CN118209651A - High performance liquid chromatography tandem mass spectrometry detection method for 10 peptides in cosmetics - Google Patents

Abstract

The invention discloses a high performance liquid chromatography tandem mass spectrometry detection method for 10 peptides in cosmetics, which comprises the following steps: firstly, precisely weighing standard substances of 10 peptides respectively, and preparing a single standard stock solution; precisely weighing a blank sample, and preparing a blank matrix extracting solution; preparing a matrix standard series solution; precisely weighing a sample, adding acetonitrile ammonia water solution for dissolution, swirling, uniformly dispersing, performing ultrasonic extraction, cooling to room temperature, fixing the volume, centrifuging, and taking supernatant as a sample solution for later use; and thirdly, detecting by adopting a high performance liquid chromatography-mass spectrometry system. The general analysis method of 10 peptides such as snake venom peptide in cosmetics is favorable for rapidly coping with the supervision of the addition of peptide components of the existing commercial products. The method can be used for measuring the polypeptide-containing raw materials in the aqueous agent, cream emulsion and gel cosmetics, and is simple to operate, rapid in analysis, strong in specificity and high in separation degree.

Description

High performance liquid chromatography tandem mass spectrometry detection method for 10 peptides in cosmetics

Technical Field

The invention relates to the technical field of detection of peptides in cosmetics, in particular to a high performance liquid chromatography tandem mass spectrometry detection method of 10 peptides in cosmetics.

Background

Peptides are a class of compounds formed by amino acids linked by peptide bonds, and are fragments of proteins with biological functions. Due to the diversity of amino acid types and the high degree of freedom of the spatial conformation of peptide chains, the peptides have the diversity of biological actions, the bioactive peptides have become component-dependent anti-aging pets in recent years, and the development and application of the bioactive peptides opens up a new field for personal care products. The method mainly researches four peptide components which are common in cosmetics, namely snake venom peptide, general oligopeptides (dipeptide-2), acetyl oligopeptides (including acetyl hexapeptide-1 and acetyl dipeptide-1), palmitoyl oligopeptides (palmitoyl pentapeptide-4, palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl hexapeptide-12 and palmitoyl tripeptide-5).

The snake venom peptide is a synthetic tripeptide, named by mimicking the mechanism of action of the snake venom WAGLERINI. Clinical trials show that the snake venom peptide can reduce the generation of wrinkles by inhibiting muscle contraction, and has excellent smoothness and rapid wrinkle removal performance.

Acetyl oligopeptides are obtained by adding acetyl groups on the basis of peptides, wherein acetyl hexapeptide-1 and acetyl dipeptide-1 are raw materials of acetyl oligopeptides with higher frequency. The peptide component has the effect of inhibiting neuron transmission and becomes a new pet of anti-wrinkle components.

Palmitoyl oligopeptides, which can emit or mimic signals during extracellular matrix protein synthesis, regulate skin tissue protein turnover, increase synthesis of extracellular matrix such as collagen, elastin and proteoglycan, tighten skin, and prevent aging by promoting extracellular matrix synthase and inhibiting metalloprotease. Wherein palmitoyl tetrapeptide-7 can stimulate the production of laminin IV and V, and collagen II and reduce the production of interleukin (IL-6), eliminate inflammation, and increase skin elasticity; palmitoyl tripeptide-1 is a collagen-newer messenger peptide with activity comparable to retinoic acid but without eliciting irritation, palmitoyl pentapeptide-3 is capable of stimulating the production of elastin, fibronectin, glycosaminoglycans and collagen (particularly types i, iii and iv), supporting extracellular matrix protein networks, and promoting wound healing. The other 3 palmitoyl oligopeptides also play roles in reducing matrix metalloproteinase or increasing collagen synthase activity through different ways, promoting synthesis of collagen or elastin, and are mainly used in anti-aging personal care products.

At present, detection methods for the snake venom peptide, the dipeptide-2, the acetyl hexapeptide-1, the palmitoyl pentapeptide-3, the palmitoyl tripeptide 38, the palmitoyl tripeptide-1, the palmitoyl tetrapeptide-7, the acetyl dipeptide-1 cetyl ester, the palmitoyl hexapeptide-12 and the palmitoyl tripeptide-5 in cosmetics are not reported in the literature.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art and provides a high performance liquid chromatography tandem mass spectrometry detection method for 10 peptides in cosmetics.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

The invention provides a high performance liquid chromatography tandem mass spectrometry detection method for 10 peptides in cosmetics, which comprises the following steps:

Precisely weighing standard substances of snake venom peptide, dipeptide-2, acetyl hexapeptide-1, palmitoyl pentapeptide-3, palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl hexapeptide-12 and palmitoyl tripeptide-5 respectively, dissolving the standard substances by using a solvent, and fixing the volume to obtain a single standard stock solution;

precisely weighing a blank sample, adding an extracting solution, and preparing a blank matrix extracting solution;

Precisely sucking the single standard stock solution respectively, and diluting the blank matrix extracting solution to obtain a matrix standard intermediate solution;

Precisely measuring the matrix standard intermediate solutions respectively, and preparing a matrix standard series solution by using the blank matrix extracting solution;

precisely weighing a sample, adding an acetonitrile-ammonia solution for dissolution, swirling, uniformly dispersing, performing ultrasonic extraction, cooling to room temperature, fixing the volume by using the acetonitrile-ammonia solution, centrifuging, and taking supernatant as a sample solution for later use;

and thirdly, detecting by adopting a high performance liquid chromatography-mass spectrometry system.

In the first step, the solvent for dissolving the standard substances of the snake venom peptide, the dipeptide-2 and the acetyl hexapeptide-1 is water.

In the first step, the palmitoyl pentapeptide-3 and palmitoyl hexapeptide-12 standard substances are dissolved by formic acid, and then dissolved by methanol and the volume is fixed.

Further, in the first step, the solvent for dissolving the palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 standard is methanol.

Further, in the second step, the acetonitrile ammonia solution comprises acetonitrile and 0.1v/v% ammonia solution in a volume ratio of 1:1.

Further, in the second step, vortex is carried out for 30-60s; ultrasonic extracting for 20-30min; centrifuging at 10000-15000r/min for 5-10min.

Further, in the third step, the chromatographic conditions are as follows:

Chromatographic column: CAPCELLPAKADME chromatographic columns, 150 mm. Times.2.1 mm,2.0 μm; mobile phase: a is 0.1v/v% formic acid aqueous solution, B is acetonitrile; flow rate: 0.15-0.25mL/min; column temperature: 25-30 ℃; sample injection amount: 1-5 mu L; gradient elution.

Further, the gradient elution procedure was as follows:

Further, in the third step, the mass spectrum conditions are:

Ion source: electrospray ion source (ESI source); capillary voltage: 3500V; cracking voltage: 380V; temperature of air curtain: 250 ℃; air curtain air flow rate: 11L/min; atomizer pressure: 20psi; sheath temperature: 250 ℃; sheath air flow rate: 14L/min; scanning mode: scanning positive ions; monitoring mode: positive ion multiple reaction monitoring mode (MRM).

Further, the monitoring ion pairs and related parameters are set as follows:

Compared with the prior art, the invention has the following technical effects:

At present, the national standard and the cosmetic safety technical Specification (2015 edition) are not accepted about the content measurement standard of the peptides in the cosmetics, and a large amount of supervision blank exists. The method can be used for measuring the polypeptide-containing raw materials in the aqueous agent, cream emulsion and gel cosmetics, and is simple to operate, rapid in analysis, strong in specificity and high in separation degree.

Drawings

FIG. 1 is a graph of multi-reaction monitoring extraction ions of a standard solution of snake venom peptide;

FIG. 2 is a graph of multi-reaction monitoring extraction ions for dipeptide-2 standard solutions;

FIG. 3 is a graph of the multi-reaction monitoring extraction ions of an acetylhexapeptide-1 standard solution;

FIG. 4 is a graph of the multi-reaction monitoring extraction ions of palmitoyl pentapeptide-3 standard solution;

FIG. 5 is a graph of the multi-reaction monitoring extraction ions of palmitoyl tripeptide 38 standard solution;

FIG. 6 is a graph of the multi-reaction monitoring extraction ions of palmitoyl tripeptide-1 standard solution;

FIG. 7 is a graph of the multi-reaction monitoring extraction ions of palmitoyl tetrapeptide-7 standard solution;

FIG. 8 is a graph of the multi-reaction monitoring extraction ions of an acetyl dipeptide-1 standard solution;

FIG. 9 is a graph of the multi-reaction monitoring extraction ions of cetyl palmitoyl hexapeptide-12 standard solution;

FIG. 10 is a graph of the multi-reaction monitoring extraction ions of palmitoyl tripeptide-5 standard solution.

Detailed Description

The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

Example 1

Instrument and reagent:

Agilent6495 high performance liquid chromatograph-mass spectrometer (Agilent company, usa); sartoriusCP224S and 225D-1CN electronic balances (Sidoris, germany); 5800 ultrasonic apparatus (Branson Corp., U.S.); MS3 vortex mixer (IKA company, germany); 5810R-type tabletop centrifuge (Eppendof, germany); milli-QREFERENCEA + type ultra-pure water instrument (Millipore Co., U.S.A.).

The purity of the snake venom peptide standard substance is 100 percent (mass fraction, m/m, dou Yunxi chemical industry Co., ltd.); dipeptide-2 standard, 99% purity (mass fraction, m/m, bi de medical); acetyl hexapeptide-1 standard with a purity of 98% (mass fraction, m/m, microphone); palmitoyl pentapeptide-3, 100% pure (mass fraction, m/m, shanghai An Spectrometry laboratory technologies Co., ltd.); palmitoyl tripeptide 38, purity 98.17% (mass fraction, m/m, dou Yunxi chemical Co., ltd.); palmitoyl tripeptide-1, 100% pure (mass fraction, m/m, shanghai An Spectrometry laboratory technologies Co., ltd.); palmitoyl tetrapeptide-7, 100% pure (mass fraction, m/m, shanghai An Spectrometry laboratory technologies Co., ltd.); acetyl dipeptide-1 cetyl ester with a purity of 99.38% (mass fraction, m/m, dou Yunxi chemical Co., ltd.); palmitoyl hexapeptide-12, 100% pure (mass fraction, m/m, shanghai An Spectrometry laboratory technologies Co., ltd.); palmitoyl tripeptide-5, 100% pure (mass fraction, m/m, shanghai An Spectrometry laboratory technologies Co., ltd.); acetonitrile, formic acid, methanol, ammonia (chromatographic purity, merk, germany); the water is ultrapure water.

The embodiment provides a high performance liquid chromatography tandem mass spectrometry detection method for 10 peptides in cosmetics, wherein the 10 peptides are snake venom peptide, dipeptide-2, acetyl hexapeptide-1, palmitoyl pentapeptide-3, palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl hexapeptide-12 and palmitoyl tripeptide-5:

1 reagent material

Except for other regulations, the reagents used in this example were all analytically pure and above, and water was primary water meeting the GB/T6682 regulations.

Acetonitrile, chromatographically pure.

1.2 Formic acid, chromatographically pure.

1.3 Methanol, chromatographically pure.

1.4 Ammonia water, chromatographically pure.

1.40.1V/v% formic acid solution: taking 1.0mL of formic acid (1.2), adding water to 1000mL, and uniformly mixing to obtain the final product.

1.50.1V/v% aqueous ammonia solution: taking 1.0mL of ammonia water (1.4), adding water to 1000mL, and uniformly mixing to obtain the product.

1.6 Acetonitrile ammonia solution: 500mL of acetonitrile is taken, 0.1v/v% ammonia water solution (1.5) to 1000mL is added, and the mixture is evenly mixed, thus obtaining the catalyst.

1.7 Standard: the purity of the snake venom peptide, dipeptide-2, acetyl hexapeptide-1, palmitoyl pentapeptide-3, palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl hexapeptide-12 and palmitoyl tripeptide-5 standard substances is more than or equal to 98 percent. The Chinese name, english name, CAS number, molecular formula, relative molecular mass, and structural formula of the standard are shown in Table 1 below.

TABLE 1

1.8 Single standard stock solution: 10mg (0.00001 g accurate) of snake venom peptide, dipeptide-2 and acetyl hexapeptide-1 standard (1.7) are weighed, respectively placed in 10mL brown volumetric flasks, dissolved with water, fixed to scale and shaken well. As single standard stock solutions of snake venom-like peptides, dipeptide-2, acetyl hexapeptide-1. 10mg (0.00001 g accurate) of palmitoyl pentapeptide-3 and palmitoyl hexapeptide-12 standard (1.7) are weighed, placed in 10mL brown volumetric flasks respectively, 1mL formic acid (1.2) is added for dissolution, methanol (1.3) is used for dissolution, the volume is fixed to the scale, and shaking is carried out uniformly. As single standard stock solutions of palmitoyl pentapeptide-3, palmitoyl hexapeptide-12. 10mg (accurate to 0.00001 g) of palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 standard (1.7) were weighed, placed in 10mL brown volumetric flasks, dissolved in methanol (1.3) and scaled up, and shaken well. As single standard stock solutions of palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5. The mass concentration of the single standard stock solution is 1000mg/L. Placing in a refrigerator at-18deg.C, and storing in dark place.

It is to be noted that:

Through a large number of tests in the selection of the constant volume solvent of the standard substance, the snake venom peptide, the dipeptide-2 and the acetyl hexapeptide-1 are found to be directly dissolved by water, so that the solubility of the target compound is good, and the peak shape of the target compound is symmetrical;

through a large number of tests in the selection of the constant volume solvent of the standard substance, the palmitoyl pentapeptide-3 and palmitoyl hexapeptide-12 are dissolved by using a small amount of formic acid, and then diluted by methanol for constant volume, so that the solubility of the target compound is good, and the peak shape of the target compound is symmetrical;

A large number of tests prove that the palmitoyl tripeptide 38, the palmitoyl tripeptide-1, the palmitoyl tetrapeptide-7, the acetyl dipeptide-1 cetyl ester and the palmitoyl tripeptide-5 are dissolved by methanol, so that the solubility of the target compound is good, and the peak shape of the target compound is symmetrical.

2 Instrument and apparatus

2.1 High performance liquid chromatography-triple quadrupole mass spectrometer.

2.2 Analytical balance: the sensing amount is 0.0001g and 0.00001g.

2.3 Ultrasonic cleaner.

2.4 Vortex mixer.

2.5 High speed centrifuge.

3 Preparation and preservation of samples

The sample should be stored according to the storage conditions identified by the tag. Before sampling, the integrity of the seal should be checked, the properties and characteristics of the sample should be observed, and the sample should be mixed well. After opening the package, the part to be assayed should be taken out as soon as possible for analysis, and after sampling, the sample should be stored in a sealed state.

4 Analysis step

4.1 Blank matrix extract

A blank sample (0.2 g, accurate to 0.0001 g) was weighed and placed in a 20mL cuvette with plug, and treated in the same manner as the sample (4.4) from "15 mL with acetonitrile aqueous ammonia solution (1.6)" to obtain a blank matrix extract.

4.2 Standard intermediate solution for matrix

Accurately sucking 0.1-10 mL of single standard stock solution (1.8), diluting to scale with blank matrix extractive solution (4.1), and shaking to obtain matrix standard intermediate solution with concentration of 10 mg/L.

4.3 Matrix Standard series solutions

Respectively precisely measuring a proper amount of matrix standard intermediate solution (4.2), and preparing 50, 100, 150, 200, 250, 500 and 1000 mug/L matrix standard series solutions (the concentration range can be adjusted according to actual conditions) by using blank matrix extracting solution (4.1). The standard matrix series solution should be prepared on site.

4.4 Sample treatment

Weighing 0.2g (accurate to 0.0001 g) of the sample, placing in a 20mL colorimetric tube with a plug, adding 15mL of acetonitrile ammonia solution (1.6), swirling for 30s, dispersing uniformly, performing ultrasonic extraction for 20min, cooling to room temperature, fixing the volume to a scale with the acetonitrile ammonia solution (1.6), centrifuging for 5min at a rotating speed of 10000r/min, and taking the supernatant as a sample solution for standby.

It is to be noted that:

In the test, the extraction recovery rates of all target compounds are compared in a blank labeling mode by taking water, 50v/v% methanol solution, 50v/v% acetonitrile solution and acetonitrile with 0.1v/v% ammonia water (1:1) as extraction solvents, and the extraction efficiency is highest when the acetonitrile with 0.1v/v% ammonia water (1:1) is adopted as the extraction solvent, and the recovery rates are all over 85% when other solutions such as water are adopted as the extraction solvents. The snake venom peptide, dipeptide-2 and acetyl hexapeptide-1 are not affected by pH value, the recovery rate can reach more than 85% in 50v/v% acetonitrile solution and acetonitrile/0.1 v/v% ammonia water (1:1), but the recovery rates of 10 peptides such as the snake venom peptide in water and 50% methanol solution are poor, and the recovery rates are all lower than 50%. Therefore, acetonitrile/0.1 v/v% aqueous ammonia (1:1), i.e., acetonitrile aqueous ammonia solution (1.6) was finally selected as the extraction solvent for the sample.

5. Detection by high performance liquid chromatography-mass spectrometry system

5.1 Chromatographic conditions

Chromatographic column: a senior hall CAPCELL PAKADME column (150 mm x 2.1mm,2.0 μm);

mobile phase: a is 0.1v/v% formic acid aqueous solution, B is acetonitrile (1.1);

flow rate: 0.15mL/min;

Column temperature: 25 ℃;

sample injection amount: 1 μl;

gradient elution: gradient elution procedure is shown in Table 2

TABLE 2

It should be noted that: through a large number of tests in the selection of a mobile phase, when 0.1v/v% formic acid aqueous solution and acetonitrile are used as the mobile phase, the obtained 10 peptides have symmetrical peak shapes and stable base lines. The total ion flow diagram of 10 polypeptides can be completely isolated.

5.2 Mass Spectrometry conditions

Ion source: electrospray ion source (ESI source); capillary voltage: 3500V; cracking voltage: 380V; temperature of air curtain: 250 ℃; air curtain air flow rate: 11L/min; atomizer pressure: 20psi; sheath temperature: 250 ℃; sheath air flow rate: 14L/min; scanning mode: and (5) positive ion scanning.

Monitoring mode: positive ion multiple reaction monitoring mode (MRM), monitoring ion pairs and related parameter settings are shown in table 3.

TABLE 3 Table 3

* The ions were quantified as recommended.

6. And (3) carrying out linear regression analysis by taking the peak area as an ordinate (y) and the concentration as an abscissa (x, mug/L) to obtain a linear equation.

6.1 Class of snake venom peptides and the like 10 peptide cream base standard curves are shown in Table 4 below:

TABLE 4 Table 4

6.2 Class snake venom peptides and the like 10 peptide gel matrix standard curves are shown in Table 5 below:

TABLE 5

6.3 Class of snake venom peptides and the like 10 peptides water based standard curves are shown in Table 6 below:

TABLE 6

Example 2

Labeling and measuring positive samples (cream base): positive cosmetic samples containing palmitoyl tetrapeptide-7, palmitoyl tripeptide-1 and palmitoyl tripeptide-5 are selected, subjected to a positive labeling test, added with a component to be detected in an amount equivalent to that of the samples, and the recovery rate results are determined.

Under the condition of high performance liquid chromatography-mass spectrometry (same as in example 1), a standard working curve solution and a sample solution are taken and respectively injected (same as in example 1), and the contents of 10 peptides such as snake venom peptides and the like in the sample solution are obtained from the standard curve. The response value of 10 peptides such as snake venom peptide in the sample solution should be within the linear range of the standard curve, and if the response value exceeds the linear range, the extract solution should be diluted and then the amount of the extract solution should be measured or increased for re-detection.

The result is calculated according to formula (1):

Wherein:

Omega-mass fraction of 10 peptides such as snake venom peptide in the sample, mg/kg;

rho-mass concentration of 10 peptides such as snake venom peptides in the sample solution, μg/L;

V-sample constant volume, mL;

m-sample sampling amount, g;

D-dilution factor (1 if undiluted).

Sample solution treatment of positive sample adding standard recovery rate:

Respectively weighing 0.1g (accurate to 0.0001 g) of each of a palmitoyl tetrapeptide-7 positive sample, a palmitoyl tripeptide-1 positive sample and a palmitoyl tripeptide-5 positive sample, respectively precisely adding palmitoyl tetrapeptide-7, palmitoyl tripeptide-1 or palmitoyl tripeptide-5 standard substances which are equivalent to the amounts in the samples, placing the samples in a 20mL colorimetric tube with a plug, adding 15mL of 50% acetonitrile ammonia solution, swirling for 30s, uniformly dispersing, ultrasonically extracting for 20min, placing the samples to room temperature, fixing the volume to a scale by using 50% acetonitrile ammonia solution, centrifuging for 5min at a rotating speed of 10000r/min, and taking the supernatant as a sample solution for standby. Each labeling level was repeated 6 times and recovery and relative standard deviation (n=6) were calculated, and the results are shown in table 7 below.

TABLE 7

Example 3

Blank sample (cream base) labelling assay: taking blank samples of 10 peptides such as non-detected snake venom peptides, performing blank labeling test, and measuring recovery rate.

Sample solution treatment of blank sample and standard recovery rate:

Respectively weighing 0.1g (accurate to 0.0001 g) of samples of 10 peptides such as brown undetected snake venom peptides, precisely adding a certain amount of mixed standard solution of the snake venom peptides, placing into a 20mL colorimetric tube with a plug, adding 15mL of 50% acetonitrile ammonia solution, swirling for 30s, dispersing uniformly, ultrasonically extracting for 20min, cooling to room temperature, fixing volume to scale with 50% acetonitrile ammonia solution, centrifuging at 10000r/min for 5min, and taking supernatant as a sample solution for standby. Each labeling level was repeated 6 times and recovery and relative standard deviation (n=6) were calculated and the results are shown in table 8.

Under the condition of high performance liquid chromatography-mass spectrometry (same as in example 1), a standard working curve solution and a sample solution are taken and respectively injected (same as in example 1), and the contents of 10 peptides such as snake venom peptides and the like in the sample solution are obtained from the standard curve. The response value of 10 peptides such as snake venom peptide in the sample solution should be within the linear range of the standard curve, and if the response value exceeds the linear range, the extract solution should be diluted and then the amount of the extract solution should be measured or increased for re-detection.

The result is calculated according to formula (1):

Wherein:

Omega-mass fraction of 10 peptides such as snake venom peptide in the sample, mg/kg;

rho-mass concentration of 10 peptides such as snake venom peptides in the sample solution, μg/L;

V-sample constant volume, mL;

m-sample sampling amount, g;

D-dilution factor (1 if undiluted).

TABLE 8

Example 4

The measurement of 10 peptides such as snake venom peptides was performed on 10 batches of common skin care cosmetics (three matrix types including cream, gel and water), and the measurement results of the samples are shown in Table 9.

TABLE 9

As described above, this test measures 10 total batches of cosmetics labeled with peptides, 6 batches of which detected the labeled ingredients. Another 3 batches of samples were labeled with palmitoyl hexapeptide-1, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester, respectively, but none was actually detected. The actual detection condition shows that the condition that the label is inconsistent with the actual addition exists in the peptide addition of cosmetics on the current market still attracts importance to the cosmetic supervision department, and the continuous attention to the use purpose and the safe use method of the bioactive peptide is recommended. Meanwhile, the study of enterprises on the cosmetic label management method is enhanced, and the product label identification is standardized.

At present, the national standard and the cosmetic safety technical Specification (2015 edition) are not accepted about the content measurement standard of peptides in cosmetics, a large number of supervision blanks exist, and the established general analysis method of 10 peptides such as snake venom peptides in cosmetics is beneficial to rapidly coping with supervision of the addition of peptide components of the existing commercial products. The method can be used for measuring polypeptide-containing raw materials in water aqua, cream emulsion and gel cosmetics.

The foregoing is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the embodiments and scope of the present invention, and it should be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the teachings and illustrations of the present invention, and that such variations are intended to be included within the scope of the present invention.

Claims (8)

1. The high performance liquid chromatography tandem mass spectrometry detection method for 10 peptides in cosmetics is characterized by comprising the following steps:

Precisely weighing standard substances of snake venom peptide, dipeptide-2, acetyl hexapeptide-1, palmitoyl pentapeptide-3, palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl hexapeptide-12 and palmitoyl tripeptide-5 respectively, dissolving the standard substances by using a solvent, and fixing the volume to obtain a single standard stock solution;

precisely weighing a blank sample, adding an extracting solution, and preparing a blank matrix extracting solution;

Precisely sucking the single standard stock solution respectively, and diluting the blank matrix extracting solution to obtain a matrix standard intermediate solution;

Precisely measuring the matrix standard intermediate solutions respectively, and preparing a matrix standard series solution by using the blank matrix extracting solution;

precisely weighing a sample, adding an acetonitrile-ammonia solution for dissolution, swirling, uniformly dispersing, performing ultrasonic extraction, cooling to room temperature, fixing the volume by using the acetonitrile-ammonia solution, centrifuging, and taking supernatant as a sample solution for later use;

and thirdly, detecting by adopting a high performance liquid chromatography-mass spectrometry system.

2. The method for detecting 10 peptides in cosmetics by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the first step, the solvent for dissolving the standard substances of snake venom peptide, dipeptide-2 and acetyl hexapeptide-1 is water.

3. The method for detecting 10 peptides in cosmetics by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the first step, formic acid is used for dissolving palmitoyl pentapeptide-3 and palmitoyl hexapeptide-12 standard substances, and then methanol is used for dissolving and fixing the volume.

4. The method for detecting 10 peptides in cosmetics by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the first step, the solvent for dissolving the standard substance of palmitoyl tripeptide 38, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 is methanol.

5. The method for detecting 10 peptides in cosmetics by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the second step, the acetonitrile ammonia solution comprises acetonitrile and 0.1v/v% ammonia solution in a volume ratio of 1:1.

6. The method for detecting 10 peptides in cosmetics by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the second step, vortexing is performed for 30-60s; ultrasonic extracting for 20-30min; centrifuging at 10000-15000r/min for 5-10min.

7. The method for detecting 10 peptides in cosmetics by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the third step, the chromatographic conditions are as follows:

Chromatographic column: CAPCELL PAKADME chromatographic columns, 150 mm. Times.2.1 mm,2.0 μm; mobile phase: a is 0.1v/v% formic acid aqueous solution, B is acetonitrile; flow rate: 0.15-0.25mL/min; column temperature: 25-30 ℃; sample injection amount: 1-5 mu L; gradient elution.

8. The method for detecting 10 peptides in cosmetics by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein in the third step, mass spectrometry conditions are as follows:

Ion source: electrospray ion source (ESI source); capillary voltage: 3500V; cracking voltage: 380V; temperature of air curtain: 250 ℃; air curtain air flow rate: 11L/min; atomizer pressure: 20psi; sheath temperature: 250 ℃; sheath air flow rate: 14L/min; scanning mode: scanning positive ions; monitoring mode: positive ion multiple reaction monitoring mode (MRM).