Method for simultaneously determining eight aminoglycoside antibiotics in cosmetics

Abstract

The invention provides a method for simultaneously determining eight aminoglycoside antibiotics in cosmetics, and belongs to the technical field of medicines. By optimizing an HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method, a method capable of simultaneously determining spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin and amikacin in cosmetics is established. The method is good in specificity, the eight aminoglycoside antibiotics can be effectively separated, interference of solvents and matrixes in cosmetics is avoided, and the accuracy of a detection result is ensured; within the range of 5-500 ng.mL <-1 >, a good linear relation is achieved, and the content of antibiotics in cosmetics can be accurately reflected; target antibiotics can still be reliably detected and quantified at low concentration; the method is good in precision and high in recovery rate and repeatability. The method provides reliable technical support for cosmetic risk monitoring, and provides scientific basis for perfecting related detection standards and supervision policies.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a method for simultaneously determining 8 aminoglycoside antibiotics in cosmetics. Background Art

[0002] Acne-removing cosmetics have attracted much attention in the market due to their claimed acne-removing effects. However, some unscrupulous merchants may illegally add prohibited antibiotic ingredients in order to pursue quick results. Aminoglycoside antibiotics are a class of drugs that exert antibacterial effects by inhibiting bacterial protein synthesis. Due to their broad-spectrum antibacterial activity, low risk of drug resistance, low price, and convenient use, topical preparations of aminoglycoside antibiotics are widely used in the treatment of skin, eye, and ear infections. However, long-term use of cosmetics containing antibiotics can not only disrupt the skin microecological balance, leading to the emergence of drug resistance, but also may cause skin problems such as acne, dermatitis, or fungal infections. In addition, some people may have allergic reactions to certain antibiotics (such as aminoglycosides and cephalosporins), manifested as skin redness, itching, rashes, and even severe allergic dermatitis, posing a potential threat to the health of consumers. It has been found that after using spectinomycin, transient dizziness, insomnia, nausea, vomiting and other symptoms may occur, and some patients may also have rashes, etc.; after using kanamycin B, gastrointestinal reactions such as nausea, vomiting, loss of appetite, abdominal distension, diarrhea, etc. may be caused.

[0003] At present, there are serious regulatory loopholes in the use of aminoglycoside antibiotics in cosmetics. Although the current legal standard "Detection Methods for Anti-infective Drugs in Cosmetics" can simultaneously detect 36 anti-infective drugs, it does not cover aminoglycoside antibiotics. At present, there are few literature reports on the determination of aminoglycoside antibiotics in cosmetic matrices, and there is a lack of relevant test standards, resulting in the illegal addition of such ingredients in cosmetics remaining in the regulatory blind spot for a long time. Therefore, developing a method for determining multiple aminoglycoside antibiotics including spectinomycin and kanamycin B in cosmetics is of great significance for the quality control of cosmetics.

[0004] The vast majority of aminoglycoside antibiotics do not have characteristic ultraviolet absorption groups and cannot be detected by conventional ultraviolet methods. Although there are research reports on the method for detecting multiple aminoglycoside antibiotics in dairy products by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) (https: / / www.instrument.com.cn / application / Solution-879713.html), on the one hand, the samples detected by this method are dairy products, not cosmetics, and the pretreatment methods of dairy products are completely different from those of cosmetics, so this method is not applicable to the detection of aminoglycoside antibiotics in cosmetics; on the other hand, this method cannot detect spectinomycin and kanamycin B in cosmetics. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the purpose of the present invention is to provide a method for simultaneously determining 8 aminoglycoside antibiotics (spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin, and amikacin) in cosmetics.

[0006] The present invention provides a method for simultaneously determining 8 aminoglycoside antibiotics in cosmetics. The 8 aminoglycoside antibiotics are spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin, and amikacin. The method is high performance liquid chromatography - mass spectrometry (HPLC - MS), and the method includes the following steps:

[0007] S1. Prepare a test sample solution;

[0008] S2. Prepare a standard solution for drawing a standard curve;

[0009] S3. Test;

[0010] The mobile phase of the high performance liquid chromatography - mass spectrometry consists of phase A and phase B. Phase A is an aqueous solution of trifluoroacetic acid, and phase B is acetonitrile.

[0011] Further, in step S1, the method for preparing the test sample solution includes the following steps: mix the test sample and a dispersant, add an extraction solvent for extraction, and then add a purification solvent for defatting to obtain the test sample solution.

[0012] In step S2, the standard solution is a series of matrix standard curve solutions. The method for preparing the series of matrix standard curve solutions includes the following steps: dissolve the reference substance in an alcohol solvent to obtain a reference substance stock solution; mix the reference substance stock solution and an extraction solvent to obtain a mixed reference substance stock solution; mix the mixed reference substance stock solution and an extraction solvent and dilute step by step to obtain a series of mixed reference substance solutions; mix the series of mixed reference substance solutions, a negative sample of cosmetics, a dispersant, and an extraction solvent, and add a solvent to obtain the series of matrix standard curve solutions.

[0013] Further, the dispersant is sea sand; the extraction solvent is a 2.5% aqueous solution of trifluoroacetic acid; the purification solvent is n - hexane; the alcohol solvent is methanol.

[0014] Further, step S3 includes the following steps:

[0015] Compare the retention time of the qualitative ion chromatogram of the sample to be tested and the relative abundance ratio of the qualitative ion pairs with the retention time of the qualitative ion chromatogram of the reference substance and the relative abundance ratio of the qualitative ion pairs, and calculate the content of 8 aminoglycoside antibiotics in the sample to be tested based on the peak area.

[0016] Further, in step S3, when the retention time of the qualitative ion chromatogram of the sample to be tested is the same as that of the reference substance qualitative ion chromatogram, the relative abundances of the qualitative ion pairs of the sample to be tested and the reference substance qualitative ion pairs are compared. When the relative deviation of the relative abundances of the qualitative ion pairs of the sample to be tested and the reference substance qualitative ion pairs is within the following range, it can be qualitatively confirmed that the sample to be tested is consistent with the reference substance: when the relative abundance > 50%, the relative deviation ≤ 20%; when the relative abundance is 20% - 50%, the relative deviation ≤ 25%; when the relative abundance is 10% - 20%, the relative deviation ≤ 30%; when the relative abundance < 10%, the relative deviation ≤ 50%.

[0017] Further, the chromatographic conditions of the high performance liquid chromatography - mass spectrometry method are as follows:

[0018] Chromatographic column: C18 column;

[0019] Mobile phase: Phase A: 0.01 - 0.2% trifluoroacetic acid aqueous solution, Phase B: acetonitrile;

[0020] Flow rate: 0.1 - 1.0 mL·min -1 ;

[0021] Column temperature: 35 - 45°C;

[0022] Injection volume: 1 - 10 μL;

[0023] Elution method: gradient elution, 0 min, A:B = 100:0, 5 min, A:B = 100:0, 5.1 min, A:B = 20:80, 9 min, A:B = 20:80, 9.1 min, A:B = 100:0, 15 min, A:B = 100:0.

[0024] Further, the flow rate is 0.5 mL·min -1 ; the column temperature is 40°C; the injection volume is 5 μL.

[0025] Further, the C18 column is YMC Triart C18, ACE Excel 3C18 - AR or ZORBAX SB - C8.

[0026] Further, the C18 column is YMC Triart C18.

[0027] Further, the specification of the YMC Triart C18 is 4.6 mm × 50 mm, 3 μm.

[0028] Further, the Phase A is 0.05% - 0.1% trifluoroacetic acid aqueous solution, preferably 0.1% trifluoroacetic acid aqueous solution.

[0029] Furthermore, the A phase also includes 1 mmol / L ammonium formate.

[0030] Furthermore, the mass spectrometry conditions for the high performance liquid chromatography - mass spectrometry method are as follows:

[0031] Ion source and scanning mode: electrospray ionization source positive ion mode, multiple reaction monitoring;

[0032] Ionization voltage: 3.0 kV;

[0033] Ion source temperature: 150 °C;

[0034] Desolvation gas temperature: 550 °C;

[0035] Desolvation gas flow rate: 700 L / Hr;

[0036] Cone gas flow rate: 150 L / Hr;

[0037] Collision gas flow rate: 0.13 mL / min;

[0038] Collision cell pressure 4.01×10 -3 mbar;

[0039] The desolvation, cone, and collision are all carried out under the protection of inert gas.

[0040] Furthermore, the inert gas for desolvation is nitrogen; the inert gases for cone and collision are argon.

[0041] The present invention has achieved the following beneficial effects:

[0042] By optimizing the HPLC - MS / MS method, the present invention has established a method for simultaneously determining 8 aminoglycoside antibiotics (including spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin, amikacin) in cosmetics. The determination method of the present invention has good specificity, can effectively separate 8 aminoglycoside antibiotics, avoids the interference of solvents and matrices in cosmetics, and ensures the accuracy of the detection results; in the range of 5 - 500 ng·mL -1 it has a good linear relationship, can accurately reflect the content of antibiotics in cosmetics; can still reliably detect and quantify the target antibiotics at low concentrations; and this method has good precision, high recovery rate and repeatability. This method provides reliable technical support for the risk monitoring of cosmetics, and at the same time provides a scientific basis for improving relevant detection standards and regulatory policies.

[0043] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0044] The following is a further detailed description of the above content of the present invention in the form of specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following embodiments. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0045] Figure 1 is the extracted ion chromatogram of 8 aminoglycoside antibiotics (100 ng·mL -1 ).

[0046] Figure 2 is the extracted ion chromatogram of aminoglycoside antibiotics measured using a Sielc Obelisc R (2.1 mm × 150 mm, 5 μm) chromatographic column.

[0047] Figure 3 is the extracted ion chromatogram of aminoglycoside antibiotics measured using a ZORBAX SB-C8 (2.1 mm × 100 mm, 1.8 μm) chromatographic column.

[0048] Figure 4 is the extracted ion chromatogram of aminoglycoside antibiotics measured using an ACE Excel 3C18-AR (4.6 mm × 150 mm, 3 μm) chromatographic column.

[0049] Figure 5 is the extracted ion chromatogram of aminoglycoside antibiotics measured using a YMC Triart C18 (4.6 mm × 50 mm, 3 μm) chromatographic column.

[0050] Figure 6 is the extracted ion chromatogram of aminoglycoside antibiotics measured using a 0.05% trifluoroacetic acid aqueous solution - acetonitrile system as the mobile phase.

[0051] Figure 7 is the extracted ion chromatogram of aminoglycoside antibiotics measured using a 0.1% trifluoroacetic acid aqueous solution - acetonitrile system as the mobile phase.

[0052] Figure 8 is the extracted ion chromatogram of aminoglycoside antibiotics measured using a 0.1% trifluoroacetic acid aqueous solution (containing 1 mmol / L ammonium formate) - acetonitrile system as the mobile phase. Specific Embodiments

[0053] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0054] The experimental materials used in the present invention are as follows:

[0055] 1. Experimental Instruments

[0056] Waters UPLC CLASS Ⅰ PLUS / Xevo TQ-S MS ultra performance liquid chromatography-triple quadrupole mass spectrometer (Waters Corporation, USA); CPA225D electronic balance (Sartorius Corporation, Germany); TGL-16M tabletop high-speed refrigerated centrifuge (Hunan Xiangyi Company).

[0057] 2. Experimental reagents

[0058] Spectinomycin reference substance (batch number 130632 - 201501, content 64.0%), Tobramycin reference substance (batch number 130527 - 200402, content 91.4%), Kanamycin reference substance (batch number 130556 - 201502, content 67.9%), Amikacin reference substance (batch number 130623 - 202101, content 65.1%) were all purchased from the National Institutes for Food and Drug Control; Kanamycin B reference substance (batch number 1123 - RB - 0001, content 96.6%), Hygromycin B reference substance (batch number 0923 - RD - 0059, content 93.3%), Streptomycin sulfate reference substance (batch number 146 - 12 - 17, content 87.3%), Dihydrostreptomycin sulfate reference substance (batch number 6C - 12 - 9 - 1, content 89.3%) were all purchased from Guangzhou Jiantu Technology Co., Ltd.; Methanol and acetonitrile, chromatographic grade (Merck KGaA, Germany); Trifluoroacetic acid, chromatographic grade (Shanghai Aladdin Co., Ltd.); The experimental water was Milli-Q ultrapure water; The sea sand was chemically pure, and the rest of the reagents were all of analytical grade. 18 batches of acne treatment cosmetics were market - purchased samples.

[0059] Example 1. A method for simultaneously determining 8 aminoglycoside antibiotics in cosmetics

[0060] 1. Condition setting and sample preparation before determination

[0061] (1) Chromatographic conditions

[0062] Use a YMC Triart C18 (4.6 mm × 50 mm, 3 μm) chromatographic column. Mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile. Elution is carried out according to Table 1. The flow rate is 0.5 mL·min -1 , the column temperature is 40 °C, the injection volume is 5 μL, and the signal acquisition time is 1 - 5 min.

[0063] Table 1 Gradient elution table

[0064]

[0065]

[0066] (2) Mass spectrometry conditions

[0067] Ion source and scanning mode: Electrospray ionization source, positive ion mode (ESI+), Multiple Reaction Monitoring (MRM); Ionization voltage: 3.0 kV; Ion source temperature: 150 °C; Desolvation gas temperature: 550 °C; Desolvation gas flow rate: 700 L / Hr (nitrogen); Cone gas flow rate: 150 L / Hr (nitrogen); Collision gas flow rate: 0.13 mL / min (argon); Collision cell pressure: 4.01×10 -3 mbar; Qualitative ion pairs, fragmentation voltage and collision voltage are shown in Table 2, and the ion pairs marked with "*" are quantitative ion pairs.

[0068] Table 2 Mass spectrometry parameters of 8 aminoglycoside antibiotics

[0069]

[0070] (3) Solution preparation

[0071] ① Test solution

[0072] Weigh 0.5 g of the sample and place it in a 10 mL plastic centrifuge tube with a stopper. Add 1 g of sea sand, vortex for 2 min, add 5 mL of 2.5% trifluoroacetic acid aqueous solution, vortex for 2 min, extract ultrasonically for 30 min, add 2 mL of n-hexane solution, vortex for 2 min, centrifuge at 10000 r / min for 10 min, and take the lower layer solution through a 0.22 μm filter membrane.

[0073] ② Blank matrix solution

[0074] Take 0.5 g of negative sample of cream type (acne cleansing milk), and treat it in the same way as "① Test solution".

[0075] ③ Reference solution

[0076] Take about 10 mg of each reference substance of 8 aminoglycoside antibiotics (spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin and amikacin), weigh accurately, quantitatively dilute with 20% methanol solution to 10 mL to obtain a reference stock solution; then accurately transfer appropriate amounts of each reference stock solution into the same 10 mL volumetric flask, dilute to the mark with 2.5% trifluoroacetic acid aqueous solution to prepare a mixed reference stock solution with a mass concentration of 50 μg·mL -1 . Gradually dilute the mixed reference stock solution with 2.5% trifluoroacetic acid aqueous solution to obtain a mixed reference solution with a mass concentration of 50 - 5000 ng·mL -1A series of mixed reference substance solutions. Weigh 0.5 g of cream-like negative sample, add 0.5 mL of the series of mixed reference substance solutions, add 1 g of sea sand, vortex for 2 min, add 4.5 mL of 2.5% trifluoroacetic acid aqueous solution, starting from "vortex for 2 min and ultrasonically extract for 30 min", and process according to the same method as "① test solution" to prepare series of matrix standard curve solutions with mass concentrations of 5 ng·mL -1 , 10 ng·mL -1 , 25 ng·mL -1 , 50 ng·mL -1 , 100 ng·mL -1 , 250 ng·mL -1 , 375 ng·mL -1 , 500 ng·mL -1 .

[0077] 2. Result analysis

[0078] The established method was used to detect 18 batches of cosmetics. Qualitative analysis was carried out based on the retention time and the relative abundance ratio of the qualitative ion pairs, and the content was calculated by the peak area according to the standard curve method. If the retention time of the chromatographic peaks detected in the test solution is consistent with that in the reference solution, and the relative abundance of the qualitative ion pairs in the chromatogram of the test solution after background subtraction is compared with the chromatogram of the reference solution obtained under the same conditions with a concentration close to it, and the relative deviation is within the following range, it can be qualitatively confirmed to be consistent with the reference substance (when the relative abundance > 50%, the relative deviation ≤ 20%; when the relative abundance is 20% - 50%, the relative deviation ≤ 25%; when the relative abundance is 10% - 20%, the relative deviation ≤ 30%; when the relative abundance < 10%, the relative deviation ≤ 50%).

[0079] As a result, spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin, and amikacin, these 8 aminoglycoside antibiotics were not detected in the 18 batches of samples (18 batches of acne-removing cosmetics). The reason for the analysis may be that the samples tested are all brand products and the product quality is basically controllable.

[0080] The following experimental examples prove the beneficial effects of the present invention.

[0081] Experimental example 1. Investigation of the effect of the method of the present invention

[0082] 1. Specificity investigation

[0083] Combined with the results in Table 2 and Figure 1 , it can be seen that under the determination method of Example 1, the 8 aminoglycoside antibiotics are well separated, and there is no interference from the blank solvent (2.5% trifluoroacetic acid aqueous solution) and the blank matrix.

[0084] 2. Investigation of linear relationship

[0085] According to the matrix standard curve solutions of the "② blank matrix solution" series in Example 1, the peak area (Y) was linearly fitted against the mass concentration (X) with a weight of 1 / X, and the linear equation and correlation coefficient were calculated. The results (see Table 3) showed that within the range of 5 - 500 ng·mL -1 , good linear relationships (r > 0.995) were obtained for each analyte.

[0086] 3. Limit of Quantitation and Limit of Detection

[0087] Referring to the determination method of Example 1, a mixed reference solution of 8 aminoglycoside antibiotics was added to the blank matrix solution, and after successive dilutions, the samples were injected for determination. The mass concentrations corresponding to 3 and 10 times the signal-to-noise ratio were taken as the limit of detection and the limit of quantitation, respectively. The results (see Table 3) indicated that the method of the present invention could ensure reliable detection and quantification of the target antibiotics even at low concentrations.

[0088] Table 3 Linear ranges, linear equations, correlation coefficients, and limits of detection of 8 aminoglycoside antibiotics

[0089]

[0090] 4. Precision

[0091] Referring to the determination method of Example 1, a mixed reference solution with a mass concentration of 250 ng·mL -1 was continuously injected 6 times for determination, and the relative standard deviations (RSD, n = 6) of the peak areas of the analytes numbered 1 - 8 were calculated, which were 1.9%, 2.7%, 1.7%, 0.9%, 3.7%, 4.2%, 1.5%, and 2.2% respectively, indicating good precision.

[0092] 5. Recovery and Repeatability

[0093] Referring to the determination method of Example 1, 0.5 g of a negative cream sample was weighed, and 2.5 μL, 10 μL, and 40 μL of a mixed reference stock solution with a mass concentration of 50 μg·mL -1 were added respectively. Each was prepared in 6 parallels, and processed in the same way as "① test solution" in Example 1, injected, and determined. The recoveries and repeatabilities of each analyte were calculated respectively. The results (Table 4) showed that the recoveries and repeatabilities of the present invention were high.

[0094] Table 4 Average recoveries and repeatabilities of 8 aminoglycoside antibiotics (n = 6)

[0095]

[0096]

[0097] The above results indicate that the determination method of the present invention has good specificity, can effectively separate 8 aminoglycoside antibiotics, avoid the interference of solvents and matrices in cosmetics, and ensure the accuracy of the detection results; in the range of 5 - 500 ng·mL -1 it has a good linear relationship and good precision, and can accurately reflect the content of antibiotics in cosmetics; it can still reliably detect and quantify the target antibiotics at low concentrations; and this method has good precision, high recovery rate and repeatability.

[0098] Experimental Example 2. Optimization of the test conditions of the present invention

[0099] 1. Selection of chromatographic column

[0100] Referring to the determination method of Reference Example 1, the present invention compared the separation effects of different chromatographic columns ①: YMC Triart C18 (4.6 mm × 50 mm, 3 μm), ②: ACE Excel 3 C18-AR (4.6 mm × 150 mm, 3 μm), ③: Sielc Obelisc R (2.1 mm × 150 mm, 5 μm), ④: ZORBAX SB-C8 (2.1 mm × 100 mm, 1.8 μm).

[0101] Retention time refers to the time required for a compound to reach the peak detected by the detector from injection. In the chromatogram, the retention time is usually in minutes and represents the position on the horizontal axis.

[0102] Peak shape describes the peak shape characteristics of a compound in the chromatogram. The ideal peak shape is a symmetric Gaussian shape. The peak shape can reflect the quality of chromatographic separation. Symmetric and sharp peaks usually indicate good separation and low system dead volume.

[0103] It was found that: multiple aminoglycoside antibiotics had very weak retention on the Sielc Obelisc R (2.1 mm × 150 mm, 5 μm) chromatographic column ( Figure 2 ), and had stronger retention on the other three chromatographic columns ( Figures 3 to 5 ). According to the separation effect, the present invention selected the YMC Triart C18 (4.6 mm × 50 mm, 3 μm) chromatographic column considering both retention time and peak shape.

[0104] 2. Selection of mobile phase

[0105] Referring to the determination method of Reference Example 1, the present invention compared different systems: ① 0.05% trifluoroacetic acid aqueous solution - acetonitrile ( Figure 6 ); ② 0.1% trifluoroacetic acid aqueous solution - acetonitrile ( Figure 7 ); ③ 0.1% trifluoroacetic acid aqueous solution - acetonitrile containing 1 mmol / L ammonium formate ( Figure 8 ).

[0106] It was found that both the retention and mass spectrometry response of the 0.05% trifluoroacetic acid aqueous solution - acetonitrile system were lower than those of the 0.1% trifluoroacetic acid aqueous solution - acetonitrile system; in addition, compared with the 0.1% trifluoroacetic acid aqueous solution - acetonitrile, the mass spectrometry response of the 0.1% trifluoroacetic acid aqueous solution - acetonitrile containing 1 mmol / L ammonium formate increased, but the retention times of multiple aminoglycoside antibiotics shortened. Therefore, the 0.1% trifluoroacetic acid aqueous solution - acetonitrile system was selected comprehensively in this invention.

[0107] The above results indicate that using YMC Triart C18 (4.6 mm × 50 mm, 3 μm) as the chromatographic column and the 0.1% trifluoroacetic acid aqueous solution - acetonitrile system as the mobile phase are the preferred determination conditions of this invention.

[0108] In summary, this invention provides a method for simultaneously determining 8 aminoglycoside antibiotics in cosmetics. By optimizing the HPLC - MS / MS method, this invention established a method that can simultaneously determine 8 aminoglycoside antibiotics (including spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin, amikacin) in cosmetics. The determination method of this invention has good specificity, can effectively separate 8 aminoglycoside antibiotics, avoids the interference of solvents and matrices in cosmetics, and ensures the accuracy of the detection results; in the range of 5 - 500 ng·mL -1 it has a good linear relationship, can accurately reflect the content of antibiotics in cosmetics; can still reliably detect and quantify the target antibiotics at low concentrations; and this method has good precision, high recovery rate and repeatability. This method provides reliable technical support for the risk monitoring of cosmetics and at the same time provides a scientific basis for improving relevant detection standards and regulatory policies.

Claims

1. A method for simultaneously determining 8 aminoglycoside antibiotics in cosmetics, characterized in that, The 8 aminoglycoside antibiotics are spectinomycin, tobramycin, kanamycin B, kanamycin, hygromycin B, streptomycin, dihydrostreptomycin and amikacin. The method is high performance liquid chromatography-mass spectrometry (HPLC-MS), and the method comprises the following steps: S1. Prepare a test solution; S2. Prepare a standard solution for drawing a standard curve; S3. Conduct a test; The mobile phase of the HPLC-MS consists of phase A and phase B, wherein phase A is an aqueous solution of trifluoroacetic acid and phase B is acetonitrile.

2. The method according to claim 1, wherein In step S1, the method for preparing the test solution comprises the following steps: mix the test sample and a dispersant, add an extraction solvent for extraction, and then add a purification solvent for defatting to obtain the test solution. In step S2, the standard solution is a series of matrix standard curve solutions. The method for preparing the series of matrix standard curve solutions comprises the following steps: dissolve the reference substance in an alcohol solvent to obtain a reference substance stock solution; mix the reference substance stock solution and an extraction solvent to obtain a mixed reference substance stock solution; mix the mixed reference substance stock solution and an extraction solvent and perform stepwise dilution to obtain a series of mixed reference substance solutions; mix the series of mixed reference substance solutions, a negative cosmetic sample, a dispersant and an extraction solvent, and add a solvent to obtain the series of matrix standard curve solutions.

3. The method according to claim 2, wherein The dispersant is sea sand; the extraction solvent is a 2.5% aqueous solution of trifluoroacetic acid; the purification solvent is n-hexane; the alcohol solvent is methanol.

4. The method according to claim 1, wherein The chromatographic conditions of the HPLC-MS are as follows: Chromatographic column: C18 column; Mobile phase: phase A: 0.01-0.2% aqueous solution of trifluoroacetic acid, phase B: acetonitrile; Flow rate: 0.1 - 1.0 mL·min -1 ; Column temperature: 35-45°C; Injection volume: 1-10 μL; Elution mode: gradient elution, at 0 min, A:B = 100:0, at 5 min, A:B = 100:0, at 5.1 min, A:B = 20:80, at 9 min, A:B = 20:80, at 9.1 min, A:B = 100:0, at 15 min, A:B = 100:

0.

5. The method according to claim 4, wherein The C18 column is YMC Triart C18, ACE Excel3C18-AR or ZORBAX SB-C8.

6. The method according to claim 5, wherein The C18 column is YMC Triart C18.

7. The method according to claim 6, wherein The specification of the YMC Triart C18 is 4.6 mm × 50 mm, 3 μm.

8. The method according to claim 4, characterized in that, Phase A is a 0.05%-0.1% aqueous solution of trifluoroacetic acid, preferably a 0.1% aqueous solution of trifluoroacetic acid.

9. The method according to claim 4, wherein Phase A further comprises 1 mmol / L ammonium formate.

10. The method according to claim 1, wherein The mass spectrometry conditions of the HPLC-MS are as follows: Ion source and scanning mode: electrospray ionization source positive ion mode, multiple reaction monitoring; Ionization voltage: 3.0 kV; Ion source temperature: 150°C; Desolvation gas temperature: 550°C; Desolvation gas flow rate: 700 L / Hr; Cone gas flow rate: 150 L / Hr; Collision gas flow rate: 0.13 mL / min; Collision cell pressure 4.01×10 -3 mbar; The desolvation, cone and collision are all carried out under the protection of an inert gas.

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