多环芳烃羟基代谢产物检测新方法及其应用研究
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摘要
多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)是环境中普遍存在的一类有机污染物,在职业高PAHs暴露环境下,易诱发肺癌、皮肤癌等癌症。由于暴露途径多样化,采用PAHs羟基代谢产物(主要有1-羟基芘、α-萘酚、β-萘酚)作为生物标志物来综合评价人体对PAHs的暴露水平已成为人们研究的热点。因而研究1-羟基芘、α-萘酚、β-萘酚的检测新方法有着重要的现实意义。
本文第二章应用同步荧光法建立了同时检测尿样中α-萘酚、β-萘酚和1-羟基芘(1-OHP)的新方法。在乙酸钠-硼砂缓冲液(pH=5.0),以β-环糊精为增敏剂,当激发单色器和发射单色器的波长差Δλ=23nm时,进行同步扫描,可得β-萘酚同步荧光特征峰为330.6nm,1-OHP为360.2nm;改Δλ为175nm,可得α-萘酚的特征荧光峰为296.4nm。三种待测物均可根据其特征峰的高度来进行定量测定。1-OHP的测定范围为0.65-218.3μg/L ,α-萘酚为3.6-1586.0μg/L ,β-萘酚为2.8-1441.0μg/L。1-OHP、α-萘酚和β-萘酚的检出限分别为0.022μg/L、1.53μg/L、和0.78μg/L,相对标准差分别1.6%-1.8%、1.5%-2.3%和1.9%-2.4%(n=9)。方法灵敏、可靠,已用于实际样品的测定,结果满意。
本文第三章应用反相高效液相色谱-紫外检测器法建立了同时检测α-萘酚、β-萘酚和1-羟基芘的新方法。采用DiamonsilTMC18(5μm,150×4.6nm)色谱柱,以甲醇-乙酸铵缓冲溶液(70:30,v:v)为流动相,在波长280nm检测,流速为0.7mL/min。结果表明,当α-萘酚、β-萘酚和1-OHP的质量浓度分别为0.3600-18.72μg/mL,??00-18.72μg/mL,0.2700-11.88μg/mL时,峰高和质量浓度呈良好的线性关系,相对标准偏差分别为0.9%-1.1%、1.3%-2.2%、1.6%-3.2%(n=9),平均回收率分别为102%、103%和99.0%。方法准确可靠,已用于尿样中α-萘酚、β-萘酚和1-OHP的同时测定。
本文第四章应用共振光散射技术建立了1-羟基芘检测的新方法。研究表明:在pH6.5的Tris-HCl缓冲溶液中,以SDBS为增敏增稳剂,灿烂绿与1-羟基芘形成离子缔合物,导致体系共振光散射强度大大增强,体系最大RLS峰位于467.6nm,其强度与1-羟基芘浓度呈良好的线性关系。方法的线性范围为4.3-1091.3μg/L,相关系数r=0.9994,检出限1.3μg/L,RSD=2.2%-5.8%,平均回收率为98.0%。已用于人尿中痕量1-羟基芘的测定,结果与常规的高效液相色谱法一致。
Polycyclic aromatic hydrocarbons (PAHs) which are ubiquitous persistent organic pollutants may induce lung and skin cancers in occupational settings. However, due to the complexity of exposure, urinary hydroxyl PAHs which are major metabolites of PAHs in human urine have been widely used as biomarkers of internal dose to evaluate the exposure to PAHs, especially 1-hydroxypyrene(1-OHP),α-naphthol andβ-naphthol. So, there is important practical significance to develop novel methods for determination ofα-naphthol,β-naphthol and 1-hydroxypyrene.
In the chapter 2, a new method for simultaneous determination ofα-naphthol,β-naphthol and 1-hydroxypyrene has been established using synchronous fluorimetry technique. The measurement was carried out in sodium acetate-boroborax buffer solution (pH=5.0)withβ-cyclodextrin enhancing fluorescence at wavelength intervalsΔλ=23nm for 1-hydroxypyrene,β-naphthol andΔλ=175nm forα-naphthol, The linear relationship was obtained between the relative fluorescence intensity and concentration of 1-hydroxypyrene,α-naphthol andβ-naphthol in the range of 0.65-218.3μg/L, 3.6-1586.0μg/L and 2.8-1441.0μg/L, respectively. The limits of detection (LOD) for 1-hydroxypyrene,α-naphthol andβ-naphthol were 0.022μg/L, 1.53μg/L and 0.78μg/L with relative standard deviations (RSD) of 1.8%-1.6%, 2.3%-1.5% and 2.4%-1.9%(n=9), respectively. The proposed method showed sensitivity and reliability for the analysis of 1-hydroxypyrene,α-naphthol andβ-naphthol in urine samples with satisfactory results.
In the chapter 3, we established a novel method for assayingα-naphthol,β-naphthol and 1-hydroxypyrene by RP-HPLC-UV. 1-OHP,α- andβ-naphthol were separated on DiamonsilTMC18(5um, 150×4.6nm)and detected at 280nm by using methanol-ammonium acetate buffer solution (70:30, v:v) as mobile phase when the column temperature was 35.0℃and the flow rate was 0.7mL/min.The calibration curves ofα-naphthol,β-naphthol and 1-OHP showed good linearities in the ranges of 0.3600-18.72μg/mL, 0.3600-18.72μg/mL, 0.2700-11.88μg/mL, respectively. The relative standard deviations (RSD) ofα-naphthol,β-naphthol and 1-OHP were 0.9%-1.1%, 1.3%-2.2%, 1.6%-3.2%(n=9)and the average recoveries were 102%, 103% and 99.0%, respectively. The proposed method was accurate and suitable for the identification and quantification of these compositions in human urine.
In the chapter 4, a new detection method for 1-hydroxypyrene has been developed using a resonance light scattering (RLS) technique. In Tris-HCl buffer solution of pH6.5 and the presence of sodium dodecylbenzene sulfonate, the intensity of RLS was significantly enhanced due to the formation of ion-associate complex between brilliant green and 1-hydroxypyrene. The maximum RLS peak was located at 467.6nm. The enhanced intensity of RLS at this wavelength was directly propotional to the concentration of 1-OHP in the range of 4.3-1091.3μg/L(r=0.9994). The detection limit was 1.3μg/L, RSD=2.2%-5.8%, and the average recovery was 98.0%. The proposed method was used??detect 1-OHP in human urine and the results were satisfactory in comparison with those obtained by the method of high performance liquid chromatography.
本文第二章应用同步荧光法建立了同时检测尿样中α-萘酚、β-萘酚和1-羟基芘(1-OHP)的新方法。在乙酸钠-硼砂缓冲液(pH=5.0),以β-环糊精为增敏剂,当激发单色器和发射单色器的波长差Δλ=23nm时,进行同步扫描,可得β-萘酚同步荧光特征峰为330.6nm,1-OHP为360.2nm;改Δλ为175nm,可得α-萘酚的特征荧光峰为296.4nm。三种待测物均可根据其特征峰的高度来进行定量测定。1-OHP的测定范围为0.65-218.3μg/L ,α-萘酚为3.6-1586.0μg/L ,β-萘酚为2.8-1441.0μg/L。1-OHP、α-萘酚和β-萘酚的检出限分别为0.022μg/L、1.53μg/L、和0.78μg/L,相对标准差分别1.6%-1.8%、1.5%-2.3%和1.9%-2.4%(n=9)。方法灵敏、可靠,已用于实际样品的测定,结果满意。
本文第三章应用反相高效液相色谱-紫外检测器法建立了同时检测α-萘酚、β-萘酚和1-羟基芘的新方法。采用DiamonsilTMC18(5μm,150×4.6nm)色谱柱,以甲醇-乙酸铵缓冲溶液(70:30,v:v)为流动相,在波长280nm检测,流速为0.7mL/min。结果表明,当α-萘酚、β-萘酚和1-OHP的质量浓度分别为0.3600-18.72μg/mL,??00-18.72μg/mL,0.2700-11.88μg/mL时,峰高和质量浓度呈良好的线性关系,相对标准偏差分别为0.9%-1.1%、1.3%-2.2%、1.6%-3.2%(n=9),平均回收率分别为102%、103%和99.0%。方法准确可靠,已用于尿样中α-萘酚、β-萘酚和1-OHP的同时测定。
本文第四章应用共振光散射技术建立了1-羟基芘检测的新方法。研究表明:在pH6.5的Tris-HCl缓冲溶液中,以SDBS为增敏增稳剂,灿烂绿与1-羟基芘形成离子缔合物,导致体系共振光散射强度大大增强,体系最大RLS峰位于467.6nm,其强度与1-羟基芘浓度呈良好的线性关系。方法的线性范围为4.3-1091.3μg/L,相关系数r=0.9994,检出限1.3μg/L,RSD=2.2%-5.8%,平均回收率为98.0%。已用于人尿中痕量1-羟基芘的测定,结果与常规的高效液相色谱法一致。
Polycyclic aromatic hydrocarbons (PAHs) which are ubiquitous persistent organic pollutants may induce lung and skin cancers in occupational settings. However, due to the complexity of exposure, urinary hydroxyl PAHs which are major metabolites of PAHs in human urine have been widely used as biomarkers of internal dose to evaluate the exposure to PAHs, especially 1-hydroxypyrene(1-OHP),α-naphthol andβ-naphthol. So, there is important practical significance to develop novel methods for determination ofα-naphthol,β-naphthol and 1-hydroxypyrene.
In the chapter 2, a new method for simultaneous determination ofα-naphthol,β-naphthol and 1-hydroxypyrene has been established using synchronous fluorimetry technique. The measurement was carried out in sodium acetate-boroborax buffer solution (pH=5.0)withβ-cyclodextrin enhancing fluorescence at wavelength intervalsΔλ=23nm for 1-hydroxypyrene,β-naphthol andΔλ=175nm forα-naphthol, The linear relationship was obtained between the relative fluorescence intensity and concentration of 1-hydroxypyrene,α-naphthol andβ-naphthol in the range of 0.65-218.3μg/L, 3.6-1586.0μg/L and 2.8-1441.0μg/L, respectively. The limits of detection (LOD) for 1-hydroxypyrene,α-naphthol andβ-naphthol were 0.022μg/L, 1.53μg/L and 0.78μg/L with relative standard deviations (RSD) of 1.8%-1.6%, 2.3%-1.5% and 2.4%-1.9%(n=9), respectively. The proposed method showed sensitivity and reliability for the analysis of 1-hydroxypyrene,α-naphthol andβ-naphthol in urine samples with satisfactory results.
In the chapter 3, we established a novel method for assayingα-naphthol,β-naphthol and 1-hydroxypyrene by RP-HPLC-UV. 1-OHP,α- andβ-naphthol were separated on DiamonsilTMC18(5um, 150×4.6nm)and detected at 280nm by using methanol-ammonium acetate buffer solution (70:30, v:v) as mobile phase when the column temperature was 35.0℃and the flow rate was 0.7mL/min.The calibration curves ofα-naphthol,β-naphthol and 1-OHP showed good linearities in the ranges of 0.3600-18.72μg/mL, 0.3600-18.72μg/mL, 0.2700-11.88μg/mL, respectively. The relative standard deviations (RSD) ofα-naphthol,β-naphthol and 1-OHP were 0.9%-1.1%, 1.3%-2.2%, 1.6%-3.2%(n=9)and the average recoveries were 102%, 103% and 99.0%, respectively. The proposed method was accurate and suitable for the identification and quantification of these compositions in human urine.
In the chapter 4, a new detection method for 1-hydroxypyrene has been developed using a resonance light scattering (RLS) technique. In Tris-HCl buffer solution of pH6.5 and the presence of sodium dodecylbenzene sulfonate, the intensity of RLS was significantly enhanced due to the formation of ion-associate complex between brilliant green and 1-hydroxypyrene. The maximum RLS peak was located at 467.6nm. The enhanced intensity of RLS at this wavelength was directly propotional to the concentration of 1-OHP in the range of 4.3-1091.3μg/L(r=0.9994). The detection limit was 1.3μg/L, RSD=2.2%-5.8%, and the average recovery was 98.0%. The proposed method was used??detect 1-OHP in human urine and the results were satisfactory in comparison with those obtained by the method of high performance liquid chromatography.
引文
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