CN106769960A - 一种地塞米松含量的检测方法 - Google Patents
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Abstract
本发明公开了一种地塞米松含量的检测方法,包括如下步骤:步骤一:取样品溶于0.06‑0.15%二甲基亚矾的不饱和有机溶液中,所述不饱和有机溶液是丙二醇、乙腈、甲醇或三乙胺,制备出不同浓度的样品溶液,浓度为500‑800mg/L;步骤二:将上述不同浓度的样品溶液放入紫外分光光度计进行检测,检测波长为200‑300nm,所得吸光度绘制标准曲线,并按照标准曲线根据比尔‑朗伯定律得出线性回归方程。本发明能克服传统的检测方法存在的检测周期长、步骤繁琐、费时费力等缺点,使检测时间大大缩短,操作步骤和劳动强度也大为缩减,达到省时省力、快速灵敏的要求。
Description
技术领域
本发明涉及一种检测方法,具体是一种地塞米松含量的检测方法。
背景技术
地塞米松(Dexamethasone,DSMS)又名氟美松、氟甲强地松龙、德沙美松,是一种人工合成的肾上腺皮质激素。是糖皮质类激素类抗炎、抗过敏药物,其药理作用主要是抗炎、抗毒、抗过敏、抗风湿,临床使用较广泛。近年来,欧盟认为食用含有雌烯二醇、黄体酮、甲羟孕酮等激素的牛肉可扰乱人体内分泌、生长发育、免疫系统、生殖系统等方面的正常功能,还可能致癌、致畸,因而禁止这类激素在动物中的使用。此外,地塞米松还作为动物生长调节剂,促进动物蛋白质合成和代谢,增加产肉量,曾经被广泛应用作为家畜、家禽、使用。但通过长期的实验室研究发现,地塞米松可以使实验动物发生癌变和基因突变,地塞米松及其残留对人、畜均有明显的毒副作用,使用高剂量的地塞米松可导致肌肉萎缩、生长抑制等副作用。并由此推断,人类长期使用该类药物或长期食用添加该类促生长剂的家畜、家禽、同样也可发生癌变和基因突变。所以此类药物禁止在治疗和饲料中使用。我国于2002年在农业部发布的235号公告的《动物性食品中兽药最高残留限量》中,地塞米松在所有食品动物肌肉中不得超过0.75ug/kg肝脏中不得超过2ug/kg因此,为确保动物源性食品的安全和对外出口贸易的发展,建立准确可靠,灵敏度高的定性定量方法是十分必要的。
发明内容
本发明的目的在于提供一种地塞米松含量的检测方法,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:
一种地塞米松含量的检测方法,包括如下步骤:步骤一:取样品溶于0.06-0.15%二甲基亚矾的不饱和有机溶液中,所述不饱和有机溶液是丙二醇、乙腈、甲醇或三乙胺,制备出不同浓度的样品溶液,浓度为500-800mg/L;步骤二:将上述不同浓度的样品溶液放入紫外分光光度计进行检测,检测波长为200-300nm,所得吸光度绘制标准曲线,并按照标准曲线根据比尔-朗伯定律得出线性回归方程;步骤三:用包被缓冲液将地塞米松半抗原与载体蛋白偶联物或抗体以0.02-0.08µg/ml浓度稀释成抗原稀释液或抗体稀释液;向酶联板的每孔中加入100µl已经稀释好的抗原稀释液或抗抗体稀释液,37℃温育2h,倾去包被液,用洗涤液洗涤4次,每次15-30s,拍干;向酶联板的每孔中加入150-200µl封闭液,37℃温育1-2h,倾去孔内液体,干燥后用铝膜真空密封保存;步骤四:将待测样品按步骤一配制后,在200-300nm的波长下进行检测吸光度,然后按以上线性回归方程计算出地塞米松磷酸钠注射液中间体的含量。
作为本发明进一步的方案:所述的缓冲液为pH值9.6、0.05mol/L、含有0.5%甲醇的碳酸盐缓冲液;所述的洗涤液为含有8%-15%吐温-20℃的去离子水或超纯水;所述的封闭液为含有2%-6%的脱脂奶和1%惰性蛋白的溶液;所述的载体蛋白为牛血清白蛋白、人血清白蛋白、人血纤维蛋白或血蓝蛋白。
作为本发明进一步的方案:所述地塞米松磷酸钠注射液中间体不饱和有机溶液的浓度为500-1000mg/L。
作为本发明进一步的方案:所述步骤三中检测吸光度在242nm的波长下进行。
作为本发明进一步的方案:所述不饱和有机溶液是丙二醇。
作为本发明再进一步的方案:所述步骤三检测之前还包括先将待测地塞米松磷酸钠注射液中间体样品稀释10-20倍,测定样品的吸光度,接着对数据进行5点三次平滑处理,将平滑处理后的数据代入线性回归方程,计算该样品中地塞米松磷酸钠注射液中间体的含量。
与现有技术相比,本发明的有益效果是:本发明能克服传统的检测方法存在的检测周期长、步骤繁琐、费时费力等缺点,使检测时间大大缩短,操作步骤和劳动强度也大为缩减,达到省时省力、快速灵敏的要求。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述。
本发明实施例中,一种地塞米松含量的检测方法,包括如下步骤:步骤一:取样品溶于0.06-0.15%二甲基亚矾的不饱和有机溶液中,所述不饱和有机溶液是丙二醇、乙腈、甲醇或三乙胺,制备出不同浓度的样品溶液,浓度为500-800mg/L;步骤二:将上述不同浓度的样品溶液放入紫外分光光度计进行检测,检测波长为200-300nm,所得吸光度绘制标准曲线,并按照标准曲线根据比尔-朗伯定律得出线性回归方程;步骤三:用包被缓冲液将地塞米松半抗原与载体蛋白偶联物或抗体以0.02-0.08µg/ml浓度稀释成抗原稀释液或抗体稀释液;向酶联板的每孔中加入100µl已经稀释好的抗原稀释液或抗抗体稀释液,37℃温育2h,倾去包被液,用洗涤液洗涤4次,每次15-30s,拍干;向酶联板的每孔中加入150-200µl封闭液,37℃温育1-2h,倾去孔内液体,干燥后用铝膜真空密封保存;步骤四:将待测样品按步骤一配制后,在200-300nm的波长下进行检测吸光度,然后按以上线性回归方程计算出地塞米松磷酸钠注射液中间体的含量。所述的缓冲液为pH值9.6、0.05mol/L、含有0.5%甲醇的碳酸盐缓冲液;所述的洗涤液为含有8%-15%吐温-20℃的去离子水或超纯水;所述的封闭液为含有2%-6%的脱脂奶和1%惰性蛋白的溶液;所述的载体蛋白为牛血清白蛋白、人血清白蛋白、人血纤维蛋白或血蓝蛋白。所述地塞米松磷酸钠注射液中间体不饱和有机溶液的浓度为500-1000mg/L。所述步骤三中检测吸光度在242nm的波长下进行。所述不饱和有机溶液是丙二醇。所述步骤三检测之前还包括先将待测地塞米松磷酸钠注射液中间体样品稀释10-20倍,测定样品的吸光度,接着对数据进行5点三次平滑处理,将平滑处理后的数据代入线性回归方程,计算该样品中地塞米松磷酸钠注射液中间体的含量。
实施例1:
本发明地塞米松含量的检测方法,包括如下步骤:步骤一:取样品溶于0.06%二甲基亚矾的不饱和有机溶液中,所述不饱和有机溶液是丙二醇、乙腈、甲醇或三乙胺,制备出不同浓度的样品溶液,浓度为500mg/L;步骤二:将上述不同浓度的样品溶液放入紫外分光光度计进行检测,检测波长为200nm,所得吸光度绘制标准曲线,并按照标准曲线根据比尔-朗伯定律得出线性回归方程;步骤三:用包被缓冲液将地塞米松半抗原与载体蛋白偶联物或抗体以0.02µg/ml浓度稀释成抗原稀释液或抗体稀释液;向酶联板的每孔中加入100µl已经稀释好的抗原稀释液或抗抗体稀释液,37℃温育2h,倾去包被液,用洗涤液洗涤4次,每次15s,拍干;向酶联板的每孔中加入150µl封闭液,37℃温育1h,倾去孔内液体,干燥后用铝膜真空密封保存;步骤四:将待测样品按步骤一配制后,在200nm的波长下进行检测吸光度,然后按以上线性回归方程计算出地塞米松磷酸钠注射液中间体的含量。所述的缓冲液为pH值9.6、0.05mol/L、含有0.5%甲醇的碳酸盐缓冲液;所述的洗涤液为含有8%吐温-20℃的去离子水或超纯水;所述的封闭液为含有2%的脱脂奶和1%惰性蛋白的溶液;所述的载体蛋白为牛血清白蛋白、人血清白蛋白、人血纤维蛋白或血蓝蛋白。所述地塞米松磷酸钠注射液中间体不饱和有机溶液的浓度为500mg/L。所述步骤三中检测吸光度在242nm的波长下进行。所述不饱和有机溶液是丙二醇。所述步骤三检测之前还包括先将待测地塞米松磷酸钠注射液中间体样品稀释10倍,测定样品的吸光度,接着对数据进行5点三次平滑处理,将平滑处理后的数据代入线性回归方程,计算该样品中地塞米松磷酸钠注射液中间体的含量。
实施例2:
本发明地塞米松含量的检测方法,包括如下步骤:步骤一:取样品溶于0.15%二甲基亚矾的不饱和有机溶液中,所述不饱和有机溶液是丙二醇、乙腈、甲醇或三乙胺,制备出不同浓度的样品溶液,浓度为800mg/L;步骤二:将上述不同浓度的样品溶液放入紫外分光光度计进行检测,检测波长为300nm,所得吸光度绘制标准曲线,并按照标准曲线根据比尔-朗伯定律得出线性回归方程;步骤三:用包被缓冲液将地塞米松半抗原与载体蛋白偶联物或抗体以0.08µg/ml浓度稀释成抗原稀释液或抗体稀释液;向酶联板的每孔中加入100µl已经稀释好的抗原稀释液或抗抗体稀释液,37℃温育2h,倾去包被液,用洗涤液洗涤4次,每次30s,拍干;向酶联板的每孔中加入200µl封闭液,37℃温育2h,倾去孔内液体,干燥后用铝膜真空密封保存;步骤四:将待测样品按步骤一配制后,在300nm的波长下进行检测吸光度,然后按以上线性回归方程计算出地塞米松磷酸钠注射液中间体的含量。所述的缓冲液为pH值9.6、0.05mol/L、含有0.5%甲醇的碳酸盐缓冲液;所述的洗涤液为含有15%吐温-20℃的去离子水或超纯水;所述的封闭液为含有6%的脱脂奶和1%惰性蛋白的溶液;所述的载体蛋白为牛血清白蛋白、人血清白蛋白、人血纤维蛋白或血蓝蛋白。所述地塞米松磷酸钠注射液中间体不饱和有机溶液的浓度为1000mg/L。所述步骤三中检测吸光度在242nm的波长下进行。所述不饱和有机溶液是丙二醇。所述步骤三检测之前还包括先将待测地塞米松磷酸钠注射液中间体样品稀释20倍,测定样品的吸光度,接着对数据进行5点三次平滑处理,将平滑处理后的数据代入线性回归方程,计算该样品中地塞米松磷酸钠注射液中间体的含量。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (6)
1.一种地塞米松含量的检测方法,其特征在于,包括如下步骤:步骤一:取样品溶于0.06-0.15%二甲基亚矾的不饱和有机溶液中,所述不饱和有机溶液是丙二醇、乙腈、甲醇或三乙胺,制备出不同浓度的样品溶液,浓度为500-800mg/L;步骤二:将上述不同浓度的样品溶液放入紫外分光光度计进行检测,检测波长为200-300nm,所得吸光度绘制标准曲线,并按照标准曲线根据比尔-朗伯定律得出线性回归方程;步骤三:用包被缓冲液将地塞米松半抗原与载体蛋白偶联物或抗体以0.02-0.08µg/ml浓度稀释成抗原稀释液或抗体稀释液;向酶联板的每孔中加入100µl已经稀释好的抗原稀释液或抗抗体稀释液,37℃温育2h,倾去包被液,用洗涤液洗涤4次,每次15-30s,拍干;向酶联板的每孔中加入150-200µl封闭液,37℃温育1-2h,倾去孔内液体,干燥后用铝膜真空密封保存;步骤四:将待测样品按步骤一配制后,在200-300nm的波长下进行检测吸光度,然后按以上线性回归方程计算出地塞米松磷酸钠注射液中间体的含量。
2.根据权利要求1所述的地塞米松含量的检测方法,其特征在于,所述的缓冲液为pH值9.6、0.05mol/L、含有0.5%甲醇的碳酸盐缓冲液;所述的洗涤液为含有8%-15%吐温-20℃的去离子水或超纯水;所述的封闭液为含有2%-6%的脱脂奶和1%惰性蛋白的溶液;所述的载体蛋白为牛血清白蛋白、人血清白蛋白、人血纤维蛋白或血蓝蛋白。
3.根据权利要求1所述的地塞米松含量的检测方法,其特征在于,所述地塞米松磷酸钠注射液中间体不饱和有机溶液的浓度为500-1000mg/L。
4.根据权利要求1所述的地塞米松含量的检测方法,其特征在于,所述步骤三中检测吸光度在242nm的波长下进行。
5.根据权利要求1所述的地塞米松含量的检测方法,其特征在于,所述不饱和有机溶液是丙二醇。
6.根据权利要求1所述的地塞米松含量的检测方法,其特征在于,所述步骤三检测之前还包括先将待测地塞米松磷酸钠注射液中间体样品稀释10-20倍,测定样品的吸光度,接着对数据进行5点三次平滑处理,将平滑处理后的数据代入线性回归方程,计算该样品中地塞米松磷酸钠注射液中间体的含量。
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