Determination of Trace Elements in Corydalis conspersa and Corydalis linarioides by ICP-AES

College of Pharmacy, Qinghai Nationalities University, Key Laboratory of Plant Resources of Qinghai-Tibet Plateau in Chemical Research, No. 3,Bayi Mid-Road, East of City Dist., Xining, Qinghai 810007, China Asia Silicon (Qinghai) Co.,Ltd., No. 1,Jingui Road, Dongchuan Industrial Park, Xining, Qinghai 810007, China Key Laboratory of Medicinal Animal and Plant Resources, Qinghai Normal University, No. 38,Wusi West Road, West of City Dist., Xining, Qinghai 810007, China Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources, 8e Chinese Academy of Sciences, No. 11A,Datun Road, Haidian Dist., Beijing 100101, China College of Chemistry and Chemical Engineering, Qinghai Nationalities University, No. 3,Bayi Mid-Road, East of City Dist., Xining, Qinghai 810007, China

scabies, etc. [2]. Corydalis linarioides is a famous medicine in Taibai Mountain and Tibetan medicine in Qinghai Province, while the Tibetan medicine is called Jiadaseva [3].
ere have the organic chemical components and inorganic chemical elements in the traditional Chinese medicine. In 1869, Russian scientist Dmitri Mendeleev created the periodic table of elements. Up to now, there are 118 kinds of chemical elements. e trace elements in the study include 13 metal elements Al, Ca, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Ti, and Zn and 4 nonmetallic elements As, B, P, and Si. Chemical elements have their own physical and chemical properties which play an important physiological role in plants and have a close relationship between the nature, taste, efficacy, and Traditional Chinese Medicine. erefore, the study of chemical elements in different collection areas and tissues of Corydalis conspersa and Corydalis linarioides can be used as an important index material basis for distinguishing the quality of medicinal materials and exploring the mechanism of curative effect related to chemical elements. e main methods for the determination of elements in Chinese medicinal materials are colorimetry [4][5][6], AAS [7], atomic fluorescence spectrometry [8], inductively coupled plasma-atomic emission spectrometry (ICP-OES/AES) [9,10], and inductively coupled plasma mass spectrometry (ICP-MS) [11]. e plasma can reach a very high temperature by the ICP-AES, which is conducive to the atoms or ions in the elements to emit photons of characteristic wavelengths decomposed into monochromatic spectra representing each element through the grating spectroscopic system. ese spectral energies are detected by the semiconductor detector, and the content of the elements to be measured in the test solution is compared to the standard solution. It has the advantages of high sensitivity, low detection limit, and wide test range to simultaneously determine the multiple elements. e content of trace elements in Corydalis conspersa and Corydalis linarioides was determined by ICP-AES to provide a certain scientific basis for the further researches regarding the meaningful functions of the elements within the plant tissues and metabolism of plant growth.

Collection and Treatment of Drug.
e medicinal materials were collected in Hainan, Huangnan, Yushu, and Haibei Tibetan Autonomous Prefecture of Qinghai Province in August 2017. After identification by Professor Lin Pengcheng (Qinghai Nationalities University), the authors compared the specimens with the China and Qinghai flora to identify them as Corydalis conspersa and Corydalis linarioides.
After cleaning the fresh plants, they were divided into dried roots, stems, leaves, and flowers in the shade and crushed using a 80-mesh sieve (Table 1 and Figure 1).

Working Parameters of the Instrument.
e working parameters of the instrument are listed in Table 3.

Selection of Analysis Line.
According to the principles of low detection limit, high sensitivity, less interference of coexisting elements, and low spectral interference, the following analytical lines (nm) were selected for analysis [12] e concentration of single standard solution of Al, As, B, Ca, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, P, Si, Ti, and Zn is 1000 μg/ml.

Preparation of Mixed Reference Solution.
Accurately suck 5.0 ml of each element standard solution in Table 2, put it into a 500.0 ml volumetric flask, and fix the volume with 5% HNO 3 to prepare 10 μg/ml element mixed standard solution; accurately absorb 10 μg/ml element mixed standard solution with appropriate amount of 1.0, 10.0, 20.0, 30.0, 40.0, and 50.0 ml, put into a 100.0 ml volumetric flask, and fix the volume of 5% HNO 3 to prepare 0.1, 1.0, 2.0, 3.0, 4.0, and 5.0 μg/ml, respectively, and 10.0 μg/ml of mixed standard solution.

Preparation of Test Solution.
Accurately weigh 0.2 g (0.01-0.02 g for flower) of Corydalis conspersa and Corydalis linarioides, put them into a conical flask with stopper, add 15.0 ml HNO 3 : HCl (3 : 1) mixed acid solution in the fume hood, shake well, and dissolve; close the plug and soak in the fume hood overnight. e acid solution in the fume hood was heated and digested on an electric furnace for 30 min-2 h until dried; 4.0 ml HNO 3 : HCl (3 : 1) mixed acid solution was added to each sample, and then the mixture was shaken and digested overnight. After overnight, the acid solution is heated and digested in an electric furnace and kept boiling for 30 min-2 h on the condition of fume hood. After the  Journal of Chemistry white smoke is exhausted, the acid is evaporated until cooled. en, each sample is diluted with 5% HCl solution into a 100.0 ml volumetric flask and placed overnight. 0.45 μm aqueous-phase microporous filter membrane is used to filter into a 50.0 ml plastic quantitative tube and then put into the refrigerator for freezing. At the same time, 5% HCl and 5% HNO 3 were used as blank control solution [13].

Development and Validation of Method.
e method for detection, separation, and quantitation of the three copies samples was developed and combined the conditions with reference. e validation parameters investigated included linearity and its range, limits of detection and quantitation, precision, stability, repeatability, and recovery.

Study of Linear Relationship and Limits of Detection and Quantitation.
Under the working conditions of ICP-AES, the mixed standard solution of 0.1, 1.0, 2.0, 3.0, 4.0, 5.0, and 10.0 μg/ml was determined and the standard curve was drawn with the concentration as the abscissa and the average value of the three intensity measurements as the ordinate. e limits of detection and quantitation were calculated by dividing the 3 and 10 times standard deviation of the measured value from the blank control solution by the slope of the corresponding element standard curve, respectively.

Precision Experiment.
Precisely, the same sample solution is drawn for injection, and each sample is detected for 6 times.

Stability Experiment.
e same sample solution was precisely absorbed, and the contents of 17 elements were determined at 0.0, 3.0, and 24.0 h after preparation under the determination conditions.

Linear Relationship and Limit.
e regression equation of trace elements has a good linear relationship with R ≥ 0.9996. e detection limits were between 0.0001 μg/g and 0.1024 μg/g. e quantitation limits were between 0.0003 μg/g and 0.3103 μg/g (Table 4).

Stability Experiment.
e results showed that the content of each element was stable within 24 h after samples preparation and was detected by the conditions listed in Table 3. e RSD values of various elements were between 0.35% and 4.70%.

Repeatability Experiment.
e RSD values were 0.20%-3.54%, indicating that the method had good repeatability.

Samples from Different Areas and Tissues.
In order to study the specific content of different elements in the collection areas and tissues, a three-dimensional characterization map was drawn for the detection of element content. It showed that the higher contents of different elements in roots, stems, leaves, and flowers in different collection areas were Al, Ca, Fe, K, Mg, Na, and P. e contents of elements in different tissues were as follows: flowers > leaves > stems > roots. e contents of elements in flowers of Maixiu Forest Farm from Henan County of Huangnan Prefecture were highest than those in other areas. However, the contents of different elements As, B, Cr, Cu, Li, Mn, Ni, Si, Ti, and Zn were lower in the plants (Table 5 and Figure 2).

Accumulation Contents from Different Areas and
Tissues. e accumulation of various elements in different parts of each collection area was the highest in flowers: Zeku County > Guide County > Nangqian County > Henan County; the stems and leaves were the second, and the roots were the lowest. e results showed that the accumulation of various elements in the flowers: Maixiu Forest Farm from Henan County of Huangnan Prefecture (185.54 μg/g), was the highest, Guide County (136.32 μg/g) and Qilian County (53.80 μg/g) were the second, and Ji Gang Mountain (43.87 μg/g) from Henan County of Huangnan Prefecture was the lowest. e accumulation of various elements in the leaves: Nangqian County (44.40 μg/g), was the highest. e accumulation of various elements in the stems: Henan County of Huangnan Prefecture (92.25 μg/g), was the highest. e accumulation of various elements in the roots: Henan County of Huangnan Prefecture (20.81 μg/g and 19.17 μg/g), was the highest (Table 5 and Figure 3).
According to the analysis of the whole element accumulation contents, the accumulation content of elements in different tissues of collection areas was higher as follows: Nangqian County of Yushu Prefecture, Henan County of Huangnan Prefecture, Qilian County of Haibei Prefecture, and Guide County of Hainan Prefecture, while those of flowers > stems > leaves > roots. e amount of flower (43.87 μg/g − 185.54 μg/g) was the highest; the root (6.19 μg/g − 11.90 μg/g) was the lowest, and the leaf (33.56 μg/g − 44.40 μg/g) and the stem (27.96 μg/g − 92.25 μg/g) were higher in the Guide County from Hainan Autonomous Prefecture of Corydalis conspersa (Table 5 and Figure 3). [14,15]

Eigenvalues and Contribution Rate of Principal
Components. In the results of principal component analysis, the KMO value is 0.647 > 0.6, which indicates that factor analysis is suitable and significance value in the sphericity test is 0.000 < 0.05 (significant level), indicating that there is correlation among variables and factor analysis. We can know the description of the original variables by the initial solution in the process of principal component analysis. e cumulative contribution rate of the total variance from three principal components is 82.46%, which means that the mathematical model established by the extracted three factors can clarify 82.46% of the experimental data. e contribution rate of principal component 1 is about 62%, which is more than half of the cumulative contribution rate of the whole principal component ( Table 6).

Factor Load Matrix of Principal Component Analysis.
e loading coefficients of Al, Ti, and Fe in the first principal component are 0.915, 0.931, and 0.893, respectively, which are all positive and significant contributions in the principal    (Table 7).
Principal components 1 and 3 were mainly from flowers and stems of Corydalis conspersa collected from Hainan, Huangnan, and Yushu Autonomous Prefectures; principal component 2 was mainly from flowers collected from Huangnan, Hainan, and Yushu Autonomous Prefectures; and the top five principal components were also from Huangnan, Hainan, and Yushu Autonomous Prefectures (Table 9 and Figure 3).

Conclusions
An suitable, precise, and selective ICP-AES method for the simultaneous determination of trace elements has been developed and validated from the TCM of Corydalis conspersa and Corydalis linarioides. According to the green industry standard for foreign trade and economic cooperation of medicinal plants and preparations issued by China in 2005 [19], the total amount of heavy metals should be less than or equal to 20.0 mg•kg −1 and the content of Pb ≤ 5.0 mg•kg −1 , Cd ≤ 0.3 mg•kg −1 , Cu ≤ 20.0 mg•kg −1 , As ≤ 2.0 mg•kg −1 , and Hg ≤ 0.2 mg•kg −1 . e content ranges of copper, arsenic, and chromium were 0.0018−1.6989 mg • kg −1 , 0.0016−0.1944 mg • kg −1 , and 0.0030−0.2063 mg • kg −1 , respectively. ey all comply with the limit range of heavy metal. It showed that the growth and ecological environment of the medicinal materials were good and no pollution. e contents of elements have the great significance within the aspects of the plant physiology, the structure and efficacy of medicinal materials, and the ecological environment [20]. is research can provide a determination basis of trace elements in Corydalis conspersa and Corydalis linarioides so as to reveal the relationship between the functions of trace elements and plant growth through the further researches.

Data Availability
All data generated or analyzed during this study are included in this article. e original data related to the method validation are included as Supplementary Data (S1-S5).

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Du Qing and Chen Zhi have made substantial direct and intellectual contribution to the work and approved it for publication. Du Qing, Cai Yanguo, and Chen Zhi contributed equally regarding the research works. All authors have made substantial contribution to the work and approved it for publication.