Screening of hormones in cosmetics by tungsten nitride sers combined with quechers

Abstract

The application discloses a screening method for hormones in cosmetics by combining tungsten nitride SERS and QuEChERS, and comprises the following steps: (1) sample pretreatment: using the QuEChERS method to extract and purify the cosmetic sample, and obtaining a to-be-tested filtrate; (2) SERS sample preparation: spin-coating the to-be-tested filtrate on the surface of a pre-prepared WN nanosheet substrate, and forming a surface-enhanced Raman scattering detection sample after solvent evaporation; and (3) constructing a double-parameter discriminant rule: using the peak position difference and relative intensity ratio of double characteristic peaks in the A-ring conjugated vibration region of steroids to construct a SERS fingerprint discriminant rule for steroid hormones. The method deeply combines efficient pretreatment, uniform sample loading and 'double characteristic peak' fingerprint information, and provides a new technical idea and reliable means for rapid, high-sensitivity and accurate qualitative screening of hormones in complex cream matrix.

Description

Technical Field

[0001] This invention relates to the field of cosmetic testing and inspection, and in particular to a method for screening hormones in cosmetics using tungsten nitride (WN) SERS combined with QuEChERS. Background Technology

[0002] Glucocorticoids possess anti-inflammatory, anti-allergic, and immunosuppressive pharmacological activities and are widely used in clinical practice. However, long-term or inappropriate exposure can lead to skin atrophy, telangiectasia, and steroid-dependent dermatitis, and in severe cases, may cause systemic adverse reactions. Therefore, they are explicitly listed as prohibited ingredients in cosmetics. Current methods for detecting hormone-like components in cosmetics primarily employ liquid chromatography-mass spectrometry (LC-MS) for qualitative and quantitative analysis of hormone-like components in cosmetics. This method is applicable to various bases such as creams, lotions, aqueous solutions, gels, and masks, and a detection framework for 63 hormone-like components has been established.

[0003] Currently, chromatography-mass spectrometry (GC-MS) remains the mainstream detection technique for glucocorticoids in cosmetics. Although GC-MS is relatively mature in terms of sensitivity, quantitative capability, and structural confirmation, its high equipment cost, cumbersome sample pretreatment, and relatively long detection cycle make it difficult to meet the needs of rapid on-site screening. Looking at the development trend of cosmetic testing technology, the integration, rapidity, and on-site application of sample pretreatment and detection methods are gradually becoming important directions. Currently, the core challenge in the analysis of glucocorticoids in cosmetics lies in the complex matrix, diverse structures, and trace amounts present; one of the key areas for future development is the combination of efficient pretreatment and rapid detection technologies. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for screening hormones in cosmetics using tungsten nitride (WN) SERS combined with QuEChERS, which is simple to operate, fast to detect, requires small sample volume, and has strong molecular fingerprint recognition ability.

[0005] This invention utilizes the dual properties of WN sheets as both SERS-enhancing substrates and hydrophobic adsorption materials to construct a novel method combining QuEChERS pretreatment with WN-SERS screening. Using dexamethasone (DEX), dexamethasone (HC), dexamethasone acetate (DEX-Ac), and clobetasol propionate (CP) as target compounds, the spectral characteristics, adsorption enhancement mechanisms, and application potential in complex cosmetic matrices were investigated.

[0006] A method for screening hormones in cosmetics using tungsten nitride (WN) SERS combined with QuEChERS includes the following steps: (1) Sample pretreatment: The cosmetic sample was extracted and purified using the QuEChERS method to obtain the filtrate to be tested; (2) SERS sample preparation: The filtrate to be tested was spin-coated onto the surface of the pre-prepared WN nanosheet substrate, and the solvent was evaporated to form a surface-enhanced Raman scattering (SERS) detection sample; (3) Constructing a two-parameter discrimination rule: Using the peak position difference (Δν) and relative intensity ratio (R) of the two characteristic peaks in the conjugate vibration region of steroid A ring, a SERS fingerprint discrimination rule for steroid hormones is constructed.

[0007] The method for screening hormones in cosmetics using tungsten nitride (WN) SERS combined with QuEChERS as described in this invention, wherein: the sample pretreatment method in step (1) specifically includes the following steps: Step 1: Accurately weigh 0.50 g of sample into a 15 mL polypropylene centrifuge tube, add 3 mL of saturated sodium chloride solution, and vortex to fully disperse the sample. Then add 5 mL of acetonitrile, vortex for 1 min, sonicate for 10 min, and then centrifuge at 4000 r / min for 5 min. Step 2: Pipette 2.0 mL of acetonitrile phase into another centrifuge tube, add 100 mg of PSA, 50 mg of C18 and 150 mg of anhydrous MgSO4, vortex to mix for 1 min, and then centrifuge at 4000 r / min for 5 min; take the supernatant and filter it through a 0.22 μm microporous membrane, take 1.0 mL of the filtrate and blow it with nitrogen to near dryness at room temperature, then redissolve it with 200 μL of 50% ethanol aqueous solution to obtain the test sample solution.

[0008] The method for screening hormones in cosmetics using tungsten nitride (WN) SERS combined with QuEChERS as described in this invention includes the following steps between the first and second steps in the sample pretreatment method, if the sample is a cream or ointment: after taking the supernatant, add 5 mL of acetonitrile-saturated n-hexane solution, mix well, centrifuge at 4000 r / min for 5 min, and take the lower acetonitrile phase as the extraction solution to be purified.

[0009] The tungsten nitride (WN) SERS combined with QuEChERS screening method for hormones in cosmetics described in this invention includes the following steps in step (2): 20 μL of the sample solution to be tested is dropped onto the surface of a pre-prepared WN substrate, and the sample solution is evenly distributed on the substrate surface by spin coating. Then, it is dried at 60 °C for 10 min. After the sample is completely dry, SERS spectrum is collected. A substrate blank and a solvent blank are set as controls during the test. The SERS detection parameters are: excitation wavelength 532 nm; laser power 0.5 mW; scanning range 400 ~ 2000 cm⁻¹ -1 The integration time for each point is 1 second, and the integration is performed once. Five different locations are selected for testing for each sample, and the average spectrum is taken as the final result.

[0010] The tungsten nitride (WN) SERS combined with QuEChERS screening method for hormones in cosmetics described in this invention, wherein: the spectral identification in step (3) further employs a two-parameter discrimination method: using 1600–1670 cm⁻¹ as the spectral density. -1 The peak position difference between two characteristic peaks within the interval is Δν = ν high - ν low With relative strength ratio R=I high / I low As an identification parameter, the structure type is determined according to the following rules: When Δν ≤ 35 cm -1 When 0.8 ≤ R ≤ 1.2, it is determined to be a hormone with a cyclohexene structure in ring A; When Δν ≥ 50 cm -1 When R > 1.5, it is determined to be a hormone with a cyclohexadiene (cross-conjugated) structure in ring A; The characteristic peak is located at 1600–1670 cm⁻¹ -1 If the interval Δν and R do not meet the above conditions, it is marked as "suspected hormone pending verification" and needs to be further confirmed by LC-MS / MS.

[0011] The present invention describes a method for screening hormones in cosmetics using tungsten nitride (WN) SERS combined with QuEChERS, wherein: the hormones having a cyclohexene structure on ring A include hydrocortisone; the hormones having a cyclohexadiene structure on ring A include dexamethasone, dexamethasone acetate, and clobetasol propionate.

[0012] The tungsten nitride (WN) SERS combined with QuEChERS method for screening hormones in cosmetics differs from existing technologies in that:

[0013] This invention establishes a screening method for hormones in cosmetics based on tungsten nitride (WN) SERS combined with QuEChERS. Using tungsten nitride (WN) tablets as an enhancing substrate and combined with QuEChERS pretreatment technology, four typical glucocorticoids, namely hydrocortisone, dexamethasone, dexamethasone acetate and clobetasol propionate, were studied.

[0014] This invention first addresses the characteristics of cosmetics (especially creams and lotions) with high lipid content and complex matrices. It introduces acetonitrile-saturated n-hexane for liquid-liquid partitioning using the QuEChERS method to effectively remove high-lipid impurities, and optimizes the adsorbent ratio (PSA 100 mg, C18 50 mg, and anhydrous MgSO4 150 mg) for deep purification. Subsequently, near-drying with nitrogen and reconstitution with a 50% ethanol aqueous solution not only minimizes matrix interference but also achieves highly efficient concentration and enrichment of the target analyte. Secondly, using WN sheets with stable backgrounds and no obvious Raman interference peaks as the enhancement substrate, SERS samples are prepared using spin-coating. This method effectively overcomes the "coffee ring effect" that easily occurs during traditional droplet natural drying, resulting in a highly uniform distribution of the sample solution on the WN substrate surface, thereby significantly improving the inter-point stability and reproducibility of the SERS signal. Then, by analyzing the common characteristics of the steroidal nucleus of four typical glucocorticoids (HC, DEX, DEX-Ac, and CP), a novel approach was taken to focus on the conjugate vibrational region of the steroid A ring (1600–1670 cm⁻¹). -1 Based on the dual characteristic peaks of clobetasol propionate, a two-parameter discrimination rule is proposed using "peak position difference (Δν)" and "relative intensity ratio (R)". This rule can distinguish glucocorticoids with different A-ring skeleton types, significantly improving the anti-interference ability and qualitative accuracy of target screening in complex systems. Finally, using clobetasol propionate as the target, quantitative analysis can be achieved through linear fitting of characteristic peak intensity and logarithmic concentration values.

[0015] The method of this invention, combined with the excellent reinforcing properties of the WN nanosheet substrate, exhibits excellent SERS responses for typical glucocorticoids of various structures. Particularly effective against high-risk substances commonly illegally added to cosmetics (such as clobetasol propionate CP), its characteristic bimodal peaks remain clearly discernible even at extremely low concentrations, fully meeting the needs of rapid screening for trace amounts of illegal additives in actual market supervision.

[0016] The method of this invention, combined with the excellent reinforcing properties of the WN nanosheet substrate, exhibits highly sensitive SERS responses to typical glucocorticoids with different A-ring backbone types (including cyclohexene and cyclohexadiene conjugated structures). Particularly targeting clobetasol propionate, a high-risk hormone with a cross-conjugated structure commonly illegally added to cosmetics, its characteristic bimodal peaks for structural typing remain clearly discernible even at extremely low concentrations. This not only verifies the extremely low detection limit of this method but also ensures that even at trace residue levels, high-risk targets can be rapidly identified using a two-parameter model, fully meeting the needs of accurate screening and risk classification of trace illegal additives in actual market supervision.

[0017] In summary, the method of this invention deeply integrates efficient pretreatment, homogenized sample loading, and a two-parameter mathematical model, providing a novel technical approach and reliable means for rapid, highly sensitive, and accurate qualitative screening of various steroid hormones in complex cream matrices.

[0018] The following description, in conjunction with the accompanying drawings, further illustrates the method for screening hormones in cosmetics using tungsten nitride (WN) SERS combined with QuEChERS according to the present invention. Attached Figure Description

[0019] Figure 1 These are the Raman spectra of the blank solvent and the WN substrate in the method of this invention; Figure 2 This is a structural diagram of the cyclopentane-polyhydrophenanthrene parent core in the method of the present invention; Figure 3 These are Raman spectra of the four glucocorticoids used in the method of this invention; Figure 4 This is a graph showing the SERS enhancement effect of four glucocorticoids in the method of this invention; Figure 5 These are characteristic peak intensity diagrams of different reconstituted sample solutions in the method of this invention; Figure 6 This is the SERS spectrum of the clobetasol propionate spiked sample in the method of the present invention. Detailed Implementation

[0020] 1. Materials and Reagents

[0021] 1.1 Reagents

[0022] Hydrocortisone, dexamethasone, dexamethasone acetate and clobetasol propionate; sodium chloride (NaCl); anhydrous magnesium sulfate (MgSO4); N-propylethylenediamine (PSA, 40 μm); octadecyl bonded silica gel (C18, 40 μm); acetonitrile, ethanol.

[0023] 1.2 Instruments and Equipment

[0024] Raman spectrometer, excitation wavelength 532 nm; laser power 0.5 mW; scanning range 400 ~ 2000 cm⁻¹ -1 The integration time for each point is 1 second, and the integration is performed once. Five different locations are selected for testing for each sample. The equipment also includes a vortex mixer, centrifuge, electronic balance, ultrasonic cleaner, nitrogen purging device, and vacuum drying oven.

[0025] 1.3 Preparation of Standard Solutions

[0026] Accurately weigh 10.0 mg of each standard and place it in a 10 mL volumetric flask. Dissolve and dilute to volume with ethanol to prepare a standard stock solution with a mass concentration of 1000 mg / L. Store at -20 ℃ protected from light. Take an appropriate amount of the standard stock solution and dilute with ethanol to prepare a standard working solution with a mass concentration of 10 mg / L. Prepare and use immediately.

[0027] 1.4 QuEChERS Preprocessing Conditions

[0028] Step 1: Accurately weigh 0.50 g of sample into a 15 mL polypropylene centrifuge tube, add 3 mL of saturated sodium chloride solution, and vortex to fully disperse the sample. Then add 5 mL of acetonitrile, vortex for 1 min, sonicate for 10 min, and then centrifuge at 4000 r / min for 5 min.

[0029] Step 2: Transfer 2.0 mL of the acetonitrile phase to another centrifuge tube, add 100 mg of PSA, 50 mg of C18, and 150 mg of anhydrous MgSO4, vortex to mix for 1 min, and then centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.22 μm microporous membrane, take 1.0 mL of the filtrate, blow it to near dryness with nitrogen at room temperature, and then redissolve it with 200 μL of 50% (v / v) ethanol aqueous solution to obtain the test sample solution.

[0030] For creams, lotions, and other cream-type cosmetics, the following steps are added between the first and second steps: After taking the supernatant, add 5 mL of acetonitrile-saturated n-hexane solution, mix well, centrifuge at 4000 r / min for 5 min, and take the lower acetonitrile phase as the extract to be purified.

[0031] 1.5 SERS Sample Preparation

[0032] 20 μL of the sample solution to be tested was dropped onto the surface of a pre-prepared WN substrate. The sample solution was then evenly distributed on the substrate surface using a spin-coating method. Subsequently, the sample was dried at 60 ℃ for 10 min. After the sample was completely dry, SERS spectra were acquired. Substrate blank and solvent blank were set up as controls during the test.

[0033] Preparation method of WN substrate: 1) WCl6 (1 mmol), Li3N (1 mmol), ethylenediamine (5 mL), and o-xylene (40 mL) were mixed and magnetically stirred at 380 r / min for 2 h at 26 °C. The solution was then transferred to a Teflon-lined stainless steel autoclave and heated to 180 °C at a heating rate of 2 °C / min, and then maintained at 180 °C for 5 h. The black product was collected by centrifugation, washed three times with deionized water and three times with anhydrous ethanol, and then dried in a vacuum drying oven at 40 °C for 3 h to obtain amorphous WN nanosheets.

[0034] 2) Place 0.3 g of amorphous WN nanosheets in a quartz boat, put it in a microwave oven in a glove box (filled with nitrogen), heat it at 1000 W for 20 s, and then let it cool naturally to obtain crystalline WN nanosheets.

[0035] 3) 20 mg of crystalline WN nanosheets were dispersed in 10 mL of deionized water, filtered, and then placed in a vacuum freeze dryer to cool to -40 °C at a rate of 0.3 °C / min. The temperature was then maintained at -40 °C for 24 h to obtain a flexible WN substrate.

[0036] 1.6 Construction of fingerprint recognition based on "dual feature peaks"

[0037] By identifying the steroid A ring in the range of 1600–1670 cm⁻¹ -1 The low-frequency peak (P1) and high-frequency peak (P2) generated by the inner conjugated double bond (C=C) and ketone group (C=O) were used for grading by extracting two core parameters: peak position difference (Δν) and relative intensity ratio (R). Discrimination rule 1: Δν ≤ 35 cm -1 Furthermore, 0.8 ≤ R ≤ 1.2 indicates that it is a glucocorticoid with a mononuclear cyclohexene structure (typically represented by hydrocortisone), with a short conjugated system and narrow peak spacing.

[0038] Judgment Rule 2 (Potential / Super-Potential Components): If Δν ≥ 50 cm -1 Furthermore, substances with an R value greater than 1.5 are identified as glucocorticoids with a mononuclear cyclohexadiene structure (typical examples being dexamethasone, dexamethasone acetate, and clobetasol propionate). These substances are high-risk prohibited ingredients, easily causing severe hormone-dependent dermatitis and systemic damage, making them a top priority for regulatory monitoring.

[0039] Discrimination rule 3: Characteristic peaks are located between 1600 and 1670 cm⁻¹ -1 If the interval Δν and R do not meet the above conditions, it is marked as "suspected glucocorticoid pending verification" and further confirmation is required by LC-MS / MS.

[0040] 2 Results and Discussion

[0041] 2.1 Blank Background Analysis

[0042] To clarify the source of the effective signal in the sample spectrum, the solvent blank and substrate blank were first analyzed. The blank spectra show that the WN substrate blank and solvent blank are located in the range of 400–2000 cm⁻¹. -1 The overall baseline was stable within the range, with no obvious sharp characteristic peaks, only a weak and gentle background response. The WN substrate blank signal was slightly higher than the solvent blank, but neither exceeded the range of 1600–1700 cm⁻¹. -1 The critical analysis region produced strong interference that overlapped with the characteristic peaks of glucocorticoids. This indicates that WN itself has a low background response under the experimental conditions, making it suitable as a basis for subsequent SERS identification of target analytes. (See...) Figure 1 .

[0043] 2.2 SERS spectral characteristics analysis of four glucocorticoids

[0044] All four target compounds belong to the steroidal glucocorticoid class and share a common cyclopentanophenenophenanthrene core structure. Figure 2 ), where ring A consists of unsaturated bonds and carbonyl conjugated systems ( Figure 3 ).Depend on Figure 3 As shown in Table 1, the four glucocorticoids were present at a depth of 1600–1670 cm⁻¹. -1 The intervals all exhibit significant characteristic responses. The Raman signals in this band are mainly attributed to the double bond stretching vibrations of the cyclohexadiene structure or the double bond stretching vibrations of the cyclohexene structure, indicating that this interval has a good characterization ability for differences in molecular structure.

[0045] Table 1 shows that hydrocortisone (HC) at 1645 cm⁻¹ -1 and 1613 cm -1 Strong characteristic peaks were observed at all locations, with similar intensities and shapes, and a narrow peak spacing, indicating a relatively balanced A-ring vibration mode. In contrast, dexamethasone (DEX) showed a similar peak at 1658 cm⁻¹. -1 The peak is clearly visible at 1605 cm. -1 Only a weak secondary peak is retained at this position; dexamethasone acetate (DEX-Ac) and clobetasol propionate (CP) continue this trend, with their main peaks shifting further towards higher wavenumbers, reaching 1664 cm⁻¹, respectively. -1 and 1666 cm -1 The secondary peaks are all located at 1608–1609 cm. -1The differences are closely related to the varying A-ring conjugated structures of the four compounds. HC exhibits a relatively simple A-ring conjugated vibrational characteristic, thus displaying two peaks of similar intensity in this region. DEX, DEX-Ac, and CP, however, have a higher degree of A-ring conjugation, leading to a redistribution of vibrational modes. The high-wavenumber peaks are significantly enhanced and become the dominant peaks, while the low-wavenumber peaks are relatively weakened. Simultaneously, local substituents and esterification modifications further affect the intramolecular electron cloud distribution and the stability of the conjugated system, causing the dominant peaks of DEX-Ac and CP to continue shifting towards higher wavenumbers. Therefore, although the four glucocorticoids share the same steroidal skeleton, differences in the unsaturated A-ring structure and substituents can significantly influence their Raman peak positions, shapes, and relative intensities.

[0046]

[0047] 2.3 Enhanced Performance Analysis

[0048] To further evaluate the enhancing performance of the WN basement membrane, this study selected the characteristic Raman peaks of four glucocorticoids as the analysis objects: HC (1645 cm⁻¹) -1 ), DEX (1658 cm) -1 ), DEX-Ac (1664 cm) -1 ) and CP (1664 cm) -1 By measuring these characteristic peaks at 10 -8 M to 10 -10 The signal response on the WN substrate within the M concentration range was calculated, and the corresponding enhancement factor (EF) was calculated. The calculation formula is shown in Equation (1).

[0049]

[0050] Wherein, ISERS and NSERS represent the peak intensity of SERS and the number of molecules on the SERS substrate, respectively, while INRS and NNRS represent the peak intensity of normal Raman spectroscopy (NRS) and the number of target molecules in the normal Raman sample, respectively.

[0051] like Figure 4 As shown, in 10 -8 M to 10 -10 Within the M concentration range, the enhancing factors of all four glucocorticoids increased with decreasing concentration. The magnitudes followed a consistent pattern across different concentrations, exhibiting the order CP > DEX-Ac > DEX > HC. This indicates a stable difference in the SERS responses of the four substances on a WN substrate, and this difference does not change significantly with varying test concentrations.

[0052] 2.4 QuEChERS conditions

[0053] 2.4.1 Optimization of Extraction Solvent

[0054] To investigate the effect of extraction solvent on target analyte extraction and SERS detection, this study selected clobetasol propionate, which is highly representative and relatively hydrophobic, as a representative analyte. Acetonitrile and methanol were used as extraction solvents, and the layering effect and characteristic peak intensity of the extracted samples were compared. The results showed that acetonitrile extraction resulted in more obvious layering and better operability than methanol extraction. Furthermore, clobetasol propionate exhibited higher and more stable SERS characteristic peak intensity. Therefore, acetonitrile was ultimately chosen as the extraction solvent.

[0055] 2.4.2 Optimization of QuEChERS purification conditions

[0056] To investigate the impact of QuEChERS purification conditions on sample purification and SERS detection, this study optimized the purification system and its dosage, using the characteristic peak intensity of the target analyte as the main evaluation index. The results showed that the purification system composed of PSA, C18, and MgSO4 significantly improved sample clarity and increased the characteristic peak intensity of the target analyte, indicating that this system effectively removed some matrix interference and improved the detection response. Specifically, PSA mainly removed polar impurities, C18 mainly adsorbed hydrophobic matrix components such as oils and waxes, and MgSO4 was used to remove residual moisture. Insufficient purifying agent dosage resulted in incomplete matrix removal and lower characteristic peak intensity of the target analyte; excessive dosage may cause non-specific adsorption loss of the target analyte, especially hydrophobic glucocorticoids, leading to a decrease in peak intensity. By comprehensively comparing the changes in characteristic peak intensity under different conditions, the optimal purification combination was determined to be 100 mg PSA, 50 mg C18, and 150 mg MgSO4.

[0057] 2.4.3 Optimization of Reconstitution Solvent

[0058] When acetonitrile is used as the final resolution solvent, it may lead to excessively rapid droplet evaporation and uneven local deposition, thus affecting the reproducibility of the SERS signal. Therefore, in this study, after nitrogen blowing concentration of the sample, different volumes of ethanol (30%, 50%, and 70%) were used for solvent replacement and resolution. The distribution of the target analyte on the WN substrate surface and the detection effect were evaluated based on the characteristic peak intensity and repeatability. Figure 5 As shown, the results indicate that when reconstituted with 50% ethanol-water, the sample solution spreads and dries more uniformly on the WN substrate surface, resulting in a stronger SERS characteristic peak response and better repeatability than other reconstitution systems. When the ethanol ratio is high, the droplet evaporation rate accelerates, easily causing uneven local enrichment, while when the ethanol ratio is low, the target analyte response decreases. Therefore, 50% ethanol-water was ultimately chosen as the reconstitution solvent.

[0059] 2.4.4 Optimization of Sample Loading Volume and Method

[0060] To investigate the effects of sample volume and loading method on the distribution of target analytes on the WN substrate surface and the detection effect, this study set up three sample volumes (10, 20, and 30 μL) and two sample loading methods: natural dripping and spin coating. The changes in characteristic peak intensity and repeatability under different conditions were examined, with five different locations selected for testing for each sample. Table 2 shows that when the sample volume was 10 μL, the target analyte loading on the substrate surface was insufficient, resulting in a weak characteristic peak response. When the sample volume increased to 30 μL, droplets tended to accumulate locally after drying, leading to uneven sample distribution and decreased detection repeatability. In contrast, 20 μL ensured sufficient target analyte loading while avoiding uneven deposition caused by excessive sample loading, thus achieving a better balance between characteristic peak intensity and repeatability. Furthermore, under natural dripping conditions, droplet spreading was mainly affected by surface tension and evaporation behavior, making local enrichment more likely after drying, resulting in larger signal fluctuations between different points. Spin coating, on the other hand, allowed for a more uniform distribution of the sample solution on the substrate surface, which is beneficial for obtaining a more stable SERS response. After comprehensively comparing the characteristic peak intensity and repeatability under different conditions, 20 μL spin coating was finally selected as the loading condition.

[0061]

[0062] 2.5 CP Concentration Gradient Map, Method Sensitivity and Methodology

[0063] Under the optimized detection conditions, no glucocorticoid residues were detected in the cosmetic sample, indicating that the sample meets national testing standards. To verify the feasibility of the SERS method, the cosmetic extract with a CP spike concentration of 1×10⁻¹⁰ mol / L was tested, and the results are as follows. Figure 6 As shown in Table 3, the recovery rate of this method is greater than 95%, and the RSD is less than 10%, indicating that the SERS detection method based on WN substrate for cyclopentane and polyhydrophenanthrene core structure residues in cosmetics has high accuracy and feasibility.

[0064]

[0065] 2.7 Actual Sample Testing

[0066] Nine different types of cosmetic samples (three lotions, three creams, and three masks) were tested. The results showed that the SERS spectra of all samples were in the range of 1600–1670 cm⁻¹. -1 No characteristic peaks of glucocorticoids were observed in any of the intervals, and the test results were all negative.

[0067]

[0068] 3. Conclusion

[0069] This invention constructed a SERS detection system for four typical hormones based on a WN substrate and explored its application potential in complex cosmetic matrices using the QuEChERS pretreatment method. The results showed that HC, DEX, DEX-Ac, and CP all obtained stable and identifiable SERS responses on the WN substrate, with the response range being 1600–1665 cm⁻¹. -1 The regions exhibit common steroid backbone characteristic response areas; different substituents and side chain modifications further affect peak distribution and relative intensity. Blank experiments show that the WN substrate and solvent background are low and do not significantly interfere with target recognition. Enhancement factor analysis indicates that the overall enhancement ability of the WN substrate for different glucocorticoids is CP > DEX-Ac > DEX > HC. Concentration gradient maps with CP as the target further demonstrate that this system has a good enhancement ability for prohibited hormones.

[0070] In summary, this invention uses WN as the SERS substrate to leverage its signal enhancement effect; combining QuEChERS with WN-SERS effectively reduces interference from the cosmetic matrix and significantly improves the SERS signal intensity and detection stability of the target analyte. Based on this, a discrimination rule was constructed using a "dual-peak" fingerprint, and a quantitative equation with good linearity was established for clobetasol propionate. These results provide a new methodological basis for the rapid screening of steroid hormones in cosmetics.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for screening hormones in cosmetics using tungsten nitride SERS combined with QuEChERS, characterized in that: Includes the following steps: (1) Sample pretreatment: The cosmetic sample was extracted and purified using the QuEChERS method to obtain the filtrate to be tested; (2) SERS sample preparation: The filtrate to be tested was spin-coated onto the surface of the pre-prepared WN nanosheet substrate, and the solvent was evaporated to form a surface-enhanced Raman scattering detection sample; (3) Constructing a two-parameter discrimination rule: Using the peak position difference and relative intensity ratio of the two characteristic peaks in the conjugate vibration region of steroid A ring, a SERS fingerprint discrimination rule for steroid hormones is constructed.

2. The method for screening hormones in cosmetics using tungsten nitride SERS combined with QuEChERS according to claim 1, characterized in that: The sample pretreatment method described in step (1) specifically includes the following steps: Step 1: Accurately weigh 0.50 g of sample into a 15 mL polypropylene centrifuge tube, add 3 mL of saturated sodium chloride solution, vortex to fully disperse the sample, then add 5 mL of acetonitrile, vortex for 1 min, sonicate for 10 min, and then centrifuge at 4000 r / min for 5 min. Step 2: Pipette 2.0 mL of acetonitrile phase into another centrifuge tube, add 100 mg of PSA, 50 mg of C18 and 150 mg of anhydrous MgSO4, vortex to mix for 1 min, and then centrifuge at 4000 r / min for 5 min; take the supernatant and filter it through a 0.22 μm microporous membrane, take 1.0 mL of the filtrate and blow it with nitrogen to near dryness at room temperature, then redissolve it with 200 μL of 50% ethanol aqueous solution to obtain the test sample solution.

3. The method for screening hormones in cosmetics using tungsten nitride SERS combined with QuEChERS according to claim 2, characterized in that: If the sample is a cream or ointment cosmetic, the sample pretreatment method includes the following steps between the first and second steps: after taking the supernatant, add 5 mL of acetonitrile-saturated n-hexane solution, mix well, centrifuge at 4000 r / min for 5 min, and take the lower acetonitrile phase as the extraction solution to be purified.

4. The method for screening hormones in cosmetics using tungsten nitride SERS combined with QuEChERS according to claim 1, characterized in that: The SERS sample preparation method described in step (2) specifically includes the following steps: 20 μL of the sample solution to be tested is dropped onto the surface of the pre-prepared WN substrate, and the sample solution is evenly distributed on the substrate surface by spin coating. Then, it is dried at 60 ℃ for 10 min. After the sample is completely dry, SERS spectrum is acquired. During the test, a substrate blank and a solvent blank are set as controls. The SERS detection parameters are: excitation wavelength 532 nm; laser power 0.5 mW; scanning range 400 ~ 2000 cm⁻¹ -1 The integration time for each point is 1 second, and the integration is performed once. Five different locations are selected for testing for each sample, and the average spectrum is taken as the final result.

5. The method for screening hormones in cosmetics using tungsten nitride SERS combined with QuEChERS according to claim 4, characterized in that: Step (3) of the spectrum identification further employs a two-parameter discrimination method: using 1600–1670 cm⁻¹ as the spectral density. -1 The peak position difference between two characteristic peaks within the interval is Δν = ν high - ν low With relative strength ratio R = I high / I low As an identification parameter, the structure type is determined according to the following rules: When Δν ≤ 35 cm -1 When 0.8 ≤ R ≤ 1.2, it is determined to be a hormone with a cyclohexene structure in ring A; When Δν ≥ 50 cm -1 When R > 1.5, it is determined to be a hormone with a cyclohexadiene structure in ring A; Characteristic peaks are located at 1600–1670 cm⁻¹ -1 If the interval Δν and R do not meet the above conditions, it is marked as "suspected hormone pending verification" and needs to be further confirmed by LC-MS / MS.

6. The method for screening hormones in cosmetics using tungsten nitride SERS combined with QuEChERS according to claim 5, characterized in that: Hormones with a cyclohexene structure on ring A include hydrocortisone; hormones with a cyclohexadiene structure on ring A include dexamethasone, dexamethasone acetate, and clobetasol propionate.

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