CN111679028B - High performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics - Google Patents
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Abstract
The invention discloses a high performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics, wherein the four peptides comprise L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8, and the method comprises the following steps: weighing a cosmetic sample, placing the cosmetic sample in a clean centrifugal tube, adding water for dissolving, uniformly mixing, whirling, centrifuging, taking a proper amount of supernatant, and filtering by using a filter membrane to obtain filtrate as sample working solution; step two, preparing a series of mixed standard solutions; and step three, respectively injecting the series of mixed standard solutions and the sample working solution into a high performance liquid chromatography tandem mass spectrometer for detection and analysis. In the high performance liquid chromatography tandem mass spectrometry detection method, water is used as a solvent of a standard substance, the peak shape of a target object is good, and the response is high; water is used as an extraction solvent of the cosmetic sample, the extraction effect is good, and the recovery rate is high; the four polypeptides obtained by using 0.1% acetic acid water solution and acetonitrile as mobile phases have symmetrical peak shapes and stable base lines.
Description
Technical Field
The invention relates to the technical field of analysis of functional components of cosmetics, in particular to a high performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics.
Background
Peptide is a protein fragment composed of amino acids with certain sequences bonded through amido bonds. Peptides with biological activity are often added into cosmetics for use as raw material drugs. L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8 are all common peptides which are adopted frequently at present.
L-Carnosine (L-Carnosine), the Chinese alias N-beta-alanyl-L histidine, is a natural anti-aging and antioxidant. Glutathione (glutamhiene), the name of which is 5-L-glutamyl-L-cysteinylglycine, can combine free radicals and peroxides in metabolism, prevent lipid peroxidation of mitochondria and the like, and play a role in protecting cells against aging. Acetyl tetrapeptide-5 (eyeseriyl), an alternative name in chinese: the N-acetyl-beta-alanyl-L-histidyl-L-seryl-L-histidine can rapidly remove eye bags, dark circles and the like. Acetyl Hexapeptide-8 (Acetyl Hexapeptide-8), also known as: acetyl hexapeptide-3, commonly known as ayurrelin. Can inhibit excessive release of catecholamine and acetylcholine from skin, relax facial muscle, and smooth fine wrinkles.
At present, the detection method of the polypeptide is mostly used in the fields of food, medicine and clinic. Such as Dailuo Yao [1] The carnosine and the Wangbaoping in the beef are determined by adopting an ion exchange chromatography method [2] The high-efficiency capillary electrophoresis method is adopted to measure the content of carnosine in the raw material medicine, and the method is Zhouxinyue [3] The content of the hexadactylon polypeptide in the traditional Chinese medicinal materials, namely the yangqing, is determined by adopting a high-efficiency capillary electrophoresis method [4] And the method for detecting the carnosine in the tissues by adopting the pre-column derivatization high performance liquid chromatography, and the polypeptide detection method in the fields of food and medicines is mostly liquid chromatography. At present, the detection method of the polypeptide in the cosmetics is rarely reported at home and abroad, and the independent determination of the acetyl hexapeptide-8 in the cosmetics by adopting the high performance liquid chromatography is reported in the literature [5] Carnosine, and pharmaceutical compositions containing them [6] And glutathione [7] No high performance liquid chromatography mass spectrometry detection method is available.
Reference documents:
[1] comparison research on determination of carnosine content in beef by using reversed phase high performance liquid chromatography and ion exchange chromatography [ J ] J of pharmaceutical analysis, 2019,39 (5): 805-812.
[2] Wangbaoping, huqingyu, yangyang, etc. research on the method for measuring the carnosine content by using a high-efficiency capillary electrophoresis method [ J ]. J.J.J.Med.Med.Med.Med.Med.Med.Med.Med.Med.No. 2011,6 (12): 1040-4041.
[3] Zhou Xin Yue, zhang Shiqi, linlu, etc. high efficiency capillary electrophoresis process to separate and detect six kinds of Gekko Swinhonis polypeptide J simultaneously, the scientific technology in the world-discussion of modernization of traditional Chinese medicine-chemical composition identification 2569-2575.
[4] The o-phthalaldehyde ex-situ derivatization high performance liquid chromatography is used for measuring the carnosine content [ J ] in tissues, china journal of Biochemical medicine, 2009,30 (4): 258-260.
[5] The content of acetyl hexapeptide-8 in the cosmetics is determined by high performance liquid chromatography (J), guangdong chemical industry, 2019,46 (20): 110-112).
[6] The carnosine content in the cosmetics is measured by high performance liquid chromatography of Zhou shou Yu, huogang, deng schoyang [ J ]. Scientific and technological Square, 2017,40 (4): 23-26.
[7]GOTTIL V.ANDRISANOL V.CAVRINI,A etc.Determination of Glutathione in Pharmaceuticals and Cosmetics by HPLC with UV and Fluorescence Detection[J].Chromatographia,1994,39(1/2):23-28.
Disclosure of Invention
The invention provides a high performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics, aiming at overcoming the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a high performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics, wherein the four peptides are L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8, and the method comprises the following steps:
step one, pretreatment of a cosmetic sample
Weighing a cosmetic sample, placing the cosmetic sample in a clean centrifugal tube, adding water for dissolving, uniformly mixing, whirling, centrifuging, taking a proper amount of supernatant, and filtering by using a filter membrane to obtain filtrate as sample working solution;
step two, preparing a series of mixed standard solutions
Respectively and precisely weighing L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8 standard substances, dissolving with water, and preparing a mixed standard solution with the concentrations of the L-carnosine, the glutathione, the acetyl tetrapeptide-5 and the acetyl hexapeptide-8 being 1.00 mu g/mL;
accurately measuring the mixed standard solutions respectively, adding water for dilution, and preparing series of mixed standard solutions with the concentrations of the L-carnosine, the glutathione, the acetyl tetrapeptide-5 and the acetyl hexapeptide-8 being 1.00ng/mL, 5.00ng/mL, 10.0ng/mL, 50.0ng/mL and 200 ng/mL;
step three, high performance liquid chromatography tandem mass spectrometry detection
And respectively injecting the series of mixed standard solutions and the sample working solution into a high performance liquid chromatography tandem mass spectrometer for detection and analysis.
Further, in the third step, the liquid chromatography conditions are as follows:
a chromatographic column: c18 column, 4.6mm × 100mm,2.7 μm; column temperature: 24-26 ℃; flow rate: 0.3-0.6mL/min; sample introduction amount: 5-10 μ L; mobile phase a was water containing 0.1% acetic acid (v/v) and mobile phase B was acetonitrile; isocratic elution 12, the volume ratio of mobile phase a to mobile phase B is 90.
Further, in step three, the mass spectrometry conditions are as follows:
an ion source: AJSIE; capillary voltage: 3400-3600V; cleavage voltage: 370-390V; air curtain air temperature: 250 ℃; air flow speed of the air curtain: 11L/min; atomizer pressure: 20psi; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 14L/min; the scanning mode comprises the following steps: scanning positive ions; the detection mode is as follows: multiple reactive ion monitoring mode (MRM).
Further preferably, the ions with higher abundance and less interference are taken as quantitative ions, and the rest are taken as qualitative ions, and the MRM optimization parameters of the L-carnosine, the glutathione, the acetyl tetrapeptide-5 and the acetyl hexapeptide-8 are as follows:
further, in the first step, the centrifugation is carried out at 12000r/min for 10min.
Further, in the first step, the filter membrane is a 0.22 μm filter membrane.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
in the high performance liquid chromatography tandem mass spectrometry detection method, water is used as a solvent of a standard substance, the peak shape of a target object is good, and the response is high; water is used as an extraction solvent of the cosmetic sample, the extraction effect is good, and the recovery rate is high; the four polypeptides obtained by using 0.1% acetic acid water solution and acetonitrile as mobile phases have symmetrical peak shapes and stable base lines.
Drawings
FIG. 1 is a total ion flow diagram of L-carnosine, glutathione, acetyl tetrapeptide-5, and acetyl hexapeptide-8, wherein peak 1 is L-carnosine, peak 2 is glutathione, peak 3 is acetyl tetrapeptide-5, and peak 5 is acetyl hexapeptide-8.
Detailed Description
The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.
Reagent and apparatus:
l-carnosine was purchased from Beijing Bailingwei science and technology Co., ltd, and had a purity of 98%; the glutathione standard is purchased from J & K company, and the purity is 99 percent; acetyl tetrapeptide-5 was purchased from Shanghai' an spectral laboratory science and technology Co., ltd, and had a purity of 97.7%; the acetyl hexapeptide-8 standard is purchased from Sichuan Jixue sanden biological medicine, inc., and has a purity of 97.7%. Acetonitrile and acetic acid are both chromatographically pure and purchased from merck, germany; the water is high purity water.
L-carnosine of formula and molecular weight C 9 H 14 N 4 O 3 226.23, the structural formula is shown as formula (1);
glutathione, molecular formula and moleculeAn amount of C 10 H 17 N 3 O 6 S/307.32, the structural formula is shown as formula (2);
acetyl tetrapeptide-5, molecular formula and molecular weight C 20 H 28 N 8 O 7 /492.49, the structural formula is shown as formula (3);
acetyl hexapeptide-8, molecular formula and molecular weight C 34 H 60 N 14 O 12 S/888.99, the structural formula is shown as formula (4);
an Agilent6495 tandem triple quadrupole mass spectrometer was equipped with an Agilent1290 liquid chromatograph, an IKA VORTEX4 VORTEX instrument, an Eppendorf 5810R centrifuge, an electronic balance of Mettler-Toriledo, switzerland, an ultrasonic instrument of EMERSON, USA, and a Milli-Q Reference A + ultrapure water instrument of Millipore, USA. 0.22 μm microporous filter membrane (nylon 6, navigator).
Example 1
The embodiment provides a high performance liquid chromatography-tandem mass spectrometry detection method for four peptides in cosmetics.
Step one, pretreatment of a cosmetic sample
Accurately weighing 0.2g (accurate to 0.0001 g) of cosmetic sample, placing in a 50mL plastic centrifuge tube, adding water to a constant volume to scale, mixing uniformly, vortex for 5min, centrifuging at 12000r/min for 10min, taking a proper amount of supernatant, filtering with a 0.22 μm filter membrane to obtain a filtrate as sample working solution for later use.
Because the four polypeptides are all water-soluble compounds, water is selected as an extraction solvent, so that the toxicity is low, and the method is environment-friendly and suitable for large-batch sample treatment in a laboratory. In addition, ultrasonic treatment is also respectively considered for 15min, 30min and 45min and vortex is also considered for 5min in the experimental process, and the result shows that the vortex 5min can completely extract four polypeptides such as L-carnosine and the like in the sample, and the extraction efficiency is consistent with that of the ultrasonic treatment for 15min to 45 min. Therefore, a simple and nontoxic pretreatment method of adding water and vortex is finally selected as a preparation method of the sample working solution.
Step two, preparing a series of mixed standard solutions
Respectively and precisely weighing 10mg of each of L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8 standard substances, respectively placing the standard substances into a 10mL volumetric flask, dissolving the standard substances with water, fixing the volume to the scale, shaking up, precisely sucking 0.10mL of each of the solutions, placing the solutions into a 100mL volumetric flask, fixing the volume to the scale with water, and shaking up to obtain a mixed standard solution with the concentrations of the L-carnosine, the glutathione, the acetyl tetrapeptide-5 and the acetyl hexapeptide-8 being 1.00 mu g/mL.
And precisely measuring the mixed standard solutions respectively, adding water for dilution, and preparing series of mixed standard solutions with the concentrations of L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8 being 1.00ng/mL, 5.00ng/mL, 10.0ng/mL, 50.0ng/mL and 200 ng/mL.
Step three, high performance liquid chromatography tandem mass spectrometry detection
And (3) respectively injecting the series of mixed standard solutions and the sample working solution into a high performance liquid chromatography tandem mass spectrometer for detection and analysis.
The liquid chromatography conditions were:
a chromatographic column: agilent Poroshell 120 EC-C18 column, 4.6mm × 100mm,2.7 μm; column temperature: 25 ℃; flow rate: 0.5mL/min; sample injection amount: 5 mu L of the solution; mobile phase a was water containing 0.1% acetic acid (v/v) and mobile phase B was acetonitrile; isocratic elution for 12min, the volume ratio of mobile phase a to mobile phase B was 90.
Wherein, regarding the optimization of the mobile phase: because L-carnosine and acetyl tetrapeptide-5 are polypeptide compounds formed by bonding amino acids and have certain dissociation capability in aqueous solution under the condition of certain pH value, 0.1% formic acid, 5mmol/L ammonium acetate, 0.1% acetic acid, methanol and acetonitrile with different pH ranges are respectively adopted as mobile phases. As a result, it was found that the baseline of the four polypeptides was not easily stabilized in the methanol system. When 5mmol/L ammonium acetate and 0.1% formic acid are taken as water phases, the peak shapes of L-carnosine, acetyl tetrapeptide-5 and acetyl hexapeptide-8 are poor, the L-carnosine is seriously trailing, and the acetyl tetrapeptide-5 has a front peak, so that the satisfactory chromatographic peak shapes cannot be obtained. And finally, when 0.1% acetic acid and acetonitrile are selected as mobile phases, the obtained L-carnosine and acetyl tetrapeptide-5 have symmetrical peak shapes and stable base lines. When the organic phase proportion is higher than 15% of volume fraction, the retention value of glutathione is very small, and when the organic phase proportion is adjusted to 10% of volume fraction, the four polypeptides have good separation degree and proper retention time. Thus, mobile phase A was finally selected to be water, containing 0.1% acetic acid (v/v), mobile phase B to be acetonitrile; the volume ratio of mobile phase a to mobile phase B was 90.
The mass spectrum conditions are as follows:
an ion source: AJS ESI; capillary voltage: 3500V; cleavage voltage: 380V; air curtain air temperature: 250 ℃; air flow speed of air curtain: 11L/min; atomizer pressure: 20psi; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 14L/min; the scanning mode comprises the following steps: scanning positive ions; the detection mode is as follows: the multi-reactive ion monitoring mode (MRM) takes ions with higher abundance and less interference as quantitative ions and the rest as qualitative ions, and the MRM optimization parameters of L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8 are as follows:
wherein, regarding the selection of the parent ion: to find parent ions, a first full scan of the four polypeptides was performed using positive ion mode in which L-carnosine, glutathione and acetyl tetrapeptide-5 were expressed as [ M + H ]] + As the parent ion, the molecular weights of L-carnosine, glutathione and acetyl tetrapeptide-5 are 226, 307 and 492 respectively, so that [ M + H ] of 227.1, 308.0 and 493.1 are obtained respectively when the compounds are subjected to primary full scan]The molecular ion peaks of (4) are referred to as parent ions thereof, respectively. And acetyl hexapeptide-8 is present [ M + H] + And [ M +2H] 2+ Two kinds of parent ions, and through investigation and comparison, [ M +2H ]] 2+ The ion response abundance is higher and more stable, so that the [ M +2H is selected] 2+ The molecular ion peak of (4) 445.3 was taken as the parent ion of acetyl hexapeptide-8.
Selection of the quantitative ions: and determining the quantifier ions by using secondary mass spectrometry, wherein fragment ion peaks of 110 appear in both the L-carnosine and acetyl tetrapeptide-5 peaks, and the ion intensities are strongest, but in order to avoid mutual interference of the quantifier ions, 156.1 with relatively strong ion intensities is finally selected as the quantifier ions of the L-carnosine, and 110.1 is selected as the quantifier ions of the acetyl tetrapeptide-5. According to the strong and weak response of the fragment ions, the quantitative ions of the glutathione and the acetyl hexapeptide-8 are finally determined to be 178.9 and 102.0 with stronger response respectively.
In this example, the linear equations, correlation coefficients, linear ranges and limits of detection for L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8 are shown in Table 1:
TABLE 1
In this example, the stability of the standard solution was also examined:
in order to further examine the stability of the standard solution, the mixed working solution containing the four polypeptides is diluted by water to 200ng/mL, and is respectively placed in brown and transparent liquid phase vials, stored in a refrigerator (4 ℃) and at room temperature (30 ℃), and the changes of the four polypeptides along with time are compared: and (4) carrying out sample injection analysis at 0, 4, 8, 12 and 24h, recording peak areas of the two components, taking the peak area of 0h as 100%, and inspecting the recovery rate of each point. The results show that: when the standard solution is placed in a brown liquid phase small bottle and refrigerated at 4 ℃, the stability of glutathione is more facilitated, and the recovery rate is 95% when the peak area is 24 h. And the glutathione content is obviously reduced within 24h when the sample is placed in a transparent liquid phase vial at room temperature. Carnosine, acetyl tetrapeptide-5 and acetyl hexapeptide-8 were stable for 24h under the above conditions. Therefore, the standard solution and the cosmetic solution to be tested are newly prepared, stored at low temperature in the dark and injected in time.
Example 2
Three commercially available cosmetic bases were used as test objects and were labeled with 2.5. Mu.g/g, 7.5. Mu.g/g, and 25. Mu.g/g, respectively, while conducting parallel tests (n = 6), and the recovery rates and RSDs were measured as shown in Table 2. The results show that the recovery rate of the four polypeptides ranges from 85.1% to 105.6% under the conditions of high, medium and low standard-adding concentration, and the relative deviation RSD (n = 6) ranges from 0.6% to 4.4%. The detection method of the invention has good recovery rate result.
TABLE 2
By applying the detection method, 20 batches of commercially available cream emulsion, liquid water base and gel cosmetics (facial masks, toner, essence, cream, gel and the like) are sequentially measured. Detecting the L-carnosine in 7 batches of samples, wherein the content is in the range of 9.6 mu g/g-1532.3 mu g/g; glutathione is detected in 1 batch of samples, and the content is 1.9 mug/g; the content of acetyl tetrapeptide-5 detected in 1 batch of samples is 1.8 mu g/g, the content of acetyl hexapeptide-8 detected in 3 batches of samples is within the range of 1.7-8.7 mu g/g, and the detected compounds are consistent with the formula marks, so that the method is further explained to be accurate and reliable and can be used for positive sample verification and actual sample detection.
The embodiments of the present invention have been described in detail, but the embodiments are only examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (4)
1. The high performance liquid chromatography tandem mass spectrometry detection method of four peptides in cosmetics is characterized by comprising the following steps of:
step one, pretreatment of a cosmetic sample
Weighing a cosmetic sample, placing the cosmetic sample in a clean centrifugal tube, adding water for dissolving, uniformly mixing, whirling, centrifuging, taking a proper amount of supernatant, and filtering by using a filter membrane to obtain filtrate as sample working solution;
step two, preparing a series of mixed standard solutions
Respectively and precisely weighing an L-carnosine standard, a glutathione standard, an acetyl tetrapeptide-5 standard and an acetyl hexapeptide-8 standard, dissolving the L-carnosine standard, the glutathione standard, the acetyl tetrapeptide-5 standard and the acetyl hexapeptide-8 standard with water, and preparing a mixed standard solution with the concentrations of the L-carnosine standard, the glutathione standard, the acetyl tetrapeptide-5 standard and the acetyl hexapeptide-8 standard being 1.00 mug/mL;
accurately measuring the mixed standard solutions respectively, adding water for dilution, and preparing series of mixed standard solutions with the concentrations of the L-carnosine, the glutathione, the acetyl tetrapeptide-5 and the acetyl hexapeptide-8 being 1.00ng/mL, 5.00ng/mL, 10.0ng/mL, 50.0ng/mL and 200 ng/mL;
step three, high performance liquid chromatography tandem mass spectrometry detection
Respectively injecting the series of mixed standard solutions and the sample working solution into a high performance liquid chromatography tandem mass spectrometer for detection and analysis;
wherein, the liquid phase chromatographic conditions are as follows:
a chromatographic column: c18 column, 4.6mm × 100mm,2.7 μm; column temperature: 24-26 ℃; flow rate: 0.3-0.6mL/min; sample introduction amount: 5-10 μ L; mobile phase a was water containing 0.1% v/v acetic acid and mobile phase B was acetonitrile; isocratic elution for 12min, wherein the volume ratio of the mobile phase A to the mobile phase B is 90;
the mass spectrum conditions are as follows:
an ion source: AJS ESI; capillary voltage: 3400-3600V; cleavage voltage: 370-390V; air curtain air temperature: 250 ℃; air flow speed of the air curtain: 11L/min; atomizer pressure: 20psi; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 14L/min; the scanning mode is as follows: scanning positive ions; the detection mode is as follows: multiple reactive ion monitoring mode MRM.
2. The HPLC-MS/MS detection method of claim 1, wherein the MRM optimization parameters of L-carnosine, glutathione, acetyl tetrapeptide-5 and acetyl hexapeptide-8 are:
ionization method for quantifying ion pair collision energy (V) and determining ion pair collision energy (V)
Carnosine ESI + 227.1/156.1 14 227.1/110.2 26
Glutathione ESI + 308.0/178.9/308.0/76.0 34
Acetyl tetrapeptide-5 ESI + 493.1/110.1 46 493.1/338 20
Acetyl hexapeptide-8 ESI + 445.3/102.0 34 445.3/572.3.
3. The HPLC-MS/MS detection method of claim 1, wherein in step one, said centrifugation is performed at 12000r/min for 10min.
4. The HPLC-MS/MS detection method of claim 1, wherein in step one, said filter is a 0.22 μm filter.
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