高效液相色谱-蒸发光散射检测法同时测定口腔护理产品中10种季铵盐类杀菌剂

doi:10.3724/SP.J.1123.2023.10013

摘要/Abstract

摘要：

建立了同时测定口腔护理产品中10种季铵盐类杀菌剂的高效液相色谱-蒸发光散射(HPLC-ELSD)检测方法。在采用无水硫酸钠脱水后的膏体样品和粉末样品中加入乙醇,常温超声提取;液体样品用乙醇稀释。采用Acclaim Surfactant色谱柱(150 mm×4.6 mm, 5 μm)进行分离,以50 mmol/L乙酸铵缓冲溶液(pH=5.5)和乙腈为流动相进行梯度洗脱,外标法定量。结果表明,10种目标化合物在25 min内完成色谱分离,检出限(信噪比(S/N)=3))和定量限(S/N=10)分别为1.42~3.31 mg/L和4.25~9.94 mg/L。十八烷基二甲基苄基氯化铵和二十二烷基三甲基氯化铵在10~200 mg/L范围内线性关系良好,其余目标物在5~100 mg/L范围内线性关系良好,10种季铵盐类杀菌剂的相关系数(R²)均不小于0.9992。以阴性样品牙膏(膏体)、漱口水(液体)和洁牙粉(粉末)为样品基质,在不同添加水平下,10种季铵盐类杀菌剂的平均回收率为87.9%~103.1%,相对标准偏差(RSD, n=6)均不大于5.5%。本方法在液相色谱分离时采用表面活性剂色谱柱,目标化合物均获得较好保留,同时采用蒸发光散射检测器,部分无紫外吸收的目标化合物也能得到较高响应。通过添加适量无水硫酸钠去除膏体样品中的水分,有利于样品前处理和保护色谱柱。本方法精密度好,准确度高,适用于各种口腔护理产品中季铵盐类杀菌剂的测定,为口腔护理产品的质量安全监控提供了参考。采用本方法对109个常用口腔护理产品进行测定,十六烷基二甲基苄基氯化铵和十八烷基二甲基苄基氯化铵的检出率较高,1个牙膏样品中十八烷基二甲基苄基氯化铵的含量超出法规要求,测定值为1511 mg/kg。

关键词: 高效液相色谱, 蒸发光散射检测, 季铵盐, 杀菌剂, 口腔护理产品

Abstract:

Quaternary ammonium salt bactericides are broad-spectrum bactericides often used in oral care products because of their high antibacterial efficacy, strong penetration, and low toxicity. However, the excessive use of quaternary ammonium salt bactericides may cause contact dermatitis, scalding poisoning, and even death. Existing methods to determine quaternary ammonium salt bactericides are unable to meet current requirements owing to the lack of determination components. Therefore, establishing a simple and accurate method for the simultaneous detection of more quaternary ammonium salt bactericides is necessary.

In this study, a method that couples sample pretreatment with high performance liquid chromatography-evaporative light-scattering detection (HPLC-ELSD) was developed for the simultaneous determination of quaternary ammonium salt bactericides in oral care products, including dodecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride, benzethonium chloride, tetradecyl trimethyl ammonium chloride, tetradecyldimethylbenzylammonium chloride, N-hexadecyltrimethylammonium chloride, benzyldimethylhexadecylammonium chloride, trimethylstearylammonium chloride, stearyldimethylbenzylammonium chloride, and docosyltrimethylammonium chloride. Some of these bactericides do not absorb ultraviolet light, so a universal evaporative light-scattering detector was used owing to testing cost and stability concerns. The paste samples contained thickening agents, which are highly soluble in water but insoluble in organic solvents; these agents can seriously affect the results of sample pretreatment and damage the chromatographic column. Hence, sample dehydration was necessary. In this study, four dehydration methods were compared. Anhydrous sodium sulfate (Na₂SO₄) was selected, and the amount of Na₂SO₄ was optimized. Based on the solubility of the 10 target compounds and extraction efficiency, three extraction solvents were compared, and ethanol was selected. Ultrasonic extraction was the primary extraction process used in this study. The effects of different ultrasonication times, temperatures, and powers on the extraction recoveries were also investigated. Ultimately, the optimized conditions were as follows: extraction of the dehydrated paste and powder samples using ethanol at room temperature (25 ℃) for 20 min under 100 W ultrasound power, and dilution of the liquid sample with ethanol.

After extraction, the samples were separated on an Acclaim Surfactant column (150 mm×4.6 mm, 5 μm) with 50 mmol/L ammonium acetate aqueous solution (pH=5.5) (A) and acetonitrile (B) as mobile phases. The gradient elution program were as follows: 0-5.0 min, 75%A-35%A, 5.0-15.0 min, 35%A-20%A, 15.0-20.0 min, 20%A, 20.0-21.0 min, 20%A-75%A, 21.0-25.0 min, 75%A. An external standard method was used for quantitative determination. The 10 compounds were analyzed within 25 min. Linear equations, correlation coefficients, and linear ranges were obtained by analyzing a series of mixed standard working solutions. The limits of detection (LODs, S/N=3) and quantification (LOQs, S/N=10) of the 10 components were determined. Stearyldimethylbenzylammonium chloride and docosyltrimethylammonium chloride showed good linear relationships in the range of 10-200 mg/L, while the other compounds demonstrated good linear relationships in the range of 5-100 mg/L. In all cases, correlation coefficients (R²) of no less than 0.9992 were obtained. The LODs and LOQs were in the range of 1.42-3.31 mg/L and 4.25-9.94 mg/L, respectively. Ten analytes were spiked in blank matrices, such as toothpaste (paste), mouthwash (liquid), and dentifrice powder (powder) at three levels, and the recoveries and precisions were calculated. The average recoveries were 87.9%-103.1%, and the corresponding relative standard deviations (RSDs) did not exceed 5.5% (n=6). The developed method was used to detect 109 oral care products. Benzyldimethylhexadecylammonium chloride and stearyldimethylbenzylammonium chloride revealed high detection rates. Moreover, the amount of stearyldimethylbenzylammonium chloride in one toothpaste sample exceeded regulatory requirements. Given its advantages of good precision and accuracy, the developed method is suitable for the quantitative analysis of the 10 aforementioned compounds in typical oral care products. The study findings can serve as a reference for the quality and safety monitoring of oral care products.

Key words: high performance liquid chromatography (HPLC), evaporative light-scattering detection (ELSD), quaternary ammonium salt, bactericides, oral care products

中图分类号:

O658

汤娟, 丁友超, 费晓庆, 吴斌, 钱志娟, 陈山丹, 李柏. 高效液相色谱-蒸发光散射检测法同时测定口腔护理产品中10种季铵盐类杀菌剂[J]. 色谱, 2024, 42(8): 783-791.

TANG Juan, DING Youchao, FEI Xiaoqing, WU Bin, QIAN Zhijuan, CHEN Shandan, LI Bai. Simultaneous determination of 10 quaternary ammonium salt bactericides in oral care products by high performance liquid chromatography-evaporative light-scattering detection[J]. Chinese Journal of Chromatography, 2024, 42(8): 783-791.

图/表 6

图1 10种目标分析物的色谱图

Fig. 1 Chromatogram of the 10 target analytes Peak identifications: 1. dodecyltrimethylammonium chloride (DTAC); 2. dodecyldimethylbenzylammonium chloride (DDBAC); 3. benzethonium chloride (BZC); 4. tetradecyl trimethyl ammonium chloride (TTAC); 5. tetradecyldimethylbenzylammonium chloride (TDBAC); 6. N-hexadecyltrimethylammonium chloride (HTAC); 7. benzyldimethylhexadecylammonium chloride (HDBAC); 8. trimethylstearylammonium chloride (STAC); 9. stearyldimethylbenzylammonium chloride (SDBAC); 10. docosyltrimethylammonium chloride (DocTAC).

图2 不同提取溶剂对10种目标分析物提取效率的影响

Fig. 2 Effects of different extraction solvents on extraction efficiencies of the 10 target analytes a. toothpaste; b. dentifrice powder. Extraction: ultrasonication; ultrasonication time: 10 min; ultrasonication temperature: room temperature. Compound Nos. 1-10 are the same as those in Fig. 1.

图3 不同超声时间对10种目标分析物提取效率的影响

Fig. 3 Effects of different ultrasonication times on extraction efficiencies of the 10 target analytes a. toothpaste; b. dentifrice powder. Compound Nos. 1-10 are the same as those in Fig. 1.

表1 10种目标分析物的线性范围、回归方程、相关系数、检出限和定量限

Table 1 Linear ranges, regression equations, correlation coefficients (R2), limits of detection (LODs) and limits of quantification (LOQs) of the 10 quaternary ammonium salt bactericides

Compound	Linear range/ (mg/L)	Regression equation	R²	LOD/ (mg/L)	LOQ/ (mg/L)
DTAC	5-100	Y=4.262X-2.017	0.9998	1.61	4.82
DDBAC		Y=4.995X-1.422	0.9992	1.46	4.39
BZC		Y=4.614X-2.336	0.9993	1.54	4.62
TTAC		Y=5.004X+12.089	0.9992	1.58	4.73
TDBAC		Y=4.996X+1.862	0.9995	1.45	4.36
HTAC		Y=4.773X-6.145	0.9993	1.63	4.90
HDBAC		Y=6.809X-3.733	0.9997	1.42	4.25
STAC		Y=4.185X-6.243	0.9999	1.64	4.93
SDBAC	10-200	Y=2.200X-5.078	0.9993	3.29	9.87
DocTAC		Y=2.293X+0.304	0.9994	3.31	9.94

表2 阴性样品中10种目标分析物的加标回收率和相对标准偏差(n=6)

Table 2 Recoveries and relative standard deviations (RSDs) of the 10 quaternary ammonium salt bactericides in negative samples (n=6)

Compound	Added/(mg/kg)	Toothpaste		Mouthwash		Dentifrice powder
Compound	Added/(mg/kg)	Recovery/%	RSD/%	Recovery/%	RSD/%	Recovery/%		RSD/%
DTAC	50	92.3	4.9	95.2	3.7	94.3	4.5
	200	93.7	3.2	97.1	3.3	95.1	4.2
	500	91.9	3.6	96.2	3.5	93.2	3.9
DDBAC	50	92.3	4.9	94.4	4.2	93.7	4.8
	200	93.0	4.4	93.2	4.5	94.0	4.8
	500	100.4	4.3	97.5	3.8	96.8	4.2
BZC	50	98.2	4.8	98.8	3.6	97.7	4.3
	200	96.3	3.5	101.4	3.9	97.9	4.5
	500	99.1	4.2	103.1	3.5	98.3	4.0
TTAC	50	90.9	4.0	97.9	4.6	92.2	4.4
	200	95.2	3.9	95.8	4.6	94.8	4.2
	500	94.9	4.7	96.7	4.2	95.1	4.7
TDBAC	50	94.1	4.3	102.3	3.6	97.3	3.9
	200	93.9	3.9	99.4	3.2	97.5	4.1
	500	98.2	4.5	100.5	3.8	95.2	3.3
HTAC	50	87.9	4.9	98.9	4.4	91.9	4.9
	200	90.6	3.3	97.3	3.2	93.0	4.3
	500	94.8	3.9	99.9	3.7	93.7	4.6
HDBAC	50	92.7	4.0	98.2	4.0	94.5	4.8
	200	95.1	4.2	97.3	3.5	93.9	4.0
	500	94.4	3.8	100.1	4.2	98.1	4.2
STAC	50	90.9	4.4	95.6	4.7	93.7	4.6
	200	92.6	3.5	97.3	4.1	94.4	3.6
	500	99.2	3.9	96.1	4.5	98.0	4.0
SDBAC	100	90.3	5.1	94.3	4.1	92.2	4.3
	500	92.2	4.9	96.0	3.4	95.2	4.5
	1000	92.7	4.5	96.4	3.2	95.6	3.8
DocTAC	100	90.9	5.5	95.2	4.5	94.9	5.3
	500	91.8	4.8	93.8	4.3	94.2	4.9
	1000	96.2	5.0	94.7	3.6	95.8	5.1

图4 10种目标分析物混合标准溶液、阴性漱口水及阴性漱口水加标样品的色谱图

Fig. 4 Chromatograms of mixed standard solution, a negative mouthwash sample and a spiked mouthwash sample of the 10 target analytes Peak Nos. 1-10 are the same as those in Fig. 1.

参考文献

[1]	Zhong Y, Lin S T, Qin F M, et al. Guangdong Chemical Industry, 2019, 46(15): 36
[1]	钟瑜, 林少婷, 覃芳敏, 等. 广东化工, 2019, 46(15): 36
[2]	Yang Y W, Zhu Y. Journal of Environment and Health, 2011, 28(10): 937
[2]	杨艳伟, 朱英. 环境与健康杂志, 2011, 28(10): 937
[3]	Chen X K, Shen C, Cao S M, et al. China Dyeing & Finishing, 2021, 47(12): 53
[3]	陈小轲, 沈婵, 曹石淼, 等. 印染, 2021, 47(12): 53
[4]	Nam M, Kim S W. J Clin Med, 2021, 10(16): 3464
[5]	QB/T 5452-2019
[6]	GB/T 30931-2014
[7]	GB/T 26369-2020
[8]	GB/T 40185-2021
[9]	GB/T 39873-2021
[10]	Liu B L, Zhao Z W, Zhan Z Y, et al. Chinese Journal of Chromatography, 2023, 41(3): 233
[10]	刘柏林, 赵紫微, 詹子悦, 等. 色谱, 2023, 41(3): 233
[11]	Mishima-Kimura S, Yonemitsu K, Ohtsu Y, et al. Legal Med-Tokyo, 2018, 32: 48
[12]	Li Z M, Lakuleswaran M, Kannan K. J Chromatogr B, 2023, 1214: 123562
[13]	Slimani K, Féret A, Pirotais Y, et al. J Chromatogr A, 2017, 1517: 86
[14]	Shi J W, Yang J Q, Li Q Q. Flavour Fragrance Cosmetics, 2019(2): 48
[14]	施敬文, 杨晋青, 李清清. 香料香精化妆品, 2019(2): 48
[15]	Liu Y Q, Wang H, Yang H M, et al. Chinese Journal of Chromatography, 2011, 29(5): 458
[15]	刘艳琴, 王浩, 杨红梅, 等. 色谱, 2011, 29(5): 458
[16]	Wang Q L, Han X P, Li Z B, et al. China Surfactant Detergent & Cosmetics, 2022, 52(9): 1023
[16]	王启林, 韩晓萍, 李镇冰, 等. 日用化学工业, 2022, 52(9): 1023
[17]	Miyauchi T, Mori M, Ito K. J Chromatogr A, 2005, 1095(1/2): 74
[18]	Xue Y Y, Hieda Y, Kimura K, et al. Legal Med-Tokyo, 2002, 4(4): 232
[19]	Song B H, Ding X J, Li J, et al. Chinese Journal of Chromatography, 2012, 30(9): 943
[19]	宋宝花, 丁晓静, 李佳, 等. 色谱, 2012, 30(9): 943
[20]	Prieto-Blanco M C, Argente-García A, Campíns-Falcó P. J Chromatogr A, 2016, 1431: 176
[21]	Mousavi Z E, Butler F, Danaher M. Food Anal Method, 2012, 6(5): 1265
[22]	Liang Y K, Guo L J, Wang B M. China Surfactant Detergent & Cosmetics, 2018, 48(10): 601
[22]	梁勇坤, 郭丽娟, 王北明. 日用化学工业, 2018, 48(10): 601
[23]	Xue J, Yan W C, Xue Z H. Industrial Microbiology, 2017, 47(5): 40
[23]	薛佳, 严万春, 薛仲华. 工业微生物, 2017, 47(5): 40
[24]	Zheng G J, Lin Y K, Yang J Q. Analytical Instrumentation, 2021(4): 49
[24]	郑国建, 林毅侃, 杨晋青,. 分析仪器, 2021(4): 49
[25]	Zhang K, Jia S D, Hu D. Modern Disease Control and Prevention, 2022, 33(9): 662
[25]	张轲, 贾松岛, 胡巅. 现代疾病预防控制, 2022, 33(9): 662
[26]	Li F, Chen B, Zhu F, et al. Jiangsu Journal of Preventive Medicine, 2017, 28(6): 622
[26]	李放, 陈蓓, 朱峰, 等. 江苏预防医学, 2017, 28(6): 622
[27]	Zheng G C, Xu Y L, Dong H R, et al. Physical Testing and Chemical Analysis (Part B: Chemical Analysis), 2020, 56(10): 1102
[27]	郑国灿, 徐亚龙, 董浩然, 等. 理化检验-化学分册, 2020, 56(10): 1102
[28]	Shen C.[MS Dissertation]Shanghai: Donghua University, 2022
[28]	沈婵.[硕士学位论文]. 上海: 东华大学, 2022
[29]	Chen J B, Huang L Q, Ma L, et al. Physical Testing and Chemical Analysis (Part B: Chemical Analysis), 2022, 58(3): 299
[29]	陈建波, 黄兰淇, 马琳, 等. 理化检验-化学分册, 2022, 58(3): 299