Detection method of black-bone chicken melanin oxidation product

Abstract

The invention provides a detection method of black-bone chicken melanin oxidation products, and belongs to the technical field of chemical detection. The detection conditions of liquid chromatography in the detection method provided by the invention are as follows: the sample size is 0.9-1 [mu] L, a mobile phase A is a formic acid aqueous solution with the volume fraction of 0.05-0.15%, a mobile phase B is methanol, gradient elution is adopted, the detection time is 1-2 hours, and the detection wavelength is 250nm. The gradient elution conditions are as follows: the volume percentage content of the mobile phase B in 0-3 min is 5%-25%, the volume percentage content of the mobile phase B in 3-4 min is 25%-95%, the volume percentage content of the mobile phase B in 4-5.5 min is 95% B, the volume percentage content of the mobile phase B in 5.5-6 min is 95%-5%, and the volume percentage content of the mobile phase B in 6-8 min is 5% B. According to the present invention, the ultra-high performance liquid chromatography-mass spectrometry detection conditions are controlled, such that the detection accuracy is improved, the sample injection amount is reduced, the elution time is shortened, and the time and the sample consumption are saved.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a method for detecting black-bone chicken melanin oxidation products. Background Art

[0002] Black-bone chicken melanin has a complex structure, primarily composed of eumelanin, a biopolymer insoluble in water and almost all solvents. Eumelanin is primarily composed of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxy-2-indolecarboxylic acid (DHICA), which, upon oxidation by strong oxidants, form 2,3-pyrrole dicarboxylate (PDCA) and 2,3,5-tricarboxypyrrole (PTCA), respectively. The melanin content can be calculated by measuring the levels of the melanin oxidation products, PDCA and PTCA. Strict control of the oxidation process is crucial for calculating melanin content. The K2CO3-H2O2 method is prone to bubbling and overflow during operation, leading to inaccurate test results. Liquid chromatography is commonly used to detect melanin oxidation products, but this requires a large injection volume of typically 20 μL and a long elution time of over 30 minutes. Therefore, improving detection accuracy while shortening detection time and reducing sample usage presents a challenge in the existing technology. Summary of the Invention

[0003] The present invention aims to provide a method for detecting oxidation products of black-bone chicken melanin. The detection method provided by the present invention improves detection accuracy while requiring a small sample volume and shortening detection time.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for detecting black-bone chicken melanin oxidation products, comprising the following steps:

[0006] (1) mixing black-bone chicken powder with acid solution and extracting to obtain crude black-bone chicken melanin;

[0007] (2) mixing the crude black chicken melanin obtained in step (1) with a potassium carbonate solution and hydrogen peroxide, performing an oxidation reaction, then adding an acid and an extractant, sequentially performing extraction, nitrogen blowing, and redissolution to obtain a test solution; the volume ratio of the crude black chicken melanin to the volume of the potassium carbonate solution and the volume of the hydrogen peroxide is 10 mg: (8-9) mL: (0.5-1) mL;

[0008] (3) The test solution obtained in the step (2) is subjected to ultra-high performance liquid chromatography-mass spectrometry to obtain the content of black-bone chicken melanin oxidation products; the detection conditions of the liquid chromatography are: the injection volume is 0.9-1 μL, the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.15%, the mobile phase B is methanol, and gradient elution is adopted, and the gradient elution conditions are: the volume percentage of the mobile phase B is from 5% to 25% in 0-3 min, the volume percentage of the mobile phase B is from 25% to 95% in 3-4 min, 95% B in 4-5.5 min, the volume percentage of the mobile phase B is from 95% to 5% in 5.5-6 min, and 5% B in 6-8 min; the column temperature is 35-45° C., the flow rate is 0.3-0.5 mL / min, and the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column, 2.1 mm×100 mm, 1.7 μm;

[0009] The mass spectrometry detection conditions are as follows: electrospray ion source, multiple reaction monitoring mode, negative ion detection mode, ionization temperature of 500-600°C, spray voltage of 5000-6000V, curtain gas volume flow rate of 30-50L / min, nebulizing gas volume flow rate of 50-60L / min, and heating auxiliary gas volume flow rate of 50-60L / min.

[0010] Preferably, the acid solution in step (1) is hydrochloric acid; the mass concentration of the acid solution is 20-30%.

[0011] Preferably, in step (1), the volume ratio of the black-bone chicken powder to the acid solution is 100 g: (200-400) mL.

[0012] Preferably, the extraction temperature in step (1) is 95-105° C., and the extraction time is 4-6 hours.

[0013] Preferably, the concentration of the potassium carbonate solution in step (2) is 0.5-1.5 mol / L, and the volume concentration of the hydrogen peroxide is 25-35%.

[0014] Preferably, the temperature of the oxidation reaction in step (2) is 90-110° C., and the time of the oxidation reaction is 10-30 minutes.

[0015] Preferably, the extractant in step (2) is methyl tert-butyl ether.

[0016] The present invention provides a method for detecting oxidation products of black-bone chicken melanin, comprising the following steps: (1) mixing black-bone chicken powder with an acid solution and performing extraction to obtain a crude black-bone chicken melanin; (2) mixing the crude black-bone chicken melanin obtained in step (1) with a potassium carbonate solution and hydrogen peroxide, performing an oxidation reaction, then adding an acid and an extractant, performing extraction, nitrogen blowing and redissolution in sequence to obtain a test solution; the ratio of the mass of the crude black-bone chicken melanin to the volume of the potassium carbonate solution and the volume of the hydrogen peroxide is 10 mg: (8-9) mL: (0.5-1) mL; (3) The test solution obtained in the step (2) is subjected to ultra-high performance liquid chromatography-mass spectrometry to obtain the content of black-bone chicken melanin oxidation products; the detection conditions of the liquid chromatography are: the injection volume is 0.9-1 μL, the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.15%, the mobile phase B is methanol, and gradient elution is adopted, and the gradient elution conditions are: the volume percentage of the mobile phase B is from 5% to 25% in 0-3 min, the volume percentage of the mobile phase B is from 25% to 95% in 3-4 min, and the volume percentage of the mobile phase B is from 4-5.5 min. The volume percentage of mobile phase B is reduced from 95% to 5% at 95% B for 5.5 to 6 minutes and then to 5% B for 6 to 8 minutes; the column temperature is 35 to 45°C, the flow rate is 0.3 to 0.5 mL / min, and the chromatographic column is a Waters ACQUITY UPLC BEHC18 column, 2.1 mm × 100 mm, 1.7 μm; the mass spectrometry detection conditions are: electrospray ionization source, multiple reaction monitoring mode, negative ion detection mode, ionization temperature of 500 to 600°C, spray voltage of 5000 to 6000 V, curtain gas volume flow rate of 30 to 50 L / min, nebulizing gas volume flow rate of 50 to 60 L / min, and heating auxiliary gas volume flow rate of 50 to 60 L / min. The present invention controls the detection conditions during ultra-high performance liquid chromatography-mass spectrometry detection, thereby improving detection accuracy while reducing the injection volume, shortening the elution time, saving time and sample consumption, overcoming the shortcomings of existing HPLC methods such as low sensitivity and long analysis time, and achieving rapid and accurate detection of trace oxidation products through the combination of UPLC-MS / MS. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 1 is the EIC graph of the black-bone chicken melanin oxidation product, PDCA standard, PTCA standard and standard solution in Example 1;

[0018] Figure 2 The EIC graphs of PTCA in black-bone chicken melanin oxidation products, PTCA standard, PTCA in standard solution, and blank solution in Example 1 are shown;

[0019] Figure 3 This is the EIC graph of PDCA in the black-bone chicken melanin oxidation product, PDCA standard, PDCA in the standard solution and blank solution in Example 1. DETAILED DESCRIPTION

[0020] The present invention provides a method for detecting black-bone chicken melanin oxidation products, comprising the following steps:

[0021] (1) mixing black-bone chicken powder with acid solution and extracting to obtain crude black-bone chicken melanin;

[0022] (2) mixing the crude black chicken melanin obtained in step (1) with a potassium carbonate solution and hydrogen peroxide, performing an oxidation reaction, then adding an acid and an extractant, sequentially performing extraction, nitrogen blowing, and redissolution to obtain a test solution; the volume ratio of the crude black chicken melanin to the volume of the potassium carbonate solution and the volume of the hydrogen peroxide is 10 mg: (8-9) mL: (0.5-1) mL;

[0023] (3) The test solution obtained in the step (2) is subjected to ultra-high performance liquid chromatography-mass spectrometry to obtain the content of black-bone chicken melanin oxidation products; the detection conditions of the liquid chromatography are: the injection volume is 0.9-1 μL, the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.15%, the mobile phase B is methanol, and gradient elution is adopted, and the gradient elution conditions are: the volume percentage of the mobile phase B is from 5% to 25% in 0-3 min, the volume percentage of the mobile phase B is from 25% to 95% in 3-4 min, 95% B in 4-5.5 min, the volume percentage of the mobile phase B is from 95% to 5% in 5.5-6 min, and 5% B in 6-8 min; the column temperature is 35-45° C., the flow rate is 0.3-0.5 mL / min, and the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column, 2.1 mm×100 mm, 1.7 μm;

[0024] The mass spectrometry detection conditions are as follows: electrospray ion source, multiple reaction monitoring mode, negative ion detection mode, ionization temperature of 500-600°C, spray voltage of 5000-6000V, curtain gas volume flow rate of 30-50L / min, nebulizing gas volume flow rate of 50-60L / min, and heating auxiliary gas volume flow rate of 50-60L / min.

[0025] Unless otherwise specified, the present invention has no particular limitation on the sources of the raw materials, and commercially available products known to those skilled in the art may be used.

[0026] The invention mixes black-bone chicken powder with acid solution and performs extraction to obtain crude black-bone chicken melanin.

[0027] The present invention has no particular limitation on the source of the black-bone chicken, and commercially available black-bone chickens known to those skilled in the art can be used. In the embodiment of the present invention, the black-bone chicken is specifically Taihe black-bone chicken provided by Taihe Aoxin Black-bone Chicken Development Co., Ltd.

[0028] The method preferably comprises the following steps: slaughtering a black-bone chicken, removing the hair, head, claws and internal organs, and sequentially washing, drying and crushing the chicken to obtain the black-bone chicken powder.

[0029] The present invention has no special limitation on the operations of slaughtering, removing hair, head, claws and viscera, cleaning, drying and crushing, and the technical solutions well known to those skilled in the art can be adopted.

[0030] The present invention has no particular limitation on the particle size of the black-bone chicken powder, and any particle size well known to those skilled in the art may be used.

[0031] In the present invention, the acid solution is preferably hydrochloric acid; the mass concentration of the acid solution is preferably 20-30%, more preferably 25%.

[0032] In the present invention, the volume ratio of the black-bone chicken powder to the acid solution is preferably 100 g: (200-400) mL, more preferably 100 g: (250-350) mL, and even more preferably 100 g: 300 mL.

[0033] In the present invention, the extraction temperature is preferably 95-105° C., more preferably 100° C.; the extraction time is preferably 4-6 h, more preferably 5 h.

[0034] The present invention controls various parameters in the extraction process within the above ranges, thereby enabling sufficient extraction of black-bone chicken melanin and further improving detection accuracy.

[0035] After the extraction is completed, the present invention preferably cools the extracted product, centrifuges it to obtain a precipitate, washes it, and dries it in sequence to obtain a crude black-bone chicken melanin product.

[0036] The present invention has no special limitation on the cooling operation, and cooling to room temperature can be performed using cooling technical solutions well known to those skilled in the art.

[0037] In the present invention, the centrifugal speed is preferably 7000-9000 rpm, more preferably 8000 rpm; the centrifugal time is preferably 0.5-1.5 h, more preferably 1 h.

[0038] In the present invention, the washing preferably includes washing with petroleum ether, washing with acetic acid and washing with water in sequence. The present invention has no particular limitation on other washing operations, and washing to neutrality can be performed using washing techniques well known to those skilled in the art.

[0039] In the present invention, the drying temperature is preferably 60-80° C., more preferably 70° C. The present invention has no particular limitation on the drying time, and the drying time may be until constant weight is achieved.

[0040] After obtaining the crude black-bone chicken melanin, the method mixes the crude black-bone chicken melanin with a potassium carbonate solution and hydrogen peroxide to carry out an oxidation reaction, then adds an acid and an extractant, and sequentially performs extraction, nitrogen blowing and redissolution to obtain a test solution.

[0041] In the present invention, the solvent of the potassium carbonate solution is preferably water; the concentration of the potassium carbonate solution is preferably 0.5 to 1.5 mol / L, more preferably 1 mol / L.

[0042] In the present invention, the volume concentration of the hydrogen peroxide solution is preferably 25-35%, more preferably 30%.

[0043] In the present invention, the volume ratio of the crude black-bone chicken melanin to the volume of potassium carbonate solution and hydrogen peroxide is 10 mg: (8-9) mL: (0.5-1) mL, preferably 10 mg: 8.6 mL: 0.8 mL.

[0044] In the present invention, the temperature of the oxidation reaction is preferably 90 to 110° C., more preferably 100° C.; the time of the oxidation reaction is preferably 10 to 30 minutes, more preferably 20 minutes. In the present invention, during the oxidation reaction, oscillation is preferably performed once every 4 to 6 minutes, more preferably once every 5 minutes. The present invention does not specifically limit the operation of the oscillation, and any oscillation technique known to those skilled in the art can be used.

[0045] In the present invention, the oxidation reaction is preferably carried out in a container having a liquid-to-volume ratio of 1:(14-16) (the liquid-to-volume ratio is the volume ratio of the oxidation reaction system to the container). Bubbles are easily generated during the oxidation reaction. Controlling the liquid-to-volume ratio of the oxidation reaction in the present invention can prevent bubbles from overflowing, thereby improving detection accuracy.

[0046] The present invention controls the various parameters of the oxidation reaction within the above ranges, which can fully oxidize the black-bone chicken melanin and further improve the detection accuracy.

[0047] After the oxidation reaction is completed, the present invention preferably cools the product of the oxidation reaction, adds a Na2SO3 aqueous solution to terminate the reaction, obtains a post-reaction solution, and then adds an acid and an extractant.

[0048] In the present invention, the cooling is preferably performed in an ice water bath; the cooling time is preferably 2 minutes.

[0049] After cooling is completed, the present invention preferably immediately adds a Na2SO3 aqueous solution to terminate the reaction. The present invention immediately adds a Na2SO3 aqueous solution to terminate the reaction to ensure a consistent yield of degradation products, thereby improving detection accuracy.

[0050] In the present invention, the mass concentration of the Na2SO3 aqueous solution is preferably 5-15%, more preferably 10%.

[0051] In the present invention, the volume ratio of the crude black-bone chicken melanin to the Na2SO3 aqueous solution is preferably 10 mg: (0.3-0.5) mL, more preferably 10 mg: 0.4 mL.

[0052] In the present invention, the acid is preferably hydrochloric acid; the concentration of the hydrochloric acid is preferably 5 to 7 mol / L, more preferably 6 mol / L.

[0053] In the present invention, after adding the acid, the pH value of the reaction solution is preferably 1 to 1.5, more preferably 1. The present invention has no particular limitation on the amount of the acid, as long as the pH value of the reaction solution after adding the acid is within the above range.

[0054] In the present invention, the solution after adding the acid is preferably centrifuged to obtain a supernatant, and then an extractant is added.

[0055] In the present invention, the centrifugal speed is preferably 9000-10000 rpm, more preferably 9500 rpm. The present invention has no particular limitation on the centrifugal time, and the supernatant can be obtained by using a centrifugal technique well known to those skilled in the art.

[0056] In the present invention, the extractant is preferably methyl tert-butyl ether.

[0057] In the present invention, the number of extractions is preferably 3 to 5 times, more preferably 4 times; the mass ratio of the volume of the extractant to the crude black chicken melanin in each extraction is preferably (40 to 60) mL:10 mg, more preferably 50 mL:10 mg.

[0058] In the present invention, the extracts after multiple extractions are preferably combined and then nitrogen blown.

[0059] The present invention has no special limitation on the operation of nitrogen blowing, and drying can be performed using nitrogen blowing technical solutions well known to those skilled in the art.

[0060] In the present invention, the solvent for re-dissolving is preferably a methanol aqueous solution; the volume concentration of the methanol aqueous solution is preferably 45-55%, more preferably 50%.

[0061] In the present invention, the volume ratio of the crude black-bone chicken melanin to the methanol aqueous solution is preferably 10 mg: (150-250) μL, more preferably 10 mg: 200 μL.

[0062] After the re-dissolution is completed, the present invention preferably filters the re-dissolved product to obtain a test solution.

[0063] In the present invention, the filtration is preferably carried out using a 0.4-0.5 μm organic membrane, more preferably a 0.45 μm organic membrane. The present invention does not particularly limit the material of the organic membrane, and any organic membrane for filtration known to those skilled in the art can be used. The present invention controls the various parameters in the preparation process of the test solution within the above ranges, which can fully oxidize the melanin in the crude black chicken melanin product and reduce the interference of other impurities, further improving the accuracy of the detection.

[0064] After obtaining the test solution, the present invention performs ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) detection on the test solution to obtain the content of black-bone chicken melanin oxidation products.

[0065] After obtaining the test solution, the present invention immediately performs ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) detection on the test solution to avoid inaccurate test results caused by long-term storage.

[0066] In the present invention, the detection conditions of the liquid chromatography are as follows: the injection volume is 0.9-1 μL, the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.15%, the mobile phase B is methanol, and gradient elution is adopted. The gradient elution conditions are as follows: the volume percentage of the mobile phase B is from 5% to 25% in 0-3 min, the volume percentage of the mobile phase B is from 25% to 95% in 3-4 min, 95% B in 4-5.5 min, 95% B in 5.5-6 min, and 5% B in 6-8 min; the column temperature is 35-45° C., the flow rate is 0.3-0.5 mL / min, and the chromatographic column is Waters ACQUITY UPLC BEH C18 chromatographic column (2.1 mm×100 mm, 1.7 μm); preferably: injection volume is 1 μL, mobile phase A is 0.1% by volume formic acid aqueous solution, column temperature is 40° C., flow rate is 0.4 mL / min.

[0067] In the present invention, the mass spectrometry detection conditions are: electrospray ionization source, multiple reaction monitoring mode, negative ion detection mode, ionization temperature of 500-600°C, spray voltage of 5000-6000V, curtain gas volume flow rate of 30-50L / min, nebulizer gas volume flow rate of 50-60L / min, and heating auxiliary gas volume flow rate of 50-60L / min; preferably: ionization temperature of 550°C, spray voltage of 5500V, curtain gas volume flow rate of 40L / min, nebulizer gas volume flow rate of 55L / min, and heating auxiliary gas volume flow rate of 55L / min. The present invention controls the chromatographic detection conditions and mass spectrometry detection conditions within the above ranges, which can reduce the injection amount and shorten the elution time while ensuring detection accuracy.

[0068] In the present invention, during mass spectrometry detection, the retention time of 2,3-pyrrole dicarboxylate (PDCA) is preferably 1.4 to 1.5 min, more preferably 1.48 min; the parent ion is preferably 198 to 198.5 Da, more preferably 198.10 Da; the quantitative daughter ion is preferably 154 to 155 Da, more preferably 154.40 Da; the collision energy is preferably -12 to -13 V, more preferably -12.06 V; the ejection voltage is preferably -15 to -16 V, more preferably is -15.97V; the retention time of the 2,3,5-tricarboxypyrrole (PTCA) is preferably 1.7 to 1.8 min, more preferably 1.73 min; the parent ion is preferably 154 to 155 Da, more preferably 154.4 Da; the quantitative daughter ion is preferably 66 to 67 Da, more preferably 66.10 Da; the collision energy is preferably -20 to -21 V, more preferably -20.98 V; the ejection voltage is preferably -11 to -12 V, more preferably -11.28 V.

[0069] The present invention preferably uses cuttlefish melanin as the true melanin standard. Cuttlefish melanin is almost pure melanin, containing about 75 wt% DHICA-melanin and 20 wt% DHI-melanin, with the remaining 5 wt% being binding proteins.

[0070] In the present invention, the preparation method of the standard solution is preferably the same as the preparation method of the test solution, which will not be described in detail here.

[0071] The present invention preferably mixes 2,3-pyrrole dicarboxylate (PDCA) and 2,3,5-tricarboxypyrrole (PTCA) with methanol to obtain a mixed reference substance mother solution with a PDCA and PTCA concentration of 1 mg / L respectively, and then the mixed reference substance mother solution is diluted stepwise with a methanol aqueous solution with a volume concentration of 50% to obtain a series of mixed reference substance solutions with different concentrations.

[0072] In the present invention, the concentrations of PDCA and PTCA in the series of mixed reference solutions are preferably 0.01-0.05 μg / mL, 0.2-0.3 μg / mL, 2-3 μg / mL, 4-6 μg / mL, 9-11 μg / mL, 20-30 μg / mL, 45-55 μg / mL, 70-80 μg / mL and 95-105 μg / mL, respectively; more preferably 0.01 μg / mL, 0.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL and 100 μg / mL, respectively.

[0073] In the present invention, 2,3-pyrrole dicarboxylate (PDCA) and methanol are preferably mixed to obtain a PDCA standard solution with a concentration of 1000 μg / mL.

[0074] In the present invention, 2,3,5-tricarboxypyrrole (PTCA) and methanol are preferably mixed to obtain a PTCA standard solution with a concentration of 1000 μg / mL.

[0075] The present invention controls the detection conditions during ultra-high performance liquid chromatography-mass spectrometry detection, thereby improving the detection accuracy while reducing the injection volume, shortening the elution time, and saving time and sample consumption.

[0076] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0077] The methanol and formic acid aqueous solution in the examples were of chromatographic grade, and methyl tert-butyl ether, hydrochloric acid, petroleum ether and acetic acid were of analytical grade, all of which were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0078] The equipment and other reagents used in the examples are shown in Table 1.

[0079] Table 1 Equipment and reagents used in the examples

[0080]

[0081] 10 mg of sepia melanin was mixed with 8.6 mL of 1 mol / L potassium carbonate aqueous solution and 0.8 mL of 30% hydrogen peroxide in a 150 mL conical flask (liquid-to-volume ratio of 1:16) and reacted in a 100°C water bath for 20 min, with shaking every 5 min. After the reaction was completed, it was cooled in an ice-water bath for 2 min, and immediately 0.4 mL of 10% mass concentration Na2SO3 aqueous solution was added to terminate the reaction. The pH value was adjusted to 1.0 with 6 mol / L hydrochloric acid. After centrifugation at 9500 rpm for 5 min, the supernatant was taken and extracted four times with 50 mL of methyl tert-butyl ether. The four extracts were combined, blown dry with nitrogen, and then redissolved with 200 μL of 50% volume concentration methanol aqueous solution. The standard solution was filtered through a 0.45 μm organic membrane.

[0082] PDCA standard solution, 1000 μg / mL.

[0083] PTCA standard solution, 1000 μg / mL.

[0084] Blank solution: methanol aqueous solution with a volume concentration of 50%.

[0085] A series of mixed reference solutions with PDCA and PTCA concentrations of 0.01 μg / mL, 0.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL and 100 μg / mL, respectively.

[0086] Example 1

[0087] A method for detecting melanin oxidation products of black-bone chickens: (1) Taihe black-bone chickens (provided by Taihe Aoxin Black-bone Chicken Development Co., Ltd.) were slaughtered, their hair, heads, claws, and internal organs removed, cleaned, dried at 90° C., and then pulverized to obtain black-bone chicken powder;

[0088] (2) 100 mg of black-bone chicken powder was placed in a round-bottom flask, 300 mL of 25% hydrochloric acid was added, and the mixture was refluxed in a water bath at 100°C for 5 h. After cooling to room temperature, the mixture was centrifuged at 8000 rpm for 1 h. The supernatant was discarded, and the precipitate was washed with petroleum ether, acetic acid, and water in sequence, and dried at 70°C to obtain crude black-bone chicken melanin BSFM.

[0089] (3) 10 mg of cuttlefish melanin was mixed with 8.6 mL of 1 mol / L potassium carbonate aqueous solution and 0.8 mL of 30% volume concentration hydrogen peroxide in a 150 mL conical flask, and reacted in a water bath at 100 ° C for 20 min, shaking once every 5 min. After the reaction was completed, it was cooled in an ice water bath for 2 min, and immediately 0.4 mL of 10% mass concentration Na2SO3 aqueous solution was added to terminate the reaction. The pH value was adjusted to 1.0 with 6 mol / L hydrochloric acid. After centrifugation at 9500 rpm for 5 min, the supernatant was taken and extracted with methyl tert-butyl ether 4 times, each time with a volume of 50 mL. The 4 extracts were combined, blown dry with nitrogen, and then redissolved with 200 μL of 50% volume concentration methanol aqueous solution. The sample solution was filtered through a 0.45 μm organic membrane to obtain the test solution.

[0090] (4) The test solution was subjected to ultra-high performance liquid chromatography-mass spectrometry to obtain the content of black-bone chicken melanin oxidation products. The liquid chromatography conditions were as follows: chromatographic column: Waters ACQUITY UPLC BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm), mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: methanol, column temperature: 40°C, flow rate: 0.4 mL / min, injection volume: 1 μL, gradient elution was adopted, and the gradient elution conditions were as follows: 0-3 min: the volume percentage of mobile phase B was from 5% to 25%, 3-4 min: the volume percentage of mobile phase B was from 25% to 95%, 4-5.5 min: 95% B, 5.5-6 min: the volume percentage of mobile phase B was from 95% to 5%, and 6-8 min: 5% B;

[0091] The mass spectrometry conditions are as follows: mass spectrometry analysis uses an electrospray ionization source (ESI), negative ion detection mode, and multiple reaction monitoring mode for quantitative analysis, ionization temperature 550°C, spray voltage 5500 V, curtain gas volume flow rate 40 L / min, nebulizer gas volume flow rate 55 L / min, and heating auxiliary gas volume flow rate 55 L / min. Other condition parameters are shown in Table 2.

[0092] Table 2 MRM ion detection information

[0093]

[0094] The blank solution, PDCA standard solution, PTCA standard solution and standard solution were tested according to the method of Example 1.

[0095] Analyst 1.7 software was used to obtain the total ion current, secondary spectrum and retention time of the compound during detection, and the primary and secondary fragments of the components were analyzed. The results are as follows Figure 1 Example 1 EIC diagram of black chicken melanin oxidation products, PDCA standard, PTCA standard and oxidation products in standard solution Figure 1 As shown, Figure 1 Where A is the PDCA standard, B is the PTCA standard, C is the oxidation product in the standard solution, and D is the oxidation product of the black-bone chicken melanin in Example 1.

[0096] Example 1 EIC of PTCA in black-bone chicken melanin oxidation product, PTCA standard, PTCA in standard solution and blank solution Figure 2 As shown, Figure 2 A is blank solution, B is PTCA standard, C is PTCA in standard solution, and D is PTCA in the black-bone chicken melanin oxidation product of Example 1.

[0097] Example 1 EIC diagram of PDCA in black-bone chicken melanin oxidation product, PDCA standard, PDCA in standard solution and blank solution Figure 3 As shown, Figure 3 A is a blank solution, B is a PDCA standard, C is PDCA in the standard solution, and D is PDCA in the black-bone chicken melanin oxidation product of Example 1.

[0098] from Figures 1 to 3 It can be proved that the oxidation products of black chicken melanin contain PTCA and PDCA.

[0099] Linear relationship investigation

[0100] A series of mixed reference solutions of different concentrations were injected according to the chromatographic conditions of Example 1, and a standard curve was drawn with the peak area as the abscissa and the concentration as the ordinate. The regression equation and detection limit are shown in Table 3.

[0101] Table 3 Linear relationship investigation table

[0102] Element Standard curve <![CDATA[R 2 ]]> Detection limit PTCA y=1,601,927,816.614x+3,164,063.586 0.992 0.0000507-0.127 μg / μL PDCA y=972,342,346.665x+4,293,044.580 0.991 0.0000078125-0.1953125μg / μL

[0103] As can be seen from Table 3, PDCA and PTCA have a good linear relationship, a wide linear range and a low detection limit.

[0104] Determination of conversion factors between melanin content and PTCA and PDCA content

[0105] The sepia melanin standard was accurately weighed, and the standard solution was prepared according to the method of step (3) of Example 1. The sample was injected according to the chromatographic conditions of Example 1, and the peak area was measured and the PTCA and PDCA contents were calculated. The conversion factors between eumelanin and PTCA and PDCA contents were established. The results are shown in Table 4.

[0106] Table 4 PTCA and PDCA content of standard products after oxidation

[0107]

[0108] As can be seen from Table 4, the conversion factor between DHI-melanin and PDCA is 30.975, and the conversion factor between DHICA-melanin and PTCA is 272.450.

[0109] Precision experiment

[0110] The mixed reference solution was accurately aspirated and injected six times continuously according to the chromatographic conditions in Example 1. The peak areas of PDCA and PTCA in the reference solution were recorded, and the relative standard deviations (RSD, n=6) of the chromatographic peak areas were calculated six times. The RSD values ​​of each component are shown in Table 5.

[0111] Table 5 Precision test results

[0112]

[0113] As can be seen from Table 5, the RSD of the peak area of ​​each component for 6 times is ≤4.7%, indicating that the instrument has good precision.

[0114] Stability test

[0115] One portion of the test solution in Example 1 was injected and tested at 0, 2, 4, 8, 10, and 12 h after preparation of the test solution according to the chromatographic conditions in Example 1. The peak areas and retention values ​​of PDCA and PTCA in the test solution were recorded, and the relative standard deviations (RSDs, n=6) were calculated. The RSD values ​​of each component are shown in Table 6.

[0116] Table 6 Stability test results

[0117]

[0118] As can be seen from Table 6, the content corresponding to each component peak is RSD≤3.6% within 12 hours, proving that the test solution is stable within 12 hours.

[0119] Repeatability experiments

[0120] Accurately weigh 6 portions of the crude black-bone chicken melanin product in Example 1, prepare a test solution according to the method of step (3) of Example 1, and inject and detect according to the chromatographic conditions of Example 1. Record the peak areas and retention times of PDCA and PTCA in the test solution, and calculate their relative standard deviations (RSD, n=6). The RSD values ​​of each component are shown in Table 7.

[0121] Table 7 Repeatability test results

[0122]

[0123] As can be seen from Table 7, the repeatability complies with the relevant provisions on "repeatability" in the 9000 Guidelines 9101 Validation Guidelines for Analytical Methods of Pharmaceutical Quality Standards in Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0124] Sample recovery experiment

[0125] Six portions of the crude black-bone chicken melanin product in Example 1 were accurately weighed, and PDCA and PTCA standard solutions of known concentrations were added respectively. The test solution was prepared according to the method of step (3) of Example 1. The test was performed according to the chromatographic conditions of Example 1. The peak areas and retention times of PDCA and PTCA in the test solution were recorded, and the recovery rate and relative standard deviation (RSD, n=6) were calculated. The results are shown in Table 8.

[0126] Table 8 Sample recovery experimental results

[0127]

[0128]

[0129] As can be seen from Table 8, the recovery rate of the method of the present invention is high and the detection result is accurate.

[0130] The black-bone chicken melanin oxidation products and black-bone chicken melanin content detected by the method of Example 1 are shown in Table 9.

[0131] Table 9 Black chicken melanin oxidation products and black chicken melanin content

[0132]

[0133] Comparative Example 1

[0134] Instruments and reagents in Comparative Example 1: high performance liquid chromatograph (Agilent 1260 Infinity II quaternary pump liquid chromatograph); high-speed refrigerated centrifuge (Thermo Fisher Scientific); HH-6 constant temperature water bath (Shanghai Heng Scientific Instrument Co., Ltd.); electronic analytical balance (Mettler Toledo, Switzerland); IQ7005 ultrapure water device (Millipore, USA); 2,3-dicarboxylic acid pyrrole (Aladdin); 2,3,5-tricarboxylic acid pyrrole (Shanghai McLean Biochemical Technology Co., Ltd.); potassium carbonate (K2CO3), 30% hydrogen peroxide (H2O2) solution, 10% sodium sulfite (Na2SO3) aqueous solution, methanol and formic acid were of chromatographic grade, and methyl tert-butyl ether, hydrochloric acid, petroleum ether and acetic acid were of analytical grade, all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0135] (1) Prepare crude black-bone chicken melanin according to the method of Example 1;

[0136] (2) 10 mg of cuttlefish melanin was mixed with 8.6 mL of 1 mol / L potassium carbonate aqueous solution and 0.8 mL of 30% volume concentration hydrogen peroxide in a 150 mL conical flask, and reacted in a water bath at 100°C for 20 min, with shaking every 5 min. After the reaction was completed, it was cooled in an ice water bath for 2 min, and immediately 0.4 mL of 10% mass concentration Na2SO3 aqueous solution was added to terminate the reaction. The pH value was adjusted to 1.0 with 6 mol / L hydrochloric acid, and after centrifugation at 9500 rpm for 5 min, the supernatant was taken and extracted with methyl tert-butyl ether 4 times, each time with a volume of 50 mL. The 4 extracts were combined, blown dry with nitrogen, and then redissolved with 200 μL of 50% volume concentration methanol aqueous solution. The sample solution was filtered through a 0.45 μm organic membrane to obtain the test solution.

[0137] (3) The test solution obtained in step (1) was subjected to high performance liquid chromatography to obtain the content of black-bone chicken melanin oxidation products. The chromatographic detection conditions were as follows: chromatographic column: Inertsil ODS-HL (4.6×250 mm, 5 μm), mobile phase A: 0.1% (volume fraction) formic acid aqueous solution; mobile phase B: methanol; injection volume 20 μL, gradient elution conditions as shown in Table 10, flow rate 0.6 mL / min, detection wavelength 270 nm, column temperature 25°C.

[0138] Table 10 Gradient elution conditions in Comparative Example 1

[0139] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 98→95 2→5 5～10 95 5 10～15 95→60 5→40 15～25 60→98 40→2

[0140] 10 mg of cuttlefish melanin was mixed with 8.6 mL of 1 mol / L potassium carbonate aqueous solution and 0.8 mL of 30% volume concentration hydrogen peroxide in a 150 mL conical flask, and the mixture was reacted in a 100°C water bath for 20 min, with shaking every 5 min. After the reaction was completed, the mixture was cooled in an ice-water bath for 2 min, and 0.4 mL of 10% mass concentration Na2SO3 aqueous solution was immediately added to terminate the reaction. The pH value was adjusted to 1.0 with 6 mol / L hydrochloric acid. After centrifugation at 9500 rpm for 5 min, the supernatant was taken and extracted four times with 50 mL of methyl tert-butyl ether. The four extracts were combined, blown dry with nitrogen, and then redissolved with 200 μL of 50% volume concentration methanol aqueous solution. The solution was filtered through a 0.45 μm organic membrane to obtain a standard solution.

[0141] PDCA standard solution, 500 μg / mL.

[0142] PTCA standard solution, 500 μg / mL.

[0143] The concentrations of PDCA and PTCA in the mixed reference solutions were 9.375 μg / mL, 18.75 μg / mL, 37.5 μg / mL, 75 μg / mL, 125 μg / mL and 250 μg / mL, respectively.

[0144] Take the mixed reference solution and inject it according to the chromatographic conditions of Comparative Example 1. Draw a standard curve with the peak area as the abscissa and the concentration as the ordinate. The regression equation is shown in Table 11.

[0145] Table 11 Linear relationship table

[0146]

[0147]

[0148] Determination of the conversion factor between melanin content and PTCA content

[0149] The standard solution was injected according to the chromatographic conditions of Comparative Example 1, the peak area was measured and the PTCA and PDCA contents were calculated to establish the conversion factors between eumelanin and PTCA and PDCA contents. The results are shown in Table 12.

[0150] Table 12 PTCA and PDCA content of standard products after oxidation

[0151]

[0152] As can be seen from Table 12, the conversion factor between DHI-melanin and PDCA is 37.225, and the conversion factor between DHICA-melanin and PTCA is 242.959.

[0153] The black-bone chicken melanin oxidation products and black-bone chicken melanin content detected by the method of Comparative Example 1 are shown in Table 13.

[0154] Table 13 Black chicken melanin oxidation products and black chicken melanin content

[0155]

[0156] It can be seen from the above examples and comparative examples that the detection method of the present invention reduces the injection volume and shortens the elution time while ensuring detection accuracy.

[0157] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting black-bone chicken melanin oxidation products, characterized in that: The following steps are involved: (1) mixing black-bone chicken powder with acid solution and extracting to obtain crude black-bone chicken melanin; (2) mixing the crude black chicken melanin obtained in step (1) with a potassium carbonate solution and hydrogen peroxide, performing an oxidation reaction, then adding an acid and an extractant, sequentially performing extraction, nitrogen blowing, and redissolution to obtain a test solution; the volume ratio of the crude black chicken melanin to the volume of the potassium carbonate solution and the volume of the hydrogen peroxide is 10 mg: (8-9) mL: (0.5-1) mL; (3) The test solution obtained in the step (2) is subjected to ultra-high performance liquid chromatography-mass spectrometry to obtain the content of black-bone chicken melanin oxidation products; the detection conditions of the liquid chromatography are: the injection volume is 0.9-1 μL, the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05-0.15%, the mobile phase B is methanol, and gradient elution is adopted, and the gradient elution conditions are: the volume percentage of the mobile phase B is from 5% to 25% in 0-3 min, the volume percentage of the mobile phase B is from 25% to 95% in 3-4 min, 95% B in 4-5.5 min, the volume percentage of the mobile phase B is from 95% to 5% in 5.5-6 min, and 5% B in 6-8 min; the column temperature is 35-45° C., the flow rate is 0.3-0.5 mL / min, and the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column, 2.1 mm×100 mm, 1.7 μm; The mass spectrometry detection conditions are as follows: electrospray ion source, multiple reaction monitoring mode, negative ion detection mode, ionization temperature of 500-600°C, spray voltage of 5000-6000V, curtain gas volume flow rate of 30-50L / min, nebulizing gas volume flow rate of 50-60L / min, and heating auxiliary gas volume flow rate of 50-60L / min.

2. The detection method according to claim 1, wherein The acid solution in step (1) is hydrochloric acid; the mass concentration of the acid solution is 20-30%.

3. The detection method according to claim 2, characterized in that In the step (1), the volume ratio of the black-bone chicken powder to the acid solution is 100 g: (200-400) mL.

4. The detection method according to claim 1, wherein The extraction temperature in step (1) is 95-105° C., and the extraction time is 4-6 hours.

5. The detection method according to claim 1, wherein In the step (2), the concentration of the potassium carbonate solution is 0.5-1.5 mol / L, and the volume concentration of the hydrogen peroxide is 25-35%.

6. The detection method according to claim 1, characterized in that The temperature of the oxidation reaction in step (2) is 90-110° C., and the time of the oxidation reaction is 10-30 minutes.

7. The detection method according to claim 1, characterized in that The extractant in step (2) is methyl tert-butyl ether.

Images