A hair matrix reference material and its preparation method

Abstract

The present invention relates to the field of biological detection, and provides a method for preparing a hair matrix reference material. The steps include: mixing a matrix with a hydrogel to obtain ink, adding a drug solution to the ink and mixing uniformly to obtain printing ink, and performing 3D printing on the printing ink to obtain the hair matrix reference material. The present invention uses 3D printing technology to prepare the hair matrix reference material, and uniformly disperses the drug solution in the ink, which not only approximates the distribution state of the target drug in the hair of a real drug-involved human body, but also makes the concentration of the drug in the hair matrix reference material more definite. The ink prepared by the present invention defines the rheological characteristic parameters, making its physical properties closer to the hair samples to be analyzed in real cases. The matrix of the present invention includes not only natural hair matrix, but also artificial hair matrix, providing more choices for users with strict ethical requirements.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to a hair matrix reference material and a preparation method thereof. Background Art

[0002] Matrix reference materials are key reagents in forensic practice and research. Since hair samples are solid biological samples, they are affected by two processes of release and dissociation and one interference factor of complex and variable biological matrices during the analysis process, so the demand for matrix reference materials is particularly strong. The International Association of Hair Analysis (SoHT) recommends that hair matrix reference materials should be used in forensic practice or research. The hair matrix reference materials and preparation methods proposed by it are as follows: Cut the hair of healthy volunteers, after washing, soak it in an organic reagent containing the drug to be detected for 1-3 months, and then take it out and dry it for use. The main disadvantages of this method are: (1) The preparation time is too long; (2) The content of the target drug in the prepared hair matrix reference material is not fixed. To address the above disadvantages, currently, most forensic laboratories internationally use the external addition method to prepare temporary hair matrix reference materials. The specific preparation method is: Cut the hair of healthy volunteers, after washing, cut it into pieces or grind it into powder, add a specified amount of the target drug, and use it directly (such as the liquid chromatography-tandem mass spectrometry test method for 15 drugs and metabolites in hair in SF / Z JD0107025—2018). The main disadvantages of this method are: (1) Each laboratory prepares its own hair matrix without unity and weak standardization ability; (2) Manual preparation and immediate use require high experience and preparation ability of the preparation personnel, and the reproducibility is weak. The National Institute of Standards and Technology (NIST) in the United States early proposed a third hair matrix reference material and its preparation method. The specific steps are: Feed the wool-producing animals such as goats and alpacas with feed containing the target drug. After a period of breeding, shave the animal hair of the wool-producing animals as the hair (substitute) matrix reference material containing the target drug. The main disadvantages of this method are: (1) The matrix (animal hair) has a large difference in properties from human hair; (2) The production method violates animal ethics. Currently, the National Institute of Standards and Technology (NIST) in the United States has stopped the preparation and sales of related products.

[0003] Therefore, there is an urgent need for a hair matrix reference material and a preparation method thereof that are simple to prepare, easy to industrialize, and have unity. Summary of the Invention

[0004] The object of the present invention is to provide a hair matrix reference material and a preparation method thereof for the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention is to provide a preparation method of a hair matrix reference material, and the steps include: mixing the matrix with a hydrogel to obtain ink, adding a drug solution to the ink and mixing evenly to obtain printing ink, and performing 3D printing on the printing ink to obtain the hair matrix reference material.

[0007] Preferably, the matrix is a natural hair matrix or an artificial hair matrix.

[0008] More preferably, the preparation method of the natural hair matrix includes: selecting the black undyed and unpermed hair of healthy volunteers, successively performing impurity removal treatment, cleaning treatment, drying treatment and hydration treatment, and the matrix with a keratin mass fraction greater than 85% is the natural hair matrix.

[0009] Preferably, the impurity removal treatment includes: placing the black undyed and unpermed hair under a microscope and picking out obvious impurities;

[0010] The cleaning treatment includes: successively placing the black undyed and unpermed hair after the impurity removal treatment in a surfactant solution, deionized water and an acetone solution for cleaning several times;

[0011] The drying treatment includes: purging the black undyed and unpermed hair after the cleaning treatment with nitrogen;

[0012] The hydration treatment includes: placing the black undyed and unpermed hair after the drying treatment in a tris(hydroxymethyl)aminomethane hydrochloride solution, slowly heating to 80 °C and maintaining for 10 min, then continuing to heat to 90 °C at a rate of 1 °C / min, maintaining for 40 min, and stirring at a low speed for 7 h.

[0013] More preferably, by weight, the artificial hair matrix includes:

[0014]

[0015] Among them, the keratin is non-human-derived keratin.

[0016] More preferably, the keratin is sheep-derived keratin.

[0017] Preferably, in the ink, the mass ratio of the matrix to the hydrogel is (0.943 - 1.213):1.

[0018] Preferably, the steps further include: before adding the drug solution, performing a rheological property test on the ink; if the rheological property test is qualified, adding the drug solution; if the rheological property test is unqualified, adding a gelling agent to modify and optimize the ink, and then repeating the rheological property test.

[0019] More preferably, the qualified criteria for the rheological characteristics test include: the viscosity is greater than 100 Pa·s and less than 2000 Pa·s, the extrusion stress is greater than the yield stress, and the Young's modulus is greater than 10 kPa and less than 100 kPa.

[0020] More preferably, the gelling agent includes at least one of gelatin or collagen.

[0021] Preferably, the solute in the drug solution includes at least one of methamphetamine, amphetamine or diazepam; the solvent of the drug solution is at least one of alcohol solvents or ether solvents.

[0022] Preferably, the parameters of the 3D printing include: the ultraviolet light intensity is 375 mW / dm 2 , and the light transmission time per 100 mL layer is 240 s.

[0023] The second aspect of the present invention is to provide a hair matrix reference material prepared by the above preparation method.

[0024] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical effects:

[0025] The present invention uses 3D printing technology to prepare a hair matrix reference material, and uniformly disperses the drug solution in the ink, which not only approximates the distribution state of the target drugs in the real drug-involved human hair, but also makes the drug concentration in the hair matrix reference material more definite; the ink prepared by the present invention defines the rheological characteristic parameters, making its physical properties more similar to the hair samples to be analyzed in real cases; the matrix of the present invention not only has natural hair matrix, but also provides artificial hair matrix, providing more choices for users with strict ethical requirements. Description of the Drawings

[0026] Figure 1 It is a physical picture of real hair;

[0027] Figure 2 It is a physical picture of the hair matrix reference material prepared in an embodiment of the present invention. Detailed Embodiments

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0029] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it is not intended to limit the present invention.

[0031] Example 1

[0032] This example provides a hair matrix reference material containing methamphetamine and a preparation method thereof;

[0033] Collect 20 g of black unpermed and uncolored hair from 10 healthy volunteers. After cutting the above-mentioned hair into fragments about 3 mm long, mix them evenly. After impurity removal treatment, cleaning treatment, and drying treatment, extract at 50 °C for 12 hours according to the ratio of 2% hair, 45% urea, 2% SDS, and 0.5% mercaptoethanol; after the extraction is completed, centrifuge the reaction solution, and take the supernatant and place it in a dialysis bag. Start dialysis with deionized water at a ratio of 1:200. Replace the deionized water every 6 hours during dialysis. Take a small amount of dialysis solution during dialysis and determine the keratin content by freeze-drying. End dialysis when the keratin content is greater than 85%. Take the dialysis solution and place it in a dialysis bag, and bury it in polyvinyl alcohol powder for concentration. The concentrated matrix solution is refrigerated at 4 °C for standby;

[0034] Take a glycerol hydrogel (purchased from EFL Company), and mix it according to the ratio of biological matrix: hydrogel = 1.017:1 to prepare a bioink. Test the rheological characteristics of the bioink. The results show that the viscosity (vis) is about 1200 Pa·s, the extrusion stress is greater than the yield stress, and the Young's modulus is about 30 kPa, indicating that the bioink meets the requirements;

[0035] Accurately weigh 4.0 mg of methamphetamine and 4.0 mg of amphetamine reference material powder respectively, add them to a 10 mL volumetric flask, dissolve and make up the volume with methanol. Accurately pipette 1.0 mL of the methanol solution containing methamphetamine and amphetamine after mixing and dissolving, add 999 mL of methanol to dilute it 1000 times. Take 1.0 mL of the diluted solution and add it to the bioink, and mix it with magnetic stirring for 5 h to prepare a printing ink containing methamphetamine and amphetamine;

[0036] Use a thiol-ene reaction-based photocurable bio-3D printer, adjust the printing parameters to be able to print a 70-μm linear material. After pouring the printing ink containing methamphetamine and amphetamine into the printer, start printing. Collect the printed linear 3D printing finished product, and package and label it in portions of 200 mg each to obtain a hair matrix reference material containing methamphetamine.

[0037] The prepared hair matrix standard substance containing methamphetamine was tested for uniformity: the 1st, 11th, 21st, 31st, 41st, 51st, 61st, 71st, 81st and 91st hair matrix standard substances were packaged for uniformity test, and the analysis was carried out using the technical standard "SF / ZJD0107025-2018 Liquid chromatography-tandem mass spectrometry test method for 15 drugs and metabolites in hair"; the uniformity of the product was analyzed by one-way analysis of variance with reference to the "CNAS GL-003 Guidelines for the Evaluation of Uniformity and Stability of Proficiency Testing Samples"; the product value was 0.2ng / mg±0.003ng / mg; the F value was less than the critical value Fα of the significance level α (α=0.05), and the product met the indicator target.

[0038] Example 2

[0039] This embodiment provides a hair matrix standard substance containing diazepam and a preparation method thereof;

[0040] Silk protein (hydrolyzed silk, CAS number: 96690-41-4), keratin (α-keratin, CAS number: 69430-36-0), palmitic acid (ethyl palmitate, CAS number: 628-97-7), stearic acid (stearic acid-13C18, CAS number: 287100-83-8), and melanin (melanin, CAS number: 8049-97-6) were purchased, and the above materials were mixed in a ratio of silk protein: keratin: palmitic acid: stearic acid: melanin = 31%: 57%: 3%: 2%: 2% to obtain an artificial hair matrix, and commercially available glycerol hydrogel was added and mixed in a ratio of biological matrix: hydrogel = 0.95: 1, and gelatin was added dropwise until the rheological characteristics of the mixed biological ink reached the following parameters: viscosity (vis) was about 800 Pa.s, extrusion stress was greater than yield stress, and Young's modulus was about 15 kPa.

[0041] Take 0.1 mL of commercially available diazepam alcohol solution standard substance (1 mg / mL), add it to a 10 mL volumetric flask, use methanol to make up to volume and dissolve it, accurately transfer 1.0 mL of the mixed and dissolved diazepam-containing methanol solution, add it to the bio-ink, and use magnetic stirring to mix for 3 hours to form a printing ink containing diazepam.

[0042] Using a thiol-ene reaction-based photocurable biological 3D printer, the printing parameters were adjusted to be able to print 70 μm linear materials, and printing began after the printer was filled with printing ink containing diazepam. The printed linear 3D printed products were collected, packaged and labeled in portions of 100 mg to obtain a hair matrix standard substance containing diazepam.

[0043] In summary, the present invention uses 3D printing technology to prepare a hair matrix reference material, dispersing the drug solution evenly in the ink, which not only approximates the distribution state of the target drugs in real drug-involved human hair, but also makes the drug concentration in the hair matrix reference material more definite; the ink prepared by the present invention defines the rheological characteristic parameters, making its physical properties closer to the hair samples to be analyzed in real cases; the matrix of the present invention includes not only natural hair matrix, but also artificial hair matrix, providing more choices for users with strict ethical requirements.

[0044] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be realized that all equivalent replacements and obvious changes made by using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of a hair matrix reference material, characterized in that the steps include: After mixing the matrix with the hydrogel to obtain ink, adding a drug solution to the ink and mixing evenly to obtain printing ink, and performing 3D printing on the printing ink to obtain the hair matrix reference material; The matrix is a natural hair matrix or an artificial hair matrix; The preparation method of the natural hair matrix includes: selecting the black undyed and unpermed hair of healthy volunteers, and successively performing impurity removal treatment, cleaning treatment, drying treatment and hydration treatment. The matrix with a keratin mass fraction greater than 85% is the natural hair matrix; By weight, the artificial hair matrix includes: Silk protein 27.3 - 32.7 parts Keratin 52.5 - 61.5 parts Palmitic acid 2.4 - 3.6 parts Stearic acid 1.6 - 2.4 parts Melanin 2.0 - 3.0 parts; Among them, the keratin is non-human-derived keratin; The solute in the drug solution includes at least one of methamphetamine, amphetamine or diazepam; the solvent of the drug solution is at least one of alcohol solvents or ether solvents.

2. The preparation method according to claim 1, characterized in that In the ink, the mass ratio of the matrix to the hydrogel is (0.943 - 1.213):

1.

3. The preparation method according to claim 1, characterized in that The steps further include: before adding the drug solution, performing a rheological property test on the ink; if the rheological property test is qualified, adding the drug solution; if the rheological property test is unqualified, adding a gelling agent to modify and optimize the ink, and then repeating the rheological property test.

4. The preparation method according to claim 3, characterized in that The qualified criteria for the rheological property test include: the viscosity is greater than 100 Pa·s and less than 2000 Pa·s, the extrusion stress is greater than the yield stress, and the Young's modulus is greater than 10 kPa and less than 100 kPa.

5. The preparation method according to claim 3, characterized in that The gelling agent includes at least one of gelatin or collagen.

6. A hair matrix reference material prepared by the preparation method according to any one of claims 1 - 5.

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