Multi-Element Analysis and Origin Discrimination of Panax notoginseng Based on Inductively Coupled Plasma Tandem Mass Spectrometry (ICP-MS/MS)

4.1. Chemicals and Reagents

Nitric acid 65% (HNO3) was purchased from Merck, USA, and hydrofluoric acid 49% (HF) was purchased from Aladdin Reagent Corporation, China. Ultrapure deionized water (ddH2O) with a resistivity of 18.2 MΩ cm was obtained from a Milli-Q Plus water purification system (Millipore, Bedford, MA, USA).
Twenty-six multielement standard solutions (Na, Mg, K, Ca, Fe (1000 μg/mL), Sr (100 μg/mL), Al, V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Ag, Cd, Sb, Sn, Ba, Pb, Tl, Th, and U (10 μg/mL)), a single-element Hg standard solution, and seventeen rare-earth elements (Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Sc, Sm, Tb, Th, Tm, Y, and Yb (10 μg/mL each)) were provided by Agilent Technologies Company.

4.2. Collection and Pretreatment of P. notoginseng

P. notoginseng samples (a total of 89 mixed samples) were collected from the main planting origins in Yunnan Province, China, in 2019 and 2020. The sampling points were shown in Table S5 (a total of 30 sampling bases), which included both the five main planting origins (WenShan, QuJing, HongHe, KunMing, and PuEr) and the cultivation model in field and forest of P. notoginseng. Each planting base was randomly sampled at three points, and eight or ten P. notoginseng samples were collected at each point as mixed samples. Then, the collected roots of P. notoginseng were washed with clean water, dried at 60 °C, coarsely crushed, ground to ultrafine powder with a Planetary Mono Mill (PULVERISETTE 6, Fritsch, Idar-Oberstein, Germany), and then passed through a 60 mesh sieve.
In addition, in order to study the accumulation dynamics of multi-element (not rare earth elements) in P. notoginseng after transplanting, two planting bases, PuEr (forest model) and HongHe (field model), were selected, and samples were collected in August 2019, November 2019, and November 2020, respectively. A three-point random sampling method was adopted for each base. Ten P. notoginseng plants were collected from each sampling point and 100 g of rhizosphere soil and edge soil was also collected during the first sampling. Then, samples from three points were mixed as the same treatment. The P. notoginseng samples were pretreated by the same method. Next, the soils were naturally dry and then passed through a 60 mesh sieve.

4.3. Microwave-Assisted Acid Digestion Procedure

All glassware and polytetrafluoroethylene (PTFE) tubes were immersed in a 10% (v/v) HNO3 solution for 48 h, followed by a minimum of three rinses with ddH2O, before being dried and finally stored ready for use [58]. A Multiwave PRO microwave digestion system (AntonPaar, Ashland, VA, USA) was used for the digestion of samples.
Soil: About 0.1 g of each soil sample was weighed and mixed with 4 mL of HNO3 and 2 mL of HF, and pre-digested at 130 °C for 30 min. The samples were then processed by microwave digestion with a ramped-up temperature from ambient to 130 °C over 10 min and held for 5 min, followed by a ramped-up temperature to 195 °C over 10 min and held for 20 min. After digestion, the solutions were evaporated to near dryness and cooled to room temperature. A negative control (no sample) was provided for each series of digestions.
P. notoginseng: About 0.4 g of each homogenized sample was weighed and mixed with 6 mL of HNO3, and pre-digested at 130 °C for 30 min. The samples were then processed by microwave digestion with a ramped-up temperature from ambient to 120 °C over 10 min and held for 2 min, followed by a ramped-up temperature to 190 °C over 4 min and held for 20 min. After digestion, the solutions were cooled to room temperature. A negative control (no sample) was provided for each series of digestions. Both digested samples and blanks were diluted to 50 mL with ddH2O and analyzed by ICP-MS/MS.

4.4. ICP-MS/MS Analysis

The concentration of 49 elements (23Na, 24Mg, 27Al, 39K, 43Ca, 44Ca, 51V, 52Cr, 53Cr, 55Mn, 56Fe, 57Fe, 59Co, 60Ni, 63Cu, 65Cu, 66Zn, 75As, 78Se, 88Sr, 89Y, 97Mo, 98Mo, 107Ag, 111Cd, 114Cd, 118Sn, 123Sb, 137Ba, 139La, 140Ce, 141Pr, 146Nd, 147Sm, 153Eu, 157Gd, 163Dy, 165Ho, 166Er, 169Tm, 172Yb, 200Hg, 202Hg, 205Tl, 206Pb, 207Pb, 208Pb, 232Th, and 238U) in P. notoginseng was determined by ICP-MS/MS (Agilent 8800, Tokyo, Japan). The calibration standard solutions were prepared in the range of 0–4 μg/mL for 23Na, 24Mg, 39K, 43Ca, 44Ca, 56Fe, and 57Fe; 0–400 ng/mL for 27Al, 66Zn, 88Sr, and 118Sn; 0–40 ng/mL for 51V, 52Cr, 53Cr, 55Mn, 59Co, 60Ni, 63Cu, 65Cu, 75As, 78Se, 89Y, 97Mo, 98Mo, 107Ag, 111Cd, 114Cd, 123Sb, 137Ba, 146Nd, 147Sm, 153Eu, 157Gd, 163Dy, 166Er, 172Yb, 205Tl, 206Pb, 207Pb, 208Pb, 232Th, and 238U; 0–12.5 ng/mL for 139La, 140Ce, 141Pr, 165Ho, and 169Tm; and 0–8 ng/mL for 200Hg and 202Hg. A mixed internal standard (ISTD) solution with a concentration of 50 ng/mL Sc, Ge, In, and Bi was used to correct changes in the sample uptake rate and plasma conditions for the ICP-MS/MS [59]. Through the tuning program, the operational mode of He and no gas mode were optimized. The instrument’s other conditions of ICP-MS/MS were shown in Table S6.
The multi-element calibration solutions were prepared at different concentration levels using 5% HNO3 media to match the sample matrix. By analyzing the experimental data, a linear fitting standard curve with the X-axis as the concentration point and the Y-axis as the response value was created. Using this standard curve, a background equivalent concentration of the analysis element was obtained by calculating the element standard deviation. LODs were calculated as (3 σ/k) and the LOQs were calculated as (10 σ/k), where standard deviation (σ) was the standard deviation of the blank signal (n = 11) and k was the slope of the calibration line [33,60]. Then, the accuracy of the method was estimated using analytical recovery, which was evaluated by adding the standard solutions with two different concentration levels (high and low) to P. notoginseng samples. These samples were both digested and analyzed in triplicate by ICP-MS/MS [22].

4.5. Statistical Analysis

All statistical analyses were conducted in the R software environment (v4.1.2;, accessed on 18 January 2022). Most of the results were visualized using the ‘ggplot2′ package [61], unless otherwise indicated. The experimental data were expressed as mean ± S.E.M, and recorded in Excel 2019 (Microsoft); then, the significance analysis was performed using the ‘agricolae’ package [62] and ‘ggpubr’ package [63] for Duncan’s test and T’test, respectively. The permutational multivariate analysis of variance (PERMANOVA), Anosim, and NMDS were performed using the ‘vegan’ package [64]. Heatmaps were illustrated based on Z-score-normalized relative abundance of taxa using the ‘pheatmap’ package [65]. Discriminative models for P. notoginseng were trained and predicted using the ‘Caret’ package [66]. In the field of data mining, supervised algorithms were used to classify samples into predefined classes. This was helpful for the establishment of models [47], such as PLS-DA, LR, LDA, RF, NB, kNNs, SVMs, and NNs.

2.1. Analytical Performance of the ICP-MS/MS for P. notoginseng

The procedure for the determination of multi-element in P. notoginseng samples by ICP-MS/MS was evaluated for its linearity, detection, and quantification limits (respectively, LODs and LOQs), as well as its accuracy and precision (Table S1, Supplementary Materials). The calibration curves for all the elements revealed a good linearity over the entire range of concentrations, with coefficients of determinations (R2) higher than 0.99, between 0.9919 and 0.9997. The instrumental LODs of ICP-MS/MS were between 0.0003 mg/kg (for 200Hg) and 7.716 mg/kg (for 44Ca); moreover, the LOQs were between 0.0011 mg/kg and 25.7202 mg/kg. The method proposed in this work showed good sensitivity for multi-element determination in P. notoginseng samples. The average recoveries of multi-element in P. notoginseng were in the range between 85.82% and 104.98% (Table S2); the relative standard deviation (RSDs) was in the range of 1.56%–9.70%, lower than 10%. Considering these results, it was concluded that this method had high accuracy and met the requirements of analyzing and measuring the content of multi-element in P. notoginseng.

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