Long ZHENG，Chunliang YANG，Zipeng SU，Jianzhi YE

Agricultural Products Processing Research Institute，Chinese Academy of Tropical Agricultural Sciences，Zhanjiang 524001，China

Determination of Heavy Metals inDendrobium candidiumWall.ex Lindl.by lCP-MS

Long ZHENG*，Chunliang YANG，Zipeng SU，Jianzhi YE

Agricultural Products Processing Research Institute，Chinese Academy of Tropical Agricultural Sciences，Zhanjiang 524001，China

［Objective］This research aimed to establish an inductively coupled plasma mass spectrometry（ICP-MS）method for determination of five kinds of heavy metals（Pb，Cd，Cu，As and Hg）in Dendrobium candidium Wall.ex Lindl. ［Method］The samples were digested in HNO3-H2O2acids system by closed vessel microwave digestion.At the same time，internal standard was added to avoid the matrix effect.［Result］The five kinds of heavy meals all showed good linear relationships，and the correlation coefficients ranged from 0.998 2 to 0.999 9.The detection limits of the five kinds of heavy metals were in the range of 0.008-0.750 μg/L，while the standard recovery rates were in the range of 90.4%-96.9%.The relative standard deviations ranged from 1.6%to 8.2%.The heavy metals in theGinsengandAstragalus national standard samples were determined by the established ICP-MS method，and the results were in conformity with the standard values.［Conclusion］The established method has simple and convenient operation and accurate and reliable results，and it meets the requirements by determination of heavy metals in Dendrobium candidium Wall.ex Lindl.

Dendrobium candidium Wall.ex Lindl.；Heavy metals；Microwave digestion；ICP-MS

Dendrobium candidium Wall.ex Lindl.（Orchidaceae:Dendrobium）is a perennial herbaceous plant，and it is named after its green skin.D.candidiumis a kind of rare Chinese medicine in China，and is described as"giant panda of medicine industry"by the international medicinal plant kingdom［1-3］. Studies have shown thatD.candidium is rich in polysaccharides，alkaloids，flavonoids，amino acids，stilbenoids，phenanthrenes and other chemical active ingredients［4-6］，and it has a variety of pharmacological effects，such as fighting fatigue，promoting digestion，fighting aging，fighting rheumatism，reducing blood sugar，reducing blood lipid and fighting tumors.Currently，the researches on D.candidium mainly focus on the determination of active ingredients and pharmacological effects［7-11］，instead of safety indices.With the continuous improvement of people’s consciousness of quality and safety and increasingly severe environmental pollution，heavy metal pollution has become of the focuses of attention.However，there have been no corresponding national standards in China for determination of heavy metals in D.candidium，and there have been also rare reports.Therefore，it is of great significance to establish a simple，convenient，efficient and accurate method for determination of heavy metals in D.candidium.

In China，the currently commonly used methods for determination of heavy metals include atomic absorption spectrophotometry（AAS）［11-13］，atomic fluorescence spectrophotometry（AFS）［14-15］，inductively coupled plasma optical emission spectrum （ICPOES）［16-17］and inductively coupled plasma mass spectrometry（ICP-MS）［18-20］. AAS and AFS can all determine only one heavy metal element at a time，and they are characterized by slow analysis speed and low efficiency. ICP-OES has faster analysis speed，and can determine simultaneously various elements.However，for the determination of trace elements in samples，the sensitivity of ECP-OES isdifficult to meet the analysis requirements.ICP-MS has advantages of high sensitivity，low detection limit and fast analysis speed，and it can also determine a variety of elements simultaneously.Therefore，in this study，ICP-MS was adopted to determine five kinds of heavy metals（Pb，Cd，Cu，As and Hg）in D.candidium，providing certain reference for researches on quality security of D.candidium.

Materials and Methods

Test materials

Main instruments and equipment

The used instruments and equipment in this study mainly included inductively coupled plasma mass spectroscopy（X seriesII，ThermoFisher，USA），high-throughput enclosed microwave digestion system （Mars，CEM，USA），ultrapure water generator（Milli-Q Element，Millipore，F(xiàn)rench）and digestion system（DV4000，Annan，China）.

Reagents and samples The used reagents included HNO3（65%，excellent level of pure，Merck，Germany），H2O2（30%，excellent level of pure，Merck，Germany），ultra-purewater（resistivity of 18.2 MΩ·cm），Pb standard stock solution（1 000 mg/L，China National Center for Quality Supervision and Testing of Iron&Steel，CISRI），Cd standard stock solution（1 000 mg/L，China National Center for Quality Supervision and Testing of Iron& Steel，CISRI），Cu standard stock solution（1 000 mg/L，China National Center for Quality Supervision and Testing of Iron&Steel，CISRI），As standard stock solution（1 000 mg/L，China National Center for Quality Supervision and Testing of Iron&Steel，CISRI），Hg standard stock solution （1 000 mg/L，China National Center for Quality Supervision and Testing of Iron&Steel，CISRI），Ginseng standard （GBW 10027，Institute of Geophysical and Geochemical Exploration，CAGS），Astragalus standard（GBW 10028，Institute of Geophysical and Geochemical Exploration，CAGS）and ICP-MS tuning solution（10 μg/L，O2si，USA）.The D.candidium was purchased from a local market.

Preparation of standard solutions The Pb，Cd，Cu，As and Hg standard stock solutions were all serially diluted with 2%HNO3for preparation of a series of mixed standard solutions.

Working conditions of lCP-MS

The working conditions of ICP-MS were shown in Table 1.

Test methods

A certain amount（0.5 g，accurate to 0.01 g）of ground D.candidium powderwas dissolved in certain amounts of HNO3（7 ml）and H2O2（1 ml）.After stood for 30-60 min，the mixture was placed in a digestion furnace.According to the preset heating program，the sample was digested.After the digestion，the sample was cooled to room temperature and diluted to 50 ml with ultra-pure water.Similarly，the blank solutions were prepared.

Results and Analysis

Selection of microwave digestion conditions

Different type of food requires different temperature and time in microwave digestion.Therefore，the optimal conditions for microwave digestion of D.candidium were determined through several tests.Under the optimal digestion conditions， samples could be digested completely.The digestion process was shown in Table 2.

lsotope selection and interference correction

In the ICP-MS analysis，the mass spectral interference can be avoided to the greatest extent by adopting appropriate isotope for analyte，equation correction or CCT technology.The isotopes for various heavy metal elements were determined as follows:

208Pb，111Cd，65Cu，75As and202Hg.In order to overcome the matrix effects of samples，Ge-Rh-In-Re mixed internal standard was added online for result correction.

Linear range，linear equation，correlation coefficient and detection limit

The contents of heavy metals in the prepared standard solutions were determined.With the counting ratio between analyte and internal standard as the vertical axis，and the mass concentration as the horizontal axis，the standard curve was drawn.The heavy metals in each blank solution were determined 11 times，and the detection limit of each heavy metal element was calculated （3S/N）.The linear range，linear equation，correlation coefficient and detection limit of each heavy metal element were shown in Table 3. As shown in Table 3，the correlation coefficients ranged from 0.998 2 to 0.999 9，and the detection limits ranged from 0.008 to 0.750 μg/L.

Accuracy and precision

The heavy metals in the Ginseng or Astragalus standard were determined 6 times by the established ICPMS method.As shown in Table 4 and Table 5，the determined results were all in the reference ranges.

Standard addition test

Standard addition test was conducted for the heavy metal contentknown samples.Each sample was determined six times by the established ICP-MS method.As shown in Table 6，the average recovery rates of the five kinds of heavy metal elements ranged from 90.4%to 96.9%，and the relative standard deviations ranged from 1.6% to 8.2%，meeting the analytical requirements by heavy metal elements in D.candidium.

Table 1 Working conditions of ICP-MS

Table 2 Microwave digestion process

Sample determination

The contents of heavy metals in the five purchased D.candidium samples were determined by the established ICP-MS method（Table 7）.According to the requirements by"the Import and Export Green-industry Criterion of the Drug Plant and Preparation"，the Pb，Cd，Cu，As and Hg contents should not be higher than 5.0，0.3，20.0，2.0 and 0.2 mg/kg，respectively.As shown in Table 7，the Pb，Cd，Cu and As contents in the five D.candidium samples were all below the indexes.The Hg contents in four out the five samples were also below the index.

Conclusions and Discussion

Combining microwave digestion and ICP-MS methods，a new method was established in this study for determination of heavy metal content in D.candidium.Under the conditions of high pressure and closed environment，the digestion will be faster and complete.Thus，dozens of samples can be processed at a time，saving time and effort.ICP-MS can determine the contents of a variety of elements at a time，and it is characterized by fast analysis speed and high sensitivity，meeting the requirements by rapid analysis of heavy metal elements in D. candidium.

Based on the edition of"Pharmacopoeia of People’s Republic of China"，the State Pharmacopoeia Commission of China intended to publish the limits of heavy metals and toxic elements in 2010.This study will not only provide a scientific method for determination of heavy metal elements in D.candidium，but also provide scientific data support for quality control，as well as preparation of heavy metal limit standards for D.candidium.

In this study，the heavy metal content differed greatly among different samples and differentelement，which was mainly related to heavy metal content in soil and selection，enrichment and accumulation ability of D.candidium.The correlation of heavy metal content between D.candidium and soil，as well as the absorption and enrichment characteristics of D.candidium for heavy metals，all require further studies.

Table 3 Linear range，linear equation，correlation coefficient and detection limit of ICP-MS for each heavy metal element

Table 4 Contents of the five kinds of heavy metals in Ginseng（GBW 10027）（n=6）

Table 5 Contents of the five kinds of heavy metals in Astragalus（GBW 10028）（n=6）

Table 6 Recovery rate of standard addition and relative standard deviation（n=6）

Table 7 Determination results of the samples（n=6）mg/kg

［1］SHAO H（邵華），ZHANG LQ（張玲琪），LI JM（李俊梅），et al.Advances in research of Dendrobium officinale（鐵皮石斛研究進(jìn)展）［J］.Chinese Traditional and Herbal Drugs（中草藥），2004，35（1）:109-112.

［2］LI GF（李桂鋒），LI JJ（李進(jìn)進(jìn)），XU JY（許繼勇），et al.A review of Dendrobium candidium Wall.ex Lindl（鐵皮石斛研究綜述）［J］.Journal of Chinese Medicinal Materials（中藥材），2010，33（1）:150-153.

［3］LI HY（李宏楊），LIU GM（劉國(guó)民），YANG ZJ（楊志娟），et al.Research and devel-opment review and prospects ofDendrobium officinale（鐵皮石斛研究開(kāi)發(fā)綜述與展望）［J］.Journal of Anhui Agricultural Sciences （安徽農(nóng)業(yè)科學(xué)），2014，（34）:12083-12086.

［4］LIU WJ（劉文杰），SUN ZR（孫志蓉），DU Y（杜遠(yuǎn)），et al.Main chemical compositions and fingerprints ofTiepishihu（Dendrobium candidium）produced from different areas（不同產(chǎn)地鐵皮石斛主要化學(xué)成分及指紋圖譜研究）［J］.Journal of Beijing University of Chinese Medicine（北京中醫(yī)藥大學(xué)學(xué)報(bào)），2013，36（2）:117-120.

［5］HU XC（胡賢春），GUO YB（郭永兵），XIANG JS（向勁松），et al.Advances in the study of polysaccharide from Dendrobium officinale（鐵皮石斛多糖的研究進(jìn)展）［J］.Journal of Anhui Agricultural Sciences（安徽農(nóng)業(yè)科學(xué)），2015，（15）:78-80，84.

［6］LI J（李娟），LI SX（李順祥），HUANG D（黃丹），et al.Advances in the research of resources，constituents and pharmacological effects ofDendrobium officinale（鐵皮石斛資源、化學(xué)成分及藥理作用研究進(jìn)展）［J］.Science&Technology Review（科技導(dǎo)報(bào)），2011，29（18）:74-79.

［7］LIU LF（柳蓮芳）.Latest research advances in caulis dendrorbii（鐵皮石斛的最新研究進(jìn)展）［J］.Journal of Anhui Agricultural Sciences（安徽農(nóng)業(yè)科學(xué)），2012，40（11）:6426-6428，6430.

［8］ZHOU J（周佳），ZHOU XL（周先麗），LIANG CQ （梁成欽），et al.Chemical constitutes ofDendrobium officinale（鐵皮石斛化學(xué)成分研究）［J］.Chinese Traditional and Herbal Drugs（中草藥），2015，46（9）:1292-1295.

［9］LI H（李昊），WU DH（呂鼎豪）.Advances in studies on chemical constituents in Dendrobium officinale（鐵皮石斛藥用成分研究進(jìn)展）［J］.Chinese Journal of Spectroscopy Laboratory（光譜實(shí)驗(yàn)室），2013，30（4）:1845-1849.

［10］LI L（李玲），DENG XL（鄧曉蘭），ZHAO XB（趙興兵），et al.Advances in studies on chemical constituents inDendrobium candidiumand their pharmacological effects（鐵皮石斛化學(xué)成分及藥理作用研究進(jìn)展）［J］.Anti-tumor Pharmacy（腫瘤藥學(xué)），2011，01（2）:90-94.

［11］RASMUSSEN RR，QIAN Y，SLOTH JJ，et al.SPE HG-AAS method for the determination of inorganic arsenic in rice-Results from method validation studies and a survey on rice products［J］.Analytical and Bioanalytical Chemistry，2013，405（24）:7851-7857.

［12］JIN L（金烈）.Atomic absorption spectrometry determination of terrace element in the oat（原子吸收分光光度法測(cè)定燕麥中微量元素）［J］.Applied Chemical Industry（應(yīng)用化工），2015，44（1）:187-189.

［13］FAN YG （范亞剛），JI QL（季秋玲），F(xiàn)ENG ZX （馮振興），et al.Application of atomic absorption spectrophotometry on content determination of metallic elements in drugs（原子吸收分光光度法在藥品金屬元素含量測(cè)定中的應(yīng)用）［J］.China Modern Medicine（中國(guó)當(dāng)代醫(yī)藥），2014，21（34）:167-169.

［14］CHEN SY（陳思穎），LAN B（蘭波），ZHU D （朱迪），et al.Determination of selenium in Gastrodia elata and Quantianma capsule by atomic fluorescence spectrometry with high pressure digestion（高壓消解-原子熒光光譜法測(cè)定天麻藥材及全天麻膠囊中硒的含量）［J］.Chinese Journal of Experimental Traditional Medical Formulae（中國(guó)實(shí)驗(yàn)方劑學(xué)雜志），2013，19（12）: 79-82.

［15］LI XF（李險(xiǎn)峰），DENG BY（鄧必陽(yáng)）. Determination of metals in Dendrobium officinale by microwave digestion with ICP-AES（微波消解-ICP-AES法測(cè)定鐵皮石斛中多種金屬元素）［J］. Hubei Agricultural Sciences（湖北農(nóng)業(yè)科學(xué)），2012，51（24）:5769-5770.

［16］HU XL（胡小玲），CHEN JG （陳劍剛），ZHANG Y （張艷），et al.Detection of cadmium in rice by inductively coupled plasma optical emission spectrometry（ICP-OES測(cè)定大米中鎘的方法研究）［J］.Practical Preventive Medicine （實(shí)用預(yù)防醫(yī)學(xué)），2015，22（8）:930-932.

［17］WANG HF（王海鳳），TANG XM（唐興敏 ）.Determination of calcium and strontium in beer by ICP-OES（電感耦合等離子體發(fā)射光譜法測(cè)定啤酒中的鈣和鍶）［J］.Resources Environment& Engineering（資源環(huán)境與工程），2015，（3）:351-353.

［18］LU MB（陸美斌），WANG BJ（王步軍），LI JM （李靜梅），et al.Research of heavy metals determination in cereals by inductively coupled plasma mass spectrometry（電感耦合等離子體質(zhì)譜法測(cè)定谷物中重金屬含量的方法研究）［J］.Spectroscopy and Spectral Analysis（光譜學(xué)與光譜分析），2012，32（8）: 2234-2237.

［19］YANG L（楊磊），AI XW （艾鑫衛(wèi)），HUANG WY（黃文鄴），et al.Determination of heavy metals in vegetables by inductively coupled plasma mass spectrometry（電感耦合等離子體質(zhì)譜法同時(shí)測(cè)定市售蔬菜中8種重金屬）［J］.Journal of Anhui Agricultural Sciences（安徽農(nóng)業(yè)科學(xué)），2015，（16）: 254-255，267.

［20］FABIEN POINTURIER，AM LIE HUBERT，ANNE-CLAIRE POTTIN，et al. Performance of laser ablation: quadrupole-based ICP-MS coupling for the analysis of single micrometric uranium particles［J］.Journal of Radioanalytical and Nuclear Chemistry:An international Journal Dealing with All Aspects and Applications of Nuclear Chemistry，2013，296（2）:609-616.

Responsible editor：Tingting XU

Responsible proofreader：Xiaoyan WU

ICP-MS測(cè)定鐵皮石斛中5種重金屬元素的研究

鄭龍*，楊春亮，蘇子鵬，葉劍芝 （中國(guó)熱帶農(nóng)業(yè)科學(xué)院農(nóng)產(chǎn)品加工研究所，廣東湛江524001）

［目的］建立電感耦合等離子體質(zhì)譜（ICP-MS）法測(cè)定鐵皮石斛中Pb、Cd、Cu、As、Hg等5種重金屬元素的方法。［方法］樣品采用硝酸、雙氧水混合體系進(jìn)行微波消解，電感耦合等離子體質(zhì)譜（ICP-MS）進(jìn)行測(cè)定。同時(shí)為避免基體效應(yīng)等影響，加入內(nèi)標(biāo)進(jìn)行校正。［結(jié)果］5種重金屬元素線性關(guān)系良好，相關(guān)系數(shù)為0.998 2～0.999 9，檢出限范圍為0.008～0.750 μg/L。加標(biāo)回收率為90.4%～96.9%，相對(duì)標(biāo)準(zhǔn)偏差為1.6%～8.2%。用該法測(cè)定國(guó)家標(biāo)準(zhǔn)物質(zhì)人參和黃芪，測(cè)定結(jié)果均在標(biāo)準(zhǔn)參考值范圍內(nèi)。［結(jié)論］建立的方法操作簡(jiǎn)便快捷，結(jié)果準(zhǔn)確可靠，滿(mǎn)足鐵皮石斛中重金屬元素的測(cè)定要求。

鐵皮石斛；重金屬元素；微波消解；ICP-MS

鄭龍（1981-），男，福建龍巖人，助理研究員，從事農(nóng)產(chǎn)品質(zhì)量與安全研究。*

2015-11-08

*Corresponding author.E-mail:zhenglong169@163.com

Received:November 8，2015 Accepted:December 10，2015

修回日期 2015-12-10