Abstract：
Various types of oxidative dyes used in hair dye products possess poor stability and have varying frequency of use. Interference problems also frequently arise in actual sample measurements. Therefore, it is necessary to establish a simple, rapid, accurate, and specific method for the determination of common dyes in hair dye products for their effective regulation. In this study, dyes were grouped according to their frequency of use. Using a C18 column that minimizes the silanol effect and influence of metals, the quantitative high performance liquid chromatography (HPLC) method for 32 dyes listed in Safety and Technical Standards for Cosmetics (2015 edition) was optimized, and a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) confirmatory method for the dyes was established. The samples were extracted using a mixed solution of ethanol-water (1∶1, v/v) with 10 g/L sodium bisulfite solution as an antioxidant, vortexed and mixed, and then extracted by ultrasonication in an ice bath for 10 min. Methanol, acetonitrile, and phosphate buffer were used as the mobile phases in the HPLC analysis. Additionally, two different elution conditions (chromatographic gradient) were used for the separation of 32 oxidative dyes, which were detected at a wavelength of 280 nm. The HPLC separations were compared using columns of particle sizes 5 μm and 2.7 μm; 5 μm C18 columns with better anti-interference and antiblocking ability were selected. Satisfactory separation was achieved for all three commercial C18 columns with a particle size of 5 μm, and the method had good general usability. In condition 1, 17 commonly used dyes and three less commonly used dyes were assigned to group Ⅰ and separated by HPLC; in condition 2, eight banned dyes and four other less commonly used dyes were assigned to group Ⅱ and separated by HPLC. The HPLC-MS/MS method used 5 mmol/L ammonium acetate aqueous solution-acetonitrile and 5 mmol/L acetic acid aqueous solution-acetonitrile as mobile phases in the positive and negative ion modes, respectively. Multiple reaction monitoring (MRM) was performed for qualitative and quantitative analyses in the electrospray ionization mode. Under the examined conditions, six pairs of isomers were well resolved. For the HPLC and HPLC-MS/MS methods, the relative standard deviations (RSDs) of the intra-day precision and 48 h stability tests were less than 10%. The recoveries were between 82.6% and 114.9% (RSD<10%). In the HPLC method, 32 dyes showed good linearity in an approximate range of 10-500 mg/L (r2>0.99), and the limits of detection (LODs) were 9.7-40.1 μg/g. The linear range of hydroquinone in the HPLC-MS/MS method was 2.0-79.7 mg/L, and the LOD was 8.0 μg/g; the linear ranges of the other components were approximately 0.1-4 mg/L, and the LODs were 0.01-0.4 μg/g. The actual samples were simultaneously measured by HPLC, HPLC-MS/MS, and the standard method. Finally, 16 of the 32 dyes were detected, and the detected contents ranged from 58 to 25160 μg/g. The RSDs of the results obtained from the three detection methods were between 1.9% and 10.1%. All detected components were within the limits of group Ⅰ of this method. In comparison with methods reported in the literature and the standard method, this method covers all components for the routine regulatory inspection of oxidative dyes in cosmetics. The method can separate the commonly used dyes under the same HPLC conditions and avoid interference from 15 other commonly used dyes during the analysis of actual samples. A suitable HPLC-MS/MS confirmatory method was also established for the identification of currently unknown substances in the statutory inspection of cosmetics. The method is simple, rapid, accurate, and specific with general usability and good operability.
摘要：
用于染发产品中的各种类型的氧化染料具有差的稳定性并且具有不同的使用频率。在实际样品测量中也经常出现干扰问题。因此,有必要建立一个简单的，快速,准确,并对染发产品中常见染料的测定方法进行了具体的研究,对其进行了有效的调控。在这项研究中,染料根据其使用频率进行分组。使用C18柱，最大限度地减少硅烷醇的影响和金属的影响，对化妆品安全技术标准（2015年版）中列出的32种染料的高效液相色谱（HPLC）定量方法进行了优化，建立了高效液相色谱-串联质谱(HPLC-MS/MS)确证方法。样品用乙醇-水(1∶1,v/v)混合溶液和10g/L亚硫酸氢钠溶液作为抗氧化剂提取,涡旋和混合，然后在冰浴中超声处理10分钟提取。甲醇,乙腈,和磷酸盐缓冲液用作HPLC分析中的流动相。此外,两种不同的洗脱条件（色谱梯度）用于分离32种氧化染料，在280nm的波长下检测。使用粒径为5μm和2.7μm的色谱柱比较HPLC分离；选择具有更好的抗干扰和抗粘连能力的5μmC18色谱柱。所有三个粒径为5μm的商业C18色谱柱都实现了令人满意的分离，该方法具有良好的通用性。在条件1中，将17种常用染料和3种不太常用的染料分配到Ⅰ组，并通过HPLC分离；在条件2中，将8种禁用染料和4种其他不太常用的染料分配到Ⅱ组，并通过HPLC分离。HPLC-MS/MS法以5mmol/L乙酸铵水溶液-乙腈和5mmol/L乙酸水溶液-乙腈为流动相,采用正负离子模式。分别。以电喷雾电离模式进行多反应监测(MRM)以进行定性和定量分析。在检查的条件下，六对异构体被很好地解析。对于HPLC和HPLC-MS/MS方法，日内精密度和48h稳定性测试的相对标准偏差（RSD）小于10％。回收率在82.6%~114.9%之间(RSD<10%)。在HPLC方法中,32种染料在约10-500mg/L（r2>0.99）范围内表现出良好的线性，检测限（LODs）为9.7-40.1μg/g。HPLC-MS/MS法测定对苯二酚的线性范围为2.0～79.7mg/L,LOD为8.0μg/g；其他成分的线性范围约为0.1-4mg/L，LOD为0.01-0.4μg/g。通过HPLC同时测量实际样品，HPLC-MS/MS,和标准方法。最后,检测到32种染料中的16种，检出量为58～25160μg/g。从三种检测方法获得的结果的RSD在1.9％至10.1％之间。检测到的所有成分均在该方法Ⅰ组的范围内。与文献报道的方法和标准方法相比，该方法涵盖了化妆品中氧化染料的常规监管检查的所有成分。该方法可以在相同的HPLC条件下分离常用染料，并且在实际样品分析过程中避免了其他15种常用染料的干扰。还建立了合适的HPLC-MS/MS确证方法，用于化妆品法定检验中目前未知物质的鉴定。方法简单,快速,准确,具有通用性和良好的可操作性。
