Determination method of collagen content on fabric

Abstract

The invention relates to the field of detection, and discloses a method for determining the content of collagen on a fabric. The determination method comprises the following steps: S1, hydrolyzing a to-be-determined sample, and adjusting the pH value to 4-8 to obtain a mixed solution; s2, mixing the mixed solution with a derivatization reagent to obtain a to-be-measured solution; the derivatization reagent contains phenyl isothiocyanate and triethylamine; s3, injecting the to-be-measured liquid into a high performance liquid chromatograph, carrying out gradient elution to measure hydroxyproline and peak area thereof, and calculating according to a standard curve equation to obtain the content of collagen in the to-be-measured sample, a chromatographic column adopted in the high performance liquid chromatography is a C18 reverse chromatographic column, a mobile phase A in an eluent adopted in the gradient elution is an acetonitrile-water solution, and a mobile phase B in the eluent adopted in the gradient elution is an acetonitrile-sodium acetate water solution. The determination method can effectively improve the detection accuracy of the collagen content on the fabric.

Description

Technical Field

[0001] The present invention relates to the field of detection, and particularly relates to a method for determining the collagen content on fabrics. Background Art

[0002] Collagen is a structural protein widely distributed in tissues such as skin, tendons, and bones. It is composed of 18 amino acids, among which glycine, proline, and hydroxyproline are the characteristic amino acids of collagen. Collagen accounts for about 1 / 3 of the total protein content and has good moisturizing effects. In addition, collagen also exhibits excellent biocompatibility and degradability.

[0003] In order to improve the softness and toughness of wool and cashmere, collagen is combined with wool and cashmere fibers during the processing process to make them have good softness and toughness. However, how to accurately quantify and detect the collagen loaded on wool fabrics is a technical problem currently faced. Traditional detection methods, such as the Kjeldahl method and formaldehyde titration method, are difficult to accurately distinguish the protein in wool fibers themselves from the loaded collagen.

[0004] At present, the high-performance liquid chromatography analysis methods for amino acids are mainly divided into two types: indirect analysis and direct analysis. The direct analysis methods include high-performance liquid chromatography-evaporative light scattering detection method and high-performance anion exchange chromatography-integrated pulsed amperometric detection method. Indirect analysis can be divided into pre-column derivatization method and post-column derivatization method. The pre-column derivatization reversed-phase high-performance liquid chromatography method has been widely used in the field of amino acid analysis in recent years due to its simple operation, fast analysis speed, high sensitivity, and a variety of derivatization reagents. However, the accuracy of determining the collagen content in fabrics by the pre-column derivatization method in the existing technology still needs to be improved. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem that the accuracy of determining the collagen content by the pre-column derivatization method in the existing technology still needs to be improved, and to provide a method for determining the collagen content on fabrics, which can effectively improve the detection accuracy of the collagen content on fabrics.

[0006] To achieve the above purpose, the present invention provides a method for determining the collagen content on fabrics, and the determination method includes the following steps:

[0007] S1. Hydrolyze the sample to be tested and adjust the pH to 4-8 to obtain a mixed solution;

[0008] S2. Mix the mixed solution with a derivatization reagent to obtain a solution to be determined;

[0009] The derivatization reagent contains phenyl isothiocyanate and triethylamine;

[0010] S3. Inject the test solution into a high performance liquid chromatograph for gradient elution to determine hydroxyproline and its peak area, and calculate the content of collagen in the test sample according to the standard curve equation;

[0011] The chromatographic column used in the high performance liquid chromatography is a C18 reverse chromatographic column. In the gradient elution, mobile phase A in the eluent is an acetonitrile - aqueous solution, and mobile phase B is an acetonitrile - sodium acetate aqueous solution.

[0012] Preferably, step S1 includes: adjusting the pH of the test sample after hydrolysis to 6 - 8 to obtain a mixed solution.

[0013] Preferably, the test sample is selected from at least one of wool fabrics, cashmere fabrics, and cotton fabrics.

[0014] More preferably, the reagent used for hydrolysis is sulfuric acid.

[0015] Preferably, the conditions for hydrolysis include: temperature of 100 - 110 °C and time of 18 - 22 h.

[0016] Preferably, the pH is adjusted using an alkaline solution.

[0017] More preferably, the alkaline solution is a sodium hydroxide solution and / or a potassium hydroxide solution.

[0018] Preferably, in step S2, relative to 1 g of the test sample, the amount of phenyl isothiocyanate used is 0.35 - 0.6 mmol, and the amount of triethylamine used is 3.5 - 6 mmol.

[0019] More preferably, the derivatization reagent further contains acetonitrile.

[0020] Preferably, the determination method further includes: before step S3, mixing and extracting the test solution with n - hexane, and taking the lower layer liquid for the operation of step S3.

[0021] Preferably, in step S3, the C18 reverse chromatographic column is an LP - C18 reverse chromatographic column. The particle size in the C18 reverse chromatographic column is 4.5 - 5.5 μm, the inner diameter of the chromatographic column is 4.4 - 4.8 mm, and the length is 230 - 270 mm.

[0022] Preferably, in step S3, the volume ratio of acetonitrile to water in the acetonitrile - aqueous solution is 3 - 5:1, the concentration of the sodium acetate aqueous solution in the acetonitrile - sodium acetate aqueous solution is 0.08 - 0.12 mol / L, and the volume ratio of acetonitrile to the sodium acetate aqueous solution is 1:9 - 19.

[0023] More preferably, the volume ratio of acetonitrile to the sodium acetate aqueous solution is 1:10 - 15.

[0024] Preferably, the elution process includes: successively injecting mobile phase B, mixture I of mobile phase B and mobile phase A, mixture II of mobile phase B and mobile phase A, mixture III of mobile phase B and mobile phase A, mixture IV of mobile phase B and mobile phase A, and mobile phase A;

[0025] In the mixture I, the volume ratio of mobile phase B to mobile phase A is 12.5 - 14:1; in the mixture II, the volume ratio of mobile phase B to mobile phase A is 6.5 - 8:1; in the mixture III, the volume ratio of mobile phase B to mobile phase A is 5 - 6.5:1; in the mixture IV, the volume ratio of mobile phase B to mobile phase A is 1.5 - 2.5:1.

[0026] Preferably, in step S3, the detector used in the measurement process is an ultraviolet dual - wavelength detector, and the column temperature is 40 - 45 °C.

[0027] More preferably, the detection wavelength of the detector is 250 - 258 nm.

[0028] Preferably, the method for calculating the content of collagen in the sample to be measured according to the standard curve equation includes:

[0029] Obtaining the content of hydroxyproline in the sample to be measured according to the standard curve equation, and then calculating the content of collagen in the sample to be measured according to formula (II),

[0030]

[0031] where Y is the amount of collagen in the sample to be measured, mg; X is the content of hydroxyproline in the sample to be measured, %; ρ1 is the concentration of hydroxyproline in the collagen solution to be measured, μg / mL; V is the volume of the collagen solution to be measured, mL; m1 is the mass of collagen, mg; m is the mass of the sample to be measured, mg.

[0032] More preferably, the method for obtaining the content of hydroxyproline in the sample to be measured according to the standard curve equation includes: calculating the concentration ρ of hydroxyproline in the solution to be measured through the standard curve, and then calculating the content of hydroxyproline in the sample to be measured according to formula (I);

[0033]

[0034] where X is the content of hydroxyproline in the sample to be measured, %; ρ is the concentration of hydroxyproline in the solution to be measured of the sample to be measured, μg / mL; V is the volume of the solution to be measured of the sample to be measured, mL.

[0035] Through the above technical solution, the method for determining the collagen content on the fabric provided by the present invention adjusts the pH to 4-8 after hydrolysis, and uses phenyl isothiocyanate and triethylamine as pre-column derivatization reagents for high-performance liquid chromatography detection. In the gradient elution, mobile phase A in the eluent is an acetonitrile-aqueous solution, and mobile phase B is an acetonitrile-sodium acetate aqueous solution, which can accurately detect the collagen content in the fabric. In addition, this method is simple to operate and has high detection sensitivity. Especially when detecting fabrics with low collagen content, this detection method shows higher sensitivity and reliability. Description of the Drawings

[0036] Figure 1 is the standard curve graph obtained in Example 1;

[0037] Figure 2 is the liquid chromatography graph obtained in Examples 1-5;

[0038] Figure 3 is the liquid chromatography graph obtained in Example 9. Detailed Embodiments

[0039] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0040] The present invention provides a method for determining the collagen content on a fabric, and the determination method includes the following steps:

[0041] S1. Hydrolyze the sample to be tested and adjust the pH to 4-8 to obtain a mixed solution;

[0042] S2. Mix the mixed solution with a derivatization reagent to obtain a solution to be determined;

[0043] The derivatization reagent contains phenyl isothiocyanate and triethylamine;

[0044] S3. Inject the solution to be determined into a high-performance liquid chromatograph for gradient elution to determine hydroxyproline and its peak area, and calculate the collagen content in the sample to be tested according to the standard curve equation;

[0045] The chromatographic column used in the high-performance liquid chromatography is a C18 reverse chromatographic column. In the gradient elution, mobile phase A in the eluent is an acetonitrile-aqueous solution, and mobile phase B is an acetonitrile-sodium acetate aqueous solution.

[0046] In the process of research, the inventor found that after the fabric was hydrolyzed, adjusting the pH to 4 - 8, and using phenyl isothiocyanate and triethylamine as pre-column derivatization reagents for high performance liquid chromatography detection, and using acetonitrile - aqueous solution as mobile phase A and acetonitrile - sodium acetate aqueous solution as mobile phase B in the eluent during gradient elution, the content of collagen in the fabric could be accurately detected. In addition, this method is simple to operate and has high detection sensitivity. Especially when detecting fabrics with low collagen content, this detection method shows higher sensitivity and reliability.

[0047] According to the present invention, in step S1, the pH can be adjusted to 4, 5, 6, 7, or any value within the range formed by these values. In order to further improve the detection accuracy of the collagen content in the fabric, preferably, step S1 includes: hydrolyzing the sample to be tested and then adjusting the pH to 6 - 8 to obtain a mixed solution.

[0048] Preferably, the sample to be tested is selected from at least one of wool fabric, cashmere fabric, and cotton fabric. This method has higher detection accuracy for wool fabric, cashmere fabric, and cotton fabric. Further preferably, the sample to be tested is wool fabric and / or cashmere fabric.

[0049] Preferably, the reagent used for hydrolysis is sulfuric acid. Using sulfuric acid during hydrolysis can improve the hydrolysis effect of collagen in the fabric, thereby further improving the detection accuracy of collagen in the fabric. To further improve the detection accuracy of collagen in the fabric, further preferably, the conditions for hydrolysis include: the temperature is 100 - 110°C, which can be 100°C, 102°C, 104°C, 106°C, 108°C, 110°C, or any value within the range formed by these temperatures; the time is 18 - 22 h, which can be 18 h, 19 h, 20 h, 21 h, 22 h, or any value within the range formed by these values.

[0050] Preferably, the reagent used for adjusting the pH is sodium hydroxide solution and / or potassium hydroxide solution. Further preferably, the concentration of the sodium hydroxide solution and / or potassium hydroxide solution is 8 - 12 mol / L.

[0051] Preferably, in step S2, relative to 1 g of the sample to be measured, the dosage of phenyl isothiocyanate is 0.35 - 0.6 mmol, which can be 0.35 mmol, 0.4 mmol, 0.45 mmol, 0.5 mmol, 0.55 mmol, 0.6 mmol, or any value between the ranges formed by these values; the dosage of triethylamine is 3.5 - 6 mmol, which can be 3.5 mmol, 4 mmol, 4.5 mmol, 5 mmol, 5.5 mmol, 6 mmol, or any value between the ranges formed by these values. Under the above conditions, the detection accuracy of collagen in the fabric can be further improved. Further preferably, the derivatizing reagent further contains acetonitrile. Preferably, the phenyl isothiocyanate and the triethylamine can be separately dissolved in acetonitrile to form a phenyl isothiocyanate-acetonitrile solution and a triethylamine-acetonitrile solution.

[0052] Preferably, the determination method further includes, before step S3, mixing and extracting the solution to be determined with n-hexane, and taking the lower-layer liquid for the operation of step S3. Extracting the solution to be determined with n-hexane can further improve the detection accuracy.

[0053] To further improve the detection accuracy, preferably, in step S3, the C18 reverse-phase chromatographic column is an LP-C18 reverse-phase chromatographic column, the particle size in the C18 reverse-phase chromatographic column is 4.5 - 5.5 μm, the inner diameter of the chromatographic column is 4.4 - 4.8 mm, and the length is 230 - 270 mm. As a specific embodiment of the present invention, in step S3, the particle size in the Welch Ultimate LP-C18 chromatographic column is 5 μm, the inner diameter of the chromatographic column is 4.6 mm, and the length is 250 mm.

[0054] Preferably, in step S3, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 3 - 5:1, the concentration of the sodium acetate aqueous solution in the acetonitrile-sodium acetate aqueous solution is 0.08 - 0.12 mol / L, and the volume of acetonitrile and the sodium acetate aqueous solution is 1:9 - 19. The above eluent can further improve the detection accuracy. Considering further improving the detection accuracy, further preferably, the volume of acetonitrile and the sodium acetate aqueous solution is 1:5 - 15.

[0055] In order to further improve the detection accuracy, preferably, the elution process is as follows: successively inject mobile phase B, mixture I of mobile phase B and mobile phase A, mixture II of mobile phase B and mobile phase A, mixture III of mobile phase B and mobile phase A, mixture IV of mobile phase B and mobile phase A, and mobile phase A; in mixture I, the volume ratio of mobile phase B to mobile phase A is 12.5 - 14:1; in mixture II, the volume ratio of mobile phase B to mobile phase A is 6.5 - 8:1; in mixture III, the volume ratio of mobile phase B to mobile phase A is 5 - 6.5:1; in mixture IV, the volume ratio of mobile phase B to mobile phase A is 1.5 - 2.5:1. Considering that the detection accuracy can be further improved, more preferably, in mixture I, the volume ratio of mobile phase B to mobile phase A is 13 - 13.5:1; in mixture II, the volume ratio of mobile phase B to mobile phase A is 7 - 7.5:1; in mixture III, the volume ratio of mobile phase B to mobile phase A is 5.5 - 6:1; in mixture IV, the volume ratio of mobile phase B to mobile phase A is 1.7 - 2.1:1.

[0056] In addition, the inventors unexpectedly found during the research process that controlling the pH of mobile phase B to be 6.2 - 6.8 can further improve the detection accuracy.

[0057] According to the present invention, the way to control the pH of mobile phase B can be to adjust the pH with acetic acid.

[0058] Preferably, in step S3, the detector used during the measurement is an ultraviolet dual-wavelength detector, and the column temperature is 40 - 45°C. The above detection conditions can further improve the detection sensitivity. Considering that the detection sensitivity can be further improved, more preferably, the detection wavelength of the detector is 250 - 258 nm.

[0059] Preferably, the method for calculating the content of collagen in the sample to be measured according to the standard curve equation includes:

[0060] Obtain the content of hydroxyproline in the sample to be measured according to the standard curve equation, and then calculate the content of collagen in the sample to be measured according to formula (II).

[0061]

[0062] Among them, Y is the amount of collagen in the sample to be tested, in mg; X is the content of hydroxyproline in the sample to be tested, in %; ρ1 is the concentration of hydroxyproline in the collagen solution to be determined, in μg / mL; V1 is the volume of the collagen solution to be determined, in mL; m1 is the mass of collagen, in mg; m is the mass of the sample to be tested, in mg. The content of collagen in the sample to be tested can be accurately calculated by the above method. Further preferably, the method for obtaining the content of hydroxyproline in the sample to be tested according to the standard curve equation includes: calculating the concentration ρ of hydroxyproline in the solution to be determined through the standard curve, and then calculating the content of hydroxyproline in the sample to be tested according to formula (I);

[0063]

[0064] Among them, X is the content of hydroxyproline in the sample to be tested, in %; ρ is the concentration of hydroxyproline in the solution to be determined of the sample to be tested, in μg / mL; V is the volume of the solution to be determined of the sample to be tested, in mL.

[0065] Preferably, the concentration of hydroxyproline in the collagen solution to be determined can be determined according to the standard curve equation.

[0066] According to a particularly preferred embodiment of the present invention, a method for determining the collagen content on a fabric is provided, including the following steps:

[0067] (1) Accurately weigh the hydroxyproline standard, dissolve it in water and make up the volume to prepare hydroxyproline standard stock solutions with different concentrations.

[0068] (2) Take part of the gradient standard working solutions and place them in 5 mL chromatographic vials respectively. Add 0.25 - 0.3 mL of each of 0.1 mol / L phenyl isothiocyanate - acetonitrile solution and 1 mol / L triethylamine - acetonitrile solution, mix well and let stand. Make up the volume to the scale with acetonitrile - aqueous solution, mix well. Precisely pipette a part of the liquid into a separating funnel, add 0.8 - 1.2 times the volume of n - hexane, mix well and let stand. Separate the lower layer solution, filter it through a 0.45 μm microporous filter membrane, take the filtrate, inject it into a high - performance liquid chromatograph to measure the hydroxyproline and its peak area. Take the concentration as the abscissa and the peak area of hydroxyproline as the ordinate to draw the standard working curve, and fit to obtain the standard curve equation.

[0069] The chromatographic column was Welch Ultimate LP-C18 (with a specification of 4.6×250 mm, 5 μm). The chromatographic conditions were as follows: acetonitrile-water (volume ratio 4:1) was used as mobile phase A, and acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (volume ratio 7:93) was used as mobile phase B. The gradient elution program was: 100 (volume)% of mobile phase B was injected within 0 - 11 min, 93 (volume)% of mobile phase B + 7 (volume)% of mobile phase A was injected within 11 - 13.9 min, 88 (volume)% of mobile phase B + 12 (volume)% of mobile phase A was injected within 13.9 - 14 min, 85 (volume)% of mobile phase B + 15 (volume)% of mobile phase A was injected within 14 - 29 min, 66 (volume)% of mobile phase B + 34 (volume)% of mobile phase A was injected within 29 - 30 min, and 100 (volume)% of mobile phase A was injected at 30 min; the detector was an ultraviolet dual-wavelength detector, the detection wavelength was 254 nm; the volumetric flow rate was 1 mL / min; the column temperature was 43°C.

[0070] (3) Weigh the sample to be tested into a hydrolysis tube, add 5 - 15 mL of sulfuric acid with a concentration of 1 - 3 mol / L, and digest at 100 - 110°C for 18 - 22 h. After cooling, add 8 - 12 mol / L sodium hydroxide solution to the digestion solution to adjust the pH to 4 - 8. After mixing evenly, transfer all the digestion solution to a volumetric flask, add water to dilute and make up to the mark. Take a part of the diluted solution and filter it with a 0.45 μm filter membrane for standby.

[0071] (4) Derivatization: Take a part of the filtered solution and place it in a 5 mL chromatographic vial. Add 0.25 - 0.3 mL each of 0.1 mol / L phenyl isothiocyanate-acetonitrile solution and 1 mol / L triethylamine-acetonitrile solution, mix well and let stand. Dilute to the mark with an acetonitrile aqueous solution with a volume fraction of 50%, mix well. Precisely pipette a part of the liquid into a separating funnel, add 0.8 - 1.2 times the volume of n-hexane, mix well and let stand. Separate the lower layer solution, filter it through a 0.45 μm microporous filter membrane, and take the filtrate for high performance liquid chromatography analysis.

[0072] (5) Inject the liquid described in step (4) into a high performance liquid chromatograph to determine the content of hydroxyproline and its peak area. Calculate the content of hydroxyproline in the sample to be tested according to the standard curve equation; and calculate the content of collagen in the sample to be tested based on the content of hydroxyproline in the sample to be tested;

[0073] The method for obtaining the content of hydroxyproline in the sample to be tested according to the standard curve equation includes: calculating the concentration ρ of hydroxyproline in the solution to be determined through the standard curve, and then calculating the content of hydroxyproline in the sample to be tested according to formula (I);

[0074]

[0075] Wherein, X is the content of hydroxyproline in the sample to be measured, %; ρ is the concentration of hydroxyproline in the solution to be measured of the sample to be measured, μg / mL; V is the volume of the solution to be measured of the sample to be measured, mL, and m is the mass of the sample to be measured, mg;

[0076] The method for calculating the content of collagen in the sample to be measured according to formula (II) includes:

[0077]

[0078] Wherein, Y is the amount of collagen in the sample to be measured, mg; X is the content of hydroxyproline in the sample to be measured, %; ρ1 is the concentration of hydroxyproline in the solution to be measured of collagen, μg / mL, which can be obtained according to the standard curve; V1 is the volume of the solution to be measured of collagen, mL, and m1 is the mass of collagen, mg.

[0079] The content of hydroxyproline in the sample to be measured can be accurately measured by the above method.

[0080] According to the present invention, in the above-provided method, the blank value needs to be deducted during calculation, and the result is retained to four significant figures.

[0081] The present invention will be described in detail below through examples.

[0082] Example 1

[0083] (1) Accurately weigh 0.1 g of hydroxyproline standard product, dissolve it with pure water and make the volume up to 100 mL to prepare a hydroxyproline standard stock solution with a concentration of 1.00 g / L. Accurately pipette the standard stock solution to prepare a series of gradient standard working solutions with concentrations of 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 15.0 μg / mL, 20.0 μg / mL, 30.0 μg / mL, and 50.0 μg / mL. Precisely pipette 1.0 mL of the above solutions.

[0084] (2) Place the gradient standard working solutions in 5 mL chromatographic vials respectively, add 0.25 mL of 0.1 mol / L phenyl isothiocyanate-acetonitrile solution and 0.25 mL of 1 mol / L triethylamine-acetonitrile solution, mix well, let stand at room temperature for 1 h, make the volume up to the mark with 50% (v / v) acetonitrile-aqueous solution, mix well, precisely pipette 1 mL, place it in a separatory funnel, add 1 mL of n-hexane, mix well, let stand for 10 min, separate the lower layer solution, filter it through a 0.45 μm microporous filter membrane, take the filtrate, inject it into a high-performance liquid chromatograph to measure hydroxyproline and its peak area, use the concentration as the abscissa and the peak area of hydroxyproline as the ordinate to draw a standard working curve, and fit to obtain the standard curve equation (see Figure 1 , y = 12.9508x - 2.3212, R 2= 0.9999), L-hydroxyproline at 1.0 μg·mL -1 -50 μg·mL -1 In the concentration range, the linear relationship is good.

[0085] The chromatographic column is Welch Ultimate LP-C18, with a specification of 4.6×250 mm, 5 μm). The chromatographic conditions are as follows: Acetonitrile-water (volume ratio 4:1) is used as mobile phase A, and acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (volume ratio 7:93) is used as mobile phase B. The gradient elution program is: Inject 100% (by volume) of mobile phase B in 0 - 11 min, 93% (by volume) of mobile phase B + 7% (by volume) of mobile phase A in 11 - 13.9 min, 88% (by volume) of mobile phase B + 12% (by volume) of mobile phase A in 13.9 - 14 min, 85% (by volume) of mobile phase B + 15% (by volume) of mobile phase A in 14 - 29 min, 66% (by volume) of mobile phase B + 34% (by volume) of mobile phase A in 29 - 30 min, and 100% (by volume) of mobile phase A at 30 min; The detector is an ultraviolet dual-wavelength detector, the detection wavelength is 254 nm; The volumetric flow rate is 1 mL / min; The column temperature is 43 °C.

[0086] (3) Digestion and dilution of the sample: Weigh collagen I into a hydrolysis tube so that each tube contains 20 mg of protein (accurate to four decimal places), add 10 mL of 3 mol / L sulfuric acid, and digest at 105 °C for 20 h. After cooling, add 10 mol / L sodium hydroxide solution to the digestion solution to adjust the pH to 5. After mixing evenly, transfer all the digestion solution to a 50 mL volumetric flask, dilute with water to the mark, and make it up to the volume. Take 1 mL of the diluted solution and filter it through a 0.45 μm filter membrane for standby.

[0087] (4) Derivatization: Take 0.5 mL of the filtered solution and place it in a 5 mL chromatographic vial. Add 0.25 mL each of 0.1 mol / L phenyl isothiocyanate-acetonitrile solution and 1 mol / L triethylamine-acetonitrile solution, mix well, let it stand at room temperature for 1 h, make it up to the mark with 50% (by volume) acetonitrile aqueous solution, mix well, accurately pipette 1 mL, place it in a separatory funnel, add 1 mL of n-hexane, mix well, let it stand for 10 min, separate the lower layer solution, filter it through a 0.45 μm microporous filter membrane, and take the filtrate for high performance liquid chromatography analysis.

[0088] (5) Inject the liquid described in step (4) into a high performance liquid chromatograph to determine hydroxyproline and its peak area. According to the standard curve equation, the content of hydroxyproline in the sample to be measured is calculated to be 14.3571 wt%.

[0089] Example 2

[0090] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with Collagen II.

[0091] The content of hydroxyproline in Collagen II was calculated to be 11.4688 wt%.

[0092] Example 3

[0093] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with gelatin.

[0094] The content of hydroxyproline in gelatin was calculated to be 12.3732 wt%.

[0095] Example 4

[0096] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with wool (raw loose fibers).

[0097] The content of hydroxyproline in wool (raw loose fibers) was calculated to be 0 wt%.

[0098] Example 5

[0099] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with cashmere (raw loose fibers).

[0100] The content of hydroxyproline in cashmere (raw loose fibers) was calculated to be 0 wt%.

[0101] From Examples 1-5 and Figure 2 It can be seen that at the retention time corresponding to hydroxyproline, no obvious characteristic peaks were detected in wool and cashmere, so they do not contain hydroxyproline; the hydroxyproline content of Collagen I is 14.36 wt%, the hydroxyproline content of Collagen II is 11.47%, and the hydroxyproline content of gelatin is 12.37 wt%.

[0102] Example 6

[0103] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with a wool fabric containing Collagen I.

[0104] The preparation method of the wool fabric containing collagen includes: taking 0.6 g of wool fabric and mixing it with an equal mass of a collagen solution with a concentration of 10.0 g / L to achieve a simulated liquor ratio of 100%, and the obtained detection graph is as Figure 3 shown.

[0105] Example 7

[0106] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with a wool fabric containing Collagen I.

[0107] The preparation method of the wool fabric containing collagen includes: taking 7.0 g of wool fabric and mixing it with an equal mass of a collagen solution with a concentration of 0.5 g / L to achieve an effect of a simulated liquor pick-up rate of 100%.

[0108] Example 8

[0109] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with a wool fabric containing Collagen I.

[0110] The preparation method of the wool fabric containing collagen includes: immersing 0.6248 g of wool fabric in a collagen solution with a concentration of 10 g / L (bath ratio 1:100), and after one dip and one nip, its liquor holding rate is 89.4526%.

[0111] Example 9

[0112] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with a wool fabric containing Collagen I.

[0113] The preparation method of the wool fabric containing collagen includes: immersing 7.0791 g of wool fabric in a collagen solution with a concentration of 0.5 g / L (bath ratio 1:100), and after one dip and one nip, its liquor holding rate is 85.0235%.

[0114] Example 10

[0115] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with a cashmere fabric containing Collagen I.

[0116] The preparation method of the cashmere fabric containing collagen includes: taking 0.6 g of cashmere fabric and mixing it with an equal mass of a collagen solution with a concentration of 10.0 g / L to achieve an effect of a simulated liquor pick-up rate of 100%.

[0117] Example 11

[0118] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with a cashmere fabric containing Collagen I.

[0119] The preparation method of the cashmere fabric containing collagen includes: taking 7.0 g of cashmere fabric and mixing it with an equal mass of a collagen solution with a concentration of 0.5 g / L to achieve an effect of a simulated liquor pick-up rate of 100%.

[0120] Example 12

[0121] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with cashmere fabric containing Collagen I.

[0122] The preparation method of the cashmere fabric containing collagen includes: immersing 0.6248 g of cashmere fabric in a 10 g / L collagen solution (bath ratio 1:100), and after one dip and one nip, its liquor pickup rate is 111.5453%.

[0123] Example 13

[0124] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with cashmere fabric containing Collagen I.

[0125] The preparation method of the cashmere fabric containing collagen includes: immersing 7.0450 g of cashmere fabric in a 0.5 g / L collagen solution (bath ratio 1:100), and after one dip and one nip, its liquor pickup rate is 149.4535%.

[0126] Example 14

[0127] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with cotton fabric containing Collagen I.

[0128] The preparation method of the cotton fabric containing collagen includes: taking 0.6 g of cotton fabric and mixing it with an equal mass of a 10.0 g / L collagen solution to achieve an effect of a simulated liquor pickup rate of 100%.

[0129] Example 15

[0130] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with cotton fabric containing Collagen I.

[0131] The preparation method of the cotton fabric containing collagen includes: taking 7.0 g of cotton fabric and mixing it with an equal mass of a 0.5 g / L collagen solution to achieve an effect of a simulated liquor pickup rate of 100%.

[0132] Example 16

[0133] The content of hydroxyproline was measured according to the method of Example 1, except that Collagen I was replaced with cotton fabric containing Collagen I.

[0134] The preparation method of the cotton fabric containing collagen includes: immersing 0.5728 g of cotton fabric in a 10 g / L collagen solution (bath ratio 1:100), and after one dip and one nip, its liquor pickup rate is 89.0538%.

[0135] Example 17

[0136] The content of hydroxyproline was measured according to the method of Example 1, except that type I collagen was replaced with a cotton fabric containing type I collagen.

[0137] The preparation method of the cotton fabric containing collagen includes: immersing 6.8156 g of cotton fabric in a collagen solution at 0.5 g / L (liquor ratio 1:100), and after one dipping and one padding, the liquor pickup rate is 117.1210%.

[0138] Example 18

[0139] The content of collagen was determined according to the method of Example 7, except that in step (4), the dosages of both 0.1 mol / L phenyl isothiocyanate - acetonitrile solution and 1 mol / L triethylamine - acetonitrile solution were 0.3 mL.

[0140] Example 19

[0141] The content of collagen was determined according to the method of Example 7, except that in step (3), the pH was adjusted to 7.

[0142] Example 20

[0143] The content of collagen was determined according to the method of Example 7, except that in step (3), the pH was adjusted to 8.

[0144] Example 21

[0145] The content of collagen was determined according to the method of Example 7, except that in steps (1) and (4), n - hexane extraction was not used.

[0146] Example 22

[0147] The content of collagen was determined according to the method of Example 7, except that the chromatographic column was Welch UltimateLP - C18 (150 mm × 4.6 mm, 3 μm).

[0148] Comparative Example 1

[0149] The content of collagen was determined according to the method of Example 7, except that in step (3), the pH was adjusted to 3.

[0150] Comparative Example 2

[0151] The content of collagen was determined according to the method of Example 7, except that in step (3), the pH was adjusted to 9.

[0152] Comparative Example 3

[0153] The content of collagen was determined according to the method of Example 7, except that mobile phase A was methanol and mobile phase B was acetic acid - 0.1 mol / L sodium acetate solution (volume ratio 7:93).

[0154] Comparative Example 4

[0155] The content of collagen was determined according to the method of Example 7, except that step (2) included: placing the gradient standard working solutions into 5 mL chromatographic vials respectively, adding 30 μL of 0.1 mol / L sodium bicarbonate solution and 60 μL of DABS-Cl derivatization reagent solution (0.013 g of DABS-Cl dissolved in 10 mL of acetonitrile), vortex mixing, derivatizing in a water bath at 70 °C for 15 min, cooling, and then adding 630 μL of reaction termination solution ((50 mmol / L sodium dihydrogen phosphate solution, adjusted to pH = 7.0 with 2 mol / L sodium hydroxide solution, and then adding an equal volume of absolute ethanol)).

[0156] The preparation method of the wool fabric containing collagen I included: taking 2.577 g of wool fabric and mixing it with an equal mass of a collagen solution with a concentration of 1.0 g / L to achieve a simulated liquor ratio of 100%.

[0157] Comparative Example 5

[0158] The content of collagen was determined according to the method of Comparative Example 4, except that the fabric mass was 1.001 g and the collagen concentration was 5.0 g / L.

[0159] Comparative Example 6

[0160] The content of collagen was determined according to the method of Comparative Example 4, except that the fabric mass was 1.102 g and the collagen concentration was 10.0 g / L.

[0161] Comparative Example 7

[0162] The content of collagen was determined according to the method of Comparative Example 4, except that the fabric mass was 1.000 g and the collagen concentration was 20.0 g / L.

[0163] Test Example

[0164] It was detected that the peak area blank value of the wool fabric was 51.133 mAU·s, the peak area blank value of the cashmere fabric was 38.093 mAU·s, and the peak area blank value of the cotton fabric was 26.398 mAU·s. The detected data are shown in Table 1.

[0165] Table 1

[0166]

[0167]

[0168] As can be seen from Table 1, the difference between the measured content of collagen in the examples and the theoretical value is smaller than the difference between the measured content of collagen in the comparative examples and the theoretical value, indicating that the method provided by the present invention has high accuracy for the detection of collagen.

[0169] At the retention time corresponding to hydroxyproline (near 6.4 min), there are also certain characteristic peaks in the blank sample fabric. As the sample mass increases and the concentration of the collagen solution decreases, it can be clearly seen that the signal of the characteristic peak of the sample becomes stronger after the concentration is decreased, and the retention time always ranges between 6.3 and 6.7 min. The recovery rate of collagen on the fabric samples treated by simulated padding of collagen measured by this method is between 98% and 105%, and the recovery rate of collagen measured on the fabric samples treated by padding of collagen is between 97% and 102%, indicating that the method of the present invention can accurately measure the content of collagen on wool fabrics.

[0170] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for determining the collagen content in a fabric, characterized in that: The determination method comprises the following steps: S1, hydrolyzing the sample to be tested and adjusting the pH to 4-8 to obtain a mixed solution; S2, mixing the mixed solution with a derivatization reagent to obtain a solution to be determined; The derivatization reagent contains phenyl isothiocyanate and triethylamine; S3, injecting the solution to be tested into a high performance liquid chromatograph for gradient elution to determine hydroxyproline and its peak area, and calculating the content of collagen in the sample to be tested according to the standard curve equation; The chromatographic column used in the high performance liquid chromatography is a C18 reverse phase chromatographic column, and the mobile phase A in the eluent used in the gradient elution is acetonitrile-water solution, and the mobile phase B is acetonitrile-sodium acetate water solution.

2. The measuring method according to claim 1, characterized in that The step S1 comprises: hydrolyzing the sample to be tested and adjusting the pH to 6-8 to obtain a mixed solution.

3. The measuring method according to claim 1, characterized in that In step S1, the sample to be tested is selected from at least one of wool fabric, cashmere fabric and cotton fabric; Preferably, the reagent used for the hydrolysis is sulfuric acid; Preferably, the hydrolysis conditions include: temperature of 100-110°C and time of 18-22h; Preferably, the reagent used to adjust the pH is sodium hydroxide solution and / or potassium hydroxide solution.

4. The measuring method according to claim 1, characterized in that In step S2, relative to 1 g of the sample to be tested, the amount of phenyl isothiocyanate used is 0.35-0.6 mmol, and the amount of triethylamine used is 3.5-6 mmol; Preferably, the derivatization reagent further contains acetonitrile.

5. The measuring method according to any one of claims 1 to 4, characterized in that The determination method further comprises: before step S3, mixing the liquid to be determined with normal hexane for extraction, and taking the lower layer of liquid to perform the operation of step S3.

6. The measuring method according to any one of claims 1 to 4, characterized in that In step S3, the C18 reverse phase chromatography column is a LP-C18 reverse phase chromatography column; Preferably, the particle size of the C18 reverse phase chromatography column is 4.5-5.5 μm, the inner diameter of the chromatography column is 4.4-4.8 mm, and the length is 230-270 mm.

7. The measuring method according to any one of claims 1 to 4, characterized in that In step S3, the volume of acetonitrile and water in the acetonitrile-water solution is 3-5:1, the concentration of the sodium acetate aqueous solution in the acetonitrile-sodium acetate aqueous solution is 0.08-0.12 mol / L, and the volume of acetonitrile and sodium acetate aqueous solution is 1:9-19, preferably 1:10-15; Preferably, the elution process comprises: sequentially injecting mobile phase B, a mixture I of mobile phase B and mobile phase A, a mixture II of mobile phase B and mobile phase A, a mixture III of mobile phase B and mobile phase, a mixture IV of mobile phase B and mobile phase A, and mobile phase A; In the mixed liquid I, the volume ratio of the mobile phase B to the mobile phase A is 12.5-14:1; in the mixed liquid II, the volume ratio of the mobile phase B to the mobile phase A is 6.5-8:1; in the mixed liquid III, the volume ratio of the mobile phase B to the mobile phase A is 5-6.5:1; in the mixed liquid IV, the volume ratio of the mobile phase B to the mobile phase A is 1.5-2.5:

1.

8. The measuring method according to any one of claims 1 to 4, characterized in that In step S3, the detector used in the determination process is an ultraviolet dual-wavelength detector, and the column temperature is 40-45°C; Preferably, the detection wavelength of the detector is 250-258 nm.

9. The measuring method according to any one of claims 1 to 4, characterized in that The method for calculating the content of collagen in the sample to be tested according to the standard curve equation comprises: The content of hydroxyproline in the sample to be tested is obtained according to the standard curve equation, and then the content of collagen in the sample to be tested is calculated according to formula (II): Wherein, Y is the amount of collagen in the sample to be tested, mg; X is the content of hydroxyproline in the sample to be tested, %; ρ1 is the concentration of hydroxyproline in the collagen solution to be tested, μg / mL; V1 is the volume of the collagen solution to be tested, mL; m1 is the mass of collagen, mg; and m is the mass of the sample to be tested, mg.

10. The measuring method according to claim 9, characterized in that The method for obtaining the content of hydroxyproline in the sample to be tested according to the standard curve equation comprises: calculating the concentration ρ of hydroxyproline in the solution to be tested by the standard curve, and then calculating the content of hydroxyproline in the sample to be tested according to formula (I); Wherein, X is the content of hydroxyproline in the sample to be tested, %; ρ is the concentration of hydroxyproline in the solution to be tested of the sample to be tested, μg / mL; V is the volume of the solution to be tested of the sample to be tested, mL.

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