Detection method for detecting pseudomonas aeruginosa metabolite based on HPLC-MS / MS and application of pseudomonas aeruginosa metabolite

Abstract

The invention discloses a method for detecting metabolite of pseudomonas aeruginosa based on HPLC-MSMS (High Performance Liquid Chromatography-Mass Spectrometry Mass Spectrometry) and application. By optimizing the pretreatment conditions of a sample solution, the pseudomonas aeruginosa metabolite in a sample can be efficiently detected by adopting high performance liquid chromatography tandem triple quadrupole mass spectrometry, and the method has the advantages of high sensitivity, strong specificity, good selectivity, good accuracy, high detection efficiency and the like. The method can be used for quality control of cosmetics and other products which may be polluted by pseudomonas aeruginosa or metabolites of pseudomonas aeruginosa; the method can also be applied to metabolism analysis of pseudomonas aeruginosa, and the content of pseudomonas aeruginosa metabolites in an enrichment culture medium and a selective culture medium can be detected only through one-loop inoculation. In addition, the screened metabolites such as pyocyanine, 2-heptyl-4-quinolinone, phenazine-1-carboxylic acid and 1-hydroxyphenazine have the potential of serving as the specific biomarkers of the pseudomonas aeruginosa, and can be used for the detection and metabonomics research of the pseudomonas aeruginosa.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic analysis, and in particular to a method and application for detecting Pseudomonas aeruginosa metabolites based on HPLC-MS / MS. Background Art

[0002] Pseudomonas aeruginosa is a widely distributed Gram-negative bacterium that produces pyocyanin. It is a common opportunistic pathogen that can cause infections in humans and animals under certain circumstances, especially in those with weakened immune systems, potentially leading to illnesses such as acute enteritis, meningitis, sepsis, and skin inflammation. Furthermore, Pseudomonas aeruginosa can liquefy gelatin and reduce nitrates to nitrites. Therefore, the presence of Pseudomonas aeruginosa and its metabolites in cosmetics not only poses a health risk to consumers but also compromises the quality of the product itself.

[0003] Currently, microbial culture is the primary method for detecting Pseudomonas aeruginosa in cosmetics. The "Safety Technical Specifications for Cosmetics" (2015) specifies methods for detecting Pseudomonas aeruginosa (Chapter 5: Microbiological Testing Methods) as including enrichment culture, isolation culture, staining microscopy, oxidase test, pyocyanin test, gelatin liquefaction test, and 42°C growth test. This method involves multiple steps, is time-consuming, and can lead to missed detections, thus consistently presenting challenges and pain points in detecting Pseudomonas aeruginosa in cosmetics. For example, Chinese patent document CN109762772 A discloses a qualitative standard sample for Pseudomonas aeruginosa in water-soluble cosmetics and its preparation method. This solution provides a pyocyanin culture medium. After the cosmetic sample is enriched with an enrichment solution, a 42°C growth test is performed directly on the pyocyanin culture medium to screen for Pseudomonas aeruginosa. The screening culture results are observed, and if colonies appear after the screening culture, further identification is performed in conjunction with a gelatin liquefaction test. Although this scheme simplifies the operating steps of the Pseudomonas aeruginosa detection method to a certain extent with the help of the provided pyocyanin culture medium and improves the detection efficiency, it basically still refers to the detection ideas of the "Technical Specifications for Safety of Cosmetics".

[0004] In addition, like all other bacteria and fungi, Pseudomonas aeruginosa can absorb various external nutrients for metabolism and produce secondary metabolites. These metabolites can be divided into antibiotics, hormones, alkaloids, toxins and vitamins according to their functions. These metabolites are not only biologically active, but also play an important role in maintaining the ecological adaptability of Pseudomonas aeruginosa. They are essential for the survival and reproduction of Pseudomonas aeruginosa in complex environments, but the relationship between microorganisms and metabolites is also very complex. With the development of technology, some methods for detecting Pseudomonas aeruginosa metabolites have gradually emerged, such as Jia Fei (F. Jia et al., Detection of pyocyanin using a new biodegradable surface enhanced raman spectroscopy (SERS) biosensor fabricated using goldcoated zein nanostructures further decorated with gold nanoparticles, J. Agr. Food. Chem. 67 (16) (2019) 4603-4610) using gold nanoparticle surface enhanced Raman spectroscopy (SERS) biosensor to detect pyocyanin in water. Fatima Al Zahra (FAa Alatraktchi et al., Fast selective detection of pyocyanin using cyclic voltammetry, Sensors-Basel. 16 (3) (2016) 408) used cyclic voltammetry to selectively detect pyocyanin. Olga et al. (Detection of Pseudomonas aeruginosa metabolite pyocyanin in water and saliva by employing the SERS technique, Sensors-Basel. 17(8)(2017)1704-1704) used a SERS-active silicon nanowire matrix to rapidly detect the bacterial biomarker pyocyanin in artificial sputum. Tanaka Yuki et al. (Aportable SERS sensor for pyocyanin detection in simulated wound fluid and through swab sampling, The Analyst. 146(22)(2021)) used a portable SERS sensor to simulate the detection of pyocyanin in wound fluid. However, these studies mainly focused on electrochemical or biosensing technologies, and the concentration detection range of metabolites was 0.5 to 100 μM.

[0005] Therefore, these methods have the limitations of low sensitivity, low specificity, and limited applicability. Furthermore, the target metabolites studied are relatively few, and the coverage of the research subjects is limited, which can easily lead to problems such as missed detection. Furthermore, due to the complex and variable chemical structures of P. aeruginosa metabolites, especially secondary metabolites, and their low content, it is necessary to develop a sensitive, accurate, reliable, and convenient method for detecting P. aeruginosa metabolites that can be used for metabolic analysis of P. aeruginosa or for quality control of products such as cosmetics that may be contaminated by P. aeruginosa or its metabolites. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method and application for detecting Pseudomonas aeruginosa metabolites based on HPLC-MS / MS (high-performance liquid chromatography tandem triple quadrupole mass spectrometry). This method has the advantages of high sensitivity, strong specificity, good selectivity, good accuracy, and convenient operation. It can be used for analyzing Pseudomonas aeruginosa metabolites and for quality control of products such as cosmetics that may be contaminated by Pseudomonas aeruginosa or its metabolites.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for detecting metabolites of Pseudomonas aeruginosa based on HPLC-MS / MS, comprising the following steps:

[0009] S1. Sample pretreatment and preparation: Weigh the sample, add saturated sodium chloride solution and mix well, add acetonitrile, shake and mix well, then extract by ultrasonic wave, then make up to volume with acetonitrile, centrifuge, clean up with C18 solid phase extraction column, and filter through 0.22 μm PTFE organic filter membrane to obtain the sample solution to be tested;

[0010] S2. Prepare standard solutions: Dissolve the standard samples of Pseudomonas aeruginosa metabolites in methanol and dilute to volume to prepare standard stock solutions; prepare mixed standard intermediate solutions by taking a single standard stock solution of each standard sample; prepare mixed standard solutions with blank matrix solution before use, and then gradually dilute with blank matrix solution to obtain a series of standard matrix solutions;

[0011] S3. (1) Liquid chromatography conditions: Column: Agilent SB-C18 RRHD (2.1 mm × 100 mm, 1.8 μm); Mobile phase: A: 0.1% formic acid in water, B: methanol; Flow rate: 0.4 mL / min; Column temperature: 40°C; Injection volume: 2 μL; Liquid chromatography was performed using gradient elution, and the gradient elution procedure was as follows:

[0012] 0-3min, 60%B-80%B;

[0013] 3-9 min, 80% B-80% B;

[0014] 9.1~12min, 60%B;

[0015] (2) Mass spectrometry conditions: electrospray ionization source (ESI source); positive ion mode electrospray voltage: 5500 V; negative ion mode electrospray voltage: -4500 V; nebulization temperature: 500°C; nebulizing gas pressure: 50 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 20 psi; collision gas pressure: 9 psi; monitoring mode: multiple reaction monitoring (MRM);

[0016] S4. Perform qualitative and quantitative analysis on the components in the sample solution.

[0017] Furthermore, the Pseudomonas aeruginosa metabolites include at least one of pyocyanin, 4-hydroxyquinoline, 2,6-dihydroxyquinoline, 2-methyl-8-hydroxyquinoline, 6,7-dimethoxy-4-hydroxyquinoline, 2-heptyl-4-hydroxyquinoline-N-oxide, 2-heptyl-4-quinolinone, 2-amino-4-hydroxyquinoline hydrate, 2-hydroxyquinoxaline, 1-hydroxyphenazine, phenazine-1-carboxylic acid, phenazine-1-carboxamide, and luteolin.

[0018] Furthermore, the mass spectrometry parameters of the target metabolites to be detected in step S3 are as follows:

[0019]

[0020] Furthermore, the qualitative analysis method in step S4 is as follows: under the same experimental conditions, the mass chromatographic peak retention time of the measured component in the sample solution is consistent with the mass chromatographic peak retention time of the corresponding component in the standard solution; and the deviation between the relative abundance ratio of the selected monitoring ion pair in the sample chromatogram and the relative abundance ratio of the ion of the standard solution of equivalent concentration is within the following range, then it can be determined that the corresponding component is present in the sample:

[0021] The maximum allowable deviations of the relative ion abundance ratio k are ±20%, ±25%, ±30% and ±50% when k≥50%, 50%>k≥20%, 20%>k≥10% and k<10%, respectively.

[0022] This deviation range complies with the requirements of the "Technical Specifications for Validation of Detection Methods for Prohibited and Restricted Substances in Cosmetics".

[0023] Furthermore, the quantitative analysis method in step S4 is: external standard quantification by matrix standardization, quantitative analysis by linear fitting of concentration and peak area, the linear correlation coefficient is greater than 0.99, the detection limit corresponding to 3 times the signal-to-noise ratio of the lowest response substance is 0.05 mg / kg, and the quantitative limit corresponding to 10 times the signal-to-noise ratio of the lowest response substance is 0.10 mg / kg.

[0024] Furthermore, in step S2, the concentration of the standard stock solution is 1.0 g / L; the concentration of the mixed standard intermediate solution is 10 mg / L; the concentration of the mixed standard solution is 1.0 mg / L; and the concentrations of the obtained series of standard matrix solutions are 1, 2, 5, 10, 20, 50, and 100 μg / L.

[0025] Furthermore, in step S1, 0.2 g of the cosmetic sample was added with 0.5 mL of saturated sodium chloride solution and mixed, and then 8 mL of acetonitrile was added. After oscillation and mixing, ultrasonic extraction was performed for 10 minutes, and then the volume of acetonitrile was adjusted to 10 mL; the centrifugation condition was: 8000 r / min for 5 minutes.

[0026] Furthermore, the method further includes, before S3, an evaluation of the thermal stability of the substance to be tested, specifically, heating the mixed standard solution in a water bath at 50° C. and 100° C. for 30 minutes, respectively, and then performing analysis and determination.

[0027] In a second aspect, the present invention provides an application of the above-described method in a metabolic analysis method for Pseudomonas aeruginosa, comprising the following steps:

[0028] S1. activating Pseudomonas aeruginosa to obtain a strain activation solution;

[0029] S2. Inoculate 90 μL of the activated solution of the strain S1 into enrichment medium respectively, and culture at 28°C and 36°C for 48 h; the enrichment medium is BHI broth;

[0030] S3. Inoculate the cultured strain into a loop of selective culture medium, and culture the sample again at the corresponding temperature for 48 hours. The selective culture medium is hexadecyltrimethylammonium bromide medium or pyocyanin assay medium;

[0031] S4. The method described above is used to respectively determine the content of metabolites of Pseudomonas aeruginosa in the enrichment medium and the selective medium.

[0032] In a third aspect, the present invention provides application of the above-described method in a method for quality control of cosmetics.

[0033] For cosmetics that may be contaminated by Pseudomonas aeruginosa or metabolites of Pseudomonas aeruginosa, the above-described method can sensitively and efficiently detect the metabolites of Pseudomonas aeruginosa contained in the cosmetics; alternatively, pyocyanin, 2-heptyl-4-quinolinone, phenazine-1-carboxylic acid and 1-hydroxyphenazine can be used as specific biomarkers of Pseudomonas aeruginosa to detect Pseudomonas aeruginosa in cosmetics through HPLC-MS / MS.

[0034] Based on a similar principle, the present invention can also be applied to quality control methods for products other than cosmetics that may be contaminated by Pseudomonas aeruginosa or metabolites of Pseudomonas aeruginosa.

[0035] The present invention has the following beneficial effects:

[0036] The present invention provides a high performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-MS / MS) method for determining Pseudomonas aeruginosa metabolites, which has the advantages of high sensitivity, strong specificity, good selectivity, good accuracy, high detection efficiency, etc., and is easier to promote and apply in practice.

[0037] This method was applied to the metabolic analysis of Pseudomonas aeruginosa. Only a single loop of inoculation was required to detect the content of Pseudomonas aeruginosa metabolites in both enrichment and selective culture media, with good sensitivity, strong specificity, and high detection efficiency. Furthermore, experiments revealed that the screened metabolites pyocyanin, 2-heptyl-4-quinolinone, phenazine-1-carboxylic acid, and 1-hydroxyphenazine have the potential to serve as specific biomarkers for Pseudomonas aeruginosa, providing technical support for the detection and metabolomics research of Pseudomonas aeruginosa. Therefore, the method of the present invention can serve as a new analytical technique for microbial metabolite analysis and biological identification, providing technical support for the application research of Pseudomonas aeruginosa in clinical and industrial fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings used in the description of the embodiments.

[0039] Figure 1 The TIC chromatograms of 13 Pseudomonas aeruginosa metabolites in the mixed standard solution are shown in Figure 2. Figure 1 (1) is the total ion current TIC diagram; Figure 1 (2) is the total ion current TIC diagram in positive ion mode; 1(3) is the total ion current TIC diagram in negative ion mode.

[0040] Figure 2 The MRM chromatograms of pyocyanin are shown in Figure 1, where (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0041] Figure 3 The MRM chromatograms of 4-hydroxyquinoline are shown in FIG1 , where (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0042] Figure 4 The MRM chromatograms of 2,6-dihydroxyquinoline are shown in FIG1 , where (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0043] Figure 5 The MRM chromatogram of 2-methyl-8-hydroxyquinoline is shown in FIG1 , wherein (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0044] Figure 6 The MRM chromatograms of 6,7-dimethoxy-4-hydroxyquinoline are shown in FIG1 , where (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0045] Figure 7 The MRM chromatogram of 2-heptyl-4-hydroxyquinoline-N-oxide, wherein (1) is the quantitative ion MRM graph, and (2) is the qualitative ion MRM graph.

[0046] Figure 8 The MRM chromatogram of 2-heptyl-4-quinolinone is shown in FIG. 1 , wherein (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0047] Figure 9 The MRM chromatograms of 2-amino-4-hydroxyquinoline hydrate are shown in FIG. 1 , wherein (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0048] Figure 10 The MRM chromatograms of 2-hydroxyquinoxaline are shown in FIG1 , wherein (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0049] Figure 11 The MRM chromatograms of 1-hydroxyphenazine are shown in FIG1 , wherein (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0050] Figure 12 The MRM chromatograms of phenazine-1-carboxylic acid are shown in FIG1 , wherein (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0051] Figure 13 The MRM chromatograms of phenazine-1-carboxamide are shown in FIG1 , wherein (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0052] Figure 14 The MRM chromatograms of luteolin are shown in Figure 1, where (1) is the quantitative ion MRM graph and (2) is the qualitative ion MRM graph.

[0053] Figure 15 This is a physical picture of the Pseudomonas selective culture medium, in which:

[0054] a-1 is a hexadecane trimethylammonium bromide medium used to culture Pseudomonas aeruginosa at 28°C; a-2 is a hexadecane trimethylammonium bromide medium used to culture three mixed Pseudomonas species at 28°C; a-3 is a hexadecane trimethylammonium bromide medium used to culture Pseudomonas aeruginosa at 36°C; a-4 is a hexadecane trimethylammonium bromide medium used to culture three mixed Pseudomonas species at 36°C; a-5 is a hexadecane trimethylammonium bromide medium used to culture Pseudomonas putida at 36°C; a-6 is a hexadecane trimethylammonium bromide medium used to culture Pseudomonas fluorescens at 36°C.

[0055] b-1 is a pyocyanin assay medium for culturing Pseudomonas aeruginosa at 28°C; b-2 is a pyocyanin assay medium for culturing three mixed Pseudomonas species at 28°C; b-3 is a pyocyanin assay medium for culturing Pseudomonas aeruginosa at 36°C; b-4 is a pyocyanin assay medium for culturing three mixed Pseudomonas species at 36°C; b-5 is a pyocyanin assay medium for culturing Pseudomonas putida at 36°C; b-6 is a pyocyanin assay medium for culturing Pseudomonas fluorescens at 36°C. DETAILED DESCRIPTION

[0056] In order to better illustrate the content of the present invention, the present invention is further verified by specific examples below. It is particularly noted that the examples are only for more direct description of the present invention, they are only a part of the present invention, and cannot constitute any limitation to the present invention.

[0057] The present invention provides a method for determining Pseudomonas aeruginosa metabolites using high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-MS / MS). This method can be applied to the metabolic analysis of Pseudomonas aeruginosa and the quality control of products such as cosmetics that may be contaminated by Pseudomonas aeruginosa metabolites. This method screens pyocyanin, 2-heptyl-4-quinolinone, phenazine-1-carboxylic acid, and 1-hydroxyphenazine as potential biomarkers specific for Pseudomonas aeruginosa, enabling the detection of Pseudomonas aeruginosa in Pseudomonas aeruginosa-contaminated products such as cosmetics.

[0058] 1. Instruments and Reagents

[0059] 1.1 Instruments: Liquid chromatography coupled with a triple quadrupole mass spectrometer (triple Quad 5500+), AB SCIEX, USA; vortex shaker (SK-1), Jintan Medical Instrument Factory, Jiangsu Province; ultrapure water analyzer (Milli-Q), Merck Millipore, Germany; high-speed centrifuge (DE-16R), Changsha Dongwang Experimental Instrument Co., Ltd.; ultrasonic cleaning machine (FRQ-1020S), Hangzhou Farangte Ultrasonic Technology Co., Ltd.; biochemical incubator (SPX-250F-II), Shanghai Longyue Instrument Equipment Co., Ltd.

[0060] 1.2 Reagents

[0061] 1.2.1 Metabolite screening

[0062] Pyocyanin is a phenazine compound with a unique color and redox activity, produced by Pseudomonas aeruginosa. It is an important virulence factor of Pseudomonas aeruginosa. Its redox activity can disrupt the redox balance of host cells, affect the function of biomacromolecules, and even cause cell death. In existing studies, Pseudomonas aeruginosa is known to release three signaling substances: 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), 4-heptyl-4(1H)-quinolone (HHQ), and 2-heptyl-4-quinolone-n-oxide (HQNO) [26,27]. In addition, Pseudomonas aeruginosa LV strain can produce the biologically active metabolites phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN). Pseudomonas aeruginosa M18 can produce a wide spectrum of secondary metabolites, including the antibiotic phenazine-1-carboxylic acid (PCA). Pseudomonas aeruginosa can also produce the extracellular metabolite 4-hydroxyquinoline. Simoska Olja achieved real-time electrochemical detection of 1-hydroxyphenazine metabolites from Pseudomonas aeruginosa and other strains. In addition to the aforementioned information on Pseudomonas aeruginosa, given the high-throughput screening capabilities of mass spectrometry, the present invention also selected additional substances with similar quinoline structures as research targets. The 13 Pseudomonas aeruginosa metabolites and their sources are listed in Table 1 below.

[0063] Table 1 13 metabolite standards of Pseudomonas aeruginosa and their sources

[0064] Serial number Metabolite name purity CAS number source 1 Pyocyanin 95％ CAS85-66-5 Bid Pharmaceuticals 2 4-Hydroxyquinoline 99.4％ CAS611-36-9 Bid Pharmaceuticals 3 2,6-Dihydroxyquinoline 99％ CAS19315-93-6 Source Leaf Bio 4 2-Methyl-8-hydroxyquinoline 99.4％ CAS826-81-3 Bid Pharmaceuticals 5 6,7-Dimethoxy-4-hydroxyquinoline 97％ CAS13425-93-9 Source Leaf Bio 6 2-Heptyl-4-hydroxyquinoline-N-oxide (HQNO) 97％ CAS341-88-8 Source Leaf Bio 7 2-Heptyl-4-quinolinone (HHQ) 98％ CAS40522-46-1 SIGMA 8 2-Amino-4-hydroxyquinoline hydrate 98％ CAS42712-64-1 damas-beta 9 2-Hydroxyquinoxaline 98.6％ CAS1196-57-2 Bid Pharmaceuticals 10 1-Hydroxyphenazine 95％ CAS528-71-2 damas-beta 11 Phenazine-1-carboxylic acid (PCA) 98％ CAS2538-68-3 BePure 12 Phenazine-1-carboxamide (PCN) 95％ CAS550-89-0 damas-beta 13 Lutein 95％ CAS25683-07-2 TargetMol

[0065] 1.2.2 Other experimental reagents: Acetonitrile (chromatographic grade), methanol (chromatographic grade), formic acid (chromatographic grade), and sodium chloride (analytical grade) were purchased from Shanghai Anpu Laboratory Technology Co., Ltd. ProElut C18 (200 mg / 3 ml), ProElut PLS (500 mg / 6 ml), QuEChERS (50 mg PSA, 150 mg MgSO4), and QuEChERS (100 mg PSA, 40 mg C18, 600 mg MgSO4) were all purchased from Dima Technology Co., Ltd.

[0066] Pseudomonas aeruginosa ATCC27853, Pseudomonas fluorescens ATCC13525, Pseudomonas putida CICC10298, brain heart infusion broth (BHI) 220107, hexadecyltrimethylammonium bromide medium 210908, and pyocyanin assay medium 211124 were all purchased from Beijing Luqiao Company.

[0067] The present invention also tested 130 batches of cosmetics, including creams, toothpastes, serums, facial masks, wipes, lotions, toners, hair dyes, bath products, shampoos, sunscreens, soothing products, conditioners, and facial cleansers, all of which were leftover samples from random inspections and commercially available samples. Sample selection was based on the "Cosmetics Classification Rules and Classification Catalog" (Announcement No. 49 of 2021 of the State Food and Drug Administration).

[0068] 2. Experimental Methods

[0069] 2.1 Sample pretreatment

[0070] In order to determine the optimal pre-treatment conditions, the present invention also studies the effects of different pre-treatment conditions on the present invention method, thereby determining and optimizing the pre-treatment conditions of the present invention scheme. The specific operation is as follows:

[0071] 2.2.1 Optimization of extraction method

[0072] Direct sample extraction using methanol and acetonitrile can easily cause some cosmetic samples to aggregate, hindering recovery. Pre-addition of saturated sodium chloride solution disperses the sample, improving extraction efficiency. Acetonitrile outperforms methanol in extraction and recovery. Therefore, we chose saturated sodium chloride solution-acetonitrile as the sample extraction method. The recoveries for different extraction methods are shown in Table 2.

[0073] Table 2 Comparison of recovery rates of 13 compounds using different extraction methods (n=3) (unit: %)

[0074]

[0075] 2.2.2 Optimization of purification methods

[0076] Cosmetics and culture medium samples have complex compositions. If the extracts are not adequately purified, impurities can easily precipitate, reducing column efficiency and contaminating the ion source, hindering experimental analysis. Therefore, the effectiveness of four purification methods was compared: C18 (200 mg / 3 mL), PLS (500 mg / 6 mL), QuEChERS (50 mg PSA, 150 mg MgSO₄), and QuEChERS (100 mg PSA, 40 mg C18, 600 mg MgSO₄). Both C18 and PLS solid-phase extraction columns improved salting-out in the extracts, with the former showing a slight advantage in recovery. The former can be assembled with a PTFE filter membrane, making it more convenient to use. Therefore, C18 (200 mg / 3 mL) was selected for the purification of the sample extracts in this experiment. The recoveries of the different purification methods are shown in Table 3.

[0077] Table 3 Comparison of recovery rates of 13 compounds using different purification methods (n=3) (unit: %)

[0078]

[0079]

[0080] 2.2.3 Effect of filtration membrane and temperature

[0081] By comparing the responses before and after filtration of the standard solution (100 μg / L), it was found that the nylon filter membrane had a strong adsorption effect on most target substances, with adsorption losses as high as 40%. Therefore, this method selected PTFE filter membrane to filter the samples. The compound recovery rates of different filter membranes are shown in Table 4.

[0082] Table 4 Comparison of recovery rates of 13 compounds using different filter membranes (n=3) (unit: %)

[0083] Serial number Compound Nylon 0.22um PTFE0.22um 1 Pyocyanin 76.8±2.3 94.2±2.5 2 4-Hydroxyquinoline 81.3±1.7 95.9±1.6 3 2,6-Dihydroxyquinoline 85.9±1.6 96.8±2.1 4 2-Methyl-8-hydroxyquinoline 78.1±2.3 95.3±1.9 5 6,7-Dimethoxy-4-hydroxyquinoline 77.2±2.4 91.5±1.7 6 2-Heptyl-4-hydroxyquinoline-N-oxide (HQNO) 82.5±1.7 95.4±2.3 7 2-Heptyl-4-quinolinone (HHQ) 70.9±1.6 96.8±1.2 8 2-Amino-4-hydroxyquinoline hydrate 78.2±1.0 99.5±1.4 9 2-Hydroxyquinoxaline 84.3±2.7 97.6±2.4 10 1-Hydroxyphenazine 100.8±2.1 100.9±1.9 11 Phenazine-1-carboxylic acid (PCA) 57.9±1.1 96±2.6 12 Phenazine-1-carboxamide (PCN) 82.1±1.6 98±2.3 13 Lutein 72.4±2.5 99.7±1.1

[0084] To evaluate the thermal stability of the test substances, a mixed standard solution (100 μg / L) was heated in a water bath at 50°C and 100°C for 30 minutes before analysis. The results showed that the responses of the 13 substances did not change much, indicating good thermal stability.

[0085] The final pretreatment conditions were as follows: 0.2 g of sample was weighed and placed in a 10 mL stoppered graduated centrifuge tube. 0.5 mL of saturated sodium chloride solution was added and mixed thoroughly. 8 mL of acetonitrile was added, and the mixture was shaken and ultrasonically extracted for 10 minutes. The acetonitrile volume was adjusted to 10 mL, and the sample was centrifuged at 8000 rpm for 5 minutes. The sample was cleaned up using a C18 solid-phase extraction column and then filtered through a 0.22 μm PTFE organic filter membrane. The sample was then subjected to LC-MS / MS analysis.

[0086] Heat the culture medium until it is liquid, mix thoroughly, and then measure. The solution to be tested can be diluted according to the content to ensure that the substance to be tested is within the concentration range of the standard curve.

[0087] 2.2 Preparation of standard solution

[0088] Of the 13 metabolites, 2,6-dihydroxyquinoline and 2-hydroxyquinoxaline were first dissolved in 0.5 mL of DMSO and then diluted to 10 mL with methanol. The remaining standards were dissolved directly in methanol and then diluted to 10 mL. The prepared standard stock solution had a concentration of 1.0 g / L and was stored at -20°C. A mixed standard intermediate solution with a concentration of 10 mg / L was prepared by taking 100 μL of the individual standard stock solutions. Before use, a 1.0 mg / L mixed standard solution was prepared using the blank matrix solution. This was then diluted to form a series of standard matrix working curves with concentrations of 1, 2, 5, 10, 20, 50, and 100 μg / L.

[0089] 2.3 Chromatographic conditions

[0090] To determine suitable chromatographic conditions, the present invention studied five different mobile phase systems: 0.1% formic acid-acetonitrile, 0.1% formic acid-methanol, water-acetonitrile, water-methanol, and 5 mmol / L ammonium formate solution-methanol. The peak shapes and responses of most substances in the methanol system were superior to those in the acetonitrile system. The peak response with 5 mmol / L ammonium formate solution as the mobile phase was comparable to that of water. Adding 0.1% formic acid to the water mobile phase improved the peak shapes of pyocyanin and 2-methyl-8-hydroxyquinoline. Due to the high sensitivity of pyocyanin, the negative ion mode still exhibited good response even under 0.1% formic acid.

[0091] In this experiment, 0.1% formic acid-methanol was selected as the mobile phase system. The specific chromatographic column conditions were: Agilent SB-C18 RRHD (2.1mm×100mm, 1.8μm); mobile phase: A was 0.1% formic acid water, B was methanol; gradient elution program: 0-3min, 60%B-80%B; 3-9min, 80%B-80%B; 9.1-12min, 60%B; flow rate: 0.4mL / min; column temperature: 40℃; injection volume: 2μL.

[0092] The total ion chromatograms (TIC) and multiple reaction monitoring chromatograms (MRM) of the 13 metabolites in the mixed standard solution are shown in Figure 2. Figures 1 to 14 shown.

[0093] 2.4 Mass spectrometry conditions

[0094] The specific mass spectrometry conditions are as follows: electrospray ion source (ESI source); positive ion mode electrospray voltage: 5500 V; negative ion mode electrospray voltage: -4500 V; nebulization temperature: 500°C; nebulizing gas pressure: 50 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 20 psi; collision gas pressure: 9 psi; monitoring mode: multiple reaction monitoring (MRM); the mass spectrometry parameters of quantitative ion pairs, qualitative ion pairs, declustering voltage and collision energy of each analyte are shown in Table 5.

[0095] A mixed standard solution with a concentration of 1.0 mg / L was continuously injected at a flow rate of 3 μL / min. Mass spectrometry parameters, including parent ion, daughter ion, declustering voltage (DP), and collision energy (CE), were scanned and optimized (see Table 5). Pyocyanin, 4-hydroxyquinoline, 2-methyl-8-hydroxyquinoline, 6,7-dimethoxy-4-hydroxyquinoline, 2-amino-4-hydroxyquinoline hydrate, 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN) showed significant advantages in positive ion mode. Lutein showed a better response in negative ion mode than in positive mode. The differences between positive and negative ion modes for the other materials were not significant. Therefore, this method employed simultaneous acquisition in positive and negative ion modes.

[0096] Table 5 Mass spectrometry parameters of 13 metabolites

[0097]

[0098]

[0099] 2.5 Matrix Effects

[0100] The matrix effects of the three methods were investigated using blank culture medium, lotion, and fragrance water as matrices. The calculation method was to divide the slope of the matrix standard curve (k1) by the slope of the solvent standard curve (k2). The lotion used in this experiment was the commercially available SeSamSu Brightening Skin Whitening Lotion, and the fragrance water was Lanxi Brightening Water.

[0101] 2.6 Method Analysis

[0102] Precision and standard recovery tests were conducted on blank culture medium, emulsion, and serum samples. Standard solutions of varying concentrations were added to the matrix samples, repeated six times. Using the lowest-responding substance in the test solution as the reference, the limit of detection (LOD) was defined as the concentration corresponding to 3 times the signal-to-noise ratio (SNR), and the limit of quantification (LOQ) was defined as the concentration corresponding to 10 times the SNR.

[0103] 2.7 Metabolic analysis

[0104] Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens from the same genus were selected for comparison to investigate their metabolism and specificity. These three strains were cultured in different culture media and temperatures. The levels of 13 metabolites in the culture media were measured, and a blank control experiment was also performed.

[0105] 2.8 Analysis of Actual Cosmetic Samples

[0106] Cosmetic raw materials contain abundant nutrients such as nitrogen and carbon sources, and inorganic salts, which are beneficial to the growth and metabolism of Pseudomonas aeruginosa. Therefore, we speculate that cosmetics may be at risk of contamination from Pseudomonas aeruginosa metabolism or raw materials. We conducted an analysis to detect residual Pseudomonas aeruginosa metabolites in cosmetics.

[0107] 3. Results Analysis

[0108] 3.1 Matrix Effect

[0109] The three typical sample types described above—culture medium, emulsion, and serum—were used as blank matrix samples. Matrix effects were assessed by comparing the slope of the blank matrix standard curve with the slope of the solvent standard curve, divided by the slope of the solvent standard curve (k1 / k2). The results showed that most substances exhibited matrix inhibition, as shown in Tables 6, 7, and 8.

[0110] 3.2 Linear range and detection limit

[0111] Three blank matrix solutions were prepared as described above. These solutions were diluted to concentrations of 1, 2, 5, 10, 20, 50, and 100 μg / L, respectively, and assayed using HPLC-MS / MS. External standard quantification was performed using matrix standardization. Linear fitting of concentration and peak area revealed good linear relationships for all 13 test substances (see Tables 6, 7, and 8). Correlation coefficients were all greater than 0.99. The detection limit for the lowest-response compound was 0.05 mg / kg at a 3-fold signal-to-noise ratio, and the quantification limit was 0.10 mg / kg at a 10-fold signal-to-noise ratio.

[0112] Table 6 Statistical results of linear equation, correlation coefficient and matrix effect of culture medium

[0113]

[0114] Table 7 Statistical results of linear equation, correlation coefficient and matrix effect of emulsion

[0115]

[0116] Table 8 Statistical results of linear equation, correlation coefficient and matrix effect of essence water

[0117]

[0118] 3.3 Recovery and precision

[0119] Recovery and precision were tested at three levels using blank culture medium, emulsion, and extract water samples: 1× the limit of quantification (LOQ), 2× the limit of quantification (2×LOQ), and 10× the limit of quantification (10×LOQ). Samples were processed and analyzed according to the experimental methods described in Part II. The results are shown in Tables 9, 10, and 11. The recovery rates for culture medium ranged from 85.1% to 114.7%, with RSDs of 1.3% to 9.8%. The recovery rates for emulsion ranged from 85.3% to 112.9%, with RSDs of 1.7% to 13.3%. The recovery rates for extract water ranged from 86.2% to 112.3%, with RSDs of 1.9% to 9.6%. The recovery and precision met the requirements of CFDA

[2010] 455 and SANTE / 11813 / 2017G7.

[0120] Table 9 Recovery and precision results of culture medium (n=6)

[0121]

[0122]

[0123] Table 10 Statistics of recovery and precision results of emulsion (n=6)

[0124]

[0125] Table 11 Recovery and precision results of essence water (n=6)

[0126]

[0127] 3.4 Metabolic analysis of Pseudomonas aeruginosa

[0128] The present invention also provides a specific embodiment of applying the above detection method to the metabolic analysis of Pseudomonas aeruginosa to analyze the factors affecting metabolism and specific metabolites. The analysis is performed using the following steps:

[0129] In the first step, Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas fluorescens are activated to obtain a bacterial activation solution.

[0130] In the second step, 90 μL of each of the three strain activation solutions was inoculated into BHI broth, and the mixed three strain activation solutions were inoculated into another portion of BHI broth. The cultures were cultured at 28°C and 36°C for 48 h.

[0131] In the third step, we inoculated the above culture medium into a loop of hexadecyltrimethylammonium bromide culture medium and pyocyanin assay culture medium. The sample was cultured again at the corresponding temperature for 48 hours, and a blank control culture medium was set up at the same time. Figure 15 shown.

[0132] A validated HPLC-MS / MS method was used to determine the levels of 13 metabolites in enrichment medium (BHI broth) and selective medium (cetyltrimethylammonium bromide medium or pyocyanin assay medium). The results are shown in Tables 12, 13, and 14.

[0133] Table 12 Content of 13 metabolites in enrichment medium (BHI broth) (unit: μg / kg)

[0134]

[0135] Table 13 Content results of 13 metabolites in hexadecyltrimethylammonium bromide culture medium (unit: μg / kg)

[0136]

[0137]

[0138] Table 14 Results of determination of the contents of 13 metabolites in the culture medium of pyocyanin (unit: μg / kg)

[0139]

[0140] Note: In Tables 12, 13, and 14 above, due to the complexity of factors affecting microbial reproduction and metabolism, the measurement results in the tables are for reference only; ND indicates that the detected content is less than the limit of quantification (0.10 mg / kg); PA indicates Pseudomonas aeruginosa; PP indicates Pseudomonas putida; PF indicates Pseudomonas fluorescens; and Mx indicates a mixture of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas putida.

[0141] Experimental results revealed that pyocyanin, 2-heptyl-4-quinolone (HHQ), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine were detected in all culture media containing Pseudomonas aeruginosa. These substances were not detected in culture media containing only Pseudomonas putida and Pseudomonas fluorescens. Pseudomonas aeruginosa can reproduce and metabolize rapidly in selective culture media. Even with only a single loop inoculation, metabolite levels were nearly 10 times higher than in the supplemental culture medium. The levels of different metabolites also varied. Pyocyanin, 2-heptyl-4-quinolone (HHQ), and phenazine-1-carboxylic acid (PCA) were high. In contrast, 1-hydroxyphenazine was relatively low. Different temperature conditions had no significant effect on the metabolism of Pseudomonas aeruginosa, nor did the introduction of other strains have a significant effect. In conclusion, pyocyanin, 2-heptyl-4-quinolinone (HHQ), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine can be used as specific biomarkers for P. aeruginosa.

[0142] 3.5 Determination of actual cosmetic samples

[0143] As a product that comes into direct contact with human skin, cosmetics have complex ingredients. In addition to being rich in water, they are also rich in glycerol, mineral oil, polyols, xanthan gum, amino acids, vitamins, plant extracts, mineral components, etc. Its abundant nitrogen sources, carbon sources, inorganic salts and other nutrients may provide favorable conditions for the metabolism of Pseudomonas aeruginosa. In view of the severity and harmfulness of current Pseudomonas aeruginosa contamination. In this embodiment, it is assumed that cosmetics may have the risk of raw material import and contamination. In order to study the residual risk of Pseudomonas aeruginosa metabolites in cosmetics, the established method is applied to the analysis of actual cosmetic samples.

[0144] Analysis of 130 actual cosmetic samples revealed that none of the 13 metabolites listed above were detected, indicating a relatively low risk of residual Pseudomonas aeruginosa metabolites in cosmetics. This suggests that, despite the presence of various nutrients in cosmetics, effective quality control for Pseudomonas aeruginosa contamination has been implemented in marketed cosmetics within the scope of the currently tested samples.

[0145] 4. Conclusion and Analysis

[0146] The present invention constructs an HPLC-MS / MS determination method for the metabolites of Pseudomonas aeruginosa. First, 13 possible metabolites were screened and the method conditions were optimized. The sample was dispersed in saturated sodium chloride, extracted with acetonitrile, purified by C18-SPE, and analyzed after PTFE membrane filtration. The results showed that within the linear range of 1 to 100 μg / L, the linear correlation coefficients (r) of the 13 metabolites were all greater than 0.99, the sample recovery rate was 85.1 to 114.7%, the relative standard deviation (RSD) was 1.3 to 13.3%, the limit of quantification was 0.05 mg / kg, and the limit of quantification was 0.1 mg / kg. The verified method was applied to the metabolic analysis of Pseudomonas aeruginosa and the detection of metabolite residues in cosmetics. In this experiment, three bacterial species of the same genus, Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas fluorescens, were cultured and the metabolite content levels in the culture medium were analyzed. Results showed that pyocyanin, 2-heptyl-4-quinolinone (HHQ), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine have the potential to serve as specific biomarkers for Pseudomonas aeruginosa. Tests on actual samples indicated a low risk of Pseudomonas aeruginosa metabolic residues in cosmetics, likely due to effective quality control of commercially available cosmetics for Pseudomonas aeruginosa contamination. The method of the present invention offers advantages such as high sensitivity, strong specificity, good selectivity, good accuracy, and ease of operation.

[0147] The above descriptions are only some preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS, characterized in that: The steps include: S1. Sample pretreatment and preparation: Weigh the sample, add saturated sodium chloride solution and mix well, add acetonitrile, shake and mix well, then extract by ultrasonic wave, then make up to volume with acetonitrile, centrifuge, clean up with C18 solid phase extraction column, and filter through 0.22 μm PTFE organic filter membrane to obtain the sample solution to be tested; S2. Prepare standard solutions: Dissolve the standard samples of Pseudomonas aeruginosa metabolites in methanol and dilute to volume to prepare standard stock solutions; prepare mixed standard intermediate solutions by taking a single standard stock solution of each standard sample; prepare mixed standard solutions with blank matrix solution before use, and then gradually dilute with blank matrix solution to obtain a series of standard matrix solutions; S3. (1) Liquid chromatography conditions: Column: Agilent SB-C18 RRHD (2.1 mm × 100 mm, 1.8 μm); Mobile phase: A: 0.1% formic acid in water, B: methanol; Flow rate: 0.4 mL / min; Column temperature: 40°C; Injection volume: 2 μL; Liquid chromatography was performed using gradient elution, and the gradient elution procedure was as follows: 0-3min, 60%B-80%B; 3-9 min, 80% B-80% B; 9.1~12min, 60%B; (2) Mass spectrometry conditions: electrospray ionization source (ESI source); electrospray voltage in positive ion mode: 5500 V; electrospray voltage in negative ion mode: -4500 V; nebulization temperature: 500°C; nebulization gas pressure: 50 psi; Auxiliary gas pressure: 50psi; Curtain gas pressure: 20psi; Collision gas pressure: 9 psi; Monitoring mode: multiple reaction monitoring (MRM); S4. Perform qualitative and quantitative analysis on the components in the sample solution.

2. The method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS according to claim 1, characterized in that The Pseudomonas aeruginosa metabolites include at least one of pyocyanin, 4-hydroxyquinoline, 2,6-dihydroxyquinoline, 2-methyl-8-hydroxyquinoline, 6,7-dimethoxy-4-hydroxyquinoline, 2-heptyl-4-hydroxyquinoline-N-oxide, 2-heptyl-4-quinolinone, 2-amino-4-hydroxyquinoline hydrate, 2-hydroxyquinoxaline, 1-hydroxyphenazine, phenazine-1-carboxylic acid, phenazine-1-carboxamide, and luteolin.

3. The method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS according to claim 1, characterized in that The mass spectrometry parameters of the target compound to be detected in step S3 are as follows:

4. The method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS according to claim 1, characterized in that The qualitative analysis method in step S4 is as follows: under the same experimental conditions, the mass chromatographic peak retention time of the measured component in the sample solution is consistent with the mass chromatographic peak retention time of the corresponding component in the standard solution; and the deviation between the relative abundance ratio of the selected monitoring ion pair in the sample chromatogram and the relative abundance ratio of the ion of the standard solution of equivalent concentration is within the following range, then it can be determined that the corresponding component is present in the sample: The maximum allowable deviations of the relative ion abundance ratio k are ±20%, ±25%, ±30% and ±50% when k≥50%, 50%>k≥20%, 20%>k≥10% and k<10%, respectively.

5. The method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS according to claim 1, characterized in that The quantitative analysis method in step S4 is: external standard quantification is performed using a matrix standard solution, and quantitative analysis is performed by linear fitting of concentration and peak area. The linear correlation coefficient is greater than 0.99, and the detection limit corresponding to 3 times the signal-to-noise ratio of the lowest response substance is 0.05 mg / kg, and the quantification limit corresponding to 10 times the signal-to-noise ratio of the lowest response substance is 0.10 mg / kg.

6. The method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS according to claim 1, characterized in that The concentration of the standard stock solution in step S2 is 1.0 g / L; the concentration of the mixed standard intermediate solution is 10 mg / L; the concentration of the mixed standard solution is 1.0 mg / L; the concentrations of the obtained series of standard matrix solutions are 1, 2, 5, 10, 20, 50, and 100 μg / L.

7. The method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS according to claim 1, characterized in that In step S1, 0.2 g of the cosmetic sample was added with 0.5 mL of saturated sodium chloride solution and mixed. Then, 8 mL of acetonitrile was added, and the mixture was shaken and mixed, followed by ultrasonic extraction for 10 min. The volume was then adjusted to 10 mL with acetonitrile. The centrifugation conditions were: 8000 rpm for 5 min.

8. The method for detecting Pseudomonas aeruginosa metabolites based on HPLC-MSMS according to claim 1, characterized in that: The method also includes, before S3, an evaluation of the thermal stability of the substance to be tested, specifically, heating the mixed standard solution in a water bath at 50° C. and 100° C. for 30 minutes, and then performing analysis and determination.

9. Use of the method according to any one of claims 1 to 8 in a metabolic analysis method of Pseudomonas aeruginosa, characterized in that: The steps include: S1. activating Pseudomonas aeruginosa to obtain a strain activation solution; S2. Inoculate 90 μL of the activated solution of the strain S1 into enrichment medium respectively, and culture at 28°C and 36°C for 48 h; the enrichment medium is BHI broth; S3. Inoculate the cultured strain into a loop of selective culture medium, and culture the sample again at the corresponding temperature for 48 hours. The selective culture medium is hexadecyltrimethylammonium bromide medium or pyocyanin assay medium; S4. The method described above is used to respectively determine the content of metabolites of Pseudomonas aeruginosa in the enrichment medium and the selective medium.

10. Use of the method according to any one of claims 1 to 8 in a method for controlling the quality of cosmetics.

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