Method for detecting heavy metals in agricultural and sideline products based on graphite rapid digestion-atomic absorption / atomic fluorescence spectrometry

Abstract

The application discloses a method for detecting heavy metals in agricultural and sideline products based on graphite rapid digestion-atomic absorption / atomic fluorescence spectrometry, and relates to the technical field of element detection. The detection method comprises the following steps: S1, adding a sample into a digestion liquid, and placing the sample on a graphite digestion instrument to perform digestion, so as to obtain a digested sample; the sample comprises grains, vegetables, fruits, livestock and poultry meat, fresh eggs and tea leaves; S2, adding the digested sample into a graphite furnace atomic absorption spectrometer (AAS) to detect the content of lead, cadmium and chromium in the sample, and adding an atomic fluorescence spectrometer (AFS) to detect the content of arsenic and mercury in the sample. The application successfully establishes and verifies a rapid detection method for five heavy metal elements, i.e., lead, cadmium, chromium, arsenic and mercury, in agricultural and sideline products based on graphite rapid digestion-AAS / AFS combined technology, and the method has strong universality; the optimized pretreatment condition is suitable for agricultural and sideline products of various matrices; the detection method is accurate, reliable, efficient, economical, green and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of elemental detection technology, specifically to a method for detecting heavy metals in agricultural and sideline products based on rapid graphite digestion-atomic absorption / atomic fluorescence spectroscopy. Background Technology

[0002] As living standards improve, people have increasingly higher demands for food quality. Agricultural and sideline products are the most important direct or indirect sources of food and important raw materials for industrial production, serving as a prerequisite and crucial guarantee for food safety. Heavy metal pollution (represented by lead, cadmium, chromium, arsenic, and mercury) is a significant issue affecting the safety of agricultural and sideline products. Long-term consumption of food with excessive heavy metal levels may damage internal organs, and in severe cases, affect the immune and reproductive systems, potentially causing birth defects and cancer. Therefore, whether heavy metal content exceeds the standard has always been a major concern for the country and society, and strengthening the testing of heavy metals in agricultural and sideline products has become an important task in the current market supervision field.

[0003] Jiujiang City, as an important agricultural and transportation hub in Jiangxi Province, boasts a rich variety of agricultural and sideline products. The quality and safety of these products are crucial to the health of local residents and the market reputation of regional agricultural products. Lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As), and mercury (Hg) are representative heavy metal elements found in agricultural and sideline products. In recent years, Jiujiang City has averaged over 4,000 batches of agricultural and sideline products requiring heavy metal testing annually, resulting in a heavy workload and demanding tasks. The current testing process involves the independent pretreatment and determination of individual elements using national standard methods, which is time-consuming, labor-intensive, and consumes a large amount of reagents. Therefore, finding a way to shorten the testing time and improve efficiency while ensuring the accuracy of sample testing is of paramount importance for enhancing food safety supervision in Jiujiang City and even Jiangxi Province, promoting green agricultural development, and strengthening the market competitiveness of agricultural products.

[0004] Currently, in the detection of heavy metal elements, samples are digested into solutions, with the pretreatment step accounting for approximately two-thirds of the total analysis time. Classic digestion methods include dry digestion, wet digestion, microwave digestion, and high-pressure digestion. Among these, microwave digestion equipment is expensive, acid removal is time-consuming, and requires personnel supervision; dry digestion involves multiple steps, is time-consuming, and the high temperature may lead to sample loss; wet digestion uses a large amount of acid. After pretreatment, the digested solution is analyzed by instruments to obtain results. Common instrumental analysis methods include atomic absorption / fluorescence spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS). While ICP-MS can determine multiple elements simultaneously, the equipment is expensive and cannot be equipped by grassroots laboratories. Therefore, developing a rapid, accurate, and simple pretreatment method combined with atomic absorption / fluorescence spectroscopy for the detection of large quantities of agricultural and sideline products is of significant practical importance for improving the capabilities of grassroots laboratories.

[0005] Graphite digestion is a new digestion technology developed in recent years based on the principle of wet digestion. It uses a graphite heating element, ensuring uniform heating speed and enabling high and low temperature digestion and acid removal. It offers advantages such as short digestion time and the ability to process large batches of samples, and is gradually replacing microwave digestion. Graphite digestion is currently used in many agricultural and sideline product applications.

[0006] Therefore, in order to overcome the limitations of the high-end "microwave digestion-ICP-MS" technical route, and based on the actual equipment level of grassroots testing institutions, it is essential to develop a rapid detection method that is universally applicable, highly accurate, economical, and environmentally friendly. Thus, developing a method that utilizes both GFAAS and AFS conventional instruments in a single digestion process to achieve rapid and simultaneous determination of five key harmful elements (Pb, Cd, Cr, As, and Hg) in agricultural and sideline products is a pressing technical problem that grassroots testing institutions need to solve. Summary of the Invention

[0007] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a method for detecting heavy metals in agricultural and sideline products based on graphite rapid digestion-atomic absorption / atomic fluorescence spectroscopy. Specifically, this invention provides a rapid detection method for five heavy metal elements in agricultural and sideline products based on graphite rapid digestion-atomic absorption / atomic fluorescence spectroscopy. This invention takes graphite digestion technology as the core, establishes a unified and efficient pretreatment method, and achieves rapid and simultaneous determination of five key harmful elements (Pb, Cd, Cr, As, and Hg) in agricultural and sideline products through a single digestion process and comprehensive use of two conventional instruments, GFAAS and AFS.

[0008] The technical route of this invention follows a systematic research path of "sample collection → pretreatment condition optimization → instrument condition optimization → methodology verification → comparison with traditional methods → application to actual samples". It focuses on solving the following key scientific problems and technical difficulties: (1) How to determine a universal graphite digestion condition suitable for agricultural and sideline products with different matrices (rich in moisture, protein, starch, fiber, etc.); (2) How to optimize the instrument parameters of GFAAS and AFS to maximize the detection sensitivity and stability of the five elements, especially solving the problem of arsenic valence reduction when AFS simultaneously determines arsenic and mercury; (3) How to systematically evaluate the various performance indicators of the established method and verify its applicability and reliability in real-world scenarios. This provides a detection method for the quality and safety of agricultural and sideline products, thereby reducing the occurrence of diseases (nervous system damage, anemia, kidney failure, cerebral edema, cirrhosis, and cancer) caused by excessive heavy metal content, and thus protecting people's health.

[0009] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for detecting heavy metals in agricultural and sideline products based on rapid graphite digestion-atomic absorption / atomic fluorescence spectroscopy, comprising the following steps: S1. Add the sample to the digestion solution and place it on a graphite digester for digestion. First, pre-digest at 85℃~95℃ for 25 min~35 min, then raise the temperature to 95℃~105℃ and hold for 25 min~35 min, and then raise the temperature to the digestion temperature for further digestion to obtain the digested sample. The digestion temperature is 105℃~140℃, and the digestion time is 45 min~90 min. The samples include grains, vegetables, fruits, meat, fresh eggs, and tea. S2. Add the digested sample to a graphite furnace atomic absorption spectrometer to determine the contents of lead, cadmium, and chromium in the sample. Add the digested sample to an atomic fluorescence spectrometer to determine the contents of arsenic and mercury in the sample.

[0010] Preferably, when the sample is a grain, in step S1, the digestion solution is a mixture of HNO3 and H2O2, with a volume ratio of HNO3 to H2O2 of 4.5~5.5:1, HNO3 concentration of analytical grade, and H2O2 mass fraction of 30%. The digestion temperature is 105℃~125℃, the digestion time is 45min~90min, and the sample to digestion solution addition ratio is 0.4 g~0.6 g:3mL~4mL. More preferably, the volume ratio of HNO3 to H2O2 is 5:1, HNO3 concentration of analytical grade, H2O2 mass fraction of 30%, the digestion temperature is 115℃, the digestion time is 60 min, and the sample to digestion solution addition ratio is 0.5 g:3mL.

[0011] Preferably, when the sample is a vegetable or fruit, in step S1, the digestion solution is a mixture of HNO3 and H2O2, with a volume ratio of HNO3 to H2O2 of 4.5~5.5:1, HNO3 concentration of analytical grade, H2O2 mass fraction of 30%, digestion temperature of 105℃~125℃, digestion time of 45min~90min, and sample to digestion solution addition ratio of 0.4 g~0.6 g:3mL~6mL. More preferably, the volume ratio of HNO3 to H2O2 is 5:1, HNO3 concentration of analytical grade, H2O2 mass fraction of 30%, digestion temperature of 115℃, digestion time of 60 min, and sample to digestion solution addition ratio of 0.2 g~1.0 g:6mL.

[0012] Preferably, when the sample is livestock or poultry meat or fresh eggs, in step S1, the digestion solution is a mixture of HNO3 and H2O2, with a volume ratio of HNO3 to H2O2 of 4.5~5.5:1, HNO3 concentration of analytical grade, H2O2 mass fraction of 30%, digestion temperature of 130℃~140℃, digestion time of 45min~60min, and sample to digestion solution addition ratio of 0.2 g~0.4 g:4mL~5mL. More preferably, the volume ratio of HNO3 to H2O2 is 5:1, HNO3 concentration of analytical grade, H2O2 mass fraction of 30%, digestion temperature of 130℃, digestion time of 45min, and sample to digestion solution addition ratio of 0.3 g:5mL.

[0013] Preferably, when the sample is tea, in step S1, the digestion solution is a mixture of HNO3 and HCl, with a volume ratio of HNO3 to HCl of 1.5 to 2.5:1, the concentration of HNO3 being of analytical grade, the mass fraction of HCl being 36% to 38%, the digestion temperature being 130°C to 140°C, the digestion time being 45 min to 60 min, and the ratio of sample to digestion solution being 0.15 g to 0.25 g: 4 mL to 5 mL.

[0014] Preferably, in S1, the food is first pre-digested at 90°C for 30 min, then heated to 100°C and held for 30 min, and then heated to the digestion temperature for further digestion.

[0015] Preferably, in S2, the conditions for determining lead include: wavelength 283.31 nm, slit width 0.7 nm, lamp current 10 mA, drying temperatures of 120 °C and 140 °C, drying time of 30 s each, ashing temperature of 750 °C, ashing time of 30 s, atomization temperature of 1700 °C, atomization time of 3 s, and a mass ratio of matrix modifier to sample of 1:2, wherein the matrix modifier is ammonium dihydrogen phosphate.

[0016] Preferably, in S2, the conditions for determining cadmium include: wavelength 228.80 nm, slit 0.7 nm, lamp current 4 mA, drying temperature of 120 °C and 140 °C, drying time of 30 s, ashing temperature of 300 °C, ashing time of 35 s, atomization temperature of 1500 °C, atomization time of 3 s, and no matrix modifier added.

[0017] Preferably, in S2, the conditions for determining chromium include: wavelength 357.87 nm, slit size 0.7 nm, lamp current 10 mA, drying temperature of 120 °C and 140 °C, drying time of 30 s, ashing temperature of 1100 °C, ashing time of 38 s, atomization temperature of 2300 °C, atomization time of 5 s, and a mass ratio of matrix modifier to sample of 1:2, wherein the matrix modifier is ammonium dihydrogen phosphate.

[0018] Preferably, in S2, the conditions for determining arsenic and mercury include: using a hydrochloric acid solution with a volume fraction of 10% to 20% as the current carrier medium, using NaBH4 with a mass concentration of 1% to 2% as the reducing agent, and a negative high voltage of 230 V to 330 V.

[0019] Preferably, in S2, the optimal instrument conditions for the simultaneous determination of total arsenic and mercury in rice using dual-channel atomic fluorescence spectrometry are: negative high voltage 270 V; As lamp current 50 mA, Hg lamp current 25 mA; carrier gas is 10% (v / v) HCl solution; reducing agent is 1.5% (m / v) NaBH4 solution (dissolved in 0.5% NaOH); carrier gas flow rate 400 mL / min, shielding gas flow rate 800 mL / min; atomizer height 8 mm.

[0020] Preferably, when the sample is a grain, the RSD of the method precision is between 1.8% and 3.5%, the RSD of the method repeatability is between 3.2% and 6.8%, and the spiked recovery rate is between 96.6% and 102.8%. When the sample is livestock or poultry meat or fresh eggs, the RSD of the method precision is between 1.8% and 3.5%, the RSD of the method repeatability is between 2.6% and 3.1%, and the spiked recovery rate is between 93.6% and 98.4%. When the sample is tea, the RSD of the method precision is between 1.8% and 3.5%, the RSD of the method repeatability is between 2.4% and 3.1%, and the spiked recovery rate is between 82.3% and 99.2%.

[0021] Secondly, this invention provides the application of the method in the rapid detection of five heavy metal elements, Pb, Cd, Cr, As, and Hg, in agricultural and sideline products.

[0022] This invention has at least one of the following beneficial effects: This invention successfully established and verified a rapid detection method for five heavy metal elements (Pb, Cd, Cr, As, and Hg) in agricultural by-products based on graphite rapid digestion-AAS / AFS coupled technology. This method has the following significant advantages: 1. High applicability: The optimized pretreatment conditions are applicable to agricultural and sideline products of various substrates such as vegetables, fruits, meat, eggs, rice, and tea.

[0023] 2. Accurate and reliable: The method has been systematically verified, and all performance indicators are excellent and equivalent to the national standard method.

[0024] 3. High efficiency and economy: It enables the determination of 5 elements by using two conventional instruments in a single digestion process, which significantly improves detection efficiency and greatly reduces costs.

[0025] 4. Green and environmentally friendly: It significantly reduces the use of toxic and harmful reagents and energy consumption, making it environmentally friendly. Attached Figure Description

[0026] Figure 1 The effect of digestion temperature on the recovery rates of lead, cadmium, and chromium when the sample is rice; Figure 2 The effect of digestion solution volume on the recovery rates of lead, cadmium, and chromium when the sample is rice; Figure 3 The effect of digestion time on the recovery rates of lead, cadmium, and chromium when the sample is rice; Figure 4 Selection of carrier concentration when the sample is rice; Figure 5 Selecting the NaBH4 concentration when the sample is rice Figure 6 The selection of negative high pressure when the sample is rice; Figure 7 The effect of digestion temperature on recovery rate when the sample is poultry meat; Figure 8 The effect of digestion solution volume on recovery rate when the sample is poultry meat; Figure 9 The effect of digestion time on recovery rate when the sample is poultry meat. Detailed Implementation

[0027] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] Experimental instruments and reagents: Main instruments: EH-20A Plus graphite digestion system (Beijing Labtech Co., Ltd.); PinAAcle 900T graphite furnace atomic absorption spectrometer (PerkinElmer Inc.), equipped with Pb, Cd, and Cr hollow cathode lamps and an ASC autosampler; AFS-2300 dual-channel atomic fluorescence spectrometer (Beijing Haiguang Instrument Co., Ltd.), equipped with As and Hg high-performance hollow cathode lamps; ME204 / 02 0.01% electronic balance (Mettler-Toledo International Ltd.); ultrapure water system (Merck Millipore).

[0029] Main reagents and standards: concentrated nitric acid (HNO3, analytical grade), concentrated hydrochloric acid (HCl, analytical grade), 30% hydrogen peroxide (H2O2, analytical grade), thiourea (CH4N2S, analytical grade), sodium borohydride (NaBH4, analytical grade), sodium hydroxide (NaOH, analytical grade). Single-element standard stock solutions of Pb, Cd, Cr, As, and Hg (all concentrations 1000 mg / L, National Institute for Nonferrous Metals Research). All glassware and PTFE digestion vessels used in the experiment were soaked in 20% nitric acid solution for 24 hours, rinsed thoroughly with ultrapure water, and air-dried before use.

[0030] Example 1 This embodiment provides a rapid detection method for five heavy metal elements in agricultural and sideline products based on graphite rapid digestion-atomic absorption / atomic fluorescence spectrometry, including the following steps: I. Sample Collection and Pretreatment Representative agricultural and sideline products from six major categories were randomly collected from major agricultural production areas, large farmers' markets, and large supermarkets in Yongxiu County, with 20 batches of each category, totaling 120 batches. Specifically, these included: Vegetables: cabbage, carrots, peppers, etc.; Fruits: apples, bananas, pears, etc.; Meat products: pork tenderloin, beef hind leg, lamb hind leg, etc.; Fresh eggs: chicken eggs, duck eggs; Grains: Rice; Tea types: local green tea and black tea.

[0031] All samples were immediately placed in a refrigerated box and transported back to the laboratory after collection. Vegetables and fruits were washed with ultrapure water, dried, and the edible parts were collected. Visible fat and connective tissue were removed from livestock and poultry meat. Fresh eggs were shelled and homogenized. Rice and tea were sampled directly. After being thoroughly homogenized, the samples were frozen at -20°C for later testing.

[0032] II. Optimization of Rapid Graphite Digestion Conditions Accurately weigh 0.2 g–1.0 g (accurate to 0.0001 g) of homogenized sample into a 50 mL polytetrafluoroethylene digestion vessel, and add four digestion solutions (nitric acid, nitric acid / perchloric acid, nitric acid / hydrochloric acid, nitric acid / hydrogen peroxide, see Tables 1, 6, and 9 for details). Then place the vessel on a graphite digestion apparatus for digestion. After digestion, cool to room temperature and transfer to a final volume. Take a portion and determine lead, cadmium, and chromium using a graphite furnace atomic absorption spectrometer. Systematically investigate the effects of the following four key factors on the digestion effect: 1. Sample weight, 2. Digestion solution system, 3. Digestion temperature, and 4. Digestion time.

[0033] The digestion procedure is as follows: Add the digestion solution at room temperature, cover with the inner lid, and pre-digest at 90 ℃ for 30 min. The next day, place the solution on a graphite digester, cover with a funnel, and digest according to the set temperature program (first raise to 100 ℃ and hold for 30 min, then raise to the target temperature and hold for the corresponding time; target temperatures are 95, 105, 115, and 125 ℃). After digestion, cool to room temperature, rinse the inner lid and the vessel wall several times with a small amount of ultrapure water, transfer all the digestion solution to a 10 mL volumetric flask, dilute to the mark with ultrapure water, and shake well before use. If there are a small amount of suspended matter in the solution, it needs to be filtered through a 0.45 μm aqueous filter membrane.

[0034] III. Optimization of Instrumental Analysis Conditions 1. Optimization of instrument conditions for graphite furnace atomic absorption spectrometry (GFAAS) determination of lead, cadmium, and chromium. The instrument conditions for determining lead, cadmium, and chromium using a graphite furnace atomic absorption spectrometer were optimized. The effects of graphite tube type, graphite furnace heating program (drying temperature 120℃ and 140℃, ashing temperature 300℃~1100℃, atomization temperature 1500℃~2300℃, etc.), and matrix modifier (ammonium dihydrogen phosphate) on the instrument signal were investigated to obtain the optimal instrument conditions.

[0035] 2. Optimization of instrument conditions for the determination of arsenic and mercury by atomic fluorescence spectrometry Since arsenic in the samples usually exists in the pentavalent form, while the optimal valence state for AFS detection is trivalent arsenic, the digestion solution needs to be pre-reduced. Take 5.0 mL of the digestion solution obtained in "Step Two" into a 10 mL colorimetric tube, add 1.0 mL of a 5% thiourea-5% ascorbic acid mixed solution, and dilute to the mark with ultrapure water. Shake well and let stand for 30 min to allow As(V) to be fully reduced to As(III). For the simultaneous determination of As and Hg in AFS, the following parameters were optimized: Negative high voltage and lamp current: Investigate their impact on signal-to-noise ratio.

[0036] Carrier and reducing agent: The effectiveness of different concentrations of hydrochloric acid (5%-25%, volume fraction) as carrier was compared; the reduction efficiency of NaBH4 concentration (0.5%-2.0%, mass concentration) at a specific acidity was optimized. Gas-liquid separation and atomization conditions: The flow rates of carrier gas (Ar) and shielding gas, as well as the atomizer height, were optimized. The final optimal instrument conditions were: negative high voltage 270 V; As lamp current 50 mA, Hg lamp current 25 mA; carrier gas 10% (v / v) HCl solution; reducing agent 1.5% (m / v) NaBH4 solution (dissolved in 0.5% NaOH); carrier gas flow rate 400 mL / min, shielding gas flow rate 800 mL / min; atomizer height 8 mm.

[0037] IV. Methodological Validation Referring to Fu Zhifeng et al. (Fu Zhifeng, Zhou He. Determination of total arsenic in rice by dilute acid microwave digestion-atomic fluorescence spectrometry [J]. Chinese Journal of Inorganic Analytical Chemistry, 2023, 13(4): 311-317), the feasibility of the graphite rapid digestion method combined with AAS / AFS for the determination of five heavy metal elements in agricultural byproducts was verified from the aspects of linearity, limit of detection (limit of quantitation), precision, repeatability, spiked recovery rate, accuracy, and interference test.

[0038] Linearity: Under optimized experimental conditions and according to the optimal instrument operating conditions, the linear range and coefficient of determination of the standard working curves of Pb, Cd, Cr, As and Hg elements were examined.

[0039] Limit of detection (limit of quantitation): Prepare 11 blank sample solutions according to the optimized processing conditions, measure them under the best instrument conditions, and multiply the result by the dilution factor by 3 times the standard deviation / the slope of the standard curve. The limit of detection is the method limit of quantitation.

[0040] Precision: The atomic absorption absorbance was measured six times for 10 μg / L Pb, 2 μg / L cadmium, and 20 μg / L chromium standard solutions, respectively, according to the instrument operating conditions and sample pretreatment conditions. The relative standard deviation of atomic absorption absorbance was calculated. The atomic fluorescence intensity was measured six times for 10 μg / L As and 1 μg / L mercury standard solutions, respectively.

[0041] Repeatability: Six parallel samples were weighed, and the signal response values ​​of the samples were measured according to the optimized sample pretreatment method and the optimal detection conditions of AAS / AFS. The relative standard deviation was then calculated.

[0042] Spiked recovery experiment: 12 samples were accurately weighed, of which 3 were used to determine the background values ​​of the contents of 5 heavy metal elements in the samples, and the remaining 9 were used for recovery tests at three standard spiking levels: high, medium and low. That is, high, medium and low concentration levels of standard solutions of each element were added to the samples, and then the contents were determined by AAS / AFS to obtain the recovery level.

[0043] Interference test: Some common coexisting ions and related interfering ions in agricultural and sideline products were selected for the experiment. A certain concentration of Na, K, Ca, Zn, Cu and Mn ion solutions were added to a mixed standard solution of lead (20 ng / mL), cadmium (2 ng / mL), chromium (20 ng / mL), arsenic (10 ng / mL) and mercury (1 ng / mL) to investigate the effect of the addition of the above ions on the determination of the five elements.

[0044] V. Comparison with traditional methods and practical applications Method Comparison: The results of the graphite rapid digestion method for positive samples were compared in parallel with the national standard method (GB 5009.268-2016 National Food Safety Standard - Determination of Multiple Elements in Food - Method I: Microwave Digestion-ICP-MS, or the corresponding microwave digestion-AAS / AFS method). t Test and compare the differences in the results obtained by the two methods.

[0045] VI. Results and Discussion (a) When the sample is rice, the test results are as follows: 1. Rapid graphite digestion-AAS determination of lead, cadmium, and chromium in rice 1.1 Optimization of rapid graphite digestion conditions (1) Selection of digestion system Four different reagents were used for rapid graphite digestion, as follows: Results showed that digestion solutions 2 (concentrated HNO3, analytical grade), 3 (HNO3 + HClO4, both concentrated HNO3 and HClO4 were analytical grade), and 4 (HNO3 + H2O2, both concentrated HNO3 and H2O2 had a mass fraction of 30%) all completely digested rice flour. However, solutions 2 and 3 used polluting acids; therefore, solution 4 (5 mL HNO3 + 1 mL H2O2) was chosen as the optimal digestion solution. Nitric acid, as a strong oxidant, effectively destroyed the organic matrix, while the addition of hydrogen peroxide increased the oxidation potential, promoted the digestion of recalcitrant substances, and made the digestion solution clearer and more transparent.

[0046] Table 1 Digestion solution system (2) Selection of digestion temperature Rice flour quality control samples were digested at preset temperatures of 95, 105, 115, and 125 °C on a graphite digester with a fixed digestion time of 60 min and 3 mL of digestion solution. The effect of digestion temperature on the recovery rate of the three elements was investigated. The results are as follows: Figure 1 As the digestion temperature increased, the recovery rates of the three heavy metal elements showed a gradual increase after exceeding 115 °C, and then tended to stabilize. Therefore, a digestion temperature of 115 °C was selected.

[0047] (3) Selection of digestion solution dosage The effects of different digestion solution addition volumes (1.0, 2.0, 3.0, 4.0 mL) on the recovery rates of lead, cadmium, and chromium in rice were investigated. Figure 2The recovery rates of each element were relatively low (88.2%–91.2%) when 1 mL and 2 mL of digestion solution were added, while no significant difference was observed in the other two addition conditions, and the appearance of the digestion solution did not change significantly. The recovery rate range of the three elements (95.0%–98.4%) was the best. In summary, a digestion solution volume of 3 mL was selected.

[0048] (4) Selection of digestion time Digestion of 0.2 g rice flour quality control samples at different time points (15, 30, 45, 60, and 90 min) was performed to investigate its effect on the recovery rate. Figure 3 As shown, after 15 min of digestion, the recovery rates of each element were relatively low, and the digestion solution contained a relatively large amount of suspended solids, ranging from 75.2% to 84.2%. After 30 min of digestion, the recovery rates of each element significantly improved. However, as the digestion time was extended (exceeding 60 min), the recovery rates did not improve significantly. This indicates that a digestion time of 60 min is sufficient to completely release the heavy metals (lead, cadmium, and chromium) from the rice flour. Therefore, the optimal digestion time was confirmed to be 60 min.

[0049] 1.2 Determination of Optimal Instrument Analysis Conditions Lead and cadmium are both relatively volatile elements, exhibiting significant ashing losses at 600 °C without matrix modifiers. Adding appropriate matrix modifiers can increase the ashing temperature and eliminate matrix interference, but it may also introduce new blank contamination. Repeated experiments revealed that adding ammonium dihydrogen phosphate significantly increased the instrument response and improved the linearity of the standard curve when determining lead in common foods. However, the instrument response was generally stable when determining cadmium, and the addition of ammonium dihydrogen phosphate did not significantly change the response; sometimes it even increased the blank value. Therefore, cadmium was analyzed directly without a matrix modifier. For chromium, the presence of phosphate ions causes chromium to react with phosphate ions to form phosphates (in the initial heating stage). After heating, chromium forms oxyphosphate complexes and adheres to the graphite tube surface, reducing chromium volatility and increasing the ashing temperature, thus promoting matrix volatilization. Therefore, it showed better results in determining chromium in rice, improving the atomization signal.

[0050] Table 2 Instrument Operating Conditions 2. Graphite rapid digestion-AFS determination of total arsenic and total mercury in rice 2.1 The preprocessing conditions are the same as above.

[0051] 2.2 Optimization of Instrument Conditions (1) Selection of carrier acidity The concentration of the carrier acid affects the hydride formation efficiency. With a fixed NaBH4 concentration of 1.5% and a negative high voltage of 270 V, the changes in fluorescence values ​​of arsenic and mercury standard solutions (As 20.0 ng / ml, Hg 2.0 ng / ml) were compared when different concentrations of hydrochloric acid (5%, 10%, 15%, 20%, 25%) were used as the carrier. Figure 4 As shown, the results indicate that when the volume fraction of the carrier medium is 10%–25%, the fluorescence intensity of arsenic and mercury does not change significantly, and is higher than that at a volume fraction of 5%. Considering that excessive acidity may corrode the instrument's sample introduction system, and also cause reagent waste and environmental pollution, this invention selects a 10% hydrochloric acid solution as the carrier medium.

[0052] (2) NaBH4 concentration NaBH4, acting as a reducing agent, reacts with arsenic to form AsH3, and its concentration is closely related to the formation of hydrides. If the NaBH4 concentration is too low, it cannot react completely with mercury and arsenic; if the NaBH4 concentration is too high, a large amount of hydrogen gas will be produced, thereby weakening the fluorescence intensity and reducing the sensitivity. This invention compares the effects of different concentrations of NaBH4 (0.5%, 1.0%, 1.5%, 2.0%) on the fluorescence values ​​of arsenic and mercury (As 20.0 ng / ml, Hg 2.0 ng / ml). Figure 5 When the NaBH4 concentration is less than 1.5%, the fluorescence intensity of arsenic and mercury first increases with the increase of NaBH4 mass concentration. When the NaBH4 mass concentration is 1.5%, the fluorescence intensity of arsenic and mercury both reach the maximum. After exceeding 1.5%, the fluorescence intensity decreases. Therefore, the experiment selected a NaBH4 mass concentration of 1.5%.

[0053] (3) Negative high voltage Appropriately increasing the negative high voltage can improve the sensitivity of arsenic and mercury detection to some extent. Figure 6 The effect of negative high voltage on the fluorescence response values ​​of arsenic and mercury standard solutions (As 20.0 ng / ml, Hg 2.0 ng / ml) was investigated. The results showed that the fluorescence values ​​of both elements increased with increasing negative high voltage in the range of 230~330 V. Since both signal and noise increase with increasing negative high voltage, and considering factors such as the sensitivity of the detection method, blank background value, signal-to-noise ratio and lamp life, this paper selected a negative high voltage of 270 V.

[0054] Through research, the optimal instrument conditions for the simultaneous determination of total arsenic and mercury in rice using dual-channel atomic fluorescence spectrometry were determined as follows: negative high voltage: 270 V; lamp current: As: 50 mA, Hg: 25 mA; carrier gas flow rate: 400 mL·min -1 Shielding gas flow rate: 800 mL·min -1Atomization height: 8 mm; Current carrier: 10% HCl; Reducing agent: 1.5% NaBH4 + 0.25% NaOH, thiourea concentration 5%.

[0055] 3. Methodological Validation Linear range and detection limit: For each element within its linear range, the standard curve... R The values ​​are all greater than 0.995, indicating a good linear relationship.

[0056] Table 3. Linearity, Limit of Detection, and Limit of Quantification for 5 Elements Precision and repeatability: The RSD (n=6) of instrument precision is between 1.8% and 3.5%. The RSD (n=6) of method repeatability is between 3.2% and 6.8%, indicating that the method has excellent stability and repeatability.

[0057] Spiked Recovery: To verify the accuracy and practicality of the method, a spiked recovery test was conducted on the same rice sample. Three concentration levels (high, medium, and low) were added for testing, using the optimal conditions described above. The results are shown in Table 4. The table shows that the spiked recovery rate of the rapid graphite digestion method ranged from 96.6% to 102.8%, with an RSD of 3.6%, which falls within the range of 80.0% to 110.0% recommended by GB / T27404-2008 "Laboratory Quality Control Standards for Physicochemical Testing of Food," indicating that the method has good accuracy.

[0058] Table 45 shows the spiked recovery results for each element. Anti-interference performance: Interference experiments were conducted on some common coexisting ions in rice and some ions that may form hydrides. 100 mg / L of Na+ was added to a 10.0 μg / L mixed standard solution of lead and arsenic. + K + Ca 2+ Zn 2+ The relative average deviation of the determination results for five heavy metal elements (Cu, Mn, and Cu ions) was within 5%, indicating that the method has good anti-interference ability.

[0059] 4. Comparison with traditional methods Method comparison: 30 positive samples t The test results showed no significant difference between the graphite rapid digestion method and the national standard microwave digestion method (ICP-MS method) in the determination of Pb, Cd, Cr, As, and Hg. P >0.05). This fully demonstrates that our method is equivalent to the national standard method in terms of accuracy.

[0060] Actual sample analysis: The graphite digestion method was used to test 20 batches of rice sampled in Yongxiu County, Jiujiang City. The average content of the three elements in the vast majority of rice samples was lower than the national standard limit. The content of Cd in a few rice samples was close to or slightly exceeded the limit, which may be related to the soil environment in which the rice was grown. In addition, the test results for arsenic and mercury showed that total mercury was not detected in all rice samples. The total arsenic in the 20 batches of rice varied from 0.031 to 0.254 mg / kg. Total arsenic was detected in all tested rice samples, but the content was not the same. The total arsenic content of a few rice samples exceeded the national standard limit, which may be related to the soil factors in which the rice variety was grown.

[0061] Table 55 shows the analysis results of the elements. In summary, the above-mentioned testing conditions can be used for all grain samples; in addition, vegetable and fruit samples are simple matrices and can be directly analyzed using the optimized analytical method for rice flour.

[0062] (ii) When the sample is livestock or poultry meat, the test results are as follows: 1. Rapid graphite digestion-AAS determination of lead, cadmium, and chromium in poultry meat 1.1 Optimization of rapid graphite digestion conditions (1) Selection of digestion system Different reagents were selected for rapid digestion. Four different digestion reagents were used for preliminary experiments, and the schemes are as follows: (1): HNO3 5 mL; (2): HNO3 4.5 mL, HClO4 0.5 mL; (3): HNO3 4 mL, HClO4 1 mL; (4): HNO3 5 mL, H2O2 1 mL. The concentrations of HNO3, HClO4, and H2O2 were the same as those of the rice digestion system.

[0063] Table 6 Comparison of Mixed Acid Systems The results showed that digestion system 4 (5 mL HNO3 + 1 mL H2O2) exhibited the best digestion effect, with a low blank value and reduced use of contaminating acid. Nitric acid, as a strong oxidant, can effectively destroy the organic matrix. The addition of hydrogen peroxide can increase the oxidation potential, promote the digestion of difficult-to-decompose substances, and make the digestion solution easier to become clear and transparent. Therefore, digestion system 4 was selected as the digestion reagent.

[0064] (2) Selection of digestion temperature The samples were digested under the preset conditions of 110, 120, 130, and 140 °C in the graphite digester. Figure 7As shown, the results indicate that with the increase of digestion temperature, the determination results of multiple elements first increase and then tend to stabilize. When the temperature exceeds 130 °C, the three heavy metal elements are all at a relatively high level (96.1%~97.9%). Considering that 130 °C is the limit temperature for mercury digestion determination, the digestion temperature for poultry meat determination is determined to be 130 °C.

[0065] (3) Selection of digestion solution dosage A fixed sample weight of 0.3 g was used, with a digestion temperature of 130 ℃ and a digestion time of 45 min. The effects of digestion solution volumes (1.0, 2.0, 3.0, 4.0, and 5.0 mL) on the recovery rates of lead, cadmium, and chromium in rice were investigated. Figure 8 As shown, when 1-4 mL of digestion solution was added, the recovery rate of each element was low. When the amount of digestion solution was 5 mL, the recovery rate of the three elements was 94.2-97.2%, which met the relevant test requirements. Therefore, the amount of digestion solution added for poultry meat was determined to be 5.0 mL.

[0066] (4) Selection of digestion time 0.2 g of poultry meat was subjected to semi-digestion at different time intervals (15, 30, 45, and 60 min) to investigate the effect of different digestion times on the recovery rate. The results are as follows: Figure 9 As shown in the figure, after 15 min of digestion, the digest contains a relatively large amount of suspended solids, and the recovery rates of each element are relatively low, ranging from 64.2% to 68.6%. After 30 min of digestion, the recovery rates of each element significantly improved. However, after 45 min, the recovery rates did not increase with further digestion time (95.4%-97.6%). Taking all factors into consideration, a digestion time of 45 min was determined.

[0067] 1.2 Instrument conditions: Same as rice substrate.

[0068] 2. Graphite rapid digestion-AFS determination of total arsenic and total mercury in braised meat 1.1 The preprocessing conditions are the same as above.

[0069] 1.2 Instrument conditions: Same as rice substrate 3. Methodological Validation Linear range and detection limit (same as rice matrix): For each element within its linear range, the standard curve... R The values ​​are all greater than 0.995, indicating a good linear relationship.

[0070] Table 7. Linearity, Limit of Detection, and Limit of Quantification for Five Elements Precision and repeatability: The RSD (n=6) of instrument precision is between 1.8% and 3.5%. The RSD (n=6) of method repeatability is between 2.6% and 3.1%, indicating that the method has excellent stability and repeatability.

[0071] Spiked Recovery: To verify the accuracy and practicality of the method, a spiked recovery test was conducted on the same poultry meat sample. Three concentration levels (high, medium, and low) were added for testing, using the optimal conditions described above. The results are shown in Table 8. Table 8 shows that the spiked recovery rate of the graphite digestion method ranged from 93.6% to 98.4%, with an RSD of 0.9% to 2.5%, which falls within the range of 80.0% to 110.0% recommended by GB / T27404-2008 "Laboratory Quality Control Standards for Physicochemical Testing of Food," indicating that the method has good accuracy.

[0072] Table 85 shows the spiked recovery results for each element. Interference resistance: Interference experiments were conducted on some common coexisting ions and ions that may form hydrides in braised meat. 100 mg / L of Na+, K+, and Ca2+ were added to a 10.0 μg / L mixed standard solution of lead and arsenic, respectively. 2+ Zn 2+ The relative average deviations of the determination results for Cu, Mn ions, lead, and total arsenic were all within 5%, indicating that the method has good anti-interference ability.

[0073] 4. Comparison with traditional methods and results of practical application Method comparison: 30 positive samples t The test results showed no significant difference between the graphite rapid digestion method and the national standard microwave digestion method in the determination of Pb, Cd, Cr, As, and Hg. p >0.05). This fully demonstrates that our method is equivalent to the national standard method in terms of accuracy.

[0074] The low detection rate of the five heavy metal elements in poultry meat samples from this region indicates that braised meat in this region is generally safe.

[0075] In summary, the above-mentioned testing conditions can be used for all livestock and poultry meat samples. In addition, fresh egg samples and livestock and poultry meat samples have similar matrices, so the analytical methods optimized for livestock and poultry meat samples can be directly used.

[0076] (III) When the sample is tea, the test results are as follows: 1. Optimization of the graphite digestion process Tea contains a high amount of organic components, rich in polyphenols, pigments, and other complex substances, making it the most difficult to digest and prone to incomplete digestion. Three digestion solutions were initially proposed for digesting green tea quality control samples, and the results are shown in Table 9. In digestion system 1 (HNO3, analytical grade), the digested state was a light black color, indicating that the sample was not completely digested. In digestion system 2 (HNO3 + H2O2, analytical grade HNO3, H2O2 mass fraction 30%), the digested state contained particles of varying sizes, indicating that a small amount of sample remained undigested. However, in digestion system 3 (HNO3 + HCl, analytical grade HNO3, HCl mass fraction 37%), the digested state was a light green, clear, and transparent liquid, indicating that the sample was completely digested. Therefore, digestion system 3 was selected.

[0077] Table 9 Comparison of Mixed Acid Systems Furthermore, the effects of digestion solutions from three different graphite digestion procedures were compared, and the specific results are shown in Table 10.

[0078] Table 10 Graphite Digestion Procedure In addition, tea contains a relatively high amount of organic components, so the optimized sampling amount of 0.2 g (reducing the sample weight) makes digestion procedure 1 prone to incomplete digestion. Digestion procedure 3 is more thorough than digestion procedure 2, but excessively high temperatures may lead to low mercury recovery. Therefore, methodological validation was conducted to examine whether digestion procedure 3 meets the relevant requirements.

[0079] 2. Methodological Examination Linear range: same as matrix rice; detection limit: same as matrix poultry meat.

[0080] Precision and repeatability: The RSD (n=6) of instrument precision is between 1.8% and 3.5%. The RSD (n=6) of method repeatability is between 2.4% and 3.1%, indicating that the method has excellent stability and repeatability.

[0081] Spiked Recovery: To verify the accuracy and practicality of the method, spiked recovery tests were conducted on tea samples using graphite digestion procedure 3. Three concentration levels (high, medium, and low) were tested under the optimal conditions described above. The results are shown in Table 11. Table 11 shows that the spiked recovery rate of tea samples using the graphite digestion method ranged from 82.3% to 99.2%, with an RSD of 1.0% to 2.9%. This falls within the range of 80.0% to 110.0% recommended by GB / T 27404-2008 "Laboratory Quality Control Standards for Physicochemical Testing of Food," indicating good accuracy. Therefore, graphite digestion procedure 3 is suitable for the determination of five heavy metal elements in tea.

[0082] Table 1. Spike recovery results for 115 elements 3. Determination of actual samples Twenty batches of green tea samples were taken and digested according to graphite digestion procedure 3, and tested according to method (III)-1. The results showed that all 20 batches of green tea samples were completely digested, resulting in a light green, clear, and transparent liquid. The lead content ranged from 0 to 0.35 mg / kg, the cadmium content from 0 to 0.052 mg / kg, the chromium content from 0 to 0.11 mg / kg, the total arsenic content from 0 to 0.11 mg / kg, and the total mercury content was undetectable. All elemental contents met the relevant requirements of the national standard limit GB 2762-2022.

[0083] Table 125 Analysis Results In summary, the key technologies for rapid graphite digestion AAS / AFS determination of agricultural products are as follows, based on the above experiments: (1) Vegetables and fruits (represented by cabbage and apples) This type of sample has a high moisture content and relatively simple fiber and organic matter composition. A rice flour digestion method can be used. Weigh 0.5 g of the sample and use a digestion system (5 mL HNO3 + 1 mL H2O2). Digestion at 115 ℃ for 60 min will completely decompose the sample, producing a clear and transparent digest. This method can then be used to determine the five heavy metal elements.

[0084] (2) Rice Weigh 0.2 g of sample, digest at 115 ℃ for 60 min, add 3 mL of digestion solution (nitric acid + hydrogen peroxide), and pre-digest for 30 min before digestion.

[0085] (3) Poultry and fresh eggs (represented by chicken and eggs) The sample was high in protein and fat, making digestion difficult. Experiments revealed that higher temperatures were necessary. The optimal conditions were: a sample weight of 0.3 g (to avoid excessive fat leading to a violent reaction), a digestion system of 5 mL (5 mL HNO3 + 1 mL H2O2), and digestion at 130 ℃ for 45 min. Under these conditions, the protein was completely decomposed, with no fat residue, and the recovery rates of Hg and As remained consistently above 90%.

[0086] (4) Tea The sample has a high organic matter content and is rich in complex components such as polyphenols and pigments, making it the most difficult to digest. An optimized enhanced oxidation system was used: HNO3:HCl = 2:1 (3 mL), with a sample weight of 0.2 g, and digestion at 140 ℃ for 60 min.

[0087] General summary: Under the premise of ensuring accuracy, 130 ℃, 60 min, system (2) can be used as general conditions for most samples except tea; while tea requires enhanced oxidation system and digestion temperature.

[0088] By integrating the digestion conditions and instrument parameters of the above four types of agricultural and sideline products, a standardized testing procedure was formed, and a complete rapid detection method was established. The optimal digestion conditions were: sample size 0.5 g, HNO3:H2O2 = 5:1 (v / v), digestion temperature 130℃, and time 60 min. Under these conditions, the first five types of samples were completely digested, and the digestion solution was clear and transparent with no residual particles. The optimal digestion system for tea was HNO3:HCl = 2:1 (3 mL), sample size 0.2 g, and digestion at 140℃ for 60 min.

[0089] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting heavy metals in agricultural and sideline products based on rapid graphite digestion-atomic absorption / atomic fluorescence spectroscopy, characterized in that, Includes the following steps: S1. Add the sample to the digestion solution and place it on a graphite digester for digestion. First, pre-digest at 85℃~95℃ for 25 min~35 min, then raise the temperature to 95℃~105℃ and hold for 25 min~35 min, and then raise the temperature to the digestion temperature for further digestion to obtain the digested sample. The digestion temperature is 105℃~140℃, and the digestion time is 45 min~90 min. The samples include grains, vegetables, fruits, meat, fresh eggs, and tea. S2. Add the digested sample to a graphite furnace atomic absorption spectrometer to determine the contents of lead, cadmium, and chromium in the sample. Add the digested sample to an atomic fluorescence spectrometer to determine the contents of arsenic and mercury in the sample.

2. The method according to claim 1, characterized in that, When the sample is a grain, in S1, the digestion solution is a mixture of HNO3 and H2O2, with a volume ratio of HNO3 to H2O2 of 4.5~5.5:

1. HNO3 is analytical grade pure nitric acid, and the mass fraction of H2O2 is 30%. The digestion temperature is 105℃~125℃, the digestion time is 45 min~90 min, and the ratio of sample to digestion solution is 0.4 g~0.6 g: 3 mL~4 mL.

3. The method according to claim 1, characterized in that, When the sample is a vegetable or fruit, in step S1, the digestion solution is a mixture of HNO3 and H2O2, with a volume ratio of HNO3 to H2O2 of 4.5~5.5:

1. The HNO3 is analytical grade pure nitric acid, and the mass fraction of H2O2 is 30%. The digestion temperature is 105℃~125℃, the digestion time is 45 min~90 min, and the ratio of sample to digestion solution is 0.4 g~0.6 g: 3 mL~6 mL.

4. The method according to claim 1, characterized in that, When the sample is livestock or poultry meat or fresh eggs, in S1, the digestion solution is a mixture of HNO3 and H2O2, with a volume ratio of HNO3 to H2O2 of 4.5~5.5:

1. HNO3 is analytical grade pure nitric acid, and the mass fraction of H2O2 is 30%. The digestion temperature is 130℃~140℃, the digestion time is 45 min~60 min, and the ratio of sample to digestion solution is 0.2 g~0.4 g: 4 mL~5 mL.

5. The method according to claim 1, characterized in that, When the sample is tea, in step S1, the digestion solution is a mixture of HNO3 and HCl, with a volume ratio of HNO3 to HCl of 1.5 to 2.5:

1. The HNO3 is analytical grade pure nitric acid, and the mass fraction of HCl is 36% to 38%. The digestion temperature is 130℃ to 140℃, the digestion time is 45 min to 60 min, and the ratio of sample to digestion solution is 0.15 g to 0.25 g: 4 mL to 5 mL.

6. The method according to claim 1, characterized in that, In S2, the conditions for lead determination included: wavelength 283.31 nm, slit width 0.7 nm, lamp current 10 mA, drying temperatures of 120 °C and 140 °C, drying time of 30 s each, ashing temperature of 750 °C, ashing time of 30 s, atomization temperature of 1700 °C, atomization time of 3 s, and a matrix modifier to sample mass ratio of 1:2, with the matrix modifier being ammonium dihydrogen phosphate. In S2, the conditions for determining cadmium include: wavelength 228.80 nm, slit 0.7 nm, lamp current 4 mA, drying temperature of 120 °C and 140 °C, drying time of 30 s, ashing temperature of 300 °C, ashing time of 35 s, atomization temperature of 1500 °C, atomization time of 3 s, and no matrix modifier added. In S2, the conditions for determining chromium included: wavelength 357.87 nm, slit size 0.7 nm, lamp current 10 mA, drying temperatures of 120 °C and 140 °C, drying time of 30 s, ashing temperature of 1100 °C, ashing time of 38 s, atomization temperature of 2300 °C, atomization time of 5 s, and a matrix modifier to sample mass ratio of 1:

2. The matrix modifier was ammonium dihydrogen phosphate.

7. The method according to claim 1, characterized in that, In S2, the conditions for determining arsenic and mercury include: using a hydrochloric acid solution with a volume fraction of 10%~20% as the current carrier medium, using NaBH4 with a mass concentration of 1%~2% as the reducing agent, and a negative high voltage of 230 V~330 V.

8. The method according to claim 1, characterized in that, In S2, the optimal instrument conditions for the simultaneous determination of total arsenic and mercury in rice using dual-channel atomic fluorescence spectrometry are: negative high voltage 270 V; As lamp current 50 mA, Hg lamp current 25 mA; carrier gas 10% (v / v) HCl solution; reducing agent 1.5% (m / v) NaBH4 solution (dissolved in 0.5% NaOH); carrier gas flow rate 400 mL / min, shielding gas flow rate 800 mL / min; atomizer height 8 mm.

9. The method according to claim 1, characterized in that, When the sample is a grain, the RSD of the method precision is between 1.8% and 3.5%, the RSD of the method repeatability is between 3.2% and 6.8%, and the spiked recovery rate is between 96.6% and 102.8%. When the sample is livestock or poultry meat or fresh eggs, the RSD of the method precision is between 1.8% and 3.5%, the RSD of the method repeatability is between 2.6% and 3.1%, and the spiked recovery rate is between 93.6% and 98.4%. When the sample is tea, the RSD of the method precision is between 1.8% and 3.5%, the RSD of the method repeatability is between 2.4% and 3.1%, and the spiked recovery rate is between 82.3% and 99.2%.

10. The application of the method according to any one of claims 1 to 9 in the rapid detection of five heavy metal elements, Pb, Cd, Cr, As and Hg, in agricultural and sideline products.

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