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GC-ECD法测定珠子参中有机氯农药残留量



全 文 :*基金项目:痛舒胶囊大品种培育示范项目(编号:2010BC007)
△通信作者:赵毅,男,云南人,正高级工程师,主要从事药品质量标准提高方面的研究,E-mail:yf_niu@126.com.
·实验研究·
GC-ECD法测定珠子参中有机氯农药残留量*
游 燕,牛延菲,杨晓燕,徐 怡,赵红伟,赵 毅△
(云南省药物研究所,云南 昆明 650111)
  摘 要:目的:建立了气相色谱一电子捕获检测(ECD)法
测定珠子参中有机氯农药残留方法。方法:采用 Agilent
DB-624毛细管柱(30m×0.32mm×1.8um);升温程序:初
始温度100℃,15℃/min升至220℃,再以 10℃/min升至
250℃,保持17min;进样量:1.0ul。结果:六六六(BHC)、滴
滴涕(DDT)、五氯硝基苯(PCNB)对照品均在0~400ug/l浓
度范围内呈现良好的线性关系。结论:该方法准确可行,重复
性好,可用于珠子参及其制剂的有机氯农残检测。
关键词:珠子参;有机氯农残;GC-ECD
中图分类号:R284 文献标识码:A
文章编号:1007-2349(2013)07-0054-03
有机氯农药是一类广谱的杀虫剂,曾经在中国最大规模
的使用,虽然有机氯农药被禁用了多年,但六六六,滴滴涕等
有机氯农药和他们的代谢产物化学性质稳定,在农作物和环
境中消解缓慢,由此造成了有机氯农药在土壤中的大量残
留[1]。中药材的种植期较长,尤其是多年生的根类药材,更易
受有机氯农药的污染,所以有机氯农药残留在中药材的检出
率相当高。而有机氯农药易在脂肪中积累,造成慢性中毒,危
害人体健康,所以有必要建立各种中药材的有机氯农残检测
方法。珠子参是一种中药药材,来源于五加科植物珠子参的
干燥根状茎,主产于甘肃、陕西、宁夏、山西及我国西南地区。
其气微,味苦、微甘,嚼之刺喉,具有补肺,养阴,活络,止血,是
我国最常用的中药之一[2]。本实验选取珠子参为研究对象,
建立了气相色谱一电子捕获检测(ECD)法测定珠子参中六六
六(BHC)、滴滴涕(DDT)和五氯硝基苯(PCNB)3种有机氯农
药残留方法,为珠子参的安全使用提供了科学的质量控制依
据,同时也为中药材有机氯农药残留检测方法提供借鉴。
1 实验部分
1.1 仪器与试药 Agilent 6890N气相色谱仪,Ni63电子捕
获检 测 器 (ECD),自 动 进 样 器,Agilent 化 学 工 作 站,
SK8200HP型超声波清洗仪,TDl-40B离心机,N-1001型
旋转蒸发仪,AG285型电子天平。
有机氯农药对照品包括六六六的4种异构体、滴滴涕的
四种异构体、五氯硝基苯,其中六六六(BHC)包括:甲体六六
六(α-BHC)、乙体六六六(β-BHC)、丙体六六六(γ-BHC)、
丁体六六六(δ-BHC)四种异构体(质量分数大于99.0%);滴
滴涕(DDT)包括PP′-DDE、PP′-DDD、OP′-DDE、PP′-
DDT四种异构体(质量分数大于99.0%);五氯硝基苯(PC-
NB,质量分数大于 98.1%)。所有对照品质量浓度均为
100μg/ml,均由农业部环境保护科研检测所提供。
丙酮(分析纯);石油醚(分析纯60~90℃);二氯甲烷(分
析纯);氯化钠和无水硫酸钠均为分析纯;硫酸(分析纯);水为
去离子水;二氯甲烷、石油醚、丙酮均经蒸馏装置重蒸馏。
10批珠子参药材样品采自珠子参主产地。
1.2 方法与结果
1.2.1 色谱条件 Agilent DB-624弹性石英毛细管柱
(30m×0.32mm×1.8um),升温程序:初始温度100℃,
15℃/min升至220℃,再以10℃/min升至250℃,保持17min;
进样口温度230℃,检测器温度300℃,采用不分流进样,进样
量:1.0ul;载气为高纯氮(质量分数>99.999%),总压力:22
kPa,总流量:53.1mL/min,电流:1nA。理论塔板数按ɑ-
BHC峰计算不低于3.9×105,两个相邻色谱峰的分离度大
于2。
1.2.2 溶液的制备
1.2.2.1 混合对照品溶液的制备 精密量取α-BHC、β-
BHC、γ-BHC、δ-BHC、PP′-DDE、PP′-DDD、OP′-DDE、
PP′-DDT、PCNB对照液各0.5mL,置25mL量瓶中,加石
油醚(60~90℃)至刻度,即得质量浓度为2μg/mL的混合对
照品贮备液。精密量取2μg/mL的混合对照品储备液适量,
加石油醚(60~90℃)制成5、10、40、100、200、400μg/L的混
合对照溶液。
1.2.2.2 供试品溶液的制备 取样品于60℃干燥4h,粉碎,
过5号筛,取约2g,精密称定,置100mL具塞锥形瓶中,加人
20mL蒸馏水浸泡过夜,精密加丙酮40mL,称定重量,超声
处理30min,放冷至室温,再称定重量,用丙酮补足减失的重
量,再加氯化钠约6g,精密加二氯甲烷30mL,称定重量,超
声处理15min,放冷至室温,再称定重量,用二氯甲烷补足减
失的重量,静置(使分层),将有机相迅速转入装有适量无水硫
酸钠的100mL具塞瓶中,放置4h。精密量取35mL,于40℃
45 云 南 中 医 中 药 杂 志          2013年第34卷第7期
水浴上减压浓缩至近干,加少量石油醚(60~90℃)如前反复
操作至二氯甲烷及丙酮除净,用石油醚(60~90℃)溶解并转
移至10ml具塞刻度离心管中,加石油醚(60~90℃)精密稀释
到5ml。小心加入浓硫酸1mL,振摇1min,3000r/min离心
10min。精密量取2.0mL上清液,置具刻度的浓缩瓶中,连
接旋转蒸发器,40℃下将溶液浓缩至适量,精密稀释至1.0
mL,即得供试品溶液[3]。
1.2.3 测定法 精密吸取供试品溶液和与之相对应浓度的
混合对照品溶液各1uL,分别连续进样3次,取3次平均值,
按外标法计算供试品中9种有机氯农药残留量。
1.3 分析方法学考察
1.3.1 系统适应性试验 按供试品溶液制备供试品溶液方
法制备供试品溶液和缺有机氯农残的阴性供试品溶液,按照
“2.1”项色谱条件分别进样对照品、阴性样品、供试品1ul,结
果见图1。由图谱可以看出供试品溶液色谱中与对照品溶液
色谱相应的位置上有相同的色谱峰出现。而阴性样品无此峰
出现,表明此处理方法对有机氯农残的含量测定无干扰。
图1 系统适应性图谱
(图A-对照品溶液,图B-阴性样品,图C-样品溶液)
1.3.2 线性关系考察 分别精密量取适量混合对照品贮备
液,用石油醚 (60~90℃)稀 释 成:0、5、10、40、100、200、
400ug/L,7个质量浓度梯度的对照品溶液。各取1uL注人
气相色谱仪,依2.1色谱条件。分别以各组分峰面积为横坐
标,质量浓度(ug/L)为纵坐标绘制标准曲线,计算标准曲线方
程和相关系数。结果表明,在上述色谱条件下,各对照品在
0~400ug/L范围内呈良好的线性关系,结果见表1。
表1 标准曲线方程
农药名称 回归方程 r 线性范围(ug/l)
ɑ-BHC  Y=213.625X+159.577  0.9999  0~400
β-BHC  Y=116.103X+388.975  0.9999  0~400
r-BHC  Y=264.810X+113.731  0.9999  0~400
-BHC  Y=155.181X-166.310  0.9999  0~400
PP′-DDE  Y=289.210X+602.864  0.9998  0~400
PP′-DDD  Y=232.476X+605.987  0.9997  0~400
OP′-DDT  Y=79.204X-475.423  0.9995  0~400
PP′-DDT  Y=52.548X-357.740  0.9994  0~400
PCNB  Y=244.523X+990.929  0.9996  0~400
1.3.3 加样回收率试验 精密称取6份供试品(样品来源云
南老君山,含量分别为:总BHC-0.019mg/kg;总-DDT未
检出;PCNB-0.005mg/kg)每份约2g,分别加入浓度为
100ug/L的混合对照品各1.0mL,混合后挥干溶剂。按
“2.2.2”项制备供试品,按照“2.1”项色谱条件,测定含量,计
算其回收率。RSD分别为:0.9%、0.8%、1.1%,表明该方法
的回收率较好,结果见表2。
表2 加样回收率测定结果
称样量
(g)
样品中农残含量(ng) 加入对照品量(ng) 加样后测得值(ng) 回收率(%)

-BHC

-DDT
PCNB

-BHC

-DDT
PCNB

-BHC

-DDT
PCNB

-BHC

-DDT
PCNB
2.0001  38 - 10.00  400  400  100  445.72 473.97 103.69 101.87 118.49 93.69
2.0063 38.12 - 10.03  400  400  100  451.73 475.84 104.44 103.37 118.96 94.41
1.9997 37.99 - 9.999  400  400  100  449.99 479.23 103.45 102.94 119.81 93.451
2.0013 38.02 - 10.01  400  400  100  452.95 483.89 104.72 103.68 120.97 94.71
2.0321 38.61 - 10.16  400  400  100  455.93 477.81 106.29 104.42 119.45 96.13
2.0330 38.63 - 10.17  100  400  100  452.64 478.55 105.44 103.6 119.64 95.27
RSD(%) - - - - - - - - - 0.9  0.8  1.1
  -为未检出
1.3.4 方法的最低检测限实验 配制质量浓度为0.5ug/L
和1.0ug/L的混合对照品溶液进样,以3倍的噪声作为方法
的最低检出量,测得α-BHC、β-BHC、γ-BHC、δ-BHC、
552013年第34卷第7期          云 南 中 医 中 药 杂 志
PP′-DDE、PP′-DDD、OP′-DDE、PP′-DDT、PCNB的最低
检测限浓度均为50ng/l。
1.3.5 精密度试验 精密吸取40ug/L混合对照品溶液,按
照“2.1”项色谱条件,重复进样6次,每次进样1uL,测定峰面
积,RSD分别为:0.5%、1.4%、0.5%,表明该方法的仪器精密
度较好。
1.3.6 重复性试验 精密称取样品(样品来源云南老君山),
按“2.2.2”项制备供试品,平行试验6份,按照“2.1”项色谱条
件,进样1uL,测定峰面积,总-BHC、PCNB的 RSD分别为:
1.9%、2.0%,表明该方法具有良好的重复性。
1.3.7 稳定性试验 取同一份供试品溶液(样品来源云南老
君山),按照“2.1”项色谱条件,于0、2、4、6、8、12h分别进样1
uL,测定峰面积,总-BHC、PCNB 的 RSD 分别为:1.6%、
1.2%,表明供试品溶液在12h内稳定。
1.3.8 样品测定 精密称取10批珠子参样品,按“2.2.2”项
制备供试品,按照“2.1”项色谱条件,进样1uL,测定含量,结
果见表3。
表3 10批珠子参药材有机氯农药测定结果
来源
残留量(mg·kg-1)
总BHC 总DDC  PCNB
云南老君山 2.08×10-2 - 5.60×10-3
陕西秦岭太白山 1.46×10-2 - 9.14×10-3
云南迪庆白雪山 1.86×10-2 - 1.42×10-2
云南丽江玉龙雪山 1.82×10-2 - 5.07×10-3
四川阿坝地区 1.59×10-2 - 9.88×10-3
云南德宏 1.82×10-2 - -
云南丽江 2.42×10-2 - -
云南大理 1.86×10-2  4.35×10-3 -
云南丽江 - - 3.36×10-3
云南香格里拉 1.06×10-2 - 1.35×10-2
  -为未检出
方法学考察结果证明,该方法灵敏度,精密度好,对珠子
参中有机氯农药残留的测定简便、结果科学可靠。
2 讨论
2.1 样品前处理的选择 样品前处理采用超声提取一硫酸
磺化法[4~5],由于每批样品形状上的差异,其有机层的色素量
均有差别,在用浓硫酸净化时,可适当增加振摇时间及硫酸
量,直至硫酸层近无色,必要时使用漩涡混悬器。
2.2 色谱柱选择 本实验对色谱柱进行了选择,使用 HP-5
及HP-1,DB-624这3种色谱柱分别进行分析,发现HP-5
柱对PP′-DDD,OP′-DDT的分离度只达到1.16,而 HP-1
不能完全分离9种农残对照品,而使用DB-624柱,可在较短
时间内完成分析,分离度达到3.91。
2010年版《中国药典》未对珠子参中的农药残留量进行限量,
参考同版药典中黄芪和甘草农药残留量限度规定[3](总六六
六和总DDT均不得过千万分之二、五氯硝基苯不得过千万分
之一)、以及国外贸行业《药用植物及制剂进出口绿色行业标
准》中关于农药残留量限度规定(总六六六和总DDT均不得
过0.2mg/kg、五氯硝基苯不得过0.1mg/kg),由表2的数据
可以看出,本文所涉及的珠子参产地中有机氯农药残留均不
超标,且远远低于参考限度,但不同产地珠子参之间有机氯农
药残留有一定的差异,如所收集的云南大理地区的样品中检
测出了DDT而其他产地均未检出,云南丽江地区的样品中未
检测到六六六而其他产地均有检出,为了使中药材能更好的
走出国门,建议规范中药材的种植,建立 GAP基地。本研究
制定的珠子参有机氯农药的检测方法,准确可行,为更好控制
珠子参质量提供了科学依据。
参考文献:
[1]成启刚,寇登民,张静,等 .气相色谱法测定人参、黄芪中有机氯农
药残留[J].南开大学学报:自然科学版,2004,37(2):125~128.
[2]段启,许冬瑾,李彩萍 .GC-ECD法测定白术中有机氯农药残
留量[J].中草药期刊,2009,40(11):1825~1827.
[3]国家药典委员会 .中华人民共和国药典[M].一部 .北京:中国医
药科技出版社,2010.
[4]赵春杰,郝桂明,李欢欣 .毛细管气相色谱法测定党参中有机氯类
农药残留量[J].中国药学杂志,2002.7:25.
[5]陈建民,张曙明,刘慧灵,等 .中药中有机氯农药残留量检测方法
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(收稿日期:2013-05-29
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殩殩


抗 击 爱 滋,关 爱 生 命!
65 云 南 中 医 中 药 杂 志          2013年第34卷第7期
Yunnan Journal of Traditional Chinese Medicine and Materia Medica
Vol. 34. No. 7 (General No. 241) July,2013
Main Content
Effect of Dange Wulingsan on the Cardiac Function and Left Ventricular Ejection Fraction of Chronic Heart Failure Patients
HUANG Xiu - xie,HUANG Jin - long,NIU Yu - jie,et al………………………………………………………………………………………
Study on TCM Physical Characteristics of Hypertension PTS and Correlation of PTS Molecular Markers in Nanning,Guangxi
LU Jian - qi,WANG Qing - gao,LI Cheng - lin,et al……………………………………………………………………………………………
GC - ECD Method for the Determination of Organochlorine Pesticides Residuesin Rhizoma Panaois Majoris
YOU Yan,NIU Yan - fei,YANG Xiao - yan,et al………………………………………………………………………………………………
Clinical Effect of One Conventional Drynaria Fortunei in Yunnan on the Treatment of Rat Osteoporosis Induced by Ovary Removal
ZHENG Yong - ren,YANG He - jin,ZHAO Chun - mei,et al……………………………………………………………………………………
Effect of Dange Wulingsan on the Cardiac Function and Left Ventricular Ejection Fraction of Chronic Heart Failure Patients
HUANG Xiu - xie,HUANG Jin - long,NIU Yu - jie,et al
(Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine,Nanning 530011,Guangxi)
Abstract:Objective:To observe the clinical effect of conventional western medicine combined with Dange Wulingsan on the treatment of chronic
heart failure patients. Methods:96 patients were randomly divided into one observation group and one control group,48 patients per group. The patients
in the control group were treated with conventional western medicine while the patients in the observation group were added with Dange Wulingsan
treatment besides western medicine treatment. 4 weeks was a course of treatment. The clinical symptoms,cardiac function and left ventricular ejection
fraction (LVEF)levels of the two groups were observed before and after the treatment. Results:After the treatment,the clinical symptoms improved
significantly,the cardiac function became better and the left ventricular ejection fraction was significantly increased. Compared with the control group,
the difference was statistically significant (P < 0. 05). Conclusion:Dange Wulingsan has a good clinical effect on the treatment of chronic heart failure
patients.
Key words:Dange Wulingsan,chronic heart failure,LVEF
Study on TCM Physical Characteristics of Hypertension PTS and Correlation of PTS Molecular Markers in Nanning,Guangxi
LU Jian - qi,WANG Qing - gao,LI Cheng - lin,et al
(The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine,Nanning 530023,Guangxi)
Abstract:Objective:To study the correlation of TCM physical constitution and PTS molecular markers through clinical studies and survey of the
physical characteristics of hypertension PTS and lay the foundation for community prevention programs. Methods:Hypertensive patients and healthy
persons were taken as subjects. 240 hypertensive patients and 30 healthy persons were investigated and their PTS hypertension molecular markers were
determined. The survey results were analyzed by statistical software. Results:1. The common constitutions of the hypertension PTS were yin deficiency,
phlegm - damp and qi deficiency. Yin deficiency and yang deficiency mainly spread above 60 - year - old persons. By chi - square test,the difference
was statistically significant (P < 0. 05). 2. The constituent ratio of gender and hypertension PTS physical constitutions had no significant difference. 3
The major TCM constitutions of the hypertension PTS had some correlation with the PTS markers. The yin deficiency,qi deficiency and phlegm - damp
types showed certain positive correlation with vWF,11 DH TXB2,GMP 140,Fib of the PTS molecular markers and negative correlation with AT. The
vWF,11 DH TXB2,GMP 140,Fib level of the PTS markers rose with the increasing age,but AT is opposite. 4. Except damp - and - hot type,the
PTS molecular standard substance of the constitution group was higher than that of the normal group. Conclusion:The hypertension PTS has its unique
physical characteristics and has some correlation with the PTS markers.
Key words:hypertension,TCM constitution,PTS,correlation
GC - ECD Method for the Determination of Organochlorine Pesticides Residues in Rhizoma Panaois Majoris
YOU Yan,NIU Yan - fei,YANG Xiao - yan,et al
(Yunnan Institute of Materia Medica,Kunming 650111,Yunnan)
Abstract:Objective:To establish a gas chromatography - electron capture detection (ECD)method for the determination of organochlorine
pesticide residues in Rhizoma Panaois Majoris. Methods:Agilent DB - 624 capillary column (30 m × 0. 32 mm × 1. 8 um)was used. Temperature
program was to rise from 100℃ initial temperature to 220℃,15℃ /min,then to 250℃,10℃ /min,keeping for 17 min. Injection volume was 1. 0ul.
Results:The reference substance of BHC,DDT and PCNB were at 0 ~ 400 ug /L concentration range and showed a good linear relationship. Conclusion:
The method is accurate and feasible with good repeatability and can be used to determine the organochlorine pesticide residues in Rhizoma Panaois
Majoris and in its preparation.
Key words:Rhizoma Panaois Majoris,organochlorine pesticides residues,GC - ECD
Clinical Effect of One Conventional Drynaria Fortunei in Yunnan on the Treatment of Rat Osteoporosis Induced by Ovary Removal
ZHENG Yong - ren,YANG He - jin,ZHAO Chun - mei,et al
(Experimental Center,Yunnan College of Traditional Chinese Medicine,Kunming 650500,Yunnan)
Abstract:Objective:To study the clinical effect of one conventional Drynaria fortunei in Yunnan on the treatment of rat osteoporosis induced by
ovary removal. Methods:Female SD rats and other rats except blank control group (sham operation group)were ovariectomized to make rat osteoporosis
models and were randomly divided into a model group,a positive group(estradiol group)and low,medium and high dose groups of Drynaria fortuni,12
rats per group. The changes of their bone mass,bone mineral density,bone histomorphometry and phosphorus,calcium,alkaline phosphatase (ALP)
in plasma and their bone biomechanical indicators were observed after Drynaria fortunei treatment 60 days later. Results:Compared with the blank
control group,the bone mineral density and the bone mass of the model group decreased in varying degrees. ALP obviously increased while Ca content
decreased,with obvious decreasing biomechanical indicators. The number of trabecular bone reduced and the thickness of cortical bone became thin.
The number of osteoblasts decreased significantly while the number of osteoclasts increased significantly. Compared with the model group,the positive
drug group and the different three - dose groups of Drynaria fortunei after the 60 - day treatment,the bone density and the bone mass of the
ovariectomized rat osteoporosis increased in varying degrees. The bone histomorphometry parameters showed significant differences. ALP in plasma
decreased significantly and Ca and P content increased. The bone biomechanical indexes significantly increased. The number of bone trabecula
increased and the thickness of cortical bone became thicker. The number of osteoblasts significantly increased while the number of osteoclasts
significantly reduced. Conclusion:Drynaria fortunei has a preventive effect on the rat osteoporosis induced by ovary removal.
Key words:osteoporosis,ovariectomized rats,Drynaria fortune,model