Method for detecting acylphosphine oxide photoinitiator in nail polish gel

Abstract

The invention relates to a method for detecting a photoinitiator in nail polish gel. Aiming at the problem of lacking of an efficient detection method for acylphosphine oxide photoinitiators in nail polish gel, the method comprises the following steps: extracting multiple photoinitiators in the nail polish gel by adopting acetonitrile, separating by utilizing high performance liquid chromatography, and carrying out multi-wavelength detection. And confirmation is carried out through a plurality of means such as liquid chromatography-tandem mass spectrometry, liquid chromatography-quadrupole / electrostatic field orbitrap mass spectrometry, gas chromatography-tandem mass spectrometry and the like. The method has the characteristics and advantages that multiple photoinitiators can be detected at the same time, the detection time is short, and the efficiency is high; acetonitrile and other solvents with low toxicity are adopted, so that harm to the environment and operators is reduced; the detection method is high in specificity, matrix interference can be effectively reduced, and the reliability and accuracy of a detection result are improved; the detection cost is low and the economy is good. The method is suitable for detecting the photoinitiator in cosmetics such as nail polish gel, and can provide powerful technical support for quality control and safety evaluation of the cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of raw material detection and confirmation in cosmetics, and relates to a method for detecting acylphosphine oxide photoinitiators in nail polish, and specifically to a quantitative determination method and a qualitative detection method for 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), 2,4,6-trimethylbenzoylphenylphosphonic acid ethyl ester (TPO-L), 2,4,6-trimethylbenzoyl-di(p-tolyl)phosphine oxide (TMO), phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide (photoinitiator 819, PB-TMBPO) and 1-hydroxycyclohexylphenyl ketone (photoinitiator 184, HCHPK). The invention specifically relates to a method for quantitatively determining acylphosphine oxide photoinitiators such as 2,4,6-trimethylbenzoyl-diphenylphosphine oxide in nail polish by using high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS), and gas chromatography-mass spectrometry (GC-MS). Background Art

[0002] In the contemporary cosmetics sector, the nail art industry is booming. As a core product, gel nail polish, with its fast-curing, vibrant colors, and long-lasting durability, is highly sought after by consumers, and the market continues to expand, becoming a vital component of the cosmetics industry. Gel nail polish offers a wide variety of products, including base coats, color coats (such as solid color coats, glitter coats, glow-in-the-dark coats, fluorescent coats, cat's eye coats, cracking coats, and temperature-sensitive coats), sealers (such as wash-free coats, scrub coats, and frosted coats), reinforcement coats, and phototherapy coats.

[0003] Photoinitiators are key components in the cosmetic curing process, efficiently absorbing radiation energy and producing reactive intermediates capable of initiating polymerization. Based on the photopolymerization mechanism, photoinitiators can be categorized as cleavage-type free radical photoinitiators (such as benzil and acylphosphine oxides), hydrogen-abstraction-type free radical photoinitiators (such as benzophenones and thioxanthones), and cationic photoinitiators (such as iodine salts and iron(III) cyclopentadienyl hexafluorophosphate). Acylphosphine oxide photoinitiators (such as 2,4,6-trimethylbenzoyldiphenylphosphine oxide, TPO) are currently the most widely used photoinitiators for nail polish due to their high curing efficiency, resistance to yellowing, and reasonable price.

[0004] However, in May 2025, European Commission Directive (EU) 2025 / 877 amended TPO to the list of banned substances, highlighting its potential safety concerns. Patented testing revealed that TPO was detected in 100% of 11 commercially available batches of gel nail polish, with the highest content reaching 5.7%, exceeding the original EU limit. The detection rate for HCHPK was also as high as 91%. Given the increasing use of gel nail polish and the increased frequency of contact between photoinitiators and human skin, the potential health risks cannot be ignored.

[0005] Currently, my country lacks testing standards for photoinitiator raw materials in cosmetics, and there is also a lack of literature on the determination of photoinitiators in nail polish. Existing photoinitiator detection methods are primarily focused on areas such as food contact materials and printing inks, using methods such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and UV spectrophotometry. For TPO, only a gas chromatography-mass spectrometry method is available for its determination in UV-curing adhesives.

[0006] However, these methods have many limitations:

[0007] 1. Lack of detection methods for acylphosphine oxide photoinitiators: Existing detection methods mostly focus on traditional photoinitiators (such as benzophenones). There is a lack of effective detection methods for the acylphosphine oxide photoinitiators (such as TPO) actually added to nail polish. Furthermore, after TPO is completely banned on September 1, 2025, cosmetics companies will inevitably use its alternatives. Existing methods cannot meet the needs of simultaneous detection of multiple photoinitiators.

[0008] 2. Conventional methods have poor detection specificity: Nail polish contains a large number of chemical additives, which can cause significant matrix interference. This is especially true for small molecules like HCHPK, which are difficult to detect against the matrix background. Some acylphosphine oxide photoinitiators, such as PB-TMBPO, exhibit severe thermal decomposition, making them unsuitable for gas chromatography detection.

[0009] 3. Low matrix compatibility with nail polish: Nail polish has a complex composition, containing a variety of resins and monomers, which differs significantly from existing matrix testing areas such as food contact materials and printing inks. Existing methods struggle to effectively extract and separate photoinitiators from nail polish, resulting in low extraction efficiency and poor accuracy.

[0010] 4. Pretreatment methods have flaws: Conventional methods use organic solvents such as toluene, which have high boiling points and are highly toxic, posing potential threats to the environment and operator health. Furthermore, extraction and detection times are long, resulting in low efficiency, making them difficult to meet the demands of rapid testing and high-throughput analysis. Photoinitiators are extremely sensitive to light, and prolonged pretreatment times can compromise the accuracy of both quantitative and qualitative analysis.

[0011] 5. Imperfect content determination and mass spectrometry confirmation system: In terms of content determination and mass spectrometry confirmation, there is a lack of a complete supporting system, which makes it difficult to accurately quantify and effectively confirm photoinitiators, and cannot meet the regulatory requirements for detection accuracy and sensitivity.

[0012] Therefore, there is an urgent need to provide a method that can simultaneously detect multiple acylphosphine oxide photoinitiators in nail polish to broaden the detection range, detection efficiency, sensitivity, accuracy, applicability and regulatory compatibility. Summary of the Invention

[0013] In order to solve the problem that the prior art lacks an efficient detection method for acylphosphine oxide photoinitiators in nail polish, the present application provides a method for detecting photoinitiators in cosmetics.

[0014] The technical solutions of the present invention are as follows:

[0015] A method for qualitative and / or quantitative detection of photoinitiators in nail polish, wherein the photoinitiator is selected from 1-hydroxycyclohexyl phenyl ketone (HCHPK) and an acylphosphine oxide photoinitiator, wherein the acylphosphine oxide photoinitiator is selected from a combination of one or more of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), ethyl 2,4,6-trimethylbenzoylphenylphosphonate (TPO-L), 2,4,6-trimethylbenzoyl-di(p-tolyl)phosphine oxide (TMO), and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (photoinitiator 819, PB-TMBPO).

[0016] The method is selected from any one of (1) to (4), or the method is selected from any one of (1) and (2) to (4):

[0017] (1)LC method:

[0018] S1. Preparation of standard solution:

[0019] Weigh HCHPK, TPO-L, TPO, TMO, and PB-TMBPO respectively, and dilute them stepwise with organic solvent to form a series of LC mixed standard solutions with at least five concentrations of the five photoinitiators ranging from 2 to 600 μg / mL.

[0020] S2. Preparation of test solution:

[0021] Take the nail polish to be tested, dilute it with an organic solvent in proportion, disperse it with ultrasonication, filter it, and determine the filtrate by HPLC; the concentration of the nail polish to be tested in the test solution is any one of 0.4 to 20 mg / mL;

[0022] S3. Chromatographic conditions:

[0023] Chromatographic column: C18 packed column, pore size: Particle size: 5μm; Specifications: 150mm×4.6mm;

[0024] Column temperature: 30°C;

[0025] Mobile phase: A is ultrapure water, B is acetonitrile;

[0026] Gradient elution program: 0-2 min, 45% A; 2-6 min, 45% A-5% A; 6-8 min, 5% A; 8-8.5 min, 5% A-45% A; 8.5-12 min,

[0027] 45% A;

[0028] Flow rate: 1 mL / min;

[0029] Injection volume: 5 μL;

[0030] Diode array detector collection wavelength: 190~400nm;

[0031] S4. Determination method:

[0032] The detection wavelength for HCHPK was 243 nm, and that for TPO-L, TPO, TMO, and PB-TMBPO was 375 nm. Standard curve and external standard methods were used for quantification.

[0033] (2) LC-MS / MS method:

[0034] S1. Preparation of standard solution:

[0035] (A) LC-MS / MS mixed standard series solution:

[0036] Weigh HCHPK, TPO-L, TPO, TMO, and PB-TMBPO separately and dilute them stepwise with organic solvent to form a series of LC-MS / MS mixed standard solutions with at least five concentrations ranging from 0.002 to 200 ng / mL for each of the five photoinitiators.

[0037] (B) LC-MS / MS qualitative standard solution:

[0038] HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted stepwise with organic solvent to prepare mixed standard solutions with a mass concentration of 50 ng / mL for each of the five photoinitiators as LC-MS / MS qualitative standard solutions;

[0039] S2. Preparation of test solution:

[0040] (A) If this Act is used alone:

[0041] Take the nail polish to be tested, dilute it with an organic solvent in proportion, disperse it with ultrasound, filter it, and determine the filtrate by HPLC; the concentration of the nail polish to be tested in the test solution is any one of 2-100 μg / ml;

[0042] (B) If it is further confirmed on the basis of (1):

[0043] Take the test solution prepared in (1) S2 and dilute it with an organic solvent until the photoinitiator content is 10-200 ng / mL;

[0044] S3. LC-MS / MS conditions:

[0045] (A) Chromatographic conditions:

[0046] Chromatographic column: C18 packed column, pore size: Particle size: 2μm; 100mm×2.1mm;

[0047] Column temperature: 40°C;

[0048] Flow rate: 0.3 mL / min;

[0049] Injection volume: 1 μL;

[0050] Mobile phase: water (A)-acetonitrile (B);

[0051] A gradient elution program was used with acetonitrile volume fraction as follows: 0-1.0 min, 40%; 1.0-4.0 min, 40%-95%; 4.0-5.5 min, 95%; 5.6-7.5 min, 40%;

[0052] (B) Mass spectrometry conditions:

[0053] The ion source was electrospray ionization (ESI);

[0054] The scanning mode was positive ion MRM mode;

[0055] The spray voltage was 5500 V;

[0056] The ion source temperature was 250 °C;

[0057] The mass spectrometry parameters are as follows:

[0058]

[0059] *Preferred monitoring window ions / ion pairs;

[0060] S4. Determination method:

[0061] Under the same experimental conditions, if the retention time of the chromatographic peak detected in the sample is consistent with that of the corresponding component in the LC-MS / MS qualitative standard solution, and the deviation between the relative abundance ratio of the selected monitoring ion pair and the ion pair abundance ratio of the standard solution of equivalent concentration does not exceed the range specified in the table below, then it can be determined that the corresponding component to be tested is present in the sample;

[0062] Ion relative abundance (k) k＞0.50 0.50≥k＞0.20 0.20≥k＞0.10 k≤0.10 Maximum allowed deviation ±20％ ±25％ ±30％ ±50％ ;

[0063] (3) LC-HRMS method:

[0064] S1. Preparation of standard solution:

[0065] (1) LC-HRMS mixed standard series solution: HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted stepwise with organic solvent to form a LC-HRMS mixed standard series solution with at least two concentrations of the five photoinitiators ranging from 1 to 1000 ng / mL;

[0066] (2) LC-HRMS qualitative standard solution: HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted stepwise with organic solvent to form a mixed standard solution with a mass concentration of 1000 ng / mL for each of the five photoinitiators as the LC-HRMS qualitative standard solution;

[0067] S2. Preparation of test solution:

[0068] (A) If this Act is used alone:

[0069] Take the nail polish to be tested, dilute it with an organic solvent in proportion, disperse it with ultrasound, filter it, and determine the filtrate by HPLC; the concentration of the nail polish to be tested in the test solution is any one of 20-1000 μg / mL;

[0070] (B) If it is further confirmed on the basis of (1):

[0071] Take the test solution prepared in (1) S2 and dilute it with an organic solvent until the photoinitiator content is 0.1-2 μg / mL;

[0072] S3, LC-HRMS conditions:

[0073] (A) Chromatographic conditions:

[0074] Chromatographic column: C18 packed column, pore size: Particle size: 2μm; 100mm×2.1mm;

[0075] Column temperature: 40°C;

[0076] Flow rate: 0.5 mL / min;

[0077] Injection volume: 2 μL;

[0078] Mobile phase: water (A) - acetonitrile (B);

[0079] Gradient elution program was used with acetonitrile volume fraction: 0-1.0 min, 40%; 1.0→3.5 min, 40%→100%; 3.5-5.5 min, 100%;

[0080] 5.6-7.5 min, 40%

[0081] (B) Mass spectrometry conditions:

[0082] The ion source was an electrospray ion source (ESI) and the scanning mode was full scan / data-dependent MS2 scan mode;

[0083] Full scan m / z range: 100-500;

[0084] The spray voltage was 3000 V;

[0085] The ion source temperature was 350 °C;

[0086] Capillary temperature: 325 °C;

[0087] Sheath gas flow: 10 Arb;

[0088] Auxiliary gas flow: 50 Arb;

[0089] Relative collision energy: 40%;

[0090] Other LC-HRMS mass spectrometry parameters were as follows:

[0091]

[0092] S4, Determination method:

[0093] The standard solution and the test sample solution were collected under full scan / data-dependent MS2 scan mode to obtain MS1 and MS2 spectrum information. Under the same experimental conditions, if the retention time of the extracted ion chromatographic peak detected in the sample was consistent with that of the corresponding component in the standard solution and the MS2 fragment ion m / z was consistent, it was determined that the corresponding component to be tested existed in the sample.

[0094] The organic solvent described in (1) to (3) is selected from a combination of one or more of acetonitrile, acetone, methanol, and ethanol;

[0095] (4) GC-MS method:

[0096] S1, Preparation of standard solution:

[0097] (A) GC-MS series mixed standard solution:

[0098] Weigh HCHPK, TPO-L, TPO, TMO, and PB-TMBPO separately and dilute them in ethyl acetate to prepare a series of GC-MS mixed standard solutions with at least two concentrations of each of the five photoinitiators ranging from 2 to 1000 ng / mL.

[0099] (B) GC-MS qualitative standard solution:

[0100] (B-1) Qualitative standard solution in full scan mode:

[0101] HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted with ethyl acetate to prepare full-scan mode qualitative standard solutions with a mass concentration of 10 μg / mL for each of the five photoinitiators.

[0102] (B-2) Qualitative standard solution for SIM and MRM modes:

[0103] HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted with ethyl acetate to prepare mixed standard solutions with a mass concentration of 500 ng / mL for each of the five photoinitiators as qualitative standard solutions for SIM and MRM modes;

[0104] S2. Preparation of test solution:

[0105] (A) If this Act is used alone:

[0106] Prepare a stock solution of the test sample with a concentration of 0.4 to 20 mg / mL of nail polish gel, dilute it with ethyl acetate in a certain proportion, and then ultrasonically disperse and filter it to obtain the test sample. Dilute it 10-fold and 50-fold with ethyl acetate to obtain GC-MS sample solution 1 and GC-MS sample solution 2. GC-MS sample solution 1 is used for detection in full scan mode; GC-MS sample solution 2 is used for detection in SIM and MRM modes.

[0107] (B) If it is further confirmed on the basis of (1):

[0108] Take the sample solution prepared in (1) S2 and dilute it with ethyl acetate to a photoinitiator content of 1-20 μg / mL (GC-MS sample solution 1) and 0.1-2 μg / mL (GC-MS sample solution 2).

[0109] S3. GC-MS conditions:

[0110] (A) Gas chromatography:

[0111] Chromatographic column: High-temperature column with 5% phenyl / 95% dimethylpolysiloxane as stationary phase, 15m×0.25mm, film thickness 0.1μm;

[0112] Injection in split mode, split ratio 10:1;

[0113] Inlet temperature: 300°C;

[0114] Carrier gas: high-purity helium (purity>99.999%);

[0115] Injection volume: 1 μL;

[0116] Flow rate: 1.2 mL / min;

[0117] Heating program: initial temperature 70 °C, hold for 1 min, increase to 180 °C at a rate of 30 °C / min, increase to 280 °C at a rate of 15 °C / min, and then increase to 320 °C at a rate of 30 °C / min, hold for 0.5 min;

[0118] (B) Mass spectrometry conditions:

[0119] An electron bombardment ion source (EI) was used, and the ion source temperature was 230°C;

[0120] Quadrupole temperature: 150°C;

[0121] MS transfer line temperature: 300°C;

[0122] Solvent delay time: 4 min;

[0123] Full scan mode (FULL SCAN): Scan range m / z 30~430;

[0124] The parameters of ion monitoring mode (SIM) and multiple reaction monitoring mode (MRM) are as follows:

[0125]

[0126]

[0127] *Preferred monitoring window ions / ion pairs;

[0128] S4. Determination method:

[0129] Under the same experimental conditions, if the retention time of the chromatographic peak detected in the sample is consistent with that of the corresponding component in the standard solution, in the FULL SCAN mode, the retention time of the extracted ion chromatogram of the primary mass spectrometer is consistent, and the m / z of the primary mass spectrometer fragment ions are consistent; in the SIM / MRM mode, the deviation of the relative abundance ratio of the selected monitoring ion pair and the ion pair abundance ratio of the standard solution of equivalent concentration does not exceed the range specified in the table below, then it can be determined that the corresponding component to be measured is present in the sample.

[0130] The maximum allowable deviation of ion relative abundance when confirming the results

[0131] Ion relative abundance (k) k＞0.50 0.50≥k＞0.20 0.20≥k＞0.10 k≤0.10 Maximum allowed deviation ±20％ ±25％ ±30％ ±50％

[0132] Furthermore, (1) the chromatographic column is a Kromasil 100-5-C18 column, (2) and (3) the chromatographic columns are ACEEXCEL 2C18 columns, and (4) the chromatographic column is a Thermofisher TG-5HT column.

[0133] Furthermore, the concentration of the nail polish to be tested in (1) S2 is 10 mg / mL.

[0134] Furthermore, (1) the concentration of the photoinitiator in the nail polish to be tested in S2 is 2 to 600 μg / mL.

[0135] Furthermore, in (2) S2(A), the dilution to 2-100 μg / mL is to prevent over-dilution and the resulting "false negatives." If the response of a particular photoinitiator in the nail polish to be tested exceeds 400% of the LC-MS / MS qualitative standard solution, the solution should be further diluted to 10% to 400% of the concentration of the LC-MS / MS qualitative standard solution and tested again for that photoinitiator.

[0136] Furthermore, the organic solvent in (1) to (3) is selected from acetonitrile, acetone or a combination of acetonitrile and acetone.

[0137] The photoinitiator structural formulas of the present invention are:

[0138] HCHPK:

[0139] TPO-L:

[0140] TPO:

[0141] TMO:

[0142] PB-TMBPO:

[0143] The beneficial effects of the present invention are:

[0144] 1. Multi-component detection and confirmation capabilities

[0145] This patent uses acetonitrile to extract five photoinitiators (TPO, TPO-L, TMO, PB-TMBPO, and HCHPK) from gel nail polish. The assays and confirmations are performed using a variety of methods, including HPLC-PDA, LC-MS / MS, LC-HRMS, and GC-MS. This enables the simultaneous detection of multiple photoinitiators. This overcomes the limitations of existing methods, which cannot simultaneously detect multiple photoinitiators, and provides strong support for the comprehensive and accurate analysis of photoinitiator components in gel nail polish.

[0146] 2. Efficient and fast testing process

[0147] This patent optimizes the detection process. The HPLC-PDA method only takes 12 minutes per injection, and confirmation methods such as LC-MS / MS, LC-HRMS, and GC-MS (full scan, selected ion detection, and multiple reaction monitoring) all take 7.5 to 13 minutes per injection, significantly improving detection efficiency. This patent selects and optimizes the extraction solvent and dilution solvent to make them more suitable for the rapid extraction of photoinitiators in nail polish, as well as compatibility with LC and GC. Compared with existing methods, this method significantly shortens the detection cycle, can meet the high-throughput requirements of actual detection, and improves the timeliness of detection work.

[0148] 3. High sensitivity and resistance to matrix interference

[0149] This patent effectively reduces matrix interference and improves detection specificity, sensitivity, and accuracy through the selection of UV wavelengths and the selection of monitoring ions for liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, and has been verified by a variety of actual samples. When detecting HCHPK based on LC-HRMS, the characteristic in-source cleavage fragment m / z 187.11174 is used as the quantitative ion, significantly improving detection sensitivity. When detecting HCHPK based on GC-MS, m / z 99 / m / z99→41 are selected as the preferred monitoring ion / ion pair. When detecting acylphosphine oxide photoinitiators based on LC-HRMS, the common characteristic in-source cleavage fragments (m / z 147.08044 and 119.08553) are monitored, significantly improving detection sensitivity (approximately 5 times).

[0150] 4. Wide applicability and regulatory compliance:

[0151] This patented method optimizes extraction, separation, and detection conditions based on the characteristics of gel nail polish, making it more compatible with the gel nail polish matrix and offering broad applicability. It can detect not only common photoinitiators such as TPO and HCHPK, but also photoinitiators in other cosmetics, providing a universal detection method for quality control and safety supervision in the cosmetics industry. Furthermore, this patented detection method can meet increasingly stringent regulatory requirements, providing technical support for the regulation of photoinitiators in gel nail polish, ensuring product safety and protecting the health rights of consumers.

[0152] 5. Detection and expansion of acylphosphine oxide photoinitiator derivatives

[0153] Based on the chromatography-mass spectrometry and sample pretreatment conditions of this patent, this patent innovatively introduces common characteristic in-source pyrolysis fragments (m / z 147.08044, m / z 119.08553) based on LC-HRMS to detect TPO derivatives; based on liquid chromatography-tandem mass spectrometry, m / z 147 (collision energy 20eV) and m / z 119 (collision energy 20eV) are simultaneously used as product ions to perform precursor ion scanning to detect TPO derivatives; based on gas chromatography-tandem mass spectrometry technology, in full scan mode, m / z 147 and m / z 119 are extracted from the total ion chromatogram, m / z 147 and m / z 119 are monitored in selected ion monitoring (SIM) mode, and m / z 147→119 and m / z 147→91 ion pairs are monitored in multiple reaction monitoring (MRM) mode to detect acylphosphine oxide photoinitiators such as TPO derivatives. This method significantly improves the detection sensitivity and versatility of acylphosphine oxide photoinitiators, and provides new ideas and methods for the structural analysis of photoinitiators and the study of unknown derivatives.

[0154] In summary, the present invention provides a method for detecting photoinitiators in cosmetics, which can detect multiple photoinitiators simultaneously, with short detection time and high efficiency; uses less toxic solvents such as acetonitrile to reduce harm to the environment and operators; the detection method has strong specificity, can effectively reduce matrix interference, and improve the reliability and accuracy of the detection results; and has low detection costs and good economy. It is suitable for the detection of photoinitiators in cosmetics such as nail polish, and can provide strong technical support for the quality control and safety assessment of cosmetics. This patent has significant advantages in terms of detection range, efficiency, sensitivity, accuracy, applicability, and regulatory compatibility, and can effectively fill the deficiencies of existing methods, providing a more advanced and reliable technical means for the determination of photoinitiators in nail polish. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1Liquid chromatograms of five photoinitiator mixed standard solutions and typical sample solutions separated by different chromatographic columns, where the red curve is the sample solution and the black curve is the standard solution.

[0156] Figure 2 Optimization of LC detection wavelength.

[0157] Figure 3 LC mixed standard liquid chromatogram (100μg / mL).

[0158] Figure 4 LC-MS / MS qualitative standard solution chart (50 ng / mL).

[0159] Figure 5 LC-HRMS primary mass spectra of five photoinitiators (10 μg / mL).

[0160] Figure 6 LC-HRMS secondary mass spectra of five photoinitiators (10 μg / mL).

[0161] Figure 7 Extracted ion chromatogram of LC-HRMS qualitative standard solution (1 μg / mL).

[0162] Figure 8 LC-HRMS extracted ion chromatogram (MC1).

[0163] Figure 9 GC-MS FULL SCAN chromatogram of qualitative mixed standard.

[0164] Figure 10 SIM qualitative mixed standard chromatogram.

[0165] Figure 11 MRM qualifier mixture chromatogram.

[0166] Figure 12 GC-MS FULL SCAN sample chromatogram.

[0167] Figure 13 SIM sample chromatogram.

[0168] Figure 14 MRM sample chromatogram.

[0169] Figure 15 Detection flow chart. DETAILED DESCRIPTION

[0170] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0171] Example 1 Determination of the Content of Five Photoinitiators in Nail Polish

[0172] 1. Reagents and instruments

[0173] 1.1 Standard substances and reagents

[0174] HCHPK (CAS No. 947-19-3, purity 99.6%), TPO (CAS No. 75980-60-8, purity 98.6%), and PB-TMBPO (CAS No. 162881-26-7, purity 98.6%) were purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.; TPO-L (CAS No. 84434-11-7, purity 97%) and TMO (CAS No. 270586-78-2, purity 98%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; methanol, ethanol, and acetonitrile were all chromatographically pure and purchased from Merck, Germany; formic acid was chromatographically pure and purchased from ACS Enko Chemicals, USA; acetone was chromatographically pure and purchased from Sinopharm Chemical Reagent Co., Ltd.; and the experimental water was ultrapure water prepared using a Milli-Q IQ 7000 ultrapure water system.

[0175] 1.2 Instruments

[0176] An LC-30AD ultrahigh-pressure liquid chromatograph (Shimadzu, Japan) was equipped with a SIL-30AC autosampler, a CTO-20AC column thermostat, and an SPD-M20A diode array detector (detection wavelength: 190–800 nm); an XP6 electronic balance (Mettler Toledo, Switzerland); an MSE224S-CE electronic balance (Sartorius, Germany); a UA22MFD ultrasonic cleaner (Wiggens, Germany); and an ultrapure water system (Millipore, USA).

[0177] 2 Experimental methods

[0178] 2.1 Preparation of LC standard solution

[0179] (1) LC mixed standard stock solution (2 mg / mL): Weigh 100 mg each of HCHPK, TPO-L, TPO, TMO, and PB-TMBPO, place them in the same 50 mL brown volumetric flask, dissolve them in acetonitrile and dilute to the mark, shake well, and obtain the stock solution.

[0180] (2) LC series mixed standard solution: Accurately measure an appropriate amount of LC mixed standard stock solution, dilute it step by step with acetonitrile to make mixed standard solutions with mass concentrations of 2, 4, 10, 40, 100, 200, 400, and 600 μg / mL, respectively, and shake well to obtain the mixed standard solution.

[0181] 2.2 Preparation of LC sample solution

[0182] The sample was mixed, 0.1 g (accurate to 0.0001 g) was weighed, and was placed in a 10 mL brown stoppered colorimetric tube. Acetonitrile was added to the mark to make the concentration of the varnish glue 10 mg / mL. The tube was tightly capped, vortexed for 1 min, and the sample was dispersed in the extraction solvent. Then, the sample was ultrasonically extracted for 20 min, and was filtered through a 0.45 μm organic filter membrane. The filtrate was used for HPLC determination. If the detected concentration of a certain photoinitiator component exceeds the linear range, the sample was diluted with acetonitrile at a certain ratio and was determined again.

[0183] 2.3 LC chromatographic conditions

[0184] Chromatographic column: Kromasil 100-5-C18 column (150 mm x 4.6 mm, 5 μm); column temperature: 30 °C; mobile phase: A is ultrapure water, B is acetonitrile; gradient elution program: 0-2 min, 45% A; 2-6 min, 45% A-5% A; 6-8 min, 5% A; 8-8.5 min, 5% A-45% A; 8.5-12 min, 45% A. Flow rate: 1 mL / min; injection volume: 5 μL; wavelength range for diode array detector: 190-400 nm.

[0185] 2.4 Determination method

[0186] Standard curve method, external standard method for quantitative determination. The detection wavelength of HCHPK is 243 nm, and the detection wavelength of TPO-L, TPO, TMO and PB-TMBPO is 375 nm.

[0187] 3. Results analysis

[0188] 3.1 Selection of mobile phase

[0189] The effects of different mobile phase combinations on the chromatographic behavior of the five target compounds were investigated. It was found that there was no difference in the retention time and peak shape of the target compounds when water and 0.1% formic acid aqueous solution were used as the aqueous phase. However, the formic acid aqueous solution caused baseline fluctuation at 243 nm wavelength. Therefore, pure water was finally selected as the aqueous phase. In the selection of organic phase, acetonitrile had stronger elution ability than methanol, which could achieve faster peak elution and better peak shape. Therefore, acetonitrile was selected as the organic phase.

[0190] 3.2 Selection of chromatographic column

[0191] Many chemical additives in nail polish have UV absorption, which can interfere with the determination of the target photoinitiator. To avoid interference with quantification, the separation performance of four chromatographic columns, Kromasil 100-5-C18, Venusil MP C18, Diamonsil plus C18, and Poroshell 120EC-C18, on the target compound and sample interferences was compared. The results showed that the Kromasil 100-5-C18 column performed best in terms of resolution, peak shape, and separation of sample interferences (see Figure 1 ).

[0192] 3.3 Selection of detection wavelength

[0193] This patent uses a diode array detector to perform a full wavelength scan of 190 to 800 nm on the five target compounds. The results show that HCHPK has the strongest absorption at 243 nm, while TPO-L, TPO, TMO and PB-TMBPO have strong absorption at 236 nm, 296 nm and 375 nm. Since the interfering substances in the sample absorb in the wavelength range of ≤300 nm, it will interfere with the determination of four compounds such as TPO-L. This patent innovatively adopts a dual-wavelength detection strategy: 243 nm is selected as the detection wavelength of HCHPK, and 375 nm is selected as the detection wavelength of TPO-L, TPO, TMO and PB-TMBPO, taking into account both detection sensitivity and specificity ( Figure 2 ).

[0194] 3.4 Optimization of sample pretreatment methods

[0195] This patent targets the complex and diverse matrix of nail polish (including base coat, sealant, etc.), innovatively selects 11 representative products for analysis, and optimizes the extraction solvent.

[0196] By examining the extraction efficiency of four solvents, methanol, acetonitrile, ethanol and acetone, it was found that although acetone had the best solubility, its use was limited.

[0197] Finally, acetonitrile was selected as the extraction solvent, which effectively overcame the extraction difficulties of the complex nail polish matrix and significantly improved the detection efficiency and accuracy. 3.5 Methodology Validation Results:

[0198] The established content determination method was validated and the experimental results showed that TPO-L, TPO, TMO, PB-TMBPO and HCHPK had good separation ( Figure 3 ).

[0199] The five initiators showed good linearity in the range of 2-600 μg / mL (correlation coefficients r were all > 0.9999). The detection concentration of the method was 3.6-45 μg / g. The recoveries at low, medium, and high spike levels were 91.6%-100.4%, and the relative standard deviations were 0.3%-4.3%.

[0200] The limits of detection (LOD) of TPO, TPO-L, TMO, PB-TMBPO, and HCHPK were 0.38, 0.45, 0.25, 0.26, and 0.036 μg / mL, and the limits of quantification (LOQ) were 0.87, 1.4, 0.65, 0.73, and 0.15 μg / mL, respectively.

[0201] Based on a sampling volume of 0.1 g and a sample extraction solvent volume of 10 mL, the detection concentrations of TPO, TPO-L, TMO, PB-TMBPO, and HCHPK were 38, 45, 25, 26, and 3.6 μg / g, respectively, and the quantitative concentrations were 87, 141, 65, 73, and 15 μg / g, respectively.

[0202] 3.6 Determination of actual samples

[0203] The method was applied to the detection of 11 batches of nail polish samples purchased online. The results showed that two photoinitiators were detected. Among them, the detection rate of TPO, which is about to be banned by the European Union, was 100%, with a content range of 1.4% to 5.7%. The detection rate of HCHPK was 91%, with a content range of 1.8% to 3.9%.

[0204] The results indicate that TPO, currently widely used in the nail polish market, urgently requires replacement and further safety evaluation. This method is simple, specific, and rapid, meeting the requirements for determining the photoinitiator content in nail polish, providing technical support for the quality control and safety assessment of nail polish products.

[0205] Example 2 Positive confirmation based on liquid chromatography-tandem mass spectrometry

[0206] Nail polish has a complex matrix and many interfering peaks in UV detection. To ensure the specificity of the assay results, this example uses "MissCandy BJ16 Painless Firming Base Gel" purchased from Tmall as an example to detail the method for confirming positive results using high-performance liquid chromatography-tandem mass spectrometry.

[0207] 1. Reagents and instruments

[0208] 1.1 Standard substances and reagents

[0209] Same as Example 1

[0210] 1.2 Instruments

[0211] Exion LCTM AC-Triple Quad 6500+Low Mass high performance liquid chromatography-triple quadrupole mass spectrometer (SCIEX, USA), the rest of the instruments are the same as in Example 1.

[0212] 2 Experimental method

[0213] 2.1 Preparation of LC-MS / MS standard solution

[0214] (1) LC-MS mixed standard intermediate solution (10 μg / mL): accurately pipette LC mixed standard stock solution (2 mg / mL) into a suitable amount, dissolve and dilute to 10 μg / mL with acetonitrile, shake well to obtain.

[0215] (2) LC-MS / MS mixed standard series solution: accurately pipette LC-MS mixed standard intermediate solution (10 μg / mL) into a suitable amount, dilute with acetonitrile to prepare mixed standard series solution containing 5 kinds of photoinitiators, each at 0.002, 0.005, 0.02, 0.2, 0.5, 1, 2, 5, 10, 50, 100 and 200 ng / mL. Among them, the 50 ng / mL mixed standard solution is used as the LC-MS / MS qualitative standard solution.

[0216] 2.2 Preparation of LC-MS / MS sample solution

[0217] Method (2) is used independently: take the sample and mix it, weigh 0.1 g of the nail polish to be tested (accurate to 0.0001 g), put it into a 10 mL brown stoppered colorimetric tube, add acetonitrile to the mark, tightly cap and vortex for 1 min to disperse the sample in the extraction solvent, then ultrasonically extract for 20 min, and pass through a 0.45 μm organic filter membrane; take an appropriate amount of the filtrate, dilute it 200 times with acetonitrile, and you get 50 μg / mL.

[0218] Scheme of (1) + (2): take the LC sample solution of "MissCandy BJ16 painless firm base glue" prepared in Example 1, according to the content determination result (HCHPK concentration is 193 μg / mL; TPO concentration is 142 μg / mL), dilute 2000 times with acetonitrile, so that the concentrations of HCHPK and TPO are 96.5 and 71 ng / mL respectively, and you get it.

[0219] 2.3 Instrument parameters

[0220] 2.3.1 Liquid chromatography:

[0221] Chromatographic column: ACE EXCEL 2C18 (100 mm × 2.1 mm, 2 μm); column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 1 μL; mobile phase: water (A)-acetonitrile (B). Gradient elution program: acetonitrile volume fraction: 0-1.0 min, 40%; 1.0-4.0 min, 40%-95%; 4.0-5.5 min, 95%; 5.6-7.5 min, 40%.

[0222] 2.3.2 Tandem mass spectrometry parameters:

[0223] Mass spectrometry

[0224] The ion source was an electrospray ionization (ESI) source, the scanning mode was positive ion MRM mode, the spray voltage was 5500 V, and the ion source temperature was 250°C. Other mass spectrometry parameters are shown in the table below.

[0225] LC-MS / MS mass spectrometry parameters

[0226]

[0227] 2.4 Determination method

[0228] 1 μL of the LC-MS / MS mixed standard series solution and sample solution were respectively drawn into a high performance liquid chromatography-triple quadrupole mass spectrometer and measured according to the parameter conditions.

[0229] Under the same experimental conditions, if the retention time of the chromatographic peak detected in the sample is consistent with that of the corresponding component in the LC-MS / MS qualitative standard solution, and the deviation between the relative abundance ratio of the selected monitoring ion pair and the ion pair abundance ratio of the standard solution of equivalent concentration does not exceed the range specified in the table below, then it can be determined that the corresponding analyte is present in the sample.

[0230] The maximum allowable deviation of ion relative abundance when confirming the results

[0231] Ion relative abundance (k) k＞0.50 0.50≥k＞0.20 0.20≥k＞0.10 k≤0.10 Maximum allowed deviation ±20％ ±25％ ±30％ ±50％

[0232] 3 Results Analysis

[0233] 3.1 Limit of Detection (LOD)

[0234] Serial number Compound Abbreviation LOD (ng / mL) 1 TPO 0.005 2 TPO-L 0.002 3 TMO 0.005 4 HCHPK 2 5 PB-TMBPO 0.5

[0235] 3.2 Confirmation of detected components of the test product

[0236] LC-MS / MS qualitative standard solution ( Figure 4 The retention times and ion abundance ratios of TPO and HCHPK detected in the test sample were consistent with those of the reference sample, confirming the presence of these two components in the test sample.

[0237]

[0238] Example 3 High-efficiency screening method based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) and confirmation of positive results

[0239] Nail polish has a complex matrix and many interfering peaks in UV detection. To ensure the specificity of the assay results, this example uses "MissCandy BJ16 Painless Firming Base Gel" purchased from Tmall as an example to detail the method for confirming positive results using liquid chromatography-high-resolution mass spectrometry.

[0240] 1. Reagents and instruments

[0241] 1.1 Standard substances and reagents

[0242] Same as Example 1

[0243] 1.2 Instruments

[0244] Ultimate 3000-Q-Exactive high-resolution liquid spectrometer (Thermo Fisher Scientific, USA) and other instruments were the same as those in Example 1.

[0245] 2 Experimental methods

[0246] 2.1 Preparation of LC-HRMS standard solution

[0247] (1) LC-HRMS mixed standard intermediate solution (10 μg / mL): Accurately pipette an appropriate amount of LC mixed standard stock solution (2 mg / mL), dissolve it in acetonitrile and dilute it to 10 μg / mL, and shake well.

[0248] (2) LC-HRMS mixed standard series solutions: Accurately measure an appropriate amount of LC-MS mixed standard intermediate solution (10 μg / mL) and dilute it with acetonitrile to prepare a series of LC-HRMS mixed standard solutions containing 1, 20, 100, 250, 500, and 1000 ng / mL of each of the five photoinitiators. The 1000 ng / mL mixed standard solution is used as the LC-HRMS qualitative standard solution.

[0249] 2.2 Preparation of LC-HRMS sample solution

[0250] Independent use method (3): Take the sample and mix it evenly. Weigh 0.1g (accurate to 0.0001g) of the nail polish to be tested and place it in a 10mL brown stoppered colorimetric tube. Add acetonitrile to the scale. Vortex the tube tightly for 1min to disperse the sample in the extraction solvent. Then ultrasonically extract for 20min and filter through a 0.45μm organic filter membrane. Take an appropriate amount of the filtrate and dilute it 20 times with acetonitrile. The concentration of the nail polish to be tested is 500μg / mL.

[0251] (1) + (3) solution: Take the "MissCandy BJ16 Painless Firm Base Glue" LC sample solution prepared in Example 1, and according to the content determination results (HCHPK concentration is 193 μg / mL; TPO concentration is 142 μg / mL), dilute it 200 times with acetonitrile to make the concentrations of HCHPK and TPO 965 ng / mL and 710 ng / mL, respectively.

[0252] 2.3 Instrument parameters

[0253] 2.3.1 Liquid chromatography:

[0254] Chromatographic column: ACE EXCEL 2C18 (100 mm × 2.1 mm, 2 μm); column temperature: 40°C; flow rate: 0.5 mL / min; injection volume: 2 μL; mobile phase: water (A)-acetonitrile (B). Gradient elution program: acetonitrile volume fraction: 0-1.0 min, 40%; 1.0-3.5 min, 40%-100%; 3.5-5.5 min, 100%; 5.6-7.5 min, 40%.

[0255] 2.3.2 High-resolution mass spectrometry parameters:

[0256] The ion source was an electrospray ionization (ESI) source, and the scanning mode was full scan / data-dependent secondary mass spectrometry mode; the full scan m / z range was 100-500; the spray voltage was 3000 V; the ion source temperature was 350°C; the capillary temperature was 325°C; the sheath gas flow was 10 Arb; the auxiliary gas flow was 50 Arb; and the relative collision energy was 40%. Other LC-HRMS mass spectrometry parameters are shown in the table below:

[0257]

[0258] 2.4 Determination method

[0259] 2 μL of the LC-HRMS mixed standard series solution and sample solution were respectively drawn into the Ultimate 3000-Q-Exactive high-resolution liquid spectrometer (Thermo Fisher Scientific, USA) and measured according to the parameter conditions.

[0260] The standard solution and the test solution are collected in full scan / data-dependent MS / MS scanning mode to obtain primary and secondary mass spectrometric information. Under the same experimental conditions, if the retention time of the extracted ion chromatographic peak detected in the sample is consistent with that of the corresponding component in the LC-HRMS qualitative standard solution, and the m / z of the secondary mass spectrometric fragment ion is consistent, the corresponding component to be tested can be determined to be present in the sample.

[0261] 3 Results Analysis

[0262] 3.1 Limit of Detection (LOD)

[0263]

[0264]

[0265] 3.2 LC-HRMS result analysis and test solution determination

[0266] The results of LC-HRMS determination of standard solution ( Figure 5 and Figure 6 ) It can be seen that all five photoinitiators have mass spectrometry primary source fragmentation, and the corresponding fragmentation ions are significantly higher than [M+H] + Peak, this patent selects the retention time of the fragmentation ion in the primary source and the extracted ion peak for qualitative analysis, which can greatly improve the detection sensitivity.

[0267] This patent selects the extracted ion chromatogram peaks of m / z 147.08044 and 119.08553 as the common characteristic peaks of acylphosphine oxides for qualitative analysis ( Figure 7 ).

[0268] When the test solution is subjected to mass spectrometry analysis, its primary mass spectrometer ( Figure 8 ) and secondary mass spectrometry detected characteristic peaks corresponding to TPO and HCHPK in the standard solution, and these characteristic peaks were highly consistent with the results of liquid chromatography.

[0269] Example 4 Parameter Optimization of Photoinitiator Detection Based on Gas Chromatography-Mass Spectrometry (GC-MS)

[0270] In order to optimize the GC-MS method for the determination of photoinitiators, four batches of different types of nail polish were used as standard solutions for method optimization.

[0271] 1. Reagents and instruments

[0272] 1.1 Standard substances and reagents

[0273] Ethyl acetate was chromatographically pure and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; toluene was chromatographically pure and purchased from Merck, Germany.

[0274] 1.2 Instruments

[0275] GC-MS Triple Quad (8890A-7010B, Agilent, USA); other procedures were the same as in Example 1.

[0276] 2 Experimental methods

[0277] 2.1 Preparation of GC-MS standard solution

[0278] (1) GC-MS mixed standard stock solution (100 μg / mL): precisely take 50 μL of LC mixed standard stock solution (2 mg / mL), dilute with ethyl acetate to 1 mL, and obtain.

[0279] (2) GC-MS single standard solution (100 μg / mL): respectively take 1 mg of HCHPK, TPO-L, TPO, TMO and PB-TMBPO, and respectively place in a 10 mL brown volumetric flask, dissolve and dilute to the mark with ethyl acetate, and shake well, and obtain.

[0280] 2.2 Preparation of GC-MS sample solution

[0281] Take the sample and mix well, take 0.1 g (accurate to 0.0001 g), place in a 10 mL brown colorimetric tube with a stopper, add ethyl acetate to dilute to the mark, tightly seal and vortex for 1 min, so that the sample is dispersed in the extraction solvent, and then ultrasonic extraction for 20 min, pass through a 0.45 μm organic filter membrane, take the filtrate and dilute 1, 20, 10, 50 and 100 times with ethyl acetate for GC-MS determination.

[0282] 2.2 Instrument parameters

[0283] 2.2.1 Gas chromatography:

[0284] Chromatographic column: Thermofisher TG-5HT (15 m x 0.25 mm, 0.1 μm); split mode injection (split ratio 10:1); injection port temperature: 300 ℃; carrier gas: high-purity helium (purity > 99.999%); injection volume: 1 μL; flow rate: 1.2 mL / min; temperature rising program: initial temperature 70 ℃, hold for 1 min, rise to 180 ℃ at a rate of 30 ℃ / min, rise to 280 ℃ at a rate of 15 ℃ / min, and then rise to 320 ℃ at a rate of 30 ℃ / min, hold for 5 min to ensure complete detection of the sample in the method exploration.

[0285] 2.2.2 Mass spectrometry conditions

[0286] Electron impact ion source (EI) is adopted, ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; MS transmission line temperature: 300 ℃; solvent delay time: 4 min. Full scan mode (FULL SCAN): scan range m / z 30-430; SIM and MRM mode parameters are shown in the table:

[0287] 2.4 Determination method

[0288] In the FULL SCAN mode, the GC-MS standard solution is collected, and the retention time and the primary mass spectrum of the standard solution are obtained.

[0289] 3 Results Analysis

[0290] 3.1 Selection of gas chromatography columns

[0291] Based on the physicochemical properties of five photoinitiators, a 6% cyanopropylphenyl-96% dimethylpolysiloxane column was selected as the stationary phase for the chromatographic column. A comparison of the retention of the five photoinitiators on a Thermofisher TG-5HT (15m×0.25mm, 0.1μm) and an Agilent HP-5MS (30m×0.25mm, 0.25μm) column revealed that acylphosphine oxides were too strongly retained on the HP-5MS (30m×0.25mm, 0.25μm) column, requiring high-temperature baking of the column, resulting in a baseline rise and significant column bleed. Therefore, a 15m-long, 0.1μm-film-thick TG-5HT (15m×0.25mm, 0.1μm) GC column was ultimately selected.

[0292] 3.2 Optimization of gas phase inlet temperature

[0293] The responses of the photoinitiators were investigated at injection port temperatures of 260, 280, 300, 320, and 340°C. The results showed that the response of HCHPK increased with increasing temperature; the response of TPO-L followed a bell-shaped curve with a peak at 300°C; and the responses of TPO and TMO decreased with increasing temperature. The sensitivity of PB-TMBPO in GC-MS analysis was poor, presumably due to its high boiling point and the tendency for thermal decomposition at high temperatures, which affected its response. Based on these considerations, 300°C was selected as the injection port temperature.

[0294] 3.3 Selection of extracted ions

[0295] The mass spectra of the single-label solution were collected by GC-MS in FULL SCAN mode. The results showed that the primary fragment ions of HCHPK were m / z 81, 99, and 77; TPO-L were m / z 147, 119, and 141; TPO were m / z 147, 119, and 201; and TMO were m / z 147, 119, 361, 381, and 229.

[0296] 3.4 Optimization of SIM and MRM modes

[0297] The two primary ions with the largest response in FULL SCAN mode were selected as monitoring ions in SIM mode, as well as the parent ions optimized in MRM mode. The system optimized the gain value, product ion, and collision energy. The results are shown in the following table.

[0298]

[0299] * is the quantitative ion / ion pair

[0300] 3.5 Optimization of extraction solvent

[0301] Four representative products were selected for analysis to optimize the extraction solvent. The extraction efficiency of five solvents, acetonitrile, methanol, ethanol, acetone, toluene, and ethyl acetate, and the dispersing of nail polish samples were investigated. The results showed that acetone, ethyl acetate, and toluene had the best dispersing properties, followed by acetonitrile, and the worst were methanol and ethanol. Considering the availability and toxicity of the solvent, ethyl acetate was preferred; considering the compatibility with liquid phase, acetone and acetonitrile were preferred.

[0302] Considering GC-MS as the confirmation method after LC quantification, acetonitrile can be used for extraction, and ethyl acetate can be used for dilution for GC-MS confirmation.

[0303] Example 5 Positive result confirmation based on gas chromatography-mass spectrometry

[0304] The nail polish matrix is complex, and there are many interfering peaks in ultraviolet detection. To ensure the specificity of the determination results, this example takes "MissCandy BJ05 plated crystal sealing layer" purchased from a Tmall online store as an example to introduce the positive result confirmation method based on gas chromatography-mass spectrometry.

[0305] 1 Reagents and instruments

[0306] 1.1 Standard substances and reagents

[0307] Ethyl acetate was chromatographically pure and purchased from Shanghai Aladdin Biochem Technology Co., Ltd. The same as in Example 1

[0308] 1.2 Instruments

[0309] The same as in Example 4.

[0310] 2 Experimental method

[0311] 2.1 Preparation of GC-MS standard solution

[0312] (1) GC-MS mixed standard stock solution (10 μg / mL): accurately measure 50 μL of LC mixed standard stock solution (2 mg / mL) and dilute to 10 mL with ethyl acetate.

[0313] (2) GC-MS mixed standard series solution: accurately measure an appropriate amount of GC-MS mixed standard stock solution (10 μg / mL) and dilute to 1 mL with ethyl acetate to obtain a series of mixed standard solutions containing 2-1000 ng of five photoinitiators per 1 mL. Used to optimize parameters and investigate the detection limit.

[0314] (3) GC-MS qualitative standard solution (FULL SCAN mode): GC-MS mixed standard stock solution (10 μg / mL).

[0315] (4) GC-MS qualitative standard solution (SIM and MRM mode): Accurately measure an appropriate amount of GC-MS mixed standard stock solution (10 μg / mL) and dilute it with ethyl acetate to obtain a 500 ng / mL mixed standard solution.

[0316] 2.2 Preparation of GC-MS sample solution

[0317] Independent use method (4): Take the sample and mix it evenly. Weigh 0.1 g (accurate to 0.0001 g) of the nail polish to be tested and place it in a 10 mL brown stoppered colorimetric tube. Add ethyl acetate to the scale. Vortex the tube tightly for 1 minute to disperse the sample in the extraction solvent. Ultrasonic extraction is then carried out for 20 minutes. The sample is filtered through a 0.45 μm organic filter membrane. Take an appropriate amount of the filtrate and dilute it 10 and 50 times with ethyl acetate to obtain GC-MS sample solution 1 and GC-MS sample solution 2. GC-MS sample solution 1 is used for detection in the full scan (FULL SCAN) mode. GC-MS sample solution 2 is used for detection in the SIM and MRM modes.

[0318] Scheme (1) + (4): Take the "MissCandy BJ16 Painless Firm Base Glue" LC sample solution prepared in Example 1, and according to the content determination results (HCHPK concentration is 193 μg / mL; TPO concentration is 142 μg / mL), dilute it 100 times with ethyl acetate to obtain GC-MS sample solution 1, which contains HCHPK and TPO concentrations of 1.93 and 1.42 μg / mL, respectively; dilute it 1000 times with ethyl acetate to obtain GC-MS sample solution 1, which contains HCHPK and TPO concentrations of 0.193 and 0.142 μg / mL, respectively.

[0319] 2.3 Instrument parameters

[0320] 2.3.1 Gas chromatography:

[0321] Chromatographic column: Thermofisher TG-5HT (15m×0.25mm, 0.1μm); split mode injection (split ratio 10:1); inlet temperature: 300°C; carrier gas: high-purity helium (purity>99.999%); injection volume: 1μL; flow rate: 1.2mL / min; heating program: initial temperature of 70°C, hold for 1min, increase to 180°C at a rate of 30°C / min, increase to 280°C at a rate of 15°C / min, and then increase to 320°C at a rate of 30°C / min and hold for 0.5min.

[0322] 2.3.2 Mass spectrometry conditions

[0323] An electron impact ion source (EI) was used with a source temperature of 230°C, a quadrupole temperature of 150°C, a MS transfer line temperature of 300°C, and a solvent delay time of 4 min. Full scan mode (FULL SCAN) was used with a scan range of m / z 30 to 430. Parameters for selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes are shown in the table below:

[0324] Table GC-MS parameters

[0325]

[0326]

[0327] *Preferred monitoring window ions / ion pairs

[0328] 2.4 Determination method

[0329] 2.4.1 Determination of GC-MS standard solution

[0330] The standard solution was collected in FULL SCAN / SIM / MRM mode to obtain the retention time of the standard solution, as well as the primary mass spectrum / SIM detection ion abundance ratio / MRM ion pair abundance ratio.

[0331] 2.4.2 GC-MS sample determination

[0332] The sample solution was collected in FULL SCAN / SIM / MRM mode; the retention time of the detected peak of the sample solution, as well as the primary mass spectrum / SIM detection ion abundance ratio / MRM ion pair abundance ratio were compared with those of the standard solution.

[0333] 2.4.3 GC-MS qualitative results determination

[0334] Under the same experimental conditions, if the retention time of the chromatographic peak detected in the sample is consistent with that of the corresponding component in the GC-MS qualitative standard solution, in the FULL SCAN mode, the retention time of the extracted ion chromatogram of the primary mass spectrometer is consistent, and the m / z of the primary mass spectrometer fragment ions are consistent; in the SIM / MRM mode, if the deviation of the relative abundance ratio of the selected monitoring ion pair and the ion pair abundance ratio of the standard solution of equivalent concentration does not exceed the range specified in the table below, then it can be determined that the corresponding component to be measured is present in the sample.

[0335] The maximum allowable deviation of ion relative abundance when confirming the results

[0336] Ion relative abundance (k) k＞0.50 0.50≥k＞0.20 0.20≥k＞0.10 k≤0.10 Maximum allowed deviation ±20％ ±25％ ±30％ ±50％

[0337] 3 Results Analysis

[0338] The results of GC-MS qualitative standard solution in FULL SCAN, SIM and MRM modes are as follows: Figure 9 、 Figure 10 、 Figure 11 PB-TMBPO is not suitable for GC-MS determination due to its high boiling point, thermal instability and poor sensitivity.

[0339] In full scan mode (FULL SCAN), the LODs of HCHPK, TPO-L, TPO, and TMO were 20, 50, 200, and 500 ng / mL, respectively; in SIM mode, they were 5, 5, 20, and 50 ng / mL; and in MRM mode, they were 5, 2, 5, and 10 ng / mL, respectively.

[0340] In full scan or SIM mode, m / z 147 and 119 were selected, and in MRM mode, 147→119 (collision energy 15 eV) and 147→91 (collision energy 25 eV) were selected to rapidly screen acylphosphine oxide photoinitiators.

[0341] The results of the "MissCandy BJ05 crystal seal layer" sample solution under FULL SCAN, SIM, and MRM modes are as follows: Figure 12 、 Figure 13 、 Figure 14 As shown in the figure, the ion peak corresponding to TPO was detected, while HCHPK, TPO-L and TMO were not detected, which was consistent with the LC detection results.

[0342] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for qualitative and / or quantitative detection of photoinitiators in nail polish, characterized in that: The photoinitiator is selected from 1-hydroxycyclohexyl phenyl ketone (HCHPK) and an acylphosphine oxide photoinitiator, and the acylphosphine oxide photoinitiator is selected from a combination of one or more of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), 2,4,6-trimethylbenzoylphenylphosphonic acid ethyl ester (TPO-L), 2,4,6-trimethylbenzoyl-di(p-tolyl)phosphine oxide (TMO), and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (PB-TMBPO). The method is selected from any one of (1) to (4), or the method is selected from any one of (1) and (2) to (4): (1)LC method: S1. Preparation of standard solution: Weigh HCHPK, TPO-L, TPO, TMO, and PB-TMBPO respectively, and dilute them stepwise with organic solvent to form a series of LC mixed standard solutions with at least five concentrations ranging from 2 to 600 μg / mL for each of the five photoinitiators. S2. Preparation of test solution: Take the nail polish to be tested, dilute it with an organic solvent in proportion, disperse it with ultrasound, filter it, and determine the filtrate by HPLC; the concentration of the nail polish to be tested in the test solution is any one of 0.4 to 20 mg / mL; S3. Chromatographic conditions: Chromatographic column: C18 as packing, pore size: Particle size: 5μm; Specifications: 150mm×4.6mm; Column temperature: 30°C; Mobile phase: A is ultrapure water, B is acetonitrile; Gradient elution program: 0-2 min, 45% A; 2-6 min, 45% A-5% A; 6-8 min, 5% A; 8-8.5 min, 5% A-45% A; 8.5-12 min, 45% A; Flow rate: 1 mL / min; Injection volume: 5 μL; Diode array detector collection wavelength: 190 ~ 400nm; S4. Determination method: The detection wavelength for HCHPK was 243 nm, and that for TPO-L, TPO, TMO, and PB-TMBPO was 375 nm; Standard curve method, external standard method for quantification; (2) LC-MS / MS method: S1. Preparation of standard solution: (A) LC-MS / MS mixed standard series solution: Weigh HCHPK, TPO-L, TPO, TMO, and PB-TMBPO separately and dilute them stepwise with organic solvent to form a series of LC-MS / MS mixed standard solutions with at least five concentrations ranging from 0.002 to 200 ng / mL for each of the five photoinitiators. (B) LC-MS / MS qualitative standard solution: HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted stepwise with organic solvent to prepare mixed standard solutions with a mass concentration of 50 ng / mL for each of the five photoinitiators as LC-MS / MS qualitative standard solutions; S2. Preparation of test solution: (A) If this Act is used alone: Take the nail polish to be tested, dilute it with an organic solvent in proportion, disperse it with ultrasound, filter it, and determine the filtrate by HPLC; the concentration of the nail polish to be tested in the test solution is any one of 2-100 μg / mL; (B) If it is further confirmed on the basis of (1): Take the test solution prepared in (1) S2 and dilute it with an organic solvent until the photoinitiator content is 10-200 ng / mL; S3. LC-MS / MS conditions: (A) Chromatographic conditions: Chromatographic column: C18 as packing, pore size: Particle size: 2μm; 100mm×2.1mm; Column temperature: 40°C; Flow rate: 0.3 mL / min; Injection volume: 1 μL; Mobile phase: water (A)-acetonitrile (B); A gradient elution program was used with the following acetonitrile volume fraction: 0–1.0 min, 40%; 1.0→4.0min, 40%→95%; 4.0~5.5min, 95%; 5.6~7.5min, 40%; (B) Mass spectrometry conditions: The ion source was electrospray ionization (ESI); The scanning mode was positive ion MRM mode; The spray voltage was 5500 V; The ion source temperature was 250 °C; The mass spectrometry parameters are as follows: *Preferred monitoring window ions / ion pairs; S4. Determination method: Under the same experimental conditions, if the retention time of the chromatographic peak detected in the sample is consistent with that of the corresponding component in the LC-MS / MS qualitative standard solution, and the deviation between the relative abundance ratio of the selected monitoring ion pair and the ion pair abundance ratio of the standard solution of equivalent concentration does not exceed the range specified in the table below, then it can be determined that the corresponding component to be tested is present in the sample; ； (3) LC-HRMS method: S1. Preparation of standard solution: (1) LC-HRMS mixed standard series solution: HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted stepwise with organic solvent to form a LC-HRMS mixed standard series solution with at least two concentrations of the five photoinitiators ranging from 1 to 1000 ng / mL; (2) LC-HRMS qualitative standard solution: HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted stepwise with organic solvent to form a mixed standard solution with a mass concentration of 1000 ng / mL for each of the five photoinitiators as the LC-HRMS qualitative standard solution; S2. Preparation of test solution: (A) If this Act is used alone: Take the nail polish to be tested, dilute it with an organic solvent in proportion, disperse it with ultrasound, filter it, and determine the filtrate by HPLC; the concentration of the nail polish to be tested in the test solution is any one of 20-1000 μg / mL; (B) If it is further confirmed on the basis of (1): Take the test solution prepared in (1) S2 and dilute it with an organic solvent until the photoinitiator content is 0.1-2 μg / mL; S3, LC-HRMS conditions: (A) Chromatographic conditions: Chromatographic column: C18 as packing, pore size: Particle size: 2μm; 100mm×2.1mm; Column temperature: 40°C; Flow rate: 0.5 mL / min; Injection volume: 2 μL; Mobile phase: water (A)-acetonitrile (B); A gradient elution program was used with the acetonitrile volume fraction as follows: 0–1.0 min, 40%; 1.0–3.5 min, 40%–100%; 3.5~5.5min, 100%; 5.6~7.5min, 40% (B) Mass spectrometry conditions: The ion source was an electrospray ionization source (ESI), and the scanning mode was full scan / data-dependent secondary mass spectrometry scanning mode; Full scan m / z range: 100-500; The spray voltage was 3000 V; The ion source temperature was 350°C; Capillary temperature: 325°C; Sheath gas flow: 10Arb; Auxiliary airflow: 50Arb; The relative collision energy is 40%; Other LC-HRMS mass spectrometry parameters are as follows: ； S4. Determination method: Collect standard solutions and test sample solutions in full scan / data-dependent secondary mass spectrometry scanning mode to obtain primary mass spectrometry and secondary mass spectrometry information; under the same experimental conditions, if the retention time of the extracted ion current chromatographic peak detected in the sample is consistent with that of the corresponding component in the LC-HRMS qualitative standard solution, and the m / z of the secondary mass spectrometry fragment ion is consistent, it can be determined that the corresponding component to be tested is present in the sample; The organic solvent in (1) to (3) is selected from a combination of one or more of acetonitrile, acetone, methanol, and ethanol; (4) GC-MS method: S1. Preparation of standard solution: (A) GC-MS series mixed standard solution: Weigh HCHPK, TPO-L, TPO, TMO, and PB-TMBPO separately and dilute them in ethyl acetate to prepare a series of GC-MS mixed standard solutions with at least two concentrations of each of the five photoinitiators ranging from 2 to 1000 ng / mL. (B) GC-MS qualitative standard solution: (B-1) Qualitative standard solution in full scan mode: HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted with ethyl acetate to prepare full-scan mode qualitative standard solutions with a mass concentration of 10 μg / mL for each of the five photoinitiators. (B-2) Qualitative standard solution for SIM and MRM modes: HCHPK, TPO-L, TPO, TMO, and PB-TMBPO were weighed separately and diluted with ethyl acetate to prepare mixed standard solutions with a mass concentration of 500 ng / mL for each of the five photoinitiators as qualitative standard solutions for SIM and MRM modes; S2. Preparation of test solution: (A) If this Act is used alone: Prepare a stock solution of the test sample with a concentration of 0.4 to 20 mg / mL of nail polish gel, dilute it with ethyl acetate in a certain proportion, and then ultrasonically disperse and filter it to obtain the test sample. Dilute it 10-fold and 50-fold with ethyl acetate to obtain GC-MS sample solution 1 and GC-MS sample solution 2. GC-MS sample solution 1 is used for detection in full scan mode; GC-MS sample solution 2 is used for detection in SIM and MRM modes. (B) If it is further confirmed on the basis of (1): Take the test solution prepared in (1) S2 and dilute it with ethyl acetate to a GC-MS sample solution 1 with a photoinitiator content of 1 to 20 μg / mL and a GC-MS sample solution 2 with a content of 0.1 to 2 μg / mL; S3. GC-MS conditions: (A) Gas chromatography: Chromatographic column: High-temperature column with 5% phenyl / 95% dimethylpolysiloxane as stationary phase, 15m×0.25mm, film thickness 0.1μm; Injection in split mode, split ratio 10:1; Inlet temperature: 300°C; Carrier gas: high-purity helium (purity>99.999%); Injection volume: 1 μL; Flow rate: 1.2 mL / min; Heating program: initial temperature 70 °C, hold for 1 min, increase to 180 °C at a rate of 30 °C / min, increase to 280 °C at a rate of 15 °C / min, and then increase to 320 °C at a rate of 30 °C / min, hold for 0.5 min; (B) Mass spectrometry conditions: An electron bombardment ion source (EI) was used, and the ion source temperature was 230°C; Quadrupole temperature: 150°C; MS transfer line temperature: 300°C; Solvent delay time: 4 min; Full scan mode (FULL SCAN): Scan range m / z 30~430; The parameters of ion monitoring mode (SIM) and multiple reaction monitoring mode (MRM) are as follows: *Preferred monitoring window ions / ion pairs; S4. Determination method: Under the same experimental conditions, if the retention time of the chromatographic peak detected in the sample is consistent with that of the corresponding component in the GC-MS qualitative standard solution, the retention time of the extracted ion chromatogram of the primary mass spectrometer in the FULL SCAN mode is consistent, and the m / z of the primary mass spectrometer fragment ions are consistent; In SIM / MRM mode, if the deviation between the relative abundance ratio of the selected monitoring ion pair and the ion pair abundance ratio of the standard solution of equivalent concentration does not exceed the specified range, it can be determined that the corresponding component to be measured exists in the sample:

2. The detection method according to claim 1, wherein (1) The chromatographic column is a Kromasil 100-5-C18 column, (2) and (3) the chromatographic columns are ACE EXCEL 2C18 columns, and (4) the chromatographic column is a Thermofisher TG-5HT column.

3. The detection method according to claim 1, wherein (1) The concentration of the nail polish to be tested in S2 is 10 mg / mL.

4. The detection method according to claim 1, wherein (1) The concentration of the photoinitiator in the nail polish to be tested in S2 is 2 to 600 μg / mL.

5. The detection method according to claim 1, wherein (2) In S2(A), if the response of a certain photoinitiator in the nail polish to be tested exceeds 400% of the LC-MS / MS qualitative standard solution after testing, it should be further diluted to 10% to 400% of the concentration of the LC-MS / MS qualitative standard solution and tested again for that photoinitiator.

6. The detection method according to claim 1, characterized in that The organic solvent in (1) to (3) is selected from acetonitrile, acetone or a combination of acetonitrile and acetone.

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