消费品检测 : 降低风险, 确保质量, 实现法规遵从性 消费品的分析检测是确保最终用户安全 保证产品质量 维护品牌形象的关键环节 沃特世 (Waters ) 公司长期以来都致力 于帮助制造商及合同检测机构解决各种科学难题和精简分析操作 制造商及其合同检测实验室必须具备提高通量 延长正常运行时间以及轻

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1 消费品检测应用文集

2 消费品检测 : 降低风险, 确保质量, 实现法规遵从性 消费品的分析检测是确保最终用户安全 保证产品质量 维护品牌形象的关键环节 沃特世 (Waters ) 公司长期以来都致力 于帮助制造商及合同检测机构解决各种科学难题和精简分析操作 制造商及其合同检测实验室必须具备提高通量 延长正常运行时间以及轻松表征各种复杂样品的能力, 与此同时还要保证目标化学物质的浓度符合国家标准和国际标准中的允许限值规定 随着我们的样品前处理 色谱柱 色谱 质谱以及数据管理软件技术不断发展, 用户可以缩短分析时间 合并多种方法 降低成本并实现法规和合同遵从性

3 目录 杀菌剂 以消费品中的杀菌剂为例, 将多种方法转换为单一分析解决方案 使用 ACQUITY UPLC PDA 分析杀菌剂 ( 第 1 部分 ) 使用 ACQUITY UPLC PDA 分析杀菌剂 ( 第 2 部分 ): 通过 / 未通过自定义计算 消费品中苯扎氯铵 (BAC) 的 UPLC 分析 分散性染料 分散性染料的分析 改善消费品中致敏和致癌染料的分析速度和定量性能 快速筛查消费品中的分散性染料 快速筛查 36 种合成染料 阻燃剂 利用 UPLC 的速度和分离度优势改善 HBCD 和 TBBP-A 非对映体的 MS-MS 检测 有害物质限制指令 (RoHS) 的法规遵从性分析中异常污染物的检测 邻苯二甲酸酯 化妆品和个人护理产品中邻苯二甲酸酯类和苯甲酸脂类的高通量分析 采用 1 分钟方法筛查玩具中法规限值水平的邻苯二甲酸酯 化学行业常规 QC 监测中的异常污染物检测 初级芳香胺 / 偶氮染料 提高初级芳香胺分析的灵敏度和选择性 油墨中初级芳香胺的分析 化妆品和个人护理产品中初级芳香胺的分析

4 杀菌剂 4

5 Transferring Multiple Methods into a Single Analytical Solution Using Biocides in Consumer Products as a Model Jane Cooper GAL To illustrate the simplicity of optimizing multiple methods for diverse biocides used in consumer products. The ACQUITY UPLC H-Class System enables a streamlined approach to multiple method analysis. BACKGRUD Waters ACQUITY UPLC H-Class System offers the flexibility of the quaternary solvent pump, with the simplicity of flow-through needle injections. With a wide range of UPLC column substrates and chemistries available, and the multi-solvent blending capabilities available with the ACQUITY UPLC H-Class System, additional scope both during method development and with routine analysis is now obtainable. Biocides are chemical substances used in a wide range of products to either control or render safe harmful organisms. Various biocides are included in restricted substances lists (RSLs) utilized by many companies in order to protect their products for consumers, their workers, and also the community/environment. Figure 1. ACQUITY UPLC H-Class System with Xevo TQD. This technology brief illustrates the flexibility users have with the ACQUITY UPLC H-Class System to combine biocide analytical suites of analyses and achieve high resolution, sensitivity, and throughput with sub-2-µm particle columns. The scope to combine suites of analyses offers the chemical industry valuable time and cost savings, faster sample turnaround times, and an associated reduction in solvent usage. Transferring Multiple Methods into a Single Analytical Solution Using Biocides in Consumer Products as a Model 5

6 THE SLUTI This investigation uses the Xevo TQD with atmospheric pressure chemical ionization (APCI), coupled to an ACQUITY UPLC H-Class System, for the detection of a range of biocide compound groups that would typically be analyzed using different chromatographic conditions. ptimum multiple reaction monitoring (MRM) or selected ion recording (SIR) conditions were developed, as shown in Table 1. Chemical substance MRM / APCI Cone MRM transitions/or Collision SIR (+/-) voltage (V) SIR mass (m/z) energy Triclosan MRM > > 215.0* 21 Tetrachlorophenol (3 isomers) MRM > > 158.7* 16 Pentachlorophenol SIR /A Tributyltin chloride MRM > > 291.1* 6 Triphenyltin chloride MRM > > 351.0* 12 2-ctyl-3-isothiazolinone MRM > > 101.9* 22 2-Methyl-3-isothiazolinone MRM > 70.9* > Table 1. Biocides, ionization mode, cone voltages, MRM transitions or SIR mass, and associated collision energy values (*refers to the quantification transition). In order to optimize chromatographic separations, there are many important factors to consider including: LC system, detection method, choice of LC column, mobile phase(s), and mobile phase buffers. The choices of mobile phase buffers depend upon the compounds to be analyzed and the associated ionization mode used for detection. Buffers affect both the ph and the ionic strength of the mobile phase, and influence such factors as selectivity and peak shape. Effects caused by the choice of mobile phase buffer with regard to the analysis of biocides are illustrated in Figure 2. The three tetrachlorophenol isomers were resolved when the basic ammonium acetate (AA) buffer was used, but the peak shape of the tin compounds, shown in Figure 2a, was very poor. The three tetrachlorophenol isomers were not resolved, however, when the acidic formic acid (FA) buffer was used, while the peak shape of the tin compounds, shown in Figure 2b, was markedly improved. Typically, to achieve improved resolution of the three tetrachlorophenol isomers, improved selectivity, and peak shape of the tin compounds, two separate LC methods would be required; one using an acidic buffer, and the other using a basic buffer. Using the quaternary pump of the ACQUITY UPLC H-Class System, this can all be achieved in one UPLC method, shown in Figure 2c, by swapping the choice of buffer during the run. Transferring Multiple Methods into a Single Analytical Solution Using Biocides in Consumer Products as a Model 6

7 a) b) c) A: 0.1 FA in water B: 0.1 FA in acetonitrile C: 5 mmol AA in water D: 5 mmol AA in acetonitrile 100 Ammonium acetate 100 Formic acid 100 Formic acid + ammonium acetate UPLC column: HSS T3 1.8 µm, 2.1 x 100 mm Triclosan Tetrachlorophenol (3 isomers) Pentachlorophenol Tributyltin chloride Triphenyltin chloride ctyl -3-isothiazolinone Methyl-3-isothiazolinone Time Time Time Figure 2. MRM / SIR chromatograms for select biocides compounds in a mixed solvent standard using different mobile phase buffer: 2a. ammonium acetate, 2b. formic acid; and 2c. a combination of ammonium acetate and formic acid. Combining methods offer time and cost savings, enabling greater sample throughput and reduced solvent consumption. Also reducing the amount of LC methods required saves time spent on switching and equilibrating LC methods in between different analytical suites of analyses. SUMMARY The flexibility offered using the quaternary solvent pump of the ACQUITY UPLC H-Class System expands the benefits of UPLC technology. It provides improved scope for method development and multi-method analyses, while maintaining high resolution, sensitivity, and throughput offered by sub-2-µm particle columns. The scope to combine suites of analyses offers the chemical industry valuable time and cost savings, faster sample turnaround times, and an associated reduction in solvent usage. Waters, The Science of What s Possible, ACQUITY UPLC, UPLC, and Xevo are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. February E TC-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: Transferring Multiple Methods into a Single Analytical Solution Using Biocides in Consumer Products as a Model 7

8 ACQUITY UPLC PDA Analysis of Biocides (Part 1) Peter J. Lee and Alice J. Di Gioia Waters Corporation, Milford, MA, USA APPLICATI BEEFITS Improves laboratory productivity by enabling the rapid and sensitive separation of six commonly used biocides. Library matching and quantification automated with Empower 3 Software increases confidence in peak confirmation and helps ensure product quality. Suitable for cosmetic and personal care product development and quality control analytical testing. WATERS SLUTIS ACQUITY UPLC System ITRDUCTI Unwanted micro-organisms such as bacteria, viruses, and molds can grow wherever there is a source of nutrition and moisture. This unwanted growth may negatively impact human health, interfere with manufacturing processes, damage building structures, and spoil consumer goods. The principal defense against deleterious micro-organisms is biocides, commonly classified as disinfectants, preservatives, antifouling products, and pest controls. Biocides are used as additives in cosmetics and personal care, household, and industrial products. To protect the environment and human health, many countries regulate biocide use. 1 In the European Union, this is done through the Directive 98/8/EC (The Biocidal Products Directive) and Regulation (EU) o 528/2012 (The Biocidal Products Regulation). In the United States, regulatory control of biocides falls under the EPA and the biocides applications in cosmetics, food, and personal health care are regulated by the U.S. FDA. Regulations impact the registration, labeling, and composition of biocides. 2 Reliable and rapid methods are therefore essential to ensure effective product quality control. This application note describes a three-minute separation of six biocides using the Waters ACQUITY UPLC PDA System with Empower 3 Software. With PDA library matching and the built-in advanced mathematical algorithms, each biocide in the mixture can be identified and quantified; the analysis is fast and reproducible. The ability to quickly and unambiguously analyze biocide content can facilitate workflow related to the quality control and regulatory compliance of biocide containing products. This methodology benefits new product development, product troubleshooting and biocide production. ACQUITY UPLC PDA Detector Empower 3 Chromatography Data Software ACQUITY UPLC BEH C 18 Column KEY WRDS Biocides, cosmetics, personal care products, consumer products, product development, quality control, regulatory compliance, library matching ACQUITY UPLC PDA Analysis of Biocides (Part 1) 8

9 E X P E R IM E TA L Sample preparation Analytes are: Kathon CG/ICP [containing 0.4 of 2-methyl-4-isothiazolin-3-one (1a), 1.2 of 5-chloro-2-methyl-4-isothiazolin-3-one (1b)]; Carbendazim (2); Benzisothiazol-3(2H)-one (3); 2-phenoxyethanol (4); RESULTS AD DISCUSSI Figure 1 shows the structures of the biocides (1a, 1b, 2-6); a 5 ppm mixture of 1 6 was separated using the Waters ACQUITY UPLC System with a three-minute linear gradient method. These compounds are frequently used in adhesives, paint and coatings, latex and sealants, inks, wood and paper products, textile and leather products, metalworking fluids, personal care products, cosmetics, laundry detergents, dishwashing liquids, and household and industrial cleaners. The acetonitrile/ water mobile phase with TFA modifier is compatible with mass spectrometry detectors, if needed. Benzoic Acid (5); Methyl paraben (6). LC conditions LC system: ACQUITY UPLC PDA Software: Empower 3 Weak wash: 95:5 Water: CH 3 C (600 μl) Strong wash: 50:50 Water: CH 3 C (200 μl) Seal wash: 90:10 Water: CH 3 C (5 min) Column temp.: 30 C Flow rate: 1 ml/min Injection: 5 μl Detection: PDA 210 to 500 nm Sampling rate: 20 pts/s Filter response: 0.1 s Column: ACQUITY UPLC BEH C x 50 mm Mobile phase A: 0.05 v Trifluoroacetic acid (TFA) in water Mobile phase B: 0.05 v TFA in CH 3 C Linear gradient: 5 to 15 B in 2.9 min H S S Cl H 1a 1b 2 H H S H H H 6 Figure 1. Chemical structures of biocides. ote: The column was equilibrated with 5 B for 2 minutes before injection, and was washed with 100 B for 2 minutes at the end of each run. ACQUITY UPLC PDA Analysis of Biocides (Part 1) 9

10 UV photodiode array (PDA) detection combined with Empower 3 Software enables a powerful range of detection and identity confirmation possibilities for chromatographic separations. PDA timed wavelength chromatograms can be plotted using the λmax of each analyte. This increases the detection limit when the analytes have very different λmax and aids quantification. Figure 2 is an overlay of nine replicate injections of PDA timed wavelength chromatograms, demonstrating that the overall reproducibilty is excellent. The three-minute linear gradient easily resolves the two active components of Kathon CG/ICP (1a and 1b) and the other five biocides. The retention time and peak area of each component observed in the above nine replicate injections are listed in Tables 1 and 2, with statistical analysis results generated using Empower 3 Software. The excellent RSD results indicate the suitability of UPLC with BEH column chemistry for biocides a 1b 2 1a (min) 1b (min) 2 (min) 3 (min) 4 (min) 5 (min) 6 (min) Mean Std. Dev RSD Minutes Figure 2. verlay PDA timed wavelength chromatograms, retention time and peak area tables of 9 replicate injections of sample containing 1.25 ppm of 1a, 3.75 ppm of 1b and 5 ppm of 2 6: (0.00 min, 275 nm; 1.40 min, 225 nm; 2.55 min, 255 nm). Table 1. Component summary for retention time for 9 replicate injections of sample containing 1.25 ppm of 1a, 3.75 ppm of 1b and 5 ppm of 2 6: (0.00 min, 275 nm; 1.40 min, 225 nm; 2.55 min, 255 nm). 1a 1b Mean Std. Dev RSD Table 2. Component summary for area for 9 replicate injections of sample containing 1.25 ppm of 1a, 3.75 ppm of 1b and 5 ppm of 2 6: (0.00 min, 275 nm; 1.40 min, 225 nm; 2.55 min, 255 nm). ACQUITY UPLC PDA Analysis of Biocides (Part 1) 10

11 Six levels of calibration standards (containing Kathon and 2 6 from 2.5 to 20 ppm) were analyzed. Empower 3 Software has built-in mathematical features and functions. Calibration curves were created from the standards and the quantity of analyte in each sample was calculated automatically. Figure 3 shows the calibration plots generated by Empower 3, using the peak areas vs the concentration. The linearity of the calibration curves is excellent with the R 2 values (residual sum of squares) above , except one with Table 3 shows a typical results analysis table for peak identification and quantification using a biocides standard mixture mix as a sample. The last column shows that amounts match well with actual values (1.25 ppm for 1a, 3.75 ppm for 1b, and 5 ppm for 2 6). The data suggest that UPLC/PDA is well suited to meet the regulatory demands for quantitative analysis of biocides. Empower 3 Software provides the capability of creating a PDA library from pure component peaks in user chromatograms. Afterwards, the library matching and peak purity features can be used with samples to confirm peak identities and to give added confidence that spectrally distinct peaks are not-coeluting. Empower 3 uses Spectral Contrast theory to quantitatively compare the shapes of UV spectra during library matching and Peak Purity analysis. 3-6 Figure 4 shows UV spectra, extracted from PDA chromatograms of standards (1a, 1b, 2-6); these spectra were used to create a library with names and retention times. Table 3 shows an example of a default Empower Report table with PDA library matching and Peak Purity results. The values of Match Angle are smaller than Match Threshold and the values of Purity Angle are smaller than Purity Threshold, indicating that the analytes were well separated and matched with PDA library of biocides. Area Calibration Plot Amount (ppm) Figure 3. Calibration curves for (1a, 1b, 2-6). ame: 1a; R 2 : ame: 1b; R 2 : ame: 2; R 2 : ame: 3; R 2 : ame: 4; R 2 : ame: 5; R 2 : ame: 6; R 2 : a 1b Figure 4. UV spectra of 1a, 1b, and 2-6 extracted from PDA data. ACQUITY UPLC PDA Analysis of Biocides (Part 1) 11

12 Component Match1 Spect. ame Match1 Angle Match1 Threshold Purity1 Angle Purity1 Threshold Amount (ppm) 1 1a 2-methy l-4-isothiazolin-3-one b 5-chloro-2-methy l-4-isothiazoline-3-one carbendazim ,2-benzisothiazol-3-one phenoxyethanol benzoic acid methyl paraben Table 3. Peak identification and quantification results shown on an Empower Report table, with additional PDA library matching and Peak Purity results. CCLUSIS Compliance with regulations that limit the type and concentration of biocides in a variety of applications necessitates analytical testing. This note illustrates that the Waters ACQUITY UPLC System with PDA detection enables rapid and sensitive separations of six commonly used biocides. With Empower 3 Software, library matching and quantification can be automated to add confidence in peak confirmation that is unavailabe with a single wavelength UV detector. This method is simple to use and suitable for quality control, new product development, and troubleshooting for both cosmetic and personal care manufacturers. References 1. D J Knight, M Cooke. The Biocides Business Regulation, Safety and Applications, WILEY-VCH 2002,ISB United States Environmental Protection Agency. Antimicrobial Pesticide Registration. [cited 2015 ovember 9]. Available from: pesticide-registration/antimicrobial-pesticide-registration. 3. M V Gorenstein, et al. Detecting Co-eluted Impurities by Spectral Comparison. LCGC. 12 (10): , Peak Identification with Waters 996 Photodiode Detector: Library Matching, Waters Corporation, WPP12, Library o Empower\Help\Interpreting PDA Peak Purity Results. 6. P J Lee, A J Di Gioia, ACQUITY UPLC/PDA: UV Filter Agents and Preservatives, Waters application note no , Waters, ACQUITY UPLC, The Science of What s Possible, and Empower are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. ovember E AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: ACQUITY UPLC PDA Analysis of Biocides (Part 1) 12

13 ACQUITY UPLC PDA Analysis of Biocides (Part 2) Pass or Fail Custom Calculation Peter J. Lee and Alice J. Di Gioia Waters Corporation, Milford, MA, USA APPLICATI BEEFITS Increases laboratory productivity by enabling the high resolution, high sensitivity separations of biocides in three minutes. Helps improve decision-making, and ensure product quality by rapidly extracting and delivering critical QC data based on user criteria. ITRDUCTI Making more informed decisions in less time is essential in today s Cosmetics, Personal Care, and Food analytical laboratories. Whether you work in method development, quality assurance, new product development, or testing for biocides, you seek a productivity edge. Waters ACQUITY UPLC System provides that edge, enabling high resolution, high sensitivity biocide separations in three minutes. 1 Better quality information on product biocide levels can now be generated far more rapidly than with traditional HPLC methods. UPLC combined with Empower 3 Software can effectively run separations, analyze data, and report results automatically. With the custom calculation function of Empower 3, raw data can be converted into the required format and the critical results can be used quickly by decision-makers to further enhance productivity. This application note describes the benefits of using a simple custom calculation created to determine if the biocide concentration in a sample passes or fails user set criteria. This type of custom calculation eliminates the need for manual calculation and prevents potential human errors. The ability to deliver critical information with speed and accuracy can help manufacturers reduce failed products, avoid product recalls and liability litigations. An example detailing the creation of a custom calculation shown in the note is provided in the Experimental section. WATERS SLUTIS ACQUITY UPLC System ACQUITY UPLC PDA Detector Empower 3 Chromatography Software KEY WRDS Biocides, cosmetics, personal care, user set criteria, custom calculations, custom field, Quality Control, pass or fail ACQUITY UPLC PDA Analysis of Biocides (Part 2): Pass or Fail Custom Calculation 13

14 E X P E R IM E TA L QC Criteria Example for Biocide Kathon amount < 4.85 ppm: Fail Kathon amount > 5.15 ppm: Fail Kathon amount from 4.85 ppm to 5.15 ppm: Pass Create a Custom Field Calculation Method with Empower 3 1. Click Configure System to open the Configuration Manager window, click Projects in the tree. 2. Select and highlight the working project, then right click the highlighted project. 3. Select Properties to open Project Properties window. 4. Click the Custom Fields tab, then click ew to open ew Custom Field Wizard: Data and Type Selection window. 5. Select the Field Type: Peak, and Data Type: Enum, then click ext to open Source Selection window. 6. Select Data Source: Calculated, Sample Type: Unknown, Peak Type: Group nly, then click ext to open Formula Entry window (Figure 1). 7. In the perations list, double-click EUM( and LT(. 8. In the Fields list, double-click Amount, in the formula workspace, enter,4.85), 9. In the perations list, double-click LTE(. 10. In the Fields list, double-click Amount, in the formula workspace, enter,5.15). 11. In the perations list, double-click GT(. 12. In the Fields list, double-click Amount, in the formula workspace, enter,5.15)). Figure 1. Formula Entry window. Create amed Groups in the Processing Method 1. From Processing Method window, select the amed Groups tab. 2. Enter Kathon in the ame text box. 3. Select the option of Sum Peaks, Curve or Sum Peaks for Quantitation. 4. Drag 1a and 1b from Single Peak Components into the tree of Kathon as shown in Figure 2. Sample preparation and UPLC Methods The methods used are the same as described previously. 1 Analytes are Kathon CG/ICP [containing methyl-4-isothiazolin-3- one (1a), chloro-2-methyl-4-isothiazolin-3-one (1b)], Carbendazim (2), Benzisothiazol-3(2H)-one (3), 2-phenoxyethanol (4), Benzoic Acid (5), and Methylparaben (6). 13. Click ext to open Translation Definition Table. 14. Enter Fail next to 0, Pass next to 1, Fail next to 2, click ext to open ame Entry window. (ote: you can enter Pass/Fail in another language). 15. Enter a name for the custom field in the Field ame text box: Pass or Fail, click Finish. Figure 2. Processing method window. ACQUITY UPLC PDA Analysis of Biocides (Part 2): Pass or Fail Custom Calculation 14

15 RESULTS AD DISCUSSI Figure 3 shows chromatograms of three biocide samples. There are seven well-resolved peaks in each sample. 1a and 1b are the two active ingredients of Kathon. The amount of 1a and 1b in each sample can be calculated automatically in Empower 3 Software from calibration curves as described previously a Kathon 1b For this discussion, a passable QC level for the Kathon biocide 0.16 is the range 4.85 ppm to 5.15 ppm. To determine Pass or Fail 0.08 biocide status of each sample, the amount of total Kathon must be calculated, that is the sum of 1a and 1b, and the result must be compared with the QC criteria Figure 4 shows the Custom Field Formula described in the Experimental section. The QC Pass or Fail criteria is based on the Kathon biocide content (5 ppm ± 3). Using both the amed Groups and Custom Field calculation functions, Empower can be set up to automatically calculate and report the quantity of Kathon in each sample and determine if the sample met the QC criteria (Table 1). In an enterprise environment, the critical Pass or Fail results can be ed to product and plant management. These advanced functions of Empower eliminate the need for manual calculations, which saves time and reduces errors Minutes Sampleame PJL07_ 85G Sampleame PJL07_ 85F_High Sampleame PJL07_ 85H_Low Figure 3. UPLC chromatograms of three biocide samples. Sampleame Component Amount (ppm) Pass/Fail 1 PJL07_85G Kathon 5.03 Pass 2 PJL07_85H_Low Kathon 2.57 Fail 3 PJL07_85F_High Kathon 7.43 Fail Table 1. Biocides Test: Pass or Fail. Figure 4. Custom Field formula. ACQUITY UPLC PDA Analysis of Biocides (Part 2): Pass or Fail Custom Calculation 15

16 CCLUSIS Hundreds of biocide containing product samples can be automatically analyzed on a daily basis using the Waters ACQUITY UPLC PDA System with Empower 3 Software. Critical product QC information can be accurately extracted and rapidly delivered based on user set criteria. The method represents a significant productivity enhancement relative to the manual verification of QC biocide data. It can be very effective for food, cosmetics and personal care manufacturers, and formulators to have a report with a simple Pass or Fail for the sample displayed. References 1. PJ Lee, AJ Di Gioia, ACQUITY UPLC/PDA Analysis of Biocides (Part 1), Waters application note no Waters, ACQUITY UPLC, UPLC, The Science of What s Possible, and Empower are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. ctober E AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: ACQUITY UPLC PDA Analysis of Biocides (Part 2): Pass or Fail Custom Calculation 16

17 UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 Christopher J. Hudalla and Kenneth J. Fountain Waters Corporation, Milford, MA, USA APPLICATI BEEFITS Accurate determination of benzalkonium chloride (BAC) content in consumer products Improved peak shape and stability relative to current USP method 95 reduction in solvents used relative to currently accepted methods 80 reduction in analysis time enables high throughput analysis ITRDUCTI Benzalkonium Chloride (BAC) refers to a series of quaternary ammonium chloride homologues with the structure shown in Figure 1. The pervasive use of BAC in consumer products results from its antiseptic and antifungal properties with widespread applications ranging from cleaning products and disinfectants to sanitizing wipes and ophthalmic solutions. Because of its extensive use, BAC has been the subject of numerous studies, including the evaluation of the reactivity of BAC with ocular tissue 1,2 and the study of worldwide municipal wastewater, which found BAC to be the most prevalent quaternary ammonium compound in wastewater, with concentrations ranging between 200 and 300 mg/l. 3,4 The USP method for the quantitation of BAC utilizes a 10 µm particle size cyano column (L10) for the separation of the BAC homologues. 5 The isocratic method uses acetonitrile and 0.1M sodium acetate (ph 5.0) as mobile phases, resulting in a separation requiring between 15 and 30 minutes. In addition to long analysis times, these separations suffer from reproducibility issues due to the traditionally poor chemical and mechanical stability of the cyano stationary phases. 6 Here we WATERS SLUTIS ACQUITY UPLC H-Class System present an alternative method employing a Charged Surface Hybrid (CSH) C 18 stationary phase under UPLC conditions, resulting in improved peak shapes with significant reductions in both analysis time and solvent consumption. Additionally, we include an example of this method that is transferred to conditions using the XSelect CSH C 18 XP 2.5 µm stationary phase (UPLC and HPLC), demonstrating the ability to transfer methods across different instrument platforms. ACQUITY UPLC CSH C 18 Column XSelect CSH C 18 XP Column ACQUITY UPLC Columns Calculator KEY WRDS Benzalkonium, quaternary ammonium, ophthalmic, disinfect, sanitize, fungicide, algaecide, antimicrobial, biocide, cationic surfactant, emulsifier, CSH, XP Figure 1. Structure of benzalkonium chloride (BAC). The C 12, C 14, and C 16 homologues are the most common homologues found in consumer products. UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 17

18 EXPERIMETAL Sample preparation Standard Solution: Prepared from a USP Reference Standard containing 10 (w/v) of BAC. Diluent was 50:50 acetonitrile/ water. Standard solutions were prepared at concentrations of 800, 500, 200, 100, 75, and 50 ppm (µg/ml). Samples Consumer products were prepared to a final concentration of approximately 80 ppm (0.008 w/v), based on label claim, with 50:50 acetonitrile/water as diluent. Products in liquid form were diluted and analyzed without additional sample preparation or filtering. UPLC conditions System: ACQUITY UPLC H-Class with PDA Detector Isocratic: 17 A / 78 B / 5 C (78 MeH, 17 mm AmAc, 10 mm TBAHS) Flow rate: 0.6 ml/min UV detection: 262 nm (40 pts/sec) Injection volume: 8 µl eedle wash: 50:50 acetonitrile/water Purge: 50:50 acetonitrile/water Seal wash: 50:50 acetonitrile/water ote: After analysis, the chromatographic system and column were flushed with 50:50 acetonitrile/water followed by an additional flush with 100 acetonitrile to prevent any precipitation of the buffer or PIC reagent in the system or column. Column: Mobile phase A: Mobile phase B: Mobile phase C: ACQUITY UPLC CSH C 18, 1.7 µm, 2.1 x 50 mm, part number mm ammonium acetate in water at ph 5.6 (adjusted with glacial acetic acid) 100 methanol 200 mm tetrabutyl ammonium hydrogen sulfate [TBAHS] in water for paired-ion chromatography (PIC) Column temp.: 35 C UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 18

19 RESULTS AD DISCUSSI Methods for the analysis of BAC, based on the USP method, rely on cyano ligand bondings on silica base particles. Employing such methods using modern stationary phases based on high purity silica can result in a significant degradation of peak shape, with increased peak tailing and propensity for overloading. This is attributed to the lack of charged impurities in the highly pure silica base particles, resulting in an increase in the undesirable interactions between the charged analyte and the stationary phase. Paired Ion Chromatography [PIC] is a technique for separating charged analytes on reversed-phase columns by exploiting electrostatic interactions between the analyte and the charged PIC reagent. Paired-ion reagents added to the mobile phase are adsorbed onto the stationary phase, where they alter the interaction between the analyte and the stationary phase surface. For oppositely charged analytes, increased stationary phase interactions can result in analyte retention, whereas similarly charged analytes can exhibit decreased interactions and result in faster elution. 7 In this application, tetrabutyl ammonium hydrogen sulfate [TBAHS], a low UV absorbing PIC reagent, is used to reduce the unwanted interaction of the charged quaternary ammonium salt with the stationary phase, producing sharper peaks with reduced tailing. Figure 2 demonstrates the separation, based on the USP method, of BAC on a traditional Spherisorb Cyano column and on the high purity silica HSS Cyano column, with and without the PIC reagent. Increased interactions between the BAC homologues and the high purity silica result in severe peak tailing. The addition of a similarly charged PIC reagent to the mobile phase reduces this interaction resulting in a decrease in retention and a significant improvement in peak shape. AU C 12 C 14 C 16 Spherisorb Cyano [no PIC] AU HSS Cyano [no PIC] C 12 C 14 C16 AU C 12 C 14 C 16 HSS Cyano [10mM PIC] Minutes Figure 2. HPLC separations of the BAC reference standard on cyano columns: 5 µm, 4.6 x 150 mm Spherisorb Cyano (top), and on a 3.5 µm, 2.1 x 100 mm XSelect HSS Cyano, without PIC reagent (middle) and with the TBAHS PIC reagent (bottom). The isocratic separations, based on the USP method, used 45:55 acetonitrile/100 mm sodium acetate (ph=5.0) at flow rates of 1.8 ml/min for the 4.6 mm I.D. column and 1.2 ml/min for the 2.1 mm I.D. column. UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 19

20 Additional improvements in peak shape and analyte loadability are realized with the use of the Charged Surface Hybrid (CSH) C 18 stationary phase, and the replacement of acetonitrile with methanol. Figure 3 demonstrates the improvement in peak shape and loading for the C 12 BAC homologue on the CSH C 18 column as a function of analyte concentration and PIC reagent concentration o PIC mm PIC mm PIC AU Minutes Minutes ppm 200 ppm 100 ppm 50 ppm Minutes Figure 3. The UPLC separation of the C 12 BAC homologue using a 1.7 µm, ACQUITY UPLC CSH C 18 column (2.1 x 50 mm) under isocratic conditions with 80:20 methanol/100 mm ammonium acetate (ph 5.6). The BAC reference standard was prepared at four concentrations (500, 200, 100, and 50 ppm). Separations are shown using no PIC reagent (left), 5 mm PIC reagent (middle), and 10 mm PIC reagent (right). Although there is an improvement in peak shape with the use of a PIC reagent, a decrease in analyte retention is also observed, as demonstrated in Figure 3. A simple adjustment in the organic concentration of the mobile phase, from 80 to 78 methanol, is all that is required to increase the retention factor, while still maintaining the improvement in peak shape. The resulting chromatography, shown in Figure 4 (bottom), under UPLC conditions, gives excellent peak shape and sensitivity for the USP reference standard for BAC, facilitating integration and quantitation. With the aid of the ACQUITY UPLC Columns Calculator, the method was also easily scaled to utilize the XSelect CSH C 18 XP, 2.5 µm column (3.0 x 75 mm) under UPLC (middle) and HPLC (top) conditions. The column dimension was chosen in order to maintain the same length to particle size ratio (L/d p ) as for the separation on the 1.7 µm particle size. When scaling methods between different column configurations, maintaining the L/d p ratio, while scaling flow rates and injection volumes accordingly, results in similar chromatography, with different time scales C 12 AU C 14 C 16 HPLC (2.5 µm) Minutes 0.06 AU AU UPLC (2.5 µm) Minutes UPLC (1.7 µm) Minutes Figure 4. The UPLC separation of BAC homologues using a 1.7 µm, 2.1 x 50 mm ACQUITY CSH C 18 column (bottom). The UPLC isocratic separation was achieved using 78 methanol at a flow rate of 0.6 ml/min. The ACQUITY UPLC Columns Calculator was used to scale the method to utilize the 2.5 µm, 3.0 x 75 mm, XSelect CSH C 18 XP column under UPLC (middle) and HPLC (top) conditions. The USP reference standard for BAC was prepared at a concentration of 100 ppm. UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 20

21 Integration of UPLC chromatograms for the C 12, C 14, and C 16 homologues in the BAC reference standard, prepared at various concentrations from 50 to 800 ppm (µg/ml), shows excellent linearity of detector response versus concentration with R 2 values greater than (Figure 5). 400,000 BAC Calibration Curve 350,000 R 2 = C ,000 Peak Area 250, , ,000 R 2 = C ,000 50,000 R 2 = C ,000 BAC Concentration (ppm) Figure 5. Calibration curve generated under UPLC conditions for the C 12, C 14, and C 16 homologues in the USP BAC reference standard. Samples were prepared at concentrations of 800, 500, 200, 100, 75, and 50 ppm. The UPLC method developed using the USP reference standard was applied to a variety of consumer products. Figure 6 shows a small sampling of products tested, each confirming the applicability of this method. C 12 e) d) c) C 14 C 16 Eye Lubricant Antiseptic Spray Household Cleaner b) a) Minutes Hand Sanitizer USP Reference Figure 6. Application of the current method to various consumer products (from bottom to top), (a) USP reference standard-100 ppm, (b) hand sanitizer, (c) household cleaner, (d) antiseptic spray, and (e) eye lubricant. Chromatograms shown were collected on the 1.7 µm ACQUITY UPLC CSH C 18 column (2.1 x 50 mm) using 78 methanol at a flow rate of 0.6 ml/min. UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 21

22 The BAC concentrations in each sample can be calculated by integration of the individual peak areas for the C 12, C 14, and C 16 homologues, and comparing those values with the peak areas from the BAC reference standard using the following equations (results shown in Table 1): ( )= =12,14,16 x A i A T x A i h h, = 100 =12,14,16 x A i ( / )= =12,14,16 x A i =12,14,16 x A k Where: W i,k = Relative molecular mass for the given homologue: 340, 368, and 396 for the C 12, C 14, and C 16 homologues, respectively. A i = Area of the peak due to the given homologue in the sample preparation. A k = Area of the peak due to the given homologue in the reference standard preparation. A T = Sum of the areas of the peaks due to all homologues in the sample preparation. Conc Std = Concentration of BAC reference standard (100 ppm) DF = Dilution Factor for sample preparation. BAC Analysis USP Tailing (C 12 ) USP Efficiency (C 12 ) ARMM C 12 C 14 C 16 Assay of BAC ( w/v) Eye Lubricant , Antiseptic Spray , Household Cleaner , Hand Sanitizer , BAC Ref Std , Table 1. Summary of BAC concentrations in consumer products. UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 22

23 CCLUSIS Analysis of BAC homologues with the ACQUITY UPLC CSH C 18 stationary phase offers a rapid, reproducible alternative to existing methods. The use of a PIC reagent reduces the undesirable interactions between the charged quaternary ammonium analyte and the stationary phase, resulting in significant improvements in peak shape. The improvement in peak shape, in combination with the excellent linearity of response, facilitates quantitation. The UPLC method reduces solvent consumption by 95 and analysis time by 80 relative to currently accepted methods, yielding significant cost savings while enabling high-throughput analyses. Additionally, scaling methods to utilize XSelect CSH C 18 XP 2.5 µm columns results in lower system operating pressures compatible with HPLC, maximizing the number of LC instruments that can be used for these analyses and facilitating the transfer of methods between facilities with combinations of UHPLC and HPLC instrumentation. References 1. B.J. Tripathi and R.C. Tripathi, Lens Eye Toxic Res., 6(3), (1989). 2. K.C. Swan, Reactivity of the cular Tissues to Wetting Agents, Am. J. phthalmol., 27, 118 (1944). 3. C. Zhang, U. Tezel, K. Li, D. Liu, R. Ren, J. Du, and S.P. Pavlostathis, Water Res., 45, (2011). 4. E. Martinez-Carballo, A. Sitka, C. Gonzalez-Barreiro,. Kreuzinger, M. Furhacker, S. Scharf and. Gans, Environ. Pollut., 145, (2007). 5. USP Monograph. Benzalkonium Chloride, USP35-F30 [1708]. The United States Pharmacopeial Convention, official from August 1, J.E. Gara, B.A. Alden,C.A. Gendreau, P.C. Iraneta, and T.H. Walter, J. Chrom. A, 893, (2000). 7. B.A. Bidlingmeyer, S.. Deming, W.P. Price, B. Sachok and M. Petrusek, J. Chrom. A, 186, (1979). Part numbers ACQUITY UPLC CSH C µm, 2.1 x 50 mm column [P: ] XSelect CSH C 18 XP, 2.5 µm, 3.0 x 75 mm column [P: ] Waters, The Science of What s Possible, ACQUITY UPLC, XSelect, and UPLC are registered trademarks of Waters Corporation. CSH is a trademark of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. March E AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: UPLC Analysis of Benzalkonium Chloride (BAC) in Consumer Products using ACQUITY UPLC CSH C 18 23

24 分散性染料 24

25 [ 应用纪要 ] 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对分散性染料进行分析 Marian Twohig 1 Michael Leary 1 和 Jane Cooper 2 1 沃特世公司 ( 美国马萨诸塞州米尔福德 ) 2 沃特世公司 ( 英国威姆斯洛 ) 应用优势 通过 PDA 和质谱检测器提高杂质分析的可靠性 通过 Empower 3 软件的单点控制功能实现易用性 双流路设计, 可模拟 HPLC 和 UHPLC 分离 简介分散性染料为低分子量合成染色剂 此类染料的结构中通常含有偶氮或蒽醌官能团 1 分散性染料主要应用于诸如纺织品 纸张 玩具等消费品中 目前已发现一些分散性染料长期接触皮肤会引起过敏反应 2 一些染料结构中存在的偶氮官能团, 可能转变为潜在或已知的致癌性芳香胺 2 消费品中这些染料的存在可能对消费者的健康造成不利影响, 因此引起了人们的极大关注 1996 年, 德国颁布了针对控制其中一些分散性染料使用的法规 研究人员根据此法规开发了 DI 标准程序, 该程序介绍了采用高效液相色谱 (HPLC) 或薄层色谱法利用紫外 (UV) 或质谱分析法 (MS) 进行分析或者利用密度测量检测法的对分析分散性染 3-5 料进行分析 沃特世解决方案 ACQUITY Arc 系统 2998 光电二极管阵列 (PDA) 检测器 ACQUITY QDa 检测器 XBridge C 18 色谱柱 Empower 3 CDS 软件 关键词分散性染料, 消费品, 纺织品, 杂质鉴别, 质谱检测 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对扩散性染料进行分析 25

26 [ 应用纪要 ] 在本应用纪要中, 我们将介绍参照 DI 标准程序利用 UV 和质谱检测器以及采用能够模拟 HPLC 或 UHPLC 分离的双流路液相色谱系统对 9 种分散性染料 ( 图 1) 进行的分析 6 质谱检测器的引入能够对特定染料样品中杂质峰确证提供更多信息 使用分散蓝 1 染料标准品进行测定时, DI 方法规定的检测限为 0.7 mg/l 使用沃特世(Waters )ACQUITY Arc 系统与 ACQUITY QDa 检测器时, 所有已评估化合物的检测限均明显高于规定的检测限 1. 分散蓝 1 C 14 H m/z 分散蓝 3 C 17 H m/z 297 H 3. 分散蓝 106 C 14 H S m/z 分散黄 3 C 15 H m/z 分散橙 3 C 12 H m/z 243 H 2 H 2 H 2 H S H H 2 H 2 H H H 6. 分散红 1 C 16 H m/z 分散蓝 35 C 15 H m/z 分散蓝 124 C 16 H S m/z 分散橙 37 C 17 H 15 Cl m/z 392 C H H 2 H H H Cl Cl S 图 1. 本研究中使用的分散性染料的经验公式 结构 和 m/z 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对扩散性染料进行分析 26

27 [ 应用纪要 ] 实验仪器和软件所有分离操作均采用配备 2998 光电二极管阵列 (PDA) 检测器的 ACQUITY Arc 系统结合 ACQUITY QDa 检测器的正离子电喷雾质谱 (MS) 进行 使用 Empower 3 软件进行数据采集和数据处理 样品前处理 将染料标准品溶于甲醇中, 并依次稀释以备用于样品分析 LC 条件 HPLC 方法 (DI 54231) LC 系统 : 分离模式 : 梯度 ACQUITY Arc 色谱柱 : XBridge C 18, 2.1 x 150 mm, 5 µm 溶剂 A: 10 mmol 醋酸铵 (ph 3.6) 溶剂 B: 乙腈 流速 : 0.30 ml/min PDA 检测条件 : 柱温 : 30 C 进样体积 : 5 µl 分析时间 : 30 min 210 至 800 nm 梯度条件 : 0 min 40 B, 7 min 60 B, 17 min 98 B, 24 min 98 B, 然后返回初始条件 MS 条件 MS 系统 ACQUITY QDa 电离模式 : ESI + 毛细管电压 : 1.2 kv 锥孔电压 : 10 V 脱溶剂气温度 : 600 C 离子源温度 : 150 C MS 扫描范围 : 100 至 600 m/z 以及选择离子记录 (SIR) 采样速率 : 5 Hz 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对扩散性染料进行分析 27

28 [ 应用纪要 ] 图 2 显示了对 9 种分散性染料标准品混合物进行分离, 在 240 nm 处所得的 PDA 色谱图 ( 下图迹线 ), 以及通过 2.1 x 150 mm, 5-µm XBridge C 18 色谱柱 ( 部件号 ) 获得的叠加 SIR 通道 ( 上图迹线 ) 4.5 x x x 10 7 ACQUITY QDa SIR 10 µg/ml 4,5 6 1 x m/z Intensity 3.0 x x x x x x x x x x x 10 7 m/z 297 m/z 336 m/z AU Min 240 nm 处的 PDA 数据 100 µg/ml A 4, Min x x x x x x x x m/z 285 m/z 378 m/z Minutes m/z 243 m/z 图 2. 使用 DI 标准方法以及 XBridge C 18, 2.1 x 150 mm, 5.0 µm 色谱柱对 9 种分散性染料标准品 (100 µg/ml,5 µl 进样体积 ) 进行分离, 在 240 nm 处所得的 ACQUITY Arc 色谱图 ( 下图 ) 图中还显示了叠加 SIR 通道 ( 上图 ) 色谱图和单个重迭 ( 右图 )SIR 通道色谱图 (10 µg/ml,5 µl 进样体积 ) 请注意, 分散黄 3( 峰 4) 和分散橙 3( 峰 5) 产生了色谱峰共洗脱, 这使得仅通过 UV 进行准确检测比较困难 如果要使用 UV 检测, 则需要对组分进行色谱分离才能实现准确检测, 但这会延长方法开发时间 如图 2 中重叠的单个 SIR 色谱图所示, 各组分具有不同的 m/z 比, 因此可以使用 ACQUITY QDa 对共流出物进行测定 使用质谱检测器可显著提高检测灵敏度 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对扩散性染料进行分析 28

29 [ 应用纪要 ] 杂质分析从保留时间 (t R )9.5 min 处的 PDA 数据中检测到主要峰 ( 峰 A) 但从 SIR 通道中未检测到该信号峰, 这是因为在实验方法中未监测该未知组分的特定 m/z 在实验中同时使用 MS 全扫描与 PDA 检测器, 能够确定混合物中所有组分的质谱图与 UV 谱图 ( 图 3) QDa PDA AU ,5 A 240 nm 处的 PDA 数据!"#$& 强度 MS TIC - QDa MS Scan - 1: QDa Positive(+) Scan ( )Da, Min Continuum, CV=15 1: 2.6x10 9 A x 10 9 ACQUITY QDa 扫描 4,5 2.2 x µg/ml 2.0 x x x x x x x x x x Min ,5 A m/z 4,5 A nm 图 3. 使用 DI 标准方法以及 XBridge C 18, 2.1 x 150 mm, 5.0 µm 色谱柱对 9 种分散性染料标准品 (100 µg/ml,5 µl 进样体积 ) 进行分离, 在 240 nm 处所得的 ACQUITY Arc 色谱图 ( 上图 ) 以及 QDa MS 扫描图 ( m/z)( 下图 ) 图中还显示了 MS 和 UV 谱图 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对扩散性染料进行分析 29

30 [ 应用纪要 ] 未知组分 A 的 MS 谱图 m/z 267 处显示有明显的谱峰 此外, 对应于分散蓝 3 的峰 2 UV 谱图以及未知组分 A 的 UV 谱图具有相似的特征, 这表明它们可能具有相同的结构特征 本文对只含有分散蓝 3 且染料含量为 20 的标准溶液进行了分析 ( 图 4) Empower 软件的质谱分析窗口通过同时显示 UV 谱图和质谱图实现了对分散蓝 3(m/z 297) 的快速确证 未知峰 A 的质谱图表明该组分的基峰为 m/z 267, 这与之前混合物的分析结果一致 此外,t R 和 UV 谱图在两次分析中是相同的 在分散蓝 3 染料标准品的分析中还检出了 t R 为 11.4 min,m/z 为 254 的第二种未知组分 (B 标记处 ) ACQUITY QDa 和 PDA 数据提供了互补信息, 由此推断, 之前在染料混合物中检测到的杂质 A 来自分散蓝 3 标准品 分散蓝 3 未知化合物 A 未知化合物 B PDA 色谱图 MS 扫描色谱图 XIC 色谱图 强度 强度 强度 强度 强度 图 4. Empower 软件的质谱分析窗口显示的 UV 和 MS 谱图 ( 上图 ) 使用 DI 标准方法所得分散蓝 3 单一标准品的 ACQUITY Arc - PDA MS 扫描 ( m/z) 以及重叠 XIC 色谱图 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对扩散性染料进行分析 30

31 [ 应用纪要 ] 结论增加质谱检测作为互补性分析检测技术, 提高化合物检测和确证的可靠性 使用质谱检测可对具有不同 m/z 比的共洗脱组分进行可靠分析 使用上述分析方法时, 所有化合物的检测限均优于 DI 方法规定的检测限 PDA 和质谱检测器联用, 有助于确认方法开发过程中检测到的杂质是否来自分散蓝 3 标准品 因此, 质谱检测可添加作为杂质分析的互补性技术 ACQUITY Arc 系统可为色谱分离提供更高的灵活性, 并且可通过适用于 HPLC 方法的 3.0~5 µm 填料最大限度提高 HPLC 分析效率, 此外采用 2.5~2.7 µm 填料还支持快速高效的 UHPLC 分离 6 参考文献 1. R M Christie. Colour Chemistry Royal Society of Chemistry. Cambridge, J Garcia-Lavandeira, E Blanco, C Salgado, R Cela. Fast throughput, highly sensitive determination of allergenic disperse dyes in textile products by use of sample composition. (2010) Talanta, 82: German Institute for Standardisation (DI). Textiles-Detection-Detection of Dispersed Dyestuffs. DI 54321: M Gay, J Chang Huang, Q Cai, Q Long Sun. Rapid Screening of 36 Synthetic Dyes using the ACQUITY UPLC H-Class System with the ACQUITY QDa Detector. Waters Technology Brief, no en, ctober J Cooper and J Marchand. Improving the speed and quantitative performance for the analysis of allergenic and carcinogenic dyes in consumer products. Waters application note no en, December Waters ACQUITY Arc System Brochure, no en, June, 扫一扫, 关注沃特世微信 沃特斯中国有限公司沃特世科技 ( 上海 ) 有限公司 Waters ACQUITY QDa Empower The Science of What s Possible 和 XBridge 是沃特世公司的注册商标 Arc 是沃特世公司的商标 其它所有商标均归各自的拥有者所有 北京 : 上海 : 广州 : 成都 : 香港 : 年沃特世公司 印刷于中国 2016 年 1 月 ZH AG-PDF 免费售后服务热线 :800 (400) 采用配备 PDA 和质谱检测器的 ACQUITY Arc 系统与 Empower 软件对扩散性染料进行分析 31

32 提高消费品中致敏和致癌染料的分析速度和定量性能 Jane Cooper 1 Jeremy Marchand 2 1 沃特世公司, 英国曼彻斯特 2 法国南斯大学 方案优势本应用纪要阐述了消费品中致敏和致癌分散染料 酸性染料 直接染料和碱性染料鉴别和定量的高通量处理方法, 并具备以下优势 : 缩短运行时间, 从而减少溶剂的用量 与现有的方法相比, 提高灵敏度 选择性和稳健性 引言最初所有的染料均是天然化合物, 但后来人们开发了各种能够快速生产的廉价合成染料 根据染色工艺中的用途, 人们对合成染料进行了分类 亲脂性分散染料用于许多合成纤维的染色, 例如聚酯 尼龙 乙酸纤维素 合成丝绒和聚氯乙烯 阴离子酸性染料 阳离子碱性染料和直接染料等水溶性染料广泛应用于天然和合成纤维中 例如, 酸性染料可以用于丝绸 羊毛 尼龙和改性丙烯酸纤维 ; 碱性染料可以用于丙烯酸纤维 羊毛 丝绸和纸张 ; 直接染料可以用于棉花 纸张 皮革 羊毛 丝绸和尼龙 为了保护消费者 员工以及社区环境, 许多公司制作了限制物质列表 (RSL) RSL 详细列出了产品供应生产链的每个环节所遵循的法规和非法规要求, 以减少和消除有害物质和工艺 在这情况下, 他们也将环境持续性融入到产品中, 确保其产品的安全性并符合法规要求 在许多消费品供应商的 RSL 中, 对许多潜在的有害分散染料 酸性染料 直接染料和碱性染料都作了详细的说明 沃特世提供的解决方案 ACQUITY UPLC H-Class 系统 Xevo TQD MassLynx 质谱软件 ACQUITY UPLC BEH C 18 柱 针对各种染料, 各国和国际组织制订了法规和非法规要求和标准, 其中包括 : 欧盟标准化委员会关于玩具安全性标准 (BS E 71 第 9 部分 ) 1 eko-tex 标准 欧盟委员会决议(2009/567/EC) 3 以及德国食品和商品法 (LFGB 30) 等 所有标准都详细规定了许多潜在的致敏 致癌 致突变或具有生殖毒性的染料为限制物质 纺织品及其组分中分散染料分析的标准方法是 DI , 该方法采用高效液相色谱 (HPLC) 或者薄层色谱 (T LC) 进行, 并使用紫外 (UV) 质谱(MS) 或光密度法检测 关键词 分散染料 酸性染料 直接染料 碱性染料 消费品 纺织品 限制物质 提高消费品中致敏和致癌染料的分析速度和定量性能 321

33 实验条件样品制备纺织品 将纺织品 (0.5 g) 剪碎, 并使用超声浴 (50 ) 以 20 ml 甲醇萃取 15 分钟 将 100 µl 萃取液转移到一个 LC 样品瓶中, 以 900 µl 水稀释 LC 条件系统 : ACQUITY UPLC H-Class 运行时间 : 7 分钟色谱柱 : ACQUITY UPLC BEH C x 50 mm, 1.7 µm 柱温 : 30 样品温度 : 10 流动相 A: 水 (5 mmol/l 乙酸铵溶液 ) 流动相 B: 乙腈 (5 mmol/l 乙酸铵溶液 ) 流速 : 0.6 ml/min 进样量 : 5 µl 详细的流动相梯度如表 1 所示 质谱条件 其他用于分散染料分析的方法包括 : 带电喷雾离子源 (ESI) 色谱质谱法 5 HPLC 和 UV/VIS 检测 6 大气压化学电离法(APCI) 和质谱检测 7 电喷 8,9 雾和质谱检测, 以及离子交换高效液相色谱 (HPIEC) 和质谱检测 本应用纪要使用与 Xevo TQD 结合 Waters ACQUITY UPLC H-Class 系统, 通过与以前的方法对比说明对消费品中分散染料 酸性染料 直接染料和碱性染料进行分析的优点 结果显示, 分析的稳健性 选择性和灵敏度都得到了增强, 并且减少了运行时间和相应的溶剂用量 如表 3 所示本方法对用于分析分散染料 酸性染料 直接染料和碱性染 料的质谱条件进行了优化 CAS 编号 分子式和结构式显示在表 2 中 如图 1 所示已经确立的染料多反应监测 (MRM) 方法使用 Xevo TQD 上的快 速极性转换操作 这样就可以使用相同的分析方法同时对正离子和负 离子染料进行分析 时间 (min) 流速 (ml/min) A B 梯度曲线 1 初值 表 1. ACQUITY UPLC H-Class 流动相梯度 质谱仪 : Xevo TQD 电离模式 : ESI 正离子和负离子 毛细管电压 : 0.7 kv 离子源温度 : 150 脱溶剂气温度 : 500 脱溶剂气体流速 :1000 L/h 锥孔气体流速 : 20 L/h 采集 : 多反应监测 (MRM) 提高消费品中致敏和致癌染料的分析速度和定量性能 33

34 1. Disperse Blue 3 2. Disperse Blue 7 3. Disperse Blue Disperse Blue 102 CAS: CAS: CAS: CAS: C 17H C 18H C 20H C 15H S H H H H H H H 2 H H H 3 C H CH 3 H CH 3 H H H H 2 H S Disperse Blue Disperse Blue Disperse Brown 1 8. Disperse range 1 CAS: CAS: CAS: CAS: C 14H 17 5 S C16H S C16H 15Cl C 18H S CH 3 H 3 C H CH 3 H 3 C CH 3 S Disperse range Disperse range Disperse range Disperse range 149 CAS: CAS: CAS: CAS: C 12H C 15H 11 2 C 17H 15Cl C 25H Cl Cl Cl Cl H H Cl H + - H 2 CH 3 CH 3 CH 3 H Disperse Red Disperse Red Disperse Red Disperse Yellow 1 CAS: CAS: CAS: CAS: C 16H C 15H C 17H C 12H H H H Cl Cl H 2 H CH 3 H 2 CH 3 CH Disperse Yellow Disperse Yellow Disperse Yellow Disperse Yellow 49 CAS: CAS: CAS: CAS: C 15H C 18H 14 4 C17H 16 2 C21H CH H H CH 3 H + - H 21. Acid Red Basic Red Basic Violet Direct Red 28 CAS: CAS: CAS: CAS: C 18H 14 2a 2 7S 2 C 19H 18 3 Cl C20H 20Cl 3 C 32H 22 6a 2 6S 2 H CH 3 CH H H 3 C CH 3 H - S S - a + a + H 2 HCl H 2 H H 2 H 3 C HCl H 2 H a + - S H 2 H 2 S - a + 表 2. 分散染料 酸性染料 直接染料和碱性染料 相关 CAS 编号 分子式和结构式 提高消费品中致敏和致癌染料的分析速度和定量性能 34

35 编号化学物质 保留时间 (min) 1 分散蓝 分散蓝 分散蓝 分散蓝 分散蓝 分散蓝 分散棕 分散橙 分散橙 分散橙 分散橙 分散橙 分散红 分散红 分散红 分散黄 分散黄 分散黄 分散黄 分散黄 酸性红 碱性红 碱性紫 直接红 ESI(+/-) 锥孔电压 (V) MRM 通道碰撞能量 > > * > * > > > 270.0* > > 208.1* > > 178.0* > > 220.1* > 197.1* > > 122.0* > > > 122.0* > 165.0* > > 133.0* > > 121.0* > > 134.0* > > 225.0* > > 164.1* > > 166.0* > > 134.0* > > 105.0* > > 130.0* > > 168.0* > > 121.1* > > 195.1* > > > 209.1* > > 152.0* 23 表 3. 分散染料 酸性染料 直接染料 碱性染料 保留时间 离子化模式 锥孔电压 多反应监测通道和相关的碰撞能量值 (* 参考定量通道 ) 提高消费品中致敏和致癌染料的分析速度和定量性能 35

36 图 种分散染料 酸性染料 直接染料和碱性染料的多反应监测方法 仪器控制 数据采集和结果处理 使用 MassLynx 软件 ( 版本 4.1) 进行数据采集 ACQUITY UPLC H-Class 系统和 Xevo TQD 的控制 数据定量使用 TargetLynx 应用管理器实现 结果与讨论 使用配有电喷雾离子源 (ESI) 的 Waters Xevo TQD 结合 ACQUITY UPLC H-Class 系统, 以多反应监测模式对 24 种分散染料 酸性染料 直接染料和碱性染料进行分析 6 开发方法优化了多反应监测条件, 并优化了基于马博士等人的工作经验得到的 HPLC 条件 ( 流动相 色谱柱 梯度 ) 利用沃特世方法开发工具, 将方法从 HPLC 转换到 UPLC, 方法开发工具包 括 : 沃特世色谱柱选择性图表, 帮助选择合适的 UPLC 色谱柱 ;ACQUITY UPLC 色谱柱计算器, 帮助开发 UPLC 梯度和流速 经过条件优化的 UPLC 使所有的化合物能够在 7 分钟的运行时间内全部洗脱 Xevo TQD 循环迅速, 极性转换时间短, 更窄的 UPLC 色谱峰同时也能够得到高效分离效果 HPLC 和 UPLC 色谱图之间的对比情况如图 2 所示, 图中表明了方法可提高灵敏度和样品处理量 提高消费品中致敏和致癌染料的分析速度和定量性能 36

37 HPLC XBridge C x 2.1 mm 3.5 µm 0.3 ml/min 17 min 5 µl UPLC ACQUITY UPLC BEH C x 2.1 mm,1.7 µm 0.6 ml/min 7 min 5 µl 图 2. HPLC 和 UPLC 重叠 1 ppm 的色谱图, 流动相 A: 水 (5 mmol/l 乙酸铵溶液 ), 流动相 B: 乙腈 (5 mmol/l 乙酸铵溶液 ) 提高消费品中致敏和致癌染料的分析速度和定量性能 37

38 制备浓度范围为 0.01 ~ 1.5 µg/ml 的混合校准标准品, 并针对所有化合物 ( 对应纺织品样品 4 ~ 600µg/g 的范围 ) 进行分析 图 3 所示为酸性红 26 的 TargetLynx Quantify 的定量结果, 图 4 所示为每种 化合物的多反应监测色谱图 图 3. TargetLynx 定量结果的浏览程序显示酸性红 26 的校准定量结果 校准曲线和多反应监测示例色谱图 提高消费品中致敏和致癌染料的分析速度和定量性能 38

39 98 酸性红 26 分散黄 1 分散红 碱性红 9 分散红 17 分散蓝 直接红 28 分散蓝 106 分散黄 碱性紫 14 分散橙 3 分散蓝 分散蓝 7 分散黄 3 分散橙 分散红 11 分散橙 11 分散橙 分散蓝 3 分散黄 39 分散黄 分散蓝 102 分散棕 1 分散橙 Time -2 Time -2 Time 图 µg/ml 校准标准品 ( 相当于纺织品样品中的 200µg/g) 中分散染料 酸性染料 直接染料 碱性染料的多反应监测色谱图 提高消费品中致敏和致癌染料的分析速度和定量性能 39

40 纺织品分析 如图 1 所示, 多反应监测质量检测方法用于相应的样品制备操作后, 以对染料进行定量分析 使用详细给出的萃取方法 ( 根据 DI 54231) 4 和仪器参数, 对以浓度 75 µg/g 和 30 µg/g 加标的合成纺织品样品进行分析得到的结果如表 4 所示 许多实验室所用的分散染料的萃取方法都是以 DI 为依据, 并将 75 µg/g 作为实际的检测限 回收率通过将加标的纺织品样品进行萃取与校准标准品进行对比来计算得到 染料分散棕 1 分散红 1 分散黄 1 分散黄 39 分散黄 49 样品 平行进样结果 (µg/g) 平均回收率 ( 空白校正 ), RSD() 空白样品 D D D µg/g µg/g 空白样品 D D D µg/g µg/g 空白样品 D D D µg/g µg/g 空白样品 µg/g µg/g 空白样品 D D D µg/g µg/g 表 4. 加入选定分散染料的纺织品样品的回收率数据 使用质谱检测得到的结果采用混合校准标准品进行定量 D= 未检出 三次平行进样得到的回收率较高, 范围是 91 ~ 110 相较于以往方法的其他优点包括 : 使用 Xevo TQD 和 ACQUITY UPLC H-Class 系统联用对染料进行分析 时, 提高了选择性和灵敏度, 缩短了运行时间, 从而节约了溶剂成本 提高消费品中致敏和致癌染料的分析速度和定量性能 40

41 结论通过 Xevo TQD 和 ACQUITY UPLC H-Class 系统联用, 开发出一种用于分散染料 酸性染料 直接染料和碱性染料分析的方法, 其具有快速 高选择性 高灵敏度的特点 Xevo TQD 配备的快速极性转换技术实现了在一次进样中同时对正离子染料和负离子染料进行 UPLC 分析 与传统的标准方法相比, 该方法具有如下优势 : 将 HPLC/UV 与 UPLC/MS 分析相比时, 使用 UPLC 分析带来更好的经济效益, 包括样品处理量提高五倍, 溶剂用量减少 86 Xevo TQD 和 ACQUITY UPLC H-Class 系统联用提升了灵敏度和选择性, 从而提高了鉴别和定量的置信度 使用沃特世方法开发工具将 HPLC 方法快速转换为 UPLC 方法, 这些工具包括 : 沃特世色谱柱选择性图表, 帮助选择合适的 UPLC 色谱柱 ;ACQUIT Y UPLC 色谱柱校准品, 帮助开发 UPLC 条件 参考文献 1. BS E 71-9:2005+A1:2007 Safety of toys. rganic chemical compounds. Requirements. 2. eko-tex Standard 1000, eko-tex International, Ausgabe/Edition 01/2012. [cited 2012 September 20]. Available from: https://www.oeko-tex.com/xdesk/ ximages/470/15540_1000-def.pdf 3. The Commission of the European Communities. Commission Decision of 9 July 2009 establishing the ecological criteria for the award of the Community Ecolabel for textile products (2009/567/EC). fficial Journal of the European Union. L 197/70: 70-86, 9th Jul [cited 2012 September 20]. Available from: LexUriServ/LexUriServ.do?uri=J:L:2009:197:0070:0086:E:PDF 4. German Institute for Standardization (DI). Textiles Detection of dispersed dyestuffs. DI54231: Lord GA, Gordon DB, Tetler LW, Carr CM. Electrochromatography-electrospray mass spectrometry of textile dyes. J Chromatography A. 1995; 700: Qiang M, Hua B, Qing Z, Wei M, et al. Determination of carcinogenic and allergenic dyestuffs in toys by LC coupled to UV/Vis spectrometry and tandem mass spectrometry. Chromatographia. 2010; 72: Morgan S, Vann B, Baguley B, Stefan A. Advances in discrimination of dyed textile fibers using capillary electrophoresis/mass spectrometry. Proceedings of the FBI Trace Evidence Symposium, Clearwater, FL, 15 August [cited 2012 September 20]. Available from: 8. Ràfols C, Barceló D. Determination of mono- and disulphonated azo dyes by liquid chromatography atmospheric pressure ionization mass spectrometry. J Chromatography A. 1997; 777: HolcÏapek M, Jandera P, PrÏikryl J. Analysis of sulphonated dyes and intermediates by electrospray mass spectrometry. Dyes and Pigments. 1999; 43: Socher G, ussbaum R, Rissler K, Lankmayr E. Analysis of sulfonated compounds by ion-exchange high performance liquid chromatography-mass spectrometry. J Chromatography A. 2001; 912: Waters reversed-phase column selectivity chart. [cited 2012 September 20]. Available from: htm?id= Craven K. HPLC to UPLC Method Migration Using Acrylate Analysis as a Model. Application ote en Sept. 沃特斯中国有限公司沃特世科技 ( 上海 ) 有限公司 Waters, ACQUITY UPLC, The Science of What s Possible, MassLynx, XBridge 和 Xevo 是沃特世公司注册商标 TargetLynx 是沃特世公司商标 其他所有商标均归各自的拥有者所有 沃特世公司中国印刷 2015 年 11 月 ZH AG-PDF 北京 : 上海 : 广州 : 成都 : 香港 : 免费售后服务热线 :800 (400) 提高消费品中致敏和致癌染料的分析速度和定量性能 41

42 [ 技术简报 ] 快速筛查消费品中的分散性染料 Jane Cooper and Jérémy Marchand 目的无需样品制备或色谱分离, 即可快速筛查消费产品中存在的分散性染料 将 Xevo TQD 与 ASAP 结合使用, 可提供一种筛查分散性染料的快速简便方法 背景分散性染料用于着色消费品中多种合成纤维, 如聚酯 尼龙 人造天鹅绒和 PVC 等 为保护产品消费者 工作人员以及社区和环境, 分散性染料被许多公司列入受限物质清单 (RSL) 一些立法和非立法的条例和标准将多种具有潜在致敏性 致癌性 致突变性或生殖毒性的分散性染料列为禁用物质, 这些条例和标准包括 : 欧洲标准化委员会玩具安全标准 (BS E 71 part 9) 1 eko-tex 标准 欧盟委员会决议 (2009/567/EC) 3 和德国食品及日用品法 (LFGB 30) 本研究介绍了如何在无需样品制备和色谱分离的条件下简单快速地筛查消费品中的分散性染料 图 1. Xevo 大气压固体分析探头 (ASAP) 这种定性筛查方法为化工行业节省了宝贵时间和运营成本, 缩短了样品运行时间, 同时减少了非环保型溶剂和化学用品的使用和相关处理 快速筛查消费品中的分散性染料 42

43 [ 技术简报 ] 解决方案本研究通过将 Waters 大气压固体分析探头 (ASAP) 与 Xevo TQD 结合使用, 在大气压电离 (API) 条件下直接快速分析筛查消费产品中的分散性染料, 并且无需样品制备或色谱分离步骤 替换现有电流探针并安装电晕放电针, 即可在 2 min 内简单快速地将 ASAP 安装到 API 源中 本研究中开发的最佳 MRM 条件如表 1 所示 在 0.5 g 合成纤维织物中加入浓度为 75 µg/g 的所选分散性染料 ( 许多实验室依据标准方法 DI 制订的分散性染料提取方案, 将 75 µg/g 作为实际检测限 ) 化学物质 锥孔电压 (V) 离子通道 碰撞能量 分散蓝 > 235.1* > 分散蓝 > 147.0* > 分散棕 > > 357.0* 37 分散橙 > > 169.0* 26 分散橙 > 92.0* > 表 1. 分散性染料名称, 锥孔电压 MRM 通道和相关碰撞能量值 (* 表示确证离子通道 ) 图 1 中,ASAP 组件分为两部分, 其中外部探头在整个使用过程中始终连接至源 ; 而内部探头可以移动以便上样, 且不会影响源条件 将封口玻璃熔点毛细管的末端在纤维样品上摩擦以进行上样 将玻璃毛细管接入内部 ASAP 探头, 直接加载到 Xevo TQD 的源部件中 样品在加热的氮气流下气化, 并在电晕放电针的气相离子 / 分子反应下发生电离 为达到影响电离机制 促使质子化离子形成的目的, 可在源中放置一瓶甲醇作为化学改性剂 图 2 所示的 MRM 色谱图揭示了对加标纤维织物样品进行 ASAP 分析的解吸特征 分散蓝 > 分散蓝 > A) 加标纤维织物 B) 空白纤维织物 Time -5 Time Time Time 分散棕 > 分散橙 > Time Time 分散橙 > Time Time Time Time 图 2. 所选分散性染料经 ASAP 分析生成的解吸特征 MRM 色谱图,A) 为加标纤维样品 ( 相当于 75 µg/g),b) 为空白纤维样品 快速筛查消费品中的分散性染料 43

44 [ 技术简报 ] 总结将 Waters Xevo TQD 与 ASAP 结合使用, 可为消费产品中分散性染料的筛查提供一种快速简便的解决方案 该方案无需进行样品制备或色谱分离, 缩短了运行时间和整个过程所需的分析时间, 为企业节约了生产力和消耗性成本 Xevo TQD 能够在 MRM 模式下采集数据, 为分散性染料的筛查提供了出色的选择性和灵敏度 该系统还能够快速切换至 UPLC/MS/MS 模式, 实现消费产品中的分散性染料的定量和确证 参考文献 1. BS E 71-9:2005+A1:2007 Safety of toys. rganic chemical compounds. Requirements. 2. eko-tex Standard 1000, eko-tex International, Ausgabe/Edition 01/2012. [cited 2012 September 20]. Available from: https://www.oekotex.com/xdesk/ ximages/470/15540_1000-def.pdf 3. The Commission of the European Communities. Commission Decision of 9 July 2009 establishing the ecological criteria for the award of the Community Ecolabel for textile products (2009/567/EC). fficial Journal of the European Union. L 197/70: 70-86, 9th Jul [cited 2012 September 20]. Available from: 2009:197:0070:0086: E:PDF 4. German Institute for Standardization (DI). Textiles Detection of dispersed dyestuffs. DI54231:2005. Waters,Xevo 和 The Science of What s Possible 是沃特世公司的注册商标 其他所有商标均归各自的拥有者所有 沃特世科技 ( 上海 ) 有限公司北京 : 上海 : 广州 : 成都 : 沃特斯中国有限公司香港 : 年沃特世公司中国印制 2013 年 1 月 ZH SC-PDF 免费售后服务热线 : 800 (400) 快速筛查消费品中的分散性染料 44

45 Rapid Screening of 36 Synthetic Dyes using the ACQUITY UPLC H-Class System with the ACQUITY QDa Detector Melvin Gay, Jin Chang Huang, Qi Cai, and Qing Long Sun GAL To selectively analyze 36 disperse, basic, acid, solvent and direct synthetic dyes to below EU legislative limits. Reduce analysis times from 17 min to <5 min. BACKGRUD Synthetic dyes are classified according to how they are used in the dyeing process. For example, disperse dyes which are mostly azo- or anthraquinone compounds are generally used for dyeing synthetic textile materials such as polyester, nylon, and PVC. The type of bonds formed between the dyes and the fabric, determine the properties of the dyes. For example, disperse dyes are not chemically bonded to the fibers of the textile, thus they can easily migrate onto the skin of the person wearing the garment, especially if the textile fastness is poor. A number of synthetic dyes are known to be allergenic when they come into contact with human skin or if they are are classified as potentially sensitizing, carcinogenic, mutagenic, or toxic to reproduction. 20 known allergenic dyes are listed by the eko-tek Standard 100 and the permitted limit is <50 mg/kg. ther legislation such as European Union (EU) 2009/567/EC has banned the use of these sensitizing dyes. Figure 1. SIR chromatogram of 36 disperse, basic, acid, solvent and direct dye standards. SIR 1 to 31 were acquired in positive polarity, and 32 to 36 were acquired in negative polarity. The standard method for the analysis of disperse dyes in textile products and components is DI using HPLC-UV-MS with an analysis time of 17 minutes. Rapid Screening of 36 Synthetic Dyes using the ACQUITY UPLC H-Class System with the ACQUITY QDa Detector 45

46 THE SLUTI The Waters ACQUITY UPLC H-Class System with the ACQUITY QDa Detector was used to monitor a total of 36 disperse, basic, acid, solvent, and direct dyes including 28 dyes listed in the eko-tex Standard 100 and 2009/567/EC. The list of the synthetic dyes considered are provided in Table 1. The time required for method development was greatly reduced using the pre-optimized source parameters in the ACQUITY QDa Detector, where the required sensitivity were achieved in both polarities for both positive and negative ionizing dyes. The low system dispersion in the ACQUITY UPLC H-Class System and the use of sub-2-µm particle columns also greatly increased peak resolution and enhanced sensitivity. Here, two methods were developed for both positive and negative ionizing dyes with analysis time of 5 and 4 minutes respectively. Dyes were monitored according to their respective retention time, ionizing polarity, and Single Ion Recording (SIR) mass-to-charge ratio (m/z), as described in Table 1. In the highly regulated inks and dyes industry, SIR provides more selectivity and sensitivity compared to HPLC-UV analysis. The SIR chromatograms, shown in Figure 1, indicate that synthetic dyes can be easily and confidently detected at low levels. Current EU legislation prohibits the use of sensitizing dyes in textiles, while allowing 5 ppm limit on the other dyes. Quantitation was also carried out with concentrations ranging from 0.3 to 2.0 ppm, and linearity of >0.997 was achieved for all the dyes. The calibration curve of Disperse Blue 3 is shown in Figure 2. Table 1. Retention times, SIR m/z, and polarity for 36 disperse, basic, acid, solvent, and direct dyes. Compounds CAS no. Retention time SIR Polarity 1 Basic Red Basic Violet Disperse Blue Acid Violet 49* Disperse Red Disperse Blue Disperse Blue Disperse Red Solvent Yellow 1* Disperse Blue Disperse range Disperse Yellow Disperse Yellow Basic Violet 1* Disperse Blue Disperse Brown Disperse Red Disperse Blue 35A Basic Violet 3* Disperse range Disperse Yellow Solvent Yellow 2* Disperse Blue Disperse Blue Solvent Yellow 3* Basic Blue 26* Disperse range 37/ Disperse Blue 35B Disperse range Disperse Yellow Disperse range Direct Blue Acid Red Direct Red Direct Brown 95* Direct Black * Compounds not listed in the eko-tek Standard 100 and EU 2009/567/EC Rapid Screening of 36 Synthetic Dyes using the ACQUITY UPLC H-Class System with the ACQUITY QDa Detector 46

47 SUMMARY The ACQUITY UPLC H-Class System with the ACQUITY QDa Detector provides a faster and more reliable analytical tool for the identification and quantification of synthetic dyes. Two methods of less than 5 minutes have been developed for both positive and negative ionizing dyes that provide increased throughput and reduced solvent usage. The ACQUITY QDa Detector can also be easily integrated into current LC-UV analysis methods which allows unsurpassed sensitivity and selectivity, with limits of detection achieved well below the EU regulatory limit of 5 ppm. Figure 2. Calibration curve of Disperse Blue 3. Waters, The Science of What s Possible, ACQUITY UPLC, ACQUITY, and QDa are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. ctober E TC-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: Rapid Screening of 36 Synthetic Dyes using the ACQUITY UPLC H-Class System with the ACQUITY QDa Detector 47

48 阻燃剂 48

49 Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers Keith Worrall, 1 Peter Hancock, 1 Alwyn Fernandes, 2 Malcolm Driffield, 2 Martin Rose 2 1 Waters Corporation, Manchester, UK; 2 Central Science Laboratory, York, UK APPLICATI BEEFITS Enables significant improvement in chromatographic resolution and run time over and above current methods. All five HBCD diastereomers and TBBP-A could be determined rapidly Increases laboratory productivity by reducing run time and acquisition-to-report time. Reduces cost and environmental impact through lower solvent usage. Results compare favorably with fully validated (IS 17025) method ensuring confidence in results. ITRDUCTI Brominated Flame Retardants (BFRs) are chemicals commonly used in many domestic and industrial appliances, equipment and textiles to increase their resistance to fire. The use of BFRs has seen an exponential rise over the last few decades with HBCD and TBBP-A being two of the most common chemicals used in the highest levels. HBCD (Figure 1) is used around the world as a flame retardant in thermal insulation foam for building and construction applications, as well as in upholstery textile coatings, to help prevent deaths and injuries from fire. The HBCD technical product is composed of a number of diasterioisomers of which the a, b, and g forms predominate. During manufacture, g-hbcd is the most dominant diastereoisomer formed, contributing approximately 80 of the technical formulation. TBBP-A (Figure 2) is used to improve the fire safety of electrical and electronic equipment. It is the largest volume BFR for this application in production today. Both HBCD and TBBP-A are currently marketed around the world without any legislative restrictions. However, as emerging contaminants or Persistent rganic Pollutants (PPs), the importance of continuous monitoring to quantify the impact of these chemicals on human health and the environment is paramount. WATERS SLUTIS ACQUITY UPLC System Tandem (Triple) Quadrupole Mass Spectrometer MassLynx Mass Spectrometry Software Br Br Br H Br Br CH 3 CH 3 Br Br H Br Br KEY WRDS Tetrabromobisphenol-A, TBBP-A, Hexabromocyclodecane, HBCD, diastereomers, Brominated Flame Retardants, BFRs, Persistent rganic Pollutants, PPs Br Figure 1. Hexabromocyclododecane. Figure 2. Tetrabromobisphenol-A. Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers 49

50 There is current concern as to the persistent nature of these chemicals and consequently, the detrimental effects they may have. Various studies have shown a presence of HBCD and TBBP-A in the environment as well as in aquatic and human tissues. There are indications that the residue levels of HBCD have increased significantly between 1969 and ,2 At their Twenty-third Meeting in December 2006, the Advisory Committee on Hazardous Substances (ACHS) highlighted the following problems with HBCD 3 : The substance is extensively monitored and found in water, sediment, soil, and most importantly biota. Levels are rising rapidly. Levels are rising in top mammals such as whale, dolphin, porpoise, and in birds. Monitoring shows a different isomeric ratio to HBCD marketed, which suggests different metabolic behavior of different diastereomers. There are robust methodologies reported in the literature 4,5 for the analysis of TBBP-A and HBCD diastereomers using HPLC-MS/MS. However, advantages can be gained by the use of Waters UltraPerformance Liquid Chromatography (UPLC ) through enhanced chromatographic resolution and throughput of the analytical method. 6 In this application note, we describe a UPLC-MS/MS analysis of all five diastereomers of HBCD and TBBP-A in samples of marine origin. The optimized separation used in this method resulted in a run time of 10 minutes, with the five HBCD diastereomers and TBBP-A analyzed being separated to <10 valley. Comparability of the UPLC data for real samples to that run using a standard HPLC-MS/MS validated method is excellent. The run time reduction of up to 15 minutes using UPLC offers a throughput improvement of up to five times, combined with a superior separation of target analytes. Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers 50

51 E X P E R IM E TA L Standards Extracts of marine origin and calibration solutions containing BFRs and 13C-Labelled BFRs (internal standards) were provided by the Central Science Laboratory, (CSL) York, UK. The a, b, g, d, e HBCD and TBBP-A single compound standards were supplied by Wellington Laboratories. The methodology used for sample preparation and HPLC-MS/MS analysis are described elsewhere. 8 UPLC conditions MS conditions LC system: ACQUITY UPLC MS system: Quattro Premier XE Column: ACQUITY UPLC BEH C x 150 mm, 1.7 µm Column temp.: 60 C Flow rate: 500 µl min -1 Mobile phase A: Mobile phase B: Water Methanol Gradient: Time 0.00 min 20 A Time 5.00 min 20 A Time 6.00 min 0 A Time 8.00 min 0 A Time 8.10 min 20 A Total run time: Injection volume: 10 min 10 µl, Full loop injection Ionization mode: Capillary voltage: Desolvation gas: Cone gas: ESI 2.5 kv Source temp.: 120 C Acquisition: itrogen, 1000 L/Hr, 400 C itrogen, 20 L/Hr Transitions: See Table 1 Collision gas: Multiple Reaction Monitoring (MRM) Argon at 3.5 x 10-3 mbar Compound Transition Cone voltage (V) Collision energy (ev) TBBP-A 542.6> TBBP-A 542.6> C-TBBP-A 544.6> C-TBBP-A 554.6> HBCD 640.4> HBCD 640.4> C-HBCD 652.4> C-HBCD 652.4> Table 1. MRM transitions used for target analytes and their internal standards. Acquisition and processing methods The data were acquired using Waters MassLynx Software v The data was processed using TargetLynx Application Manager. This quantification package is capable of automating quality control checks such as calculating ion ratios, flagging analytical results above/below thresholds set by the user, and many other features. Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers 51

52 RESULTS AD DISCUSSI Current HPLC based methods are becoming more isomer specific 7,8 to enable more specific toxicological studies to be performed. Until recently, published HBCD concentration data have been derived by Gas Chromatographic (GC) techniques. However, GC analysis is currently limited as it is unable to chromatographically resolve the different diastereoisomers using standard GC parameters. The diastereomers are thermally labile, with degradation or interconversion observed at temperatures >160 C. Thus, values have been reported as total HBCD. During development, two UPLC methods were assessed with comparisons being made to the original HPLC based method. The initial evaluation was a direct transfer of the separation achieved with HPLC. The chromatogram for the four target compounds (TBBP-A, a, b and g HBCD) using HPLC separation is shown in Figure 3. It was possible to achieve slightly improved chromatographic resolution, while reducing the total run time from 25 minutes to 5 minutes by using an ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 x 50 mm, part no The chromatograms for the optimized UPLC separation are shown in Figure 4. It can be seen that the UPLC separation would allow the laboratory to increase throughput from 2.4 samples per hour to 12 samples per hour. After optimization of the rapid separation, a mixed standard containing the five HBCD diastereomers and TBBP-A was analyzed. To achieve the required separation, the UPLC BEH C x 2.1 mm, 1.7 µm column was required, which resulted in a total run time of 10 minutes (6 samples per hour) Figure 3. HPLC separation of TBBP-A (6.49 mins), a-hbcd (11.25 mins), b-hbcd (12.08 mins), and g-hbcd (12.55 mins) Figure 4. UPLC separation of TBBP-A (0.59 mins), a-hbcd (1.26 mins), b-hbcd (1.47 mins), and g-hbcd (1.60 mins). Time Time Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers 52

53 Acquisition of the five single component HBCD standards resulted in the elution order of a, b, g, d, and e being deduced, with peak widths of 0.15 minutes. The chromatogram for the eluting peaks, including TBBP-A is presented in Figure 5, where the valley of <10 between d and e HBCD can be observed. Following this analysis, a bracketed calibration curve was acquired with a number of sample extracts. This curve contained both native and 13 C-labelled a, b, g-hbcds and TBBP-A, with native concentrations from 5 ng ml -1 to 600 ng ml -1. The linearity of measurement over the calibration curve range was good for the four compounds determined quantitatively (TBBP-A, a, b, g-hbcds), with all coefficients of determination (r 2 ) being >0.999 for the un-weighted curves. 0 Time Figure 5. ptimized separation of TBBP-A (1.50 mins) and the five HBCD diastereomers, with the final peak eluting in less than 4.25 mins. The reduction in peak widths achieved using UPLC separation resulted in significant sensitivity gains when compared with HPLC separation, with the TBBP-A peak width being reduced from 0.67 minutes to 0.15 minutes and the HBCDs peak widths being reduced from 0.53 minute to 0.16 minute. The chromatograms in Figure 6 show the comparative signals for the target compounds for a 10 ng ml -1 solvent standard. TBBP-A was detected in all of the marine origin samples analyzed, with a, b, g-hbcds being detected in most extracts, d and e-hbcd were not detected in any. The a-enantiomer dominated in all the samples analyzed, as observed in other reports. 5,7 This profile is characteristic of marine biota and probably arises as a result of selective metabolism of the different enantiomers and/or biotransformation processes S/:PtP= S/:PtP= Time Time Figure 6. Comparison of HPLC and UPLC 10 ng/ml -1 solvent standard injections. Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers 53

54 A typical marine origin extract with concentrations of 0.38, 0.056, and ng g -1 for a, b, g-hbcd respectively is given in Figure 7. All results were compared between the two methods. The mean deviation between HPLC and UPLC was <20. a-hbcd results across the batch are shown in Table 2, with all data shown with 15 error bars, conforming with a typical measurement uncertainty. QPXE_280307_ mm column, 5 min isocratic Fish 764, Blank Alpha-HBCD 3.24 F2:MRM of 4 channels, ES > e Beta-HBCD 3.67 Gamma-HBCD min QPXE_280307_ mm column, 5 min isocratic Fish 764, Blank Alpha-HBCD 3.23 F2:MRM of 4 channels, ES > e+003 Beta-HBCD Gamma-HBCD min Figure 7. TargetLynx view of a typical extract of marine origin showing low levels of TBBP-A and HBCDs concentration (ng/g) HPLC UPLC Sample number HPLC UPLC Table 2. Comparison of TargetLynx determined concentrations in samples of marine origin for a-hbcd using HPLC and UPLC. Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers 54

55 CCLUSIS The use of the ACQUITY UPLC System with tandem (triple) quadrupole mass spectrometry enabled a significant improvement in chromatographic resolution and a reduction of run time over current HPLC methods. All five HBCD diastereomers and TBBP-A could be determined rapidly, with added confidence given through TargetLynx data processing. This vastly increases a cost conscious laboratory s productivity by reducing both run time and acquisition-to-report time. Also, cost and environmental impact will be reduced through lower solvent usage required with UPLC. Final results compare favorably with an established fully validated (IS 17025) method ensuring confidence in results. This methodology carried out on the Waters ACQUITY UPLC System with tandem (triple) quadrupole mass spectrometry can enable laboratories to achieve increased sample capacity, flexibility in workflow, and improved lab efficiency, leading to maximized asset utilization and a faster return on investment. References 1. Morris S. et al. Environmental Science & Technology. 2004, 38 (21), Sjodin, A.; Patterson, J. Bergman, A., Environment International. 2003, 29 (6), DEFRA website, The Advisory Committee on Hazardous Substances (ACHS) 5th Dec 2006: environment/chemicals/achs/061205/achs0628.pdf 4. Covaci A, Voorspoels S, Ramos L, eels H, Blust R. J. Chromatography A / J.Chroma Cariou R, Antignac J-P, Marchand P, Berrebi A, Zalko D, Andre F, Le Bizec B. J. Chromatography A. 2005; : Jin J, Peng H, Wang Y, Yang R, Cui J. rganohalogen Comp 2006; 68: Fernandes A, Driffield M et al. Molecular utrition and Food Research (in press). 8. Fernandes A, Driffield M et al. Submitted to Food Addit. Contam Alaee, M, Arias, P, Sjodin, A. Bergman, A, Environment International. 2003, 29 (6), Waters, ACQUITY UPLC, MassLynx, The Science of What s Possible, and UPLC are registered trademarks of Waters Corporation. Quattro Premier and TargetLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. February E AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: Utilizing the Speed and Resolution of UPLC to Enhance the MS/MS Detection of HBCD and TBBP-A Diastereomers 55

56 [ 技术简报 ] 支持有害物质限制指令 (RoHS) 法规, 检测消费者权益保护分析中的异常污染物 目的采用带 APGC 源的 Xevo TQ-S MS 系统, 展示并行采集多重反应监测 (MRM) 和 MS 扫描数据 (RADAR 采集 ) 的优势, 为消费者保护法的执行提供支持 背景溴化阻燃剂 (BFRs) 是用于降低起火可能性以及减弱火势的化合物 这些化合物存在于包括电子器件 服装以及家具在内的各类消费品中 根据有害物质限制指令 (RoHS)(2002/95/EC), 这类化合物被禁止用于多种电子设备中 与此密切相关的还有废弃电子电机设备指令 (WEEE,2002/96/EU), 该指令是一项旨在解决大量有毒电子废弃物的立法措施 PBDE MRM 通道 人们最常利用选择离子 (SIR) 或 MRM 模式的目标的 GC/MS 方法对 BFR 进行分析 而仅使用目标方法的问题是, 它忽略了其他相关化合物或基质背景 Waters Xevo TQ-S MS 系统可通过使用 RADAR, 在单次采集中并行采集 MRM 和 MS 扫描数据 MS 扫描 BPI 迹线 图 1. MRM 通道的重叠迹线 ( 上图 ) 以及 MS 扫描数据的 BPI 迹线 ( 下图 ) 支持有害物质限制指令 (RoHS) 法规, 检测消费者权益保护分析中的异常污染物 56

57 [ 技术简报 ] 解决方案将 Xevo TQ-S MS 系统与带有 APGC 源的 GC 联用, 在 RADAR 模式下运行 针对不同溴化程度选择两个 MRM 通道, 对一系列多溴联苯醚 (PBDE) 进行分析 同时, 采集 50 至 1050 Da 质量范围内的 MS 扫描数据 对 2006 年 7 月立法禁止 BFR 之前生产的一份计算机键盘提取样品进行了 PBDE 分析 Br H H Br Br H H H H Br Br H H Br 阻燃剂 Firemaster 680 的分子式 使用采集到的 MRM 通道数据定量样品中的 PBDE 质谱扫描功能可以获取相关化合物或可能干扰物的信息 图 1 显示了 MRM 通道迹线 ( 上图 ) 以及 MS 扫描数据的 BPI 迹线 ( 下图 ) 图 1 中的 MS 扫描数据十分密集且非常复杂 利用 MassLynx 软件中的聚集 / 抽离功能提取间距为 2 Da 的离子对, 并标出光谱的卤代同位素模型 在 IST08 质谱库中查找样品中疑似具有这种性质的化合物的谱图 图 2 显示了在 min 处的谱峰以及放大的分子离子簇 此外, 还显示了将从疑似峰提取的同位素簇与 C14H8Br62 的理论同位素模型进行比较的结果 建议的分子式与一种商业产品 FireMaster 680 中的活性成分三溴苯氧基乙烷一致 此外, 碎片簇 (m/z 357 和 359) 表明, 分子离子发生了裂解 (C6H2Br3+CH3 丢失 ), 这也进一步支持了该假设 要更有力地确认该化合物, 还需要更多的后续工作 同位素模型 实测结果 图 2. 在峰 Rt 处 ( 分子离子簇被放大 ) 的质谱图, 以及测定结果与理论同位素模型之间的对比 总结 利用 RADAR 功能通过 APGC 和 Xevo TQ-S, 成功地采集了分析计算机键盘样品中 PBDE 所需的 MRM 和全扫描数据 RADAR 可以对目标化合物进行准确定量, 同时还能采集 MS 扫描数据, 以便监测基质成分或其他化合物 该过程对 MRM 数据只有极少或完全没有影响 非目标阻燃化合物在使用质谱扫描功能获取数据后得到初步鉴定 只有当仪器能在 MS 和 MS/MS 模式之间进行无损性能的快速转换时, 才能使用 RADAR 模式 Waters ACQUITY UPLC UPLC MassLynx 和 The Science of What s Possible 是沃特世公司的注册商标 RADAR 和是沃特世公司的商标 其他所有商标均归各自的拥有者所有 沃特世科技 ( 上海 ) 有限公司北京 : 上海 : 广州 : 成都 : 沃特斯中国有限公司香港 : 年沃特世公司 印制于中国 2011 年 7 月 ZH A-PDF 免费售后服务热线 : 800 (400) 支持有害物质限制指令 (RoHS) 法规, 检测消费者权益保护分析中的异常污染物 57

58 邻苯二甲酸酯 58

59 High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products Using UPLC with Mass Detection and Empower 3 Software Jane Cooper Waters Corporation, Wilmslow, UK APPLICATI BEEFITS The ACQUITY QDa Detector linked to the ACQUITY UPLC H-Class System provides improved confidence in the identification and quantification of phthalates and parabens in cosmetics and personal care products offering: Increased sample throughput and a reduction of solvent usage due to reduced run times. Improved sensitivity, selectivity, and robustness, compared with existing methodologies. Cost effective, reliable mass confirmation. WATERS SLUTIS ACQUITY UPLC H-Class System ACQUITY UPLC BEH Column ACQUITY QDa Detector Empower 3 CDS Software KEY WRDS Phthalates, parabens, triclocarban, consumer products, cosmetics, personal care products ITRDUCTI Phthalates are esters of phthalic acid that have extensively been used as plasticizers to increase flexibility, transparency, durability, and longevity in a wide variety of consumer and household products, such as children s toys, electronics, clothes, flooring, wallpaper, and paints. Phthalates are also used, as plasticizers, solubilizers, or denaturants in cosmetics and personal care products, such as perfumes, nail polishes, and hair sprays. Parabens are esters of parahydroxybenzoic acid, which due to their low volatility, high stability, antibacterial and antifungal properties, have been used as preservatives in cosmetics, personal care, pharmaceutical, food, and industrial products. Triclocarban is an antibacterial and antifungal agent that is used in many cosmetic and personal care products, including soap, toothpaste, deodorant, shampoo and shaving cream. Triclocarban is also used in several consumer products including kitchen cutting boards, shoes, towels, and clothing, as well as in medical disinfectants and medical products. But there are several health concerns related to the use of triclocarban, including potential hormone and endocrine disruption, and also its potential to contribute to the development of antibiotic resistance. Many phthalates are classified as hazardous because of their effects on the reproductive system and their association with an increased risk of cancer. Parabens are associated with allergenic contact dermatitis and rosecea. Studies 1,2 have also suggested parabens may be carcinogenic and possess estrogenic disrupting activities. Due to these properties phthalates, parabens, and triclocarban are either banned or restricted, as regulated by the Cosmetic Directive 1223/ In order to accommodate consumer demands for higher standards, many manufacturers are developing, and labeling cosmetic and personal care products free from phthalates and parabens. Previous example methodologies for the analysis of phthalates include GC-MS, 4 and HPLC-UV 4 ; GC-FID, 5 HPLC-UV, 4,6 HPLC-MS, 7 GC-MS, 4 and capillary electrophoresis 6 for the analysis of parabens; and HPLC-MS 8 for the analysis of triclocarban. High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products 59

60 Accessible and intuitive as an optical detector, the ACQUITY QDa Detector has been designed for chromatographers with ease of use in mind. Mass detection can be used to achieve reliable analytical methods to unequivocally identify and quantify compounds such as phthalates, parabens, and triclocarban, during both method development stages, and during routine regulatory analysis. This application note considers the method development, sample extraction, and mass spectral analysis of parabens, phthalates, and triclocarban using Waters ACQUITY UPLC H-Class System, coupled to the ACQUITY QDa Detector. E X P E R IM E TA L Sample preparation Cosmetic and personal care sample analysis Add 2.5 ml water and 2.5 ml methanol to 0.2 g sample. Vortex mixture for 2 minutes (1600 rpm). Further extract mixture in an ultrasonic bath for 30 minutes. Centrifuge approximately 1 ml of extract for 5 min (10,000 rpm). Transfer centrifuge extract to LC vials for analysis. LC conditions LC system: Runtime: ACQUITY UPLC H-Class 5.00 min Column: ACQUITY UPLC BEH C 18, 1.7 µm, 2.1 x 50 mm Column temp.: 40 C Sample temp.: 10 C Mobile phase A: Mobile phase B: Flow rate: Water formic acid Methanol formic acid 0.6 ml/min Injection volume: 5.0 µl Mobile phase gradient is detailed in Table 1. Time Flow rate (min) (ml/min) A B Curve 1 Initial MS conditions MS system: ACQUITY QDa Ionization mode: ESI + and - Capillary voltage: 0.8 kv Probe temp.: 450 C Acquisition: Cone voltage: Selected Ion Recording (SIR) 15 V The list of compounds considered, including phthalates, parabens, and triclocarban, along with their expected retention times are detailed in Table 2. ESI ionization mode (-/+) SIR (m/z) Retention time (minutes) Diethyl phthalate Dipropyl phthalate Dibutyl phthalate Benzylbutyl phthalate Bis(2-ethylhexyl) phthalate Diisobutyl phthalate Di-n-pentyl phthalate Di-n-hexyl phthalate Dicyclohexyl phthalate Di-(2-methoxyethyl) phthalate Di-n-octyl phthalate Methylparaben Ethylparaben Propylparaben Butylparaben Hydroxybenzoic acid Benzylparaben Triclocarban Table 2. Phthalates, parabens, and triclocarban; ionization mode, SIR m/z, and expected retention times. Table 1. ACQUITY UPLC H-Class mobile phase gradient. High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products 60

61 The empirical formulas and structures are detailed in Tables 3 and 4. Instrument control, data acquisition, and result processing Empower 3 Software was used to control the ACQUITY UPLC H-Class System and the AQCUITY QDa Detector, as well as for data acquisition and quantitation. Table 3. Phthalates, associated CAS numbers, empirical formulas, and structures. Table 4. Parabens and triclocarban, associated CAS numbers, empirical formulas, and structures. High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products 61

62 RESULTS AD DISCUSSI A fast, selective, and sensitive LC-MS method for the detection of a selection of phthalates, parabens, and triclocarban in cosmetic and personal care products has been developed. The ACQUITY QDa Detector s SIR parameters were optimized, considering both negative and positive electrospray ionization modes, in order to ensure full coverage of all the compounds being analyzed (as detailed in Table 2.) Method development was carried out using reversed phase UPLC, where different gradient conditions, columns, and mobile phases were considered. The objective was to separate the isomeric phthalate compounds considered: di-n-octyl phthalate (DiP), and diisobutyl phthalate (DiBP); bis(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DnP) while maintaining sample throughput. This was achieved by optimizing the mobile phases and the gradient eluting conditions used. The final LC conditions used are detailed in the methods section. The method was established over the calibration ranges of 0.01 µg/ml to 10 µg/ml for phthalates and triclocarban, and 0.05 µg/ml to 25 µg/ml for parabens, equivalent to 0.25 to 250 mg/kg, and 1.25 to 625 mg/kg in the extracted samples respectively. Good linearity was achieved for all the compounds considered (R 2 >0.99). SIR chromatograms for phthalates, parabens, and triclocarban in a mixed 1.0 µg/ml calibration standard are shown in Figure 1. The developed five-minute UPLC method, is more than seven times faster than existing HPLC and GC methods, with an excess of 90 less solvent usage than existing HPLC methods. Figure 1. SIR chromatograms for phthalates, parabens, and triclocarban in a mixed 1.0 µg/ml calibration standard. High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products 62

63 The SIR mass detection conditions detailed in Table 2 were used after appropriate sample preparation to screen for phthalates, parabens, and triclocarban in cosmetic and personal care samples. Cosmetic and personal care sample analysis Samples were fortified at various levels with selected phthalates and parabens, then prepared for analysis as detailed in the experimental section. Example SIR chromatograms achieved are shown in Figure 2. Figure 2. SIR chromatograms for selected phthalates and parabens in hair conditioner. High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products 63

64 CCLUSIS A fast, robust, and sensitive method was developed for the combined analysis of phthalates, parabens, and triclocarban in cosmetic and personal care samples. The ACQUITY QDa Detector provides cost effective reliable mass confirmation, during both method development and routine analysis. Combining the ACQUITY UPLC H-Class System with the ACQUITY QDa Detector offers accurate and reproducible quantification. Empower Chromatography Data Software provides confidence in data management, data processing, and reporting. The developed 5-minute UPLC method is more than 7 times faster than existing HPLC and GC methods, with an excess of 90 less solvent usage than existing HPLC methods. The ACQUITY H-Class System, a quarternary system based on UPLC Technology, offers the best in chromatographic resolution, and sensitivity. References 1. Darbe P D. Environmental oestrogens, cosmetics and breast cancer. Best Practice & Research Clinical Endocrinology & Metabolism. 20(1): , Golden R, Gandy J, Vollmer G. A review of the Endocrine activity of parabens and implications for potential risks to human health. Critical reviews in toxicology. 35(5): , The European Parliament and the Council of the European Union. Regulations (EC) o 1223/2009 of The European Parliament and of the Council of 30 ovember 2009 on Cosmetic Products. fficial Journal of the European Union. L 342: , 30th ovember [cited 2015 August 25]. Available from: eur-lex.europa.eu/lexuriserv/lexuriserv. do?uri=j:l:2009:342:0059:0209:en:pdf 4. Shen H Y, Jiant H L, Mao H L, et al. Simultaneous determination of seven phthalates and four parabens in cosmetic products using HPLC-DAD and GC-MS methods. J Sep Sci. 30: 48 54, Farajzadeh M A, Djozan Dj, et al. Use of a capillary tube for collecting an extraction solvent lighter than water after dispersive liquid-liquid microextraction and its application in the determination of parabens in different samples by gas chromatography-flame ionization detection. Talanta. 81: 1360, Labat L, Kummer E, Dallet P, Dubost JP. Comparison of high-performance liquid chromatography and capillary zone electrophoresis for the determination of parabens in a cosmetic product. J Pharm Biomed Anal. 23:763, González-Mariño I, Quintana J B, Rodríguez, Cela R. Evaluation of the occurrence and biodegradation of parabens and halogenated by-products in wastewater by accurate-mass liquid chromatography-quadrupole-timeof-flight-mass spectrometry (LC-QTF-MS). Water Research. 20: , Wang Y, Li P, Liu Y, et al. Determination of Triclocarban, Triclosan and Methyl-Triclosan in Environmental Water by Silicon Dioxide/Polystyrene Composite Microspheres Solid- Phase Extraction Combined with HPLC-ESI-MS. Journal of Geoscience and Environment Protection. 1(2): Waters, ACQUITY, ACQUITY UPLC, QDa, UPLC, Empower, and The Science of What s Possible are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. ctober E AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products 64

65 ne-minute Method for the Screening of Phthalates in Toys at Regulatory Limits Using UPLC-MS and Empower Software Dimple Shah, Jennifer Burgess Waters Corporation, Milford, MA, USA APPLICATI BEEFITS ne-minute screening method for phthalates in toys at legislative limits Benefits of MS, such as selectivity and sensitivity, can be brought to existing Empower users, without the need for additional training Users can define specific limits and quickly identify samples that exceed permitted levels with Empower Software s system suitability functionality. WATERS SLUTIS ACQUITY SQD Empower 3 Software KEY WRDS P hthalates, DEHP, BBP, DIP, DIDP, DnP, phthalic acid, plastics ITRDUCTI Phthalates, esters of phthalic acid, are widely used to modify the physical properties of plastics. They are added to products to increase flexibility, transparency, softness, durability, and longevity. wing to their unique properties, phthalates are widely used in toys, childcare items, food packages, raincoats, shower curtains, paints, lubricants, detergents, and personal care products. Since there is no chemical bond between phthalates and the plastics, phthalates can migrate into the environment. Upon use by children or adults, they have the potential to cause serious side effects such as hormone malfunctioning, reproductive defects, and cancer. 1 According to the Consumer Product Safety Improvement Act (CPSIA, August 2008),2 some phthalates are restricted in particular products. Since February 2009, children s toys and childcare articles cannot contain concentrations of more than 0.1 of Bis (2-ethylhexyl) phthalate (DEHP), Di-n-butyl phthalate (DBP), and Benzyl butyl phthalate (BBP). Temporary restrictions were also placed on children s toys and childcare articles that contain more than 0.1 Diisononyl phthalate (DIP), Di-isodecyl phthalate (DIDP), or Di-n-octyl phthalate (DnP). The European Union and Japanese toy safety standards also enforce the same legislative limits for all six phthalates listed above. 3,4 This application note describes a screening method for 14 phthalates (six of which are legislated) in one minute, using the Waters ACQUITY UPLC with SQ Detector and Empower 3 Software. A typical quantitative phthalate analysis using GC-MS takes approximately 30 minutes per sample. 5 Using this one-minute screening method dramatically increases the sample throughput. Those samples that show positive results can then be submitted for confirmatory analysis. SAMPLE PREPARATI The sample was prepared as described previously. 6 Briefly, a child s teething toy was finely chopped. Two grams of the sample was sonicated with 200 ml of methanol for 10 min. The supernatant was collected and filtered through a 0.2 µm nylon filter. The filtrate was diluted 10-fold in methanol and placed into a Waters certified vial for analysis. ne-minute Method for the Screening of Phthalates in Toys at Regulatory Limits Using UPLC-MS and Empower Software 65

66 EXPERIMETAL LC conditions LC system: Runtime: ACQUITY UPLC 1.0 min IntelliStart Technology was used to tune all the phthalates in this application note. IntelliStart, a standard feature of Waters MS systems automates system calibration, sample tuning, and daily checks, so that non-expert users can acquire data with confidence that the system is operating optimally. The resulting tuning parameters are shown in Table 1. Column: ACQUITY UPLC BEH C µm, 2.1 x 50 mm Mobile phase A: Isocratic flow rate: Injection volume: 2 µl Weak needle wash: Strong needle wash: MS conditions MS system: Ionisation mode: Capillary voltage: Methanol formic acid 0.6 ml/min 98:2 Water: Methanol formic acid Methanol formic acid ACQUITY SQ Detector ESI+ 3.5 kv Source temp.: 150 C Desolvation temp.: 450 C Desolvation gas: Acquisition: 800 L/H Selected Ion Recording (SIR) Phthalate Parent ion Dwell time(s) Cone voltage (V) Peak name Dimethyl phthalate a Diethyl phthalate b Dipropyl phthalate c Di-n-butyl phtahlate d Diisobutyl phthalate e Bis (methylglycol) f phthalate Dipentyl phthalate g Benzyl butyl phthalate h Dihexyl phthalae i Butyl phthylyl phthalate j Bis (2-Ethylhexyl) phthalate k Di-n-ctyl phthalate l Diisononyl phthalate m Di-isodecyl phthalate n Table 1. MS tuning parameters for phthalates obtained using IntelliStart. ne-minute Method for the Screening of Phthalates in Toys at Regulatory Limits Using UPLC-MS and Empower Software 66

67 RESULTS AD DISCUSSI Using the ACQUITY SQD, 14 phthalates were analyzed within 1 min. The chromatograms of each of the phthalates analyzed at 1000 ng/ml are shown in Figure 1. a b c d e f g h i j k l m n Minutes Figure 1. Chromatograms showing separation of 14 phthalates. The results for each of the phthalates showed good injection-to-injection reproducibility. Table 2 shows the RSD on the area count from six injections of a toy extract that was spiked at 1000 ng/ml with each of the phthalates. ne-minute Method for the Screening of Phthalates in Toys at Regulatory Limits Using UPLC-MS and Empower Software 67

68 Compound name RSD Dimethyl phthalate 10.6 Diethyl phthalate 13.4 Dipropyl phthalate 4.1 Di-n-butyl phtahlate 3.7 Diisobutyl phthalate 8.9 Bis methylglycol 6.1 phthalate Dipentyl phthalate 8.5 Benzyl butyl phthalate 7.1 Dihexyl phthalae 4.7 Butyl phthaylyl phthalate 10.0 bis 2 Ethylhexyl phthalate 7.5 Di-n- ctyl phthalate 8.4 Di-isononyl phthalate 6.4 Di-isodecyl phthalate 3.9 Table 2. Relative standard deviation from 6 injections of a spiked toy extract at 1000 ng/ml. According to the CPSIA legislation, 1 the concentration of phthalates should not exceed more than 0.1 of the total mass. Due to the sample preparation and dilution in the sample extraction, the legislative limit is equivalent to 1000 ng/ml. Figure 2 shows an example 5-point calibration curve of one of the phthalate standards (diisononyl phthalate) around the concentration that corresponds to the legislated level. The calibration curve showed an r 2 value of Figure 2. Five-point calibration curve for Diisononyl phthalate from 500 ng/ml to 2000 ng/ml. ne-minute Method for the Screening of Phthalates in Toys at Regulatory Limits Using UPLC-MS and Empower Software 68

69 The data were acquired using Empower 3 Software and processed using the system suitability function. Empower system suitability software monitors the chromatographic system automatically and provides a summary based on parameters and limits set by the user. This feature was used to set minimum and maximum values in summary charts and to flag out-of-range values. The target limit for the phthalates is 1000 ng/ml (taking into account the sample preparation), so any sample that exceeded this limit was flagged and reported in a different color and font. A typical system suitability report is shown in Table 3. The table shows the results for diisononyl phthalate from the extracted toy sample as well as two spiked extracts from the toy sample at 500 and 1000 ng/ml. As shown in Table 3, the 1000 ng/ml spiked toy extract had a calculated concentration of 1042 ng/ml, which was flagged as being over the maximum concentration of 1000 ng/ml. The first sample (the toy extract that was not spiked) does not show a reported amount, as the detected peak for this compound was below the minimum reporting level (which was set at the lowest concentration standard, 500 ng/ml). Component summary table ame Diisonoyl phthalate Sample name ame RT Concentration Units 1 TY extracted blank ng/ml 2 TY spiked 500 ng/ml ng/ml 3 TY spiked 1000 ng/ml ng/ml Table 3. Report table generated for the unspiked and spiked toy extracts using the system suitability function of Empower 3 Software. ne-minute Method for the Screening of Phthalates in Toys at Regulatory Limits Using UPLC-MS and Empower Software 69

70 CCLUSIS The results presented in this application note show how the ACQUITY SQD can be used to rapidly screen for the presence of phthalates in toy samples at the regulated limits. The ACQUITY SQD can easily be added to existing laboratories using Empower Software, circumventing the requirement of dedicated MS software. The benefits of MS, such as selectivity and sensitivity, can be brought to existing Empower users without the need for additional training. IntelliStart Technology ensures ease-of-use and consistent performance. Empower s system suitability functionality enables users to define specific limits and quickly identify samples that exceed the permitted levels. References 1. L J Schierow. CRS Report for Congress: Phthalates in Plastics and Possible Human Health Effects, July 29, American Chemistry, phthalates and children toys: Consumer Product Safety Improvement Act: Phthalates provision for Toys, August phthalate/sec.asp?cid=2103&did= do?uri=j:l:2005:344:0040:0043:e:pdf 4. Safeguards: SGS consumer testing services: Japan update part 3: toy safety standard-eight edition of ST2002, February S C Rastogi. Gas chromatographic analysis of phthalate esters in plastic toys. ational Environmental Research Institute, Department of Environmental Chemistry. Chromatographia. 47:784, J Morphet. Rapid phthalate screening in consumer products, increasing profits with greater sample throughput, Waters Application ote no en, April, Waters, The Science of What s Possible, Empower, ACQUITY, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. IntelliStart is a trademark of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. ovember E AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: ne-minute Method for the Screening of Phthalates in Toys at Regulatory Limits Using UPLC-MS and Empower Software 70

71 Detection of Unexpected Contaminants During Routine Chemical Industry QC Monitoring Using Tandem Quadrupole with RADAR Functionality GAL To carry out QC monitoring for the manufacture of Diethylhexyl Phthalate (DEHP) using tandem quadrupole MS with RADAR functionality to scan for unexpected process contamination. The Xevo TQD with RADAR functionality provides MS full scan acquisition without compromising MRM data quality. BACKGRUD Bulk chemical manufacturing processes, such as DEHP, need to have quick and robust QC checks in place to ensure the purity of both the starting materials and the final product. Producing materials that are out of specification is not only expensive in terms of raw materials, but the potential cost of a plant shutdown and start up can run into millions of dollars. Early detection of contamination while still at a low level may allow corrective action to be taken rather than requiring a full shutdown. Waters Xevo TQD with the ACQUITY UPLC System allows the monitoring of known compounds using Multiple Reaction Monitoring (MRM), which delivers high sensitivity and selectivity to eliminate the risk of misidentification. Waters RADAR functionality simultaneously acquires full scan data while maintaining the quality of MRM data and allowing post analysis interrogation to identify unexpected contamination if required. This aids in complete understanding of the manufacturing process and sample purity. Figure 1. Xevo TQD RADAR full scan Total Ion Chromatogram (TIC) with MRM chromatograms for DEHP (plasticizer) and Irgafos 168 (stabilizer). Detection of Unexpected Contaminants During Routine Chemical Industry QC Monitoring 71

72 THE SLUTI Waters Xevo TQD MS utilizing Atmospheric Pressure Photo Ionization (APPI), coupled to an ACQUITY UPLC System were used to monitor the plasticizer DEHP and the stabilizer Irgafos 168. Compounds of interest can be identified with complete confidence by operating the tandem quadrupole instrument in MRM mode. Many plasticizers, antistatic agents, UV absorbers, stabilizers, and optical brighteners commonly used in the polymer industry can be monitored in this way. During the monitoring of DEHP and Irgafos 168, RADAR scan functionality was used. RADAR completes a full mass spectral scan every duty cycle during the analytical run without affecting the quality of the MRM data, as shown in Figure 1. RADAR full scan data were interrogated post analysis and a low level of contamination was found. Figure 2 shows RADAR and the extracted ion chromatogram of mass m/z 587, with the mass spectra for the peak as an insert. Investigation into the mass m/z 587 shows it is likely to be the precursor ion of Irganox 245, a stabilizer commonly used in the polymer industry. The added information that RADAR provides allows plant managers to make knowledgeable decisions if the product falls out of specification. Contamination that is considered to be benign and have no implication on the use of the product could allow an out of specification batch to be blended with other batches. There are clear financial and environmental advantages where this is possible. Figure 2. RADAR scan showing DEHP, Irgafos 168, and Irganox 245 (contamination), extracted ion chromatogram of m/z 587 with an insert of the full mass spectrum of the peak. (Chromatograms are not to the same scale). SUMMARY Waters Xevo TQD provides high-quality quantitative MRM data while simultaneously acquiring full mass spectral scans without compromising data quality. Quantification by MRM allows high sensitivity and selectivity, which eliminates the risk of misidentification. The extra information provided by RADAR functionality offers a better understanding of how the manufacturing process is running. The identification of unknown contamination allows better business and process decisions to be made. If contamination is detected, action can be taken to address the problem and possibly save the batch, which offers significant cost savings to the business. Waters, The Science of What s Possible, Xevo, and ACQUITY UPLC are registered trademarks of Waters Corporation. RADAR is a trademark of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. May E IH-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: Detection of Unexpected Contaminants During Routine Chemical Industry QC Monitoring 72

73 初级芳香胺 / 偶氮染料 73

74 [ 技术简报 ] 使用 ACQUITY QDa 质谱检测器提升初级芳香胺分析的灵敏度和选择性 Jane Cooper 目的证明与现有类似检测方法相比, 使用 Waters ACQUITY QDa 质谱检测器分析初级芳香胺可获得更高的灵敏度和选择性 配备 ACQUITY QDa 质谱检测器的 Waters ACQUITY UPLC H-Class 系统为油墨和染料行业中初级芳香胺的鉴定和定量提供了更高的可靠性 背景油墨和染料行业具有严格的法规控制, 这就要求油墨和染料的制造商必须监控和调整各种参数, 包括是否存在初级芳香胺 (PAA) PAA 可用于生产多种产品, 例如药品 农药 炸药 环氧聚合物 橡胶 芳香族聚氨酯和偶氮染料 它们可能由于不完全反应或是作为杂质 副产品或降解产物而存在于最终产物中 AU 2.75e-2 2.5e e-2 2.0e e-2 1.5e e-2 1.0e-2 7.5e-3 5.0e-3 2.5e e ,4,5- 三甲基苯胺,UV 286 nm S/: ,4,5- 三甲基苯胺,m/z 136 S/: 7939 H 3 C H 2 CH 3 CH 3 许多 PAA 是可疑致癌物, 存在多种潜在健康风险, 因此在世界范围内对其进行了严格规定 美国 FDA 法规 (21 CFR 和 21 CFR ) 限制了可降解为 PAA 的偶氮染料的用量, 而欧盟法规 ( 欧盟指令 2002/72/EC 和指令 19/2007/EC) 也声明了相关法定限值 分析实验室需要精确 耐用的技术以确保可靠性和多样性, 同时满足法规要求 配备 ACQUITY QDa 质谱检测器的 Waters ACQUITY UPLC H-Class 系统便是针对此行业而设计的解决方案 -0 Time 图 1. 2,4,5- 三甲基苯胺的 UV 和提取离子色谱图 解决方案使用配备 ACQUITY QDa 检测器的 ACQUITY UPLC H-Class 系统监控 PAA ACQUITY QDa 检测器是一款经过专门设计的质谱检测器, 可作为色谱分离系统的完美补充 ACQUITY QDa 检测器利用更高质量的质谱定性分析数据作为光学数据的有效补充, 对成分进行准确鉴定 ACQUITY QDa 检测器拓宽了光学检测的样品范围, 可对 UV 无响应的化合物以及光学检测不适用或是无法确定的化合物进行定量分析 使用 ACQUITY QDa 质谱检测器提升初级芳香胺分析的灵敏度和选择性 74

75 [ 技术简报 ] 分析方法需要建立相应的开发和验证方法使其具有更高的灵敏度 选择性和方法可靠性以满足法规要求, 这一过程昂贵且耗时 而配备 ACQUITY QDa 质谱检测器的 ACQUITY UPLC H-Class 系统可实现多种商业优势 它通过更快的运行速度 更少的溶剂消耗量和更高的样品通量节省了成本 专为 PAA 而开发的分析方法的运行时间只需 10 分钟, 与众多现有方法相比快达七倍 此外, 无需经历耗时的衍生化阶段, 因而在样品准备阶段进一步节省了时间 与 PAA 2,4,5- 三甲基苯胺的 UV 和质谱数据相比时, 可通过对比信噪比 (S/) 来证实选择性的提升 这种 PAA 具有 m/z 136 的母离子, 最大紫外吸收波长为 286 nm 图 1 显示了使用质谱数据时 S/ 的增加量 为了观察选择性的提升, 需要考虑在复杂基质中精确且特异性地测量目标分析物的能力 这可以通过考虑 PAA 2,4,5- 三甲基苯胺来进行证实, 当其加标到油墨中时, 由于存在其它 UV 吸收化合物, 因此无法区分, 如图 2 所示 但质谱检测具有足够的灵敏度和选择性, 可在油墨基质中进行 2,4,5- 三甲基苯胺的可靠检测和定量 AU AU AU 图 2. 2,4,5- 三甲基苯胺加标油墨 (0.5 mg/ml) 溶剂标准品 (5.0 µg/ml, 等同于加标油墨中的 0.5 mg/ml) 和空白油墨基质的 UV 和提取离子色谱图 同时采集 UV 和 MS 数据 总结 3.0e-1 2.5e-1 2.0e-1 1.5e-1 1.0e-1 5.0e e-1 2.5e-1 2.0e-1 1.5e-1 1.0e-1 5.0e e-1 2.5e-1 2.0e-1 1.5e-1 1.0e-1 5.0e 空白油墨基质 UV:286 nm 加标油墨 UV:286 nm 溶剂标准品 UV:286 nm Time 配备 ACQUITY QDa 检测器的 Waters ACQUITY H-Class 系统与常用分析技术相比, 提高了油墨中 PAA 的鉴定和定量分析的可靠性 空白油墨基质 m/z : 加标油墨 m/z : 溶剂标准品 m/z :136 Time 由于缩短了运行时间,PAA 的 UPLC 分析提高了样品通量并减少了溶剂用量, 从而节省了宝贵的业务时间和成本 ACQUITY QDa 检测器可实现更优的质谱检测的选择性和灵敏度, 提高了定量结果报告的可靠性 Waters, ACQUITY, ACQUITY UPLC 和 The Science of W hat s Possible 是沃特世公司的注册商标 其他所有商标均归各自的拥有者所有 2013 沃特世公司 中国印制 2013 年 10 月 ZH TC-PDF 沃特世科技 ( 上海 ) 有限公司北京 : 上海 : 广州 : 成都 : 沃特斯中国有限公司香港 : 免费售后服务热线 : 800 (400) 使用 ACQUITY QDa 质谱检测器提升初级芳香胺分析的灵敏度和选择性 75

76 The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System with the SQ Detector 2 and MassLynx Software Jane Cooper, Eleanor Riches, and Kate Williams Waters Corporation, Manchester, UK APPLICATI BEEFITS This application provides improved confidence in the identification and quantification of Primary Aromatic Amines (PAAs) offering: Increased sample throughput and a reduction of solvent usage due to reduced run times. Improved sensitivity, selectivity, and robustness, compared with existing methodologies. The ultimate in chromatographic resolution and sensitivity. Cost-effective, reliable mass confirmation. ITRDUCTI PAAs are widely used in high amounts as a chemical feed stock within the chemical industry, and many of them are highly toxic to humans. 1,2,3 PAAs can be used to produce many commodities, such as pharmaceuticals, pesticides, explosives, epoxy polymers, rubber, aromatic polyurethane products, and azo-dyes. They can be found in final products due to incomplete reactions, as impurities, by-products, or as degradation products. PAAs can be produced as by-products of azo dyes, which are a diverse and widely used group of organic dyes. Azo dyes have a wide range of uses including special paints, printing inks, varnishes, and adhesives; and can be found in many products such as textiles, cosmetics, plastics, and also in food contact material. The inks and dyes industry is highly legislated and manufacturers that use these materials must monitor and quantify various regulated parameters, such as the presence or absence of PAAs. Previous example methodologies for the analysis of PAAs include: GC/MS analysis following ion-pair extraction with bis-2-ethyl phosphate followed by derivatization with isobutyl chloroformate; 4,5 UPLC analysis following a solid phase extraction (SPE) using cation-exchange cartridges; 6 and reduction by liquid phase sorbent trapping followed by thermal desorption GC/MS analysis. 7 Many previously used methods for PAA analysis lack robustness, selectivity, and sensitivity, and require lengthy, costly and time-consuming pre-treatments (derivatization, SPE). WATERS SLUTIS ACQUITY UPLC H-Class System SQ Detector 2 MassLynx Software KEY WRDS Primary Aromatic Amines, PAAs, azo dyes, ink Many PAAs have either a proven or suspected carcinogenic nature and are highly toxic, so there are a range of potential health risks that have led to strict worldwide regulations. U.S. FDA regulations (21 CFR and 21 CFR ) restrict the use of azo dyes that could degrade to PAAs; whereas EU regulations (commission directive 2002/72/EC and the amendment 2007/19/EC) set legislative limits for the release of total PAAs from food contact material. Analytical laboratories require accurate and robust techniques to ensure confidence and versatility in meeting these legislative requirements. The SQ Detector 2 offers a flexible solution for the ink and dyes industry. The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 76

77 E X P E R IM E TA L Sample preparation Ink analysis eat ink diluted 1:100 with 5 methanol/95 water. Diluted ink samples were transferred to LC vials for analysis. Paper analysis For each experiment 10 cm x 10 cm pieces of paper were cut up and extracted with 100 ml of water for over 24 hours. Aliquots were transferred into LC vials for analysis. LC conditions LC system: Runtime: ACQUITY UPLC H-Class min This application note describes the use of Waters ACQUITY UPLC H-Class coupled with the SQ Detector 2 for the rapid analysis of PAAs in ink. Time (min) Flow rate (ml/min) A B Curve 1 Initial Table 1. ACQUITY UPLC H-Class mobile phase gradient. Variables such as cone voltages, desolvation gas (temperature and flow rate), and cone gas flow rate were optimized using solvent standards. The list of PAAs, associated CAS number, expected retention times, and cone voltages are detailed in Table 2. The established SIR MS method is illustrated in Figure 1. Column: ACQUITY UPLC BEH C 18, 1.7 mm, 2.1 x 50 mm Column temp.: 40 C Mobile phase A: Mobile phase B: Flow rate: 10 ml of 1 M aqueous ammonium acetate solution and 990 ml water 10 ml of 1 M aqueous ammonium acetate solution and 990 ml methanol 0.5 ml/min Figure 1. PAAs SIR method. 34 compounds covered over 31 channels (including 3 isomer pairs). Injection volume: 10.0 µl Mobile phase gradient is detailed in Table 1. MS conditions MS system: SQ Detector 2 Ionization mode: Capillary voltage: ESI+ 3.0 kv Source temp.: 150 C Desolvation temp.: 350 C Desolvation gas: Cone gas: Acquisition: 650 L/hr 20 L/hr Selected Ion Recording (SIR) The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 77

78 PAA number Primary Aromatic Amines (PAAs) CAS umber m/z Retention time (minutes) 1 Aniline o-toluidine ,3-Phenylenediamine ,4-Phenylenediamine ,4-Dimethylaniline ,6-Dimethylaniline ,4-Toluenediamine ,6-Toluenediamine o-anisidine Chloroaniline ,4,5-Trimethylaniline Methoxy-5-methylaniline Methoxy-m-phenylenediamine aphtylamine Amino-4-methylbenzamide Chloro-4-methoxyaniline Chloro-2-methoxyaniline ,5-Diaminonaphtalene Methoxy-4-nitroaniline Aminobiphenyl Aminobiphenyl Benzidine Chloro-2,5-dimethoxyaniline Aminoazobenzol ,4'-Methylenedianiline ,4'-Diaminodiphenylether ,3'-Dimethylbenzidine ,4'-Thioaniline o-aminoazotoluene ,4'-Diamino-3,3'-dimethylbiphenylmethane Amino-p-anisanilide o-dianisidine ,3'-Dichlorobenzidine ,4'-Diamino-3,3'-dichlorobiphenylmethane Cone Voltage (V) Table 2. PAAs, associated CAS number, m/z, expected retention times, and cone voltages. The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 78

79 Instrument control, data acquisition, and result processing MassLynx Software was used to control the ACQUITY UPLC H-Class and the SQ Detector 2 and also for data acquisition. Data quantitation was achieved using TargetLynx Application Manager. The advantages of mass spectral detection over core detectors Many gains can be accomplished using an ACQUITY UPLC System for chromatographic separation, due to the reduced column particle size (sub-2-µm), which results in improvements in speed and peak capacity, with superior sensitivity and resolution efficiently achievable over HPLC analysis. During method development, considerations need to be given to the appropriate detector to use in order to meet the analytical requirements. The use of mass spectral detection over core detectors (e.g. UV or fluorescence) offers advantages in areas such as sensitivity and selectivity, especially where complex matrices are present. Matrix effects can be greatly reduced by using mass spectral detection over DAD (UV) detection and this can be demonstrated by considering many of the PAAs detailed within this application. Examples can be seen considering the PAAs, 2-Aminobiphenyl and 3,3'-Dichlorobenzidine. When using the current UPLC conditions the two compounds are not completely resolved giving retention times of 7.71 and 7.76 minutes respectively. Using mass spectral detection, the resulting efficient selectivity is illustrated in Figure 2. m/z 170 (4-Aminobiphenyl) 2-Aminobiphenyl m/z 253 3,3'-Dichlorobenzidine Figure 2. Extracted ion chromatograms for 2-Biphenylamine and 3,3'-Dichlorobenzidine in fortified ink (containing 4.6 µg/ml PAAs). The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 79

80 In this example when using UV detection due to the UV absorbing nature of the solvents used, the ink matrix, and other PAAs present this level of selectivity is very hard to achieve. This reduced selectivity can be demonstrated by again considering the PAAs, 2-Aminobiphenyl and 3,3'-Dichlorobenzidine in solvent standards. When considering individual solvent standards for 2-Aminobiphenyl and 3,3'-Dichlorobenzidine, maximum UV absorbance can be found at 295 and 284 nm respectively. When comparing individual solvent standards against mixed solvent standards, the reduction in selectivity is demonstrated in Figures 3a and 3b, which could potentially lead to misidentification, poor integration, and false positive results. a Individual 2 ppm solvent standards b Mixed 1 ppm solvent standard 2-Aminobiphenyl UV 295 nm 2-Aminobiphenyl UV 295 nm 3,3'-Dichlorobenzidine UV 284 nm 3,3'- Dichlorobenzidine UV 284 nm Figure 3. a) UV chromatograms for 2-Aminobiphenyl and 3,3'-Dichlorobenzidine in individual solvent standards; b) UV chromatograms for 2-Aminobiphenyl and 3,3'-Dichlorobenzidine in a mixed solvent standard. Improvements in selectivity in this example could only be made by changing the chromatographic separation by altering the UPLC conditions to reducing the solvent gradient, which would result in longer run times and associated increases in solvent usage. The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 80

81 RESULTS AD DISCUSSI The analysis of 34 PAAs was achieved using Waters SQ Detector 2 with an electrospray ionization (ESI) source, coupled to an ACQUITY UPLC H-Class System in SIR mode. ptimum UPLC and SIR conditions were developed, with the elution of all compounds within a 10-minute run. Mixed calibration standards were prepared and analyzed for all the PAAs considered. The TargetLynx Quantify results for aniline are shown in Figure 4, and the SIR chromatograms for each PAA are shown in Figure 5. Figure 4. TargetLynx Quantify results browser showing the calibration quantitation results, calibration curve, and example SIR chromatogram for aniline. The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 81

82 PAA-4 PAA-3 PAA-26 PAA-31 PAA-8 PAA-19 PAA-14 PAA-13 PAA-10 PAA-28 PAA-7 PAA-6 PAA-5 PAA-11 PAA-1 PAA-12 PAA-30 PAA-15 PAA-18 PAA-25 PAA-20 PAA-21 PAA-23 PAA-33 PAA-9 PAA-2 PAA-32 PAA-24 PAA-16 PAA-27 PAA-34 PAA-22 PAA-17 PAA-29 Figure 5. SIR chromatograms for 34 PAAs in a mixed 1 µg/ml calibration standard. The SIR mass detection method detailed in Figure 1 was used after appropriate sample preparation to screen for PAAs in ink (containing PAAs) and paper (applied with ink containing PAAs). The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 82

83 Ink analysis eat ink diluted 1:100 with 5 methanol/95 water was fortified at various levels with selected PAAs, and analyzed without any further cleanup or concentration steps. The results obtained are detailed in Table 3. Amine Sample Aniline o-toluidine 2,4-Dimethylaniline 2,6-Dimethylaniline o-anisidine 4-Chloroaniline 2,4,5-Trimethlaniline 3-Chloro-4- methoxyaniline 5-Chloro-2-methoxyaniline 2-Aminobiphenyl 4-Chloro-2,5- dimethoxyaniline Replicate injection results (µg/ml) Average recovery (blank corrected) Ink blank Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml Ink blank D Ink µg/ml Ink µg/ml Ink 4.60 µg/ml RSD () Table 3. Ink fortified with PAAs recovery data. Results quantified against mixed calibration standards. The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 83

84 The efficient recoveries obtained (ranging between 83 to 108) demonstrated that minimal signal enhancement/suppression was observed using ESI ionization for the analysis of PAAs within an ink matrix. Paper analysis Within the food packaging industry great efforts are made to reduce food contamination in order to guarantee consumer safety and comply with regulations. The design of the packaging and the products used ideally afford minimal leaching and hence reduce potential contamination of the food product. Such packaging leachables have a large number of potential sources including PAAs from the ink used within the packaging. In order to consider the EU regulations with regard to the release of total PAAs from food contact material, a cold water paper extraction based on the European standard (E 646:1993) was used. Three pieces of paper (10 cm x 10 cm) were taken, one kept as a blank and two applied with 100 µl ink previously fortified with selected PAAs. The paper was left to dry and then cut up and extracted in sealed containers with 100 ml of water and left for over 24 hours prior to analysis. The results obtained are detailed in Table 4. Amine Aniline o-toluidine 2,4-Dimethylaniline o-anisidine 3-Chloro-4-methoxyaniline 5-Chloro-2-methoxyaniline Sample Replicate injection results (µg/ml) Average µg aniline equivalents /kg of food* Leachability () A D B C D D E A D B C D D E A D B C D D E A D B C D D E A D B C D D E A D B C D D E RSD () Table 4. Leachability results for paper previously applied with ink containing selected PAAs. A = water blank, B = water containing 0.1 µg/ml PAAs, C = paper blank with no ink, D = paper applied with ink containing 10 µg PAAs, E = paper applied with ink containing 5 µg PAAs. *Calculated using a conventional surface area/volume conversion factor of 6 dm2/kg as established in the EU commission a 2007/19/EC. The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 84

85 Sample A results demonstrate that there were no residual PAAs in the water used or as background within the system. Sample B shows the efficacy of the extraction method used, as demonstrated by the high leachability recovery values observed (90 to 104) when PAAs were added to the water with no paper present. The results most relevant to the food packaging industry were obtained for Samples C and D, which revealed the different extents to which the selected PAAs were being absorbed and not leached from paper. CCLUSIS A fast, robust, and sensitive method has been developed for the analysis of PAAs in ink. SQ Detector 2 linked to the ACQUITY UPLC H-Class System offers improved confidence in identification and quantification. Business benefits include increased sample throughput and a reduction of solvent usage with no time-consuming derivatization or pre-concentration stages and reduced run times. References 1. Benigni R, Passerini L. Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment. Mutation Research. 511: , Anirban M P, Cote R J. Molecular Pathogenesis and Diagnosis of Bladder Cancer. Annual Review of Pathology. 4: , Ward E, Carpenter A, Markowitz S, et al. Excess Cancers in Workers Exposed to rtho-toluidine and Aniline. ational Cancer Institute. 83(7): , Akyuz M, Ata S. Determination of aromatic amines in hair dye and henna samples by ion-pair extraction and gas chromatography-mass spectrometry. J Pharm Biomed Anal. 47: 68, Ekladius L, King H K. A colorimetric method for the determination of aliphatic amines in the presence of ammonia. Chromatography. A. 1129(1), Epub 2006 Jul Aznar M, Canallas E, erin J. Quantitative determination of 22 primary aromatic amines by cation-exchange solid-phase extraction and liquid chromatography-mass spectrometry. J Chromatography A. 1216: , Zhang Q, Wang C, et al. Determination of aromatic amines from azo dyes reduction by liquid-phase sorbent trapping and thermal desorption-gas chromatography-mass spectrometry. J Sep Sci. 32: , The ACQUITY UPLC H-Class System, a quarternary system based on UPLC, offers the best in chromatographic resolution and sensitivity. The SQ Detector 2 offers cost-effective, reliable mass confirmation. Waters, The Science of What s Possible, MassLynx, ACQUITY UPLC, UPLC, and ACQUITY are registered trademarks of Waters Corporation. TargetLynx is a trademark of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. December en AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: The Analysis of Primary Aromatic Amines in Ink Using the ACQUITY UPLC H-Class System 85

86 采用配备 ACQUITY QDa 检测器的 ACQUITY UPLC H-Class 系统和 Empower 软件对化妆品和个人护理产品中的初级芳香胺进行合规性分析 Jane Cooper 沃特世公司 ( 英国威姆斯洛 ) 应用优势将 ACQUITY QDa 与 ACQUITY UPLC H-Class 系统联用, 提升对化妆品和个人护理产品中初级芳香胺 (PAA) 鉴定与定量的可信度 : 极佳的色谱分离度和灵敏度 通过缩短运行时间提高样品通量, 减少溶剂用量 相较于现有方法, 灵敏度 选择性和 稳定性更好 经济有效并且可靠的质量数确认 简介初级芳香胺 (PAA) 作为一种化工原料被大量广泛地用于化学行业 许 1,2,3 多 PAA 被证实或疑似具有致癌性并且评级为剧毒, 存在多种潜在健康风险, 因此在世界范围内对其进行了严格规定 欧盟化妆品法 (EC)1223/2009 号法规 4 禁止多种 PAA 用于化妆品 尽管具有毒性和致癌性,PAA 仍然是许多商品的重要生产原料, 诸如药品 农药 炸药 环氧聚合物 橡胶 芳香族聚氨酯产品和偶氮染料 虽然并不期望最终产品中含有 PAA, 但 PAA 还是会因为不完全反应 杂质 副产物等原因或作为降解产物存在于最终产品中 例如,PAA 能够以偶氮染料副产物的形式产生, 而偶氮染料种类繁多, 属于广泛使用的有机染料 偶氮染料可用于特种漆 印刷油墨 清漆和粘合剂, 而且存在于多种产品中, 例如纺织品 化妆品 个人护理产品 塑料制品, 还存在于食品接触材料中 为确保公共安全和产品功能, 已对化妆品和个人护理产品严格立法 自此, 产品生产过程中使用诸如 PAA 等原料的生产商必须对各种管制参数进行监测和定量, 例如是否存在 PAA 此前进行 PAA 分析的示例方法包括 : 沃特世解决方案 ACQUITY UPLC H-Class 系统 ACQUITY QDa 检测器 Empower 3 色谱数据软件 使用双 (2- 乙基 ) 磷酸酯进行离子对萃取, 再用氯甲酸异丁酯衍生, 5,6 然后进行 GC/MS 分析 ; 使用阳离子交换小柱完成固相萃取 (SPE) 后进行 UPLC 分析 7 ; 通过液相吸附浓缩还原, 然后进行热解吸 GC/MS 分析 8 但此前所用的许多 PAA 分析方法在稳定性 选择性和灵敏度方面都有所欠缺, 并且需要进行繁琐耗时且成本高的前处理 ( 衍生化,SPE) 关键词初级芳香胺 PAA 偶氮染料 化妆品 个人护理产品 化妆品和个人护理产品中初级芳香胺的合规性分析 86

87 实验 LC 条件 LC 系统 : ACQUITY UPLC H-Class 运行时间 : min 色谱柱 : ACQUITY BEH C 18,1.7 µm, 2.1 x 50 mm 柱温 : 40 C 样品温度 : 10 C 流动相 A: 水 +0.1 甲酸 流动相 B: 甲醇 +0.1 甲酸 流速 : 0.4 ml/min 进样体积 : 10.0 µl 流动相梯度详见表 1 时间 流速 A B 曲线 (min) (ml/min) 1 初始 表 1. ACQUITY UPLC H-Class 流动相梯度 化妆品和个人护理产品行业中理想的 PAA 分析解决方案应能突破之前方法的限制, 同时确保可靠性和通用性, 以满足法规要求 本应用资料介绍了一种准确 快速并且稳定的替代方法, 用以实现化妆品和个人护理产品中 PAA 的快速分析, 该方法采用配备 ACQUITY QDa 检测器的 Waters ACQUITY UPLC H-Class 系统, 通过 Empower 3 软件进行控制 仪器控制 数据采集和结果处理 Empower 3 软件用于控制 ACQUITY UPLC H-Class 系统和 ACQUITY QDa 检测器以及数据采集和定量分析 样品制备化妆品和个人护理产品样品分析 ( 眼影 腮红 洗发水 ) 0.5 g( 固体样品 ) 或 0.5 ml( 液体样品 ), 加 8 ml 水和 2 ml 甲醇 涡旋混合 2 分钟 (1600 rpm) 取 1 ml 提取物离心约 5 分钟 (10,000 rpm) 在 LC 样品瓶中用甲醇稀释离心后的提取物, 以供分析 (250 µl 提取物 +750 µl 甲醇 ) MS 条件 质谱检测器 : ACQUITY QDa 电离模式 : ESI+ 毛细管电压 : 0.8 kv 探头温度 : 450 采集模式 : 选择离子监测 (SIR) 锥孔电压 : 15 V PAA 列表 对应 CAS 号 预期保留时间和锥孔电压详见表 2 化妆品和个人护理产品中初级芳香胺的合规性分析 87

88 PAA 编号 初级芳香胺 (PAAs) CAS 编号 m/z 保留时间 (min) 1 苯胺 (Aniline) 邻甲苯胺 (o-toluidine) 间苯二胺 (1,3-Phenylenediamine) ,4- 二甲基苯胺 (2,4-Dimethylaniline) ,6- 二甲基苯胺 (2,6-Dimethylaniline) ,4- 二氨基甲苯 (2,4-Toluenediamine) ,6- 二氨基甲苯 (2,6-Toluenediamine) 邻甲氧基苯胺 (o-anisidine) 氯苯胺 (4-Chloroaniline) 甲氧基 -5- 甲基苯胺 (2-Methoxy-5-methylaniline) 甲氧基间苯二胺 (4-Methoxy-m-phenylenediamine) 萘胺 (2-aphtylamine) 氨基 -4- 甲基苯甲酰胺 (3-Amino-4-methylbenzamide) 氯 -4- 甲氧基苯胺 (3-Chloro-4-methoxyaniline) 氯 -2- 甲氧基苯胺 (5-Chloro-2-methoxyaniline) ,5- 萘二胺 (1,5-Diaminonaphtalene) 甲氧基 -4- 硝基苯胺 (2-Methoxy-4-nitroaniline) 氨基联苯 (4-Aminobiphenyl ) 氨基联苯 (2-Aminobiphenyl) 联苯胺 (Benzidine) 氯 -2,5- 二甲氧基苯胺 (4-Chloro-2,5-dimethoxyaniline) 氨基偶氮苯 (4-Aminoazobenzol) ,4'- 二氨基二苯甲烷 (4,4'-Methylenedianiline) ,3'- 二甲基联苯胺 (3,3'-Dimethylbenzidine) ,4- 二氨基二苯硫醚 (4,4'-Thioaniline) 邻氨基偶氮甲苯 (o-aminoazotoluene) ,4'- 二氨基 -3,3'- 二甲基二苯甲烷 (4,4'-Diamino-3,3'-dimethylbiphenylmetha) 氨基 -4- 甲氧基苯甲酰苯胺 (3-Amino-p-anisanilide) 邻联茴香胺 (o-dianisidine) ,4'- 二氨基 -3,3'- 二氯二苯甲烷 (4,4'-Diamino-3,3'-dichlorobiphenylmethane) 表 2. PAA 对应 CAS 号 m/z 和预期保留时间 化妆品和个人护理产品中初级芳香胺的合规性分析 88

89 结果与讨论建立最佳的 UPLC 和 SIR 条件, 使所有化合物均在 10 分钟内被洗脱 使用 ACQUITY QDa 检测器代替 UV 检测显著提升了方法开发速度 在方法开发过程中, 通常要考虑不同的条件 / 参数, 例如色谱柱 流动相和梯度的选择 这些选择可能使目标化合物的洗脱顺序发生改变 仅通过 UV 检测进行峰追踪需要分析各个确证标准品以确认洗脱顺序 (Rt) 但使用质谱检测时, 可轻松跟踪色谱峰移动并轻松识别存在的共洗脱峰 图 1 为共洗脱峰识别的示例, 所示为最佳波长相近的两种 PAA(4,4'- 二氨基二苯甲烷和 2- 甲氧基 -5- 甲基苯胺 ) A) 单个标准品 B) 混合标准品 AU AU UV 谱图 1.5e-1 1.0e-1 5.0e e-1 1.0e-1 5.0e ,4'- 二氨基二苯甲烷 (PAA-23) 二甲氧基 -5- 甲基苯胺 (PAA-10) nm nm AU UV 谱图 8.0e-2 6.0e-2 4.0e-2 2.0e-2 UV 谱图 PAA-10 or 23? 240 SIR 色谱图 289 nm 质谱 (PAA-10) (PAA-23) 图 1. 利用两种 PAA(4,4'- 二氨基二苯甲烷和 2- 甲氧基 -5- 甲基苯胺 ) 说明在方法开发过程中通过质谱检测识别共洗脱峰的优势 ;a) 单个标准品的 UV 谱图,b) 混合标准品的 UV 谱图 质谱图和 SIR 色谱图 Time m/z 配制范围在 µg/ml-1.0 µg/ml 内的混标, 对所有目标 PAA 进行分析 ( 使用所开发方法, 相当于 mg/kg 范围内的提取样品, 提取物被稀释时则更大 ) 图 2 所示为各种 PPA 的 SIR 色谱图 PAA-3 PAA-7 PAA-11 PAA-2 PAA-14 PAA-9 PAA-25 PAA-17 PAA-15 表 2 中详细列出了化妆品和个人护理产品中 PAA 筛查的 SIR 质谱检测条件, 可对通过制备后的样品进行筛查 PAA-6 PAA-24 PAA-21 PAA-20 PAA-10 PAA-28 PAA-1 PAA-4 PAA-18 PAA-16 PAA-29 PAA-19 PAA-23 PAA-5 PAA-22 PAA-13 PAA-27 PAA-30 PAA-8 PAA-12 PAA-26 图 种 PAA 的 SIR 色谱图, 混合校准标准品浓度为 0.5 µg/ml 化妆品和个人护理产品中初级芳香胺的合规性分析 89

90 化妆品和个人护理产品样品分析 向样品中添加不同水平的目标 PAA, 然后按照实验部分所述进行制备, 以供分析 洗发水 腮红 眼影的结果详见表 3 4 和 5, 所选样品的 SIR 色谱图如图 3 所示 胺类物质 mg/kg 回收率 ()* 苯胺 /A 表 3. 添加不同浓度苯胺的洗发水 定量结果基于混合校准标准品 * 空白校正后的回收率数据 胺类物质 添加浓度 mg/kg 添加浓度 mg/kg mg/kg 回收率 ()* 2,6- 二甲基苯胺 /A 氯苯胺 /A 萘胺 0 D /A 胺类物质 添加浓度 mg/kg mg/ Kg 回收率 ()* 2,6- 二甲基苯胺 0 D /A 氯苯胺 /A 氯 -2- 甲氧基苯胺 /A 表 5. 添加了不同浓度的所选 PAA 的眼影 定量结果基于混合校准标准品 * 空白校正后的回收率数据 a) 洗发水中的苯胺 (Fortified at 0.25 mg/kg) mg/kg (80.5) b) 腮红中的 2- 萘胺 (Fortified at 0.25 mg/kg) mg/kg (101.6) c) 眼影中的 2,6- 二甲基苯胺 (Fortified at 0.25 mg/kg) mg/kg (82.8) 表 4. 添加了不同浓度的所选 PAA 的腮红 定量结果基于混合校准标准品 * 空白校正后的回收率数据 (Fortified at 0.5 mg/kg) mg/kg (71.8) (Fortified at 0.5 mg/kg) mg/kg (80.8) (Fortified at 0.5 mg/kg) mg/kg (70.6) 得到的回收率 ( 介于 范围内 ) 表明, 采用 UPLC 色谱分离和 ESI 电离分析目标化妆品和个人护理产品中的 PAA 时, 观察到信号增强 / 抑制很小 (Fortified at 1.0 mg/kg) mg/kg (81.8) (Fortified at 1.0 mg/kg) mg/kg (86.5) (Fortified at 1.0 mg/kg) mg/kg (77.5) 图 3. 以下基质中所选 PPA 的 SIR 色谱图 :a) 洗发水 b) 腮红和 c) 眼影 化妆品和个人护理产品中初级芳香胺的合规性分析 90

91 结论 针对化妆品和个人护理产品样品开发了一种快速 稳定 灵敏度 高的 PAA 分析方法 ACQUITY QDa 检测器可实现更加经济有效且可靠的质量数确认, 在方法开发和常规分析中表现出优于 UV 检测的实验可靠性 ACQUITY UPLC H-Class 系统与 ACQUITY QDa 检测器相结合可实现准确且 可重现的定量分析 Empower 3 色谱数据软件能够为数据管理 数据处理和报告操作提 供保证 相较于先前的方法, 本方法的商业优势体现在 : 增加了样品通量 无需进行耗时的衍生化或预富集步骤, 减少了溶剂用量 缩短了运行时间 ACQUITY H-Class 系统是基于 UPLC 技术的四元系统, 拥有最佳的 色谱分离度和灵敏度 参考文献 1. Benigni R, Passerini L. Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment. Mutation Research. 511: ; Anirban M.P, Cote R.J. Molecular Pathogenesis and Diagnosis of Bladder Cancer. Annual Review of Pathology. 4: ; Ward E, Carpenter A, Markowitz S, et al. Excess Cancers in Workers Exposed to rtho-toluidine and Aniline. ational Cancer Institute. 83(7): ; The European Parliament and the Council of the European Union. Regulations (EC) o 1223/2009 of the European Parliament and of the Council of 30 ovember 2009 on Cosmetic Products. fficial Journal of the European Union. L 342/59: , 22nd Dec [cited 2015 January 15]. Available from: europa.eu/lexuriserv/lexuriserv.do?uri=j:l:2009:342:0 059:0209:en:PDF 5. Akyuz M, Ata S. Determination of aromatic amines in hair dye and henna samples by ion-pair extraction and gas chromatography-mass spectrometry. J Pharm Biomed Anal. 47, 68; Ekladius L, King H K. A colorimetric method for the determination of aliphatic amines in the presence of ammonia. J Chrom A. 1129(1). Epub 2006 Jul Aznar M, Canallas E, erin J. Quantitative determination of 22 primary aromatic amines by cation-exchange solid-phase extraction and liquid chromatography-mass spectrometry. J Chrom A. 2009; 1216: ; Zhang Q, Wang C, et al. Determination of aromatic amines from azo dyes reduction by liquid-phase sorbent trapping and thermal desorption-gas chromatography-mass spectrometry. J Sep Sci. 32: ; 沃特斯中国有限公司沃特世科技 ( 上海 ) 有限公司 Waters, ACQUITY, QDa, ACQUITY UPLC, UPLC, Empower 和 The Science of What s Possible 是沃特世公司的注册商标 其它所有商标均归各自的拥有者所有 2015 年沃特世公司中国印刷 2015 年 3 月 ZH AG-PDF 北京 : 上海 : 广州 : 成都 : 香港 : 免费售后服务热线 :800 (400) 化妆品和个人护理产品中初级芳香胺的合规性分析 91

92 沃特世科技 ( 上海 ) 有限公司地址 : 上海市浦东新区金海路 1000 号金领之都 13 栋邮编 : 电话 : 传真 : 北京分公司地址 : 北京市朝阳区铜牛国际大厦光华路 15 号院 2 号楼 9 层邮编 : 电话 : 传真 : 广州分公司地址 : 广州市荔湾区中山七路 50 号西门口广场 室邮编 : 电话 : 传真 : 成都分公司地址 : 成都市高新区科园南路 88 号天府生命科技园孵化楼 C1 栋 411 室邮编 : 电话 : 传真 : 沃特斯中国有限公司 地址 : 香港新界沙田香港科学园 科技大道西 2 号生物资讯中心 6 楼 608 室 电话 : 传真 : 扫一扫, 关注沃特世微信 全国免费售后服务热线 : 800(400) Waters, The Science of What s Possible, ACQUITY, ACQUITY UPLC, UPLC, Empower, MassLynx, QDa, XBridge, 和 Xevo 是沃特世公司的注册商标 Arc, BEH Technology, XSelect, CSH, Quattro Premier, RADAR, TargetLynx 和 IntelliStart 是沃特世公司的商标 其它所有商标均归各自的拥有者所有 2016 沃特世公司中国印刷 2016 年 1 月 ZH LM-PDF