.041.4 ± 1.9450.7 ± 21.91629.1 ± 2.5336.8 ± 10.6122.5 ± 14.3454.0 ± 30.61744.4 ± 2.9391.4 ± 14.9116.2 ± 9.6480.3 ± 50.01822.3 ± 2.5393.7 ± 11.426.6 ± 0.3499.6 ± 45.21927.1 ± 3.2235.1 ± 9.9129.7 ± 16.9476.9 ± 47.520168.8 ± 8.6264.8 ± 10.068.1 ± 5.2595.3 ± 58.22119.1 ± 0.7478.3 ± 17.5104.0 ± 1.8363.9 ± 23.02230.3 ± 1.4389.6 ± 23.697.6 ± 6.9392.1 ± 8.52359.3 ± 1.4532.1 ± 26.7116.1 ± 5.9454.2 ± 7.92436.8 ± 2.0563.2 ± 46.1115.5 ± 5.9371.4 ± 9.92555.22.1457.0 ± 22.8167.8 ± 3.3327.2 ± 5.42646.0 ± 1.0434.8 ± 26.8202.1 ± 17.5508.5 ± 22.22728.7 ± 0.8398.5 ± 18.8136.6 ± 2.4417.4 ± 18.22829.8 ± 1.0436.3 ± 33.4119.3 ± 2.9378.2 ± 6.52922.2 ± 1.7407.4 ± 26.0102.5 ± 2.7373.1 ± 6.13042.3 ± 1.0273.4 ± 14.4150.5 ± 5.3449.5 ± 13.2average41.7349.2139.0399.357.0364.827.9289.7.

[0127]

[0128] Carnosine content (mg / 100g) Spring Summer Fall Winter Korean Japanese Korean Japanese Korean Japanese Korean Japanese Korean Japanese 10.08 ± 0.02 4.36 ± 0.84 50.42 ± 3.6 3.62 ± 0.21 2.20 ± 0.15 2.73 ± 0.06 0.24 ± 0.01 1.97 ± 0.43 20.94 ± 0.18 3.99 ± 0.64 3.83 ± 0.12 3.46 ± 0.35 2.05 ± 0.43 3.04 ± 0.28 0.23 ± 0.03 3.63 ± 0.69 31.70 ± 0.09 3.18 ± 0.22 39.01 ± 2.58 2.07 ± 0.16 1.07 ± 0.132.37 ± 0.120.21 ± 0.011.67 ± 0.1040.54 ± 0.083.13 ± 0.1460.77 ± 3.563.29 ± 0.331.13 ± 0.031.99 ± 0.050.20 ± 0.042.01 ± 0.0450.62 ± 0.033.30 ± 0.1866.58 ± 4.604.42 ± 0.220.33 ± 0.031.73 ± 0.150.16 ± 0.032.62 ± 0.2960.60 ± 0.074.81 ± 0.4550.72 ± 4.293.38 ± 0.385.62 ± 0.244.07 ± 0.340.22 ± 0.031.69 ± 0.0670.16 ± 0.033.65 ± 0.583.45 ± 0.413.59 ± 0.376.25 ± 0.615.23 ± 0.300.23 ± 0.013.44 ± 0.0480.59 ± 0.062.89 ± 0.1638.20 ± 2.821.87 ± 0.392.87 ± 0.155.20 ± 0.270.20 ± 0.011.79 ± 0.0890.08 ± 0.013.43 ± 0.4158.80 ± 4.862.98 ± 0.342.92 ± 0.484.41 ± 0.190.21 ± 0.011.96 ± 0.32100.08 ± 0.011.80 ± 0.2263.68 ± 6.164.31 ± 0.624.15 ± 0.374.71 ± 0.140.21 ± 0.022.42 ± 0.06117.79 ± 0.481.88 ± 0.180.64 ± 0.083.64 ± 0.330.51 ± 0.053.24 ± 0.176.76 ± 0.307.18 ± 0.331214.01 ± 1.531.85 ± 0.191.18 ± 0.183.20 ± 0.141.31 ± 0.073.10 ± 0.340.60 ± 0.055.39 ± 0.141312.18 ± 1.291.74 ± 0.262.71 ± 0.133.13 ± 0.052.66 ± 0.186.59 ± 0.172.16 ± 0.024.42 ± 0.231412.89 ± 1.731.79 ± 0.120.60 ± 0.092.61 ± 0.040.46 ± 0.043.12 ± 0.241.22 ± 0.194.85 ± 0.11154.49 ± 0.311.34 ± 0.110.84 ± 0.085.36 ± 0.590.99 ± 0.073.22 ± 0.241.53 ± 0.123.23 ± 0.22161.25 ± 0.064.25 ± 0.331.75 ± 0.173.45 ± 0.52171.87 ± 0.054.94 ± 0.251.78 ± 0.143.85 ± 0.05180.73 ± 0.034.95 ± 0.170.44 ± 0.024.66 ± 0.18190.82 ± 0.052.78 ± 0.122.15 ± 0.322.96 ± 0.41202.03 ± 0.062.89 ± 0.180.53 ± 0.063.23 ± 0.42210.58 ± 0.098.71 ± 0.745.68 ± 0.233.54 ± 0.17220.82 ± 0.088.17 ± 0.816.43 ± 0.263.23 ± 0.13231.26 ± 0.1210.27 ± 0.917.14 ± 0.323.37 ± 0.05240.77 ± 0.0712.93 ± 1.3210.01 ± 0.701.88 ± 0.24251.01 ± 0.099.37 ± 0.571.90 ± 0.043.44 ± 0.16260.30 ± 0.078.87 ± 0.692.43 ± 0.303.88 ± 0.13270.55 ± 0.088.53 ± 0.663.27 ± 0.023.19 ± 0.09280.66 ± 0.127.01 ± 0.706.96 ± 0.163.61 ± 0.09290.29 ± 0.059.66 ± 1.087.35 ± 0.092.72 ± 0.13300.88 ± 0.053.46 ± 0.269.97 ± 0.483.86 ± 0.14Average2.355.0016.973.392.303.650.963.22.

[0129]

[0130] The average anserine content of domestic red sea bream was 41.7 mg / 100 g in spring, 139.0 mg / 100 g in summer, 57.0 mg / 100 g in fall, and 27.9 mg / 100 g in winter, while the average anserine content of Japanese red sea bream was 349.2 mg / 100 g in summer, 399.3 mg / 100 g in fall, and 289.7 mg / 100 g in winter. A t-test was performed to compare the average anserine content of domestic and Japanese red sea breams. The results are shown in Figure 24. It was confirmed that the anserine content of Japanese red sea bream was significantly higher than that of domestic red sea bream across all seasons (p< 0.0001).

[0131] The average carnosine content of domestic red sea bream was 2.35 mg / 100 g in spring, 16.97 mg / 100 g in summer, 2.30 mg / 100 g in fall, and 0.96 mg / 100 g in winter, while the average carnosine content of Japanese red sea bream was 5.00 mg / 100 g in spring, 3.39 mg / 100 g in summer, 3.65 mg / 100 g in fall, and 3.22 mg / 100 g in winter. The carnosine content of Japanese red sea breams collected in spring, fall, and winter was confirmed to be higher than that of domestic red sea breams. However, in red sea breams collected in summer, the carnosine content of domestic red sea breams was higher than that of Japanese red sea breams. A t-test was performed to compare the average carnosine content of domestic and Japanese red sea breams. The performance results are shown in Figure 25. When samples from all seasons were analyzed together, no significant difference was found between the two groups, and the p-value was greater than 0.05.

[0132] The above results confirmed that anserine can be used to distinguish between domestic and Japanese red sea bream, and that carnosine is not suitable for determining the country of origin.

[0133]

[0134] 2-5. Receiver operating characteristic (ROC) curve analysis

[0135] Receiver operating characteristic (ROC) curve analysis was performed based on the concentration of anserine, the concentration of carnosine, the ratio of anserine and carnosine, and the country of origin calculated through Example 2-4. The results of the ROC curve analysis are shown in Figures 12 to 26.

[0136] Figures 12 to 15 show the results of ROC curve analysis for anserine in red sea bream by season. Examining the results of seasonal ROC curve analysis, the AUC values ​​for all ROC curves were 1, confirming that the discrimination criteria were reliable. For red sea breams collected in spring, 173 mg / 100 g was the discrimination criteria for domestic and Japanese red sea breams, for red sea breams collected in summer, 235 mg / 100 g was the discrimination criteria for domestic and Japanese red sea breams, for red sea breams collected in fall, 160 mg / 100 g was the discrimination criteria for domestic and Japanese red sea breams, and for red sea breams collected in winter, 144 mg / 100 g was the discrimination criteria for domestic and Japanese red sea breams.

[0137] The results of the ROC curve analysis for anserine in all red sea breams are shown in Figure 24. 227 mg / 100 g was found to be the discrimination criterion for domestic and Japanese red sea breams, and the AUC value was 0.995, confirming the reliability of the discrimination criterion.

[0138]

[0139] Additionally, Figures 16 to 19 show the results of ROC curve analysis for carnosine in red sea bream by season.

[0140] The AUC values ​​were 0.569 to 0.924, confirming that the reliability was lower than when anserine was used as the discrimination standard. In the case of red sea bream collected in spring, 1.72 mg / 100g was shown to be the discrimination standard for domestic and Japanese red sea bream, in the case of red sea bream collected in summer, 5.52 mg / 100g was shown to be the discrimination standard for domestic and Japanese red sea bream, in the case of red sea bream collected in fall, 2.98 mg / 100g was shown to be the discrimination standard for domestic and Japanese red sea bream, and in the case of red sea bream collected in winter, 1.6 mg / 100g was shown to be the discrimination standard for domestic and Japanese red sea bream.

[0141] The results of the ROC curve analysis for carnosine in all red sea breams are shown in Figure 25, and 2.72 mg / 100g was found to be the discrimination standard for domestic and Japanese red sea breams.

[0142]

[0143] In addition, Figures 20 to 23 show the results of ROC curve analysis on the ratio of anserine and carnosine in red sea bream by season.

[0144] The AUC values ​​were 0.776 to 0.93, confirming that the reliability was lower than when anserine was used as the discrimination standard. In the case of red sea bream collected in spring, 54.7 was shown to be the discrimination standard for domestic and Japanese red sea bream, in the case of red sea bream collected in summer, 73.8 was shown to be the discrimination standard for domestic and Japanese red sea bream, in the case of red sea bream collected in fall, 59.9 was shown to be the discrimination standard for domestic and Japanese red sea bream, and in the case of red sea bream collected in winter, 85.3 was shown to be the discrimination standard for domestic and Japanese red sea bream.

[0145] The results of ROC curve analysis for the ratio of anserine and carnosine in all red sea breams are shown in Figure 26, and 61.2 was found to be the discrimination standard for domestic and Japanese red sea breams.

[0146]

[0147] Through the above results, it was confirmed that anserine had significantly higher reliability when used as a criterion for determining the origin of red sea bream compared to when the ratio of carnosine and anserine / carnosine was used as a criterion for determining the origin of red sea bream. Therefore, it was confirmed that anserine can be used as a biomarker for determining the origin of red sea bream.

[0148]

[0149] <Example 3> Determining the origin of red sea bream

[0150] Twenty samples were randomly selected from among the red sea bream samples, and the country of origin was determined based on the anserine content (227 mg / 100 g). The results are shown in Table 7 below.

[0151] Sample Red sea bream 1Red sea bream 2Red sea bream 3Red sea bream 4Red sea bream 5Identification rateAnserine (mg / 100 g)354.03 ± 40.69401.44 ± 57.34367.36 ± 11.08284.26 ± 14.35481.70 ± 40.53100%EstimatedFrom JapanFrom JapanFrom JapanFrom JapanIdentification○○○○○Sample Red sea bream 6Red sea bream 7Red sea bream 8Red sea bream 9Red sea bream 10Anserine (mg / 100 g)613.67 ± 8.11499.63 ± 45.22546.55 ± 51.01288.06 ± 7.05290.67 ± 27.02 Estimated from Japan, ... 2.64 Estimated domestic domestic domestic domestic domestic domestic domestic domestic determination ○○○○○

[0152]

[0153] When the anserine content in red sea bream was set at 227 mg / 100 g to determine the origin of each red sea bream, the origin of all red sea breams could be accurately determined, resulting in a 100% identification rate. These results confirmed that anserine can be used as a biomarker for determining the origin of red sea bream.

[0154]

[0155] Through the results of the above examples, it was confirmed that the analysis method of the present invention, unlike existing analysis methods, can detect anserine and carnosine separately, is accurate, and significantly shortens the analysis time, and that anserine can be used as a biomarker for determining the origin of red sea bream.

Claims

1. A biomarker composition for determining the origin of red sea bream (Pagrus major) containing anserine as an active ingredient.

2. In paragraph 1, A composition wherein the above origin is domestic or Japanese.

3. In paragraph 1, A composition characterized in that the above anserine concentration is lower in domestic red sea bream than in Japanese red sea bream.

4. A kit for determining the origin of red sea bream, including a preparation for measuring the concentration of anserine in red sea bream. 5.1) Step of measuring the concentration of anserine in red sea bream; and 2) A method for determining the origin of red sea bream, including a step of comparing the concentration of anserine measured in step 1) with a reference value to determine the origin.

6. In paragraph 5, A method of determining the concentration of the above anserine, wherein the step of measuring the concentration of the above anserine uses an ultra-high performance liquid chromatography-tandem mass spectrometer.

7. In paragraph 5, A method for determining the origin of the product, characterized in that the step of determining the origin of the product is such that when the measured concentration of anserine exceeds 227 mg / 100g, the product is determined to be from Japan, and when it is less than 227 mg / 100g, the product is determined to be from Korea.

8. In paragraph 5, A method for determining the origin of the red sea bream, characterized in that when the red sea bream is collected from March to May, the step of determining the origin is such that when the measured concentration of anserine exceeds 173 mg / 100g, it is determined to be from Japan, and when it is 173 mg / 100g or less, it is determined to be from Korea.

9. In paragraph 5, A method for determining the origin of the red sea bream, characterized in that when the red sea bream is collected from June to August, the step of determining the origin is such that when the measured concentration of anserine exceeds 235 mg / 100g, it is determined to be from Japan, and when it is less than 235 mg / 100g, it is determined to be from Korea.

10. In paragraph 5, A method for determining the origin of the red sea bream, characterized in that when the red sea bream is collected from September to November, the step of determining the origin is such that when the measured concentration of anserine exceeds 160 mg / 100g, it is determined to be from Japan, and when it is 160 mg / 100g or less, it is determined to be from Korea.

11. In paragraph 5, A method for determining the origin of the red sea bream, characterized in that when the red sea bream is collected in December to February, the step of determining the origin is to determine that it is from Japan when the measured concentration of anserine exceeds 144 mg / 100g, and to determine that it is from Korea when it is 144 mg / 100g or less. 12.1) Step of preparing a standard solution including anserine and carnosine as standard substances, and preparing an internal standard solution including an internal standard substance; 2) Step of preprocessing red sea bream samples; 3) A step of obtaining a chromatogram of a pretreated red sea bream sample using an ultra-high performance liquid chromatography-tandem mass spectrometer; and 4) A method for quantitative analysis of anserine, a biomarker, comprising: a step of measuring the content of anserine through the peak and area of ​​the obtained chromatogram.

13. In paragraph 12, A method wherein the internal standard substance is phenylephrine hydrochloride.

14. In paragraph 12, The above ultra-high performance liquid chromatography-tandem mass spectrometer uses a BEH HILIC column, uses a 50% acetonitrile and 10 mM ammonium acetate mixture solution as mobile phase A, and uses a 75% acetonitrile and 10 mM ammonium acetate mixture solution as mobile phase B, the pH of mobile phases A and B is 5 in the aqueous phase, the mobile phase flow rate is 0.2 mL / min, the injection volume is 2 μL, the temperature of the column oven is 35°C, the ion mode is positive mode, the spray voltage is 3500 V, the temperature of the ion transfer tube is 350°C, and the temperature of the vaporizer is 275°C.

15. In paragraph 14, A method characterized in that the gradient elution of the above mobile phase A and mobile phase B is performed under the following mobile phase concentration gradient conditions. - Start ~ 10 minutes - Mobile phase A: 0%, Mobile phase B: 100% - 10.1 to 15 minutes: Mobile phase A: 100%, Mobile phase B: 0% - 15.1 min ~ 25 min: Mobile phase A: 0%, Mobile phase B: 100%

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