Kawaguchi Migaku, Ito Rie, Hayatsu Yoshio, Nakata Hisao, Sakui Norihiro, Okanouchi Noriya, Saito Koichi, Yokota Hiroshi, Izumi Shun-ichiro, Makino Tsunehisa, Nakazawa Hiroyuki
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Tokyo 142-8501, Japan.
J Pharm Biomed Anal. 2006 Jan 23;40(1):82-7. doi: 10.1016/j.jpba.2005.05.024. Epub 2005 Jul 12.
4-Nonylphenol glucuronide (NP-G) in human urine samples was analyzed using stir bar sorptive extraction (SBSE) with in situ de-conjugation by beta-glucuronidase and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Distilled water (1 ml), 1.0 M ammonium acetate solution (100 microl) and beta-glucuronidase (10,000 units ml(-1), 10 microl) were added to human urine sample (1 ml), and extraction was commenced for 90 min at 37 degrees C while stirring at 250 rpm with a stir bar coated with a 500-microm-thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was subjected to TD-GC-MS in the selected ion monitoring (SIM) mode. The calibration curve was made by SBSE method using 4-nonylphenol (NP) as the standard solution. The method showed good linearity and the correlation coefficients were 0.999 over the concentration range of 5-500 nM. Moreover, to optimize the conditions for SBSE with in situ de-conjugation and the recovery test, NP-G was synthesized by a biochemical technique in our laboratory. The limits of detection (S/N = 3) and quantitation (S/N > 10) for NP were 0.2 ng ml(-1) (1.0 nM) and 1.1 ng ml(-1) (5.0 nM), respectively. The average recoveries in the human urine samples (n = 6) spiked with NP-G at levels of 20 and 100 nM were 104.1 (R.S.D. 7.1%) and 100.6% (R.S.D. 9.2%), respectively, with correction using the added internal standard, 4-(1-methyl) octylphenol-d(5). The method enabled the precise determination of the standard and was applicable to the detection of trace amounts of NP-G in human urine samples.
采用搅拌棒吸附萃取（SBSE）结合β-葡萄糖醛酸酶原位去结合及热脱附（TD）-气相色谱-质谱联用（GC-MS）法分析人尿液样本中的4-壬基酚葡萄糖醛酸苷（NP-G）。向1 ml人尿液样本中加入1 ml蒸馏水、100 μl 1.0 M醋酸铵溶液和10 μl β-葡萄糖醛酸酶（10000单位ml⁻¹），在37℃下以250 rpm转速搅拌，同时用涂有500 μm厚聚二甲基硅氧烷（PDMS）层的搅拌棒萃取90分钟。然后，将搅拌棒置于选定离子监测（SIM）模式下进行TD-GC-MS分析。以4-壬基酚（NP）为标准溶液，采用SBSE法绘制校准曲线。该方法线性良好，在5 - 500 nM浓度范围内相关系数为0.999。此外，为优化原位去结合SBSE条件及回收率测试，我们实验室采用生化技术合成了NP-G。NP的检测限（S/N = 3）和定量限（S/N > 10）分别为0.2 ng ml⁻¹（1.0 nM）和1.1 ng ml⁻¹（5.0 nM）。在添加了20 nM和100 nM NP-G的人尿液样本（n = 6）中，使用添加的内标4-(1-甲基)辛基酚-d(5)进行校正后，平均回收率分别为104.1%（相对标准偏差7.1%）和100.6%（相对标准偏差9.2%）。该方法能够精确测定标准品，适用于人尿液样本中痕量NP-G的检测。
