Sample pretreatment method for measuring content of heavy metals in honey

Abstract

The invention discloses a sample pretreatment method for measuring the content of heavy metals in honey, and belongs to the technical field of analytical chemistry and food detection.The sample pretreatment method comprises the following steps that S1, honey and nitric acid are subjected to vortex mixing and pre-oxidation, and a premixed solution is obtained; s2, adding perchloric acid into the premixed liquid obtained in the step S1, installing a reflux condensing device, and digesting to obtain a digesting solution; s3, cooling the digestion solution to room temperature, and fixing the volume to obtain a to-be-detected sample solution. According to the sample pretreatment method for measuring the content of the heavy metals in the honey, a mixed acid system, an acid adding mode and digestion conditions are optimized, and a reflux condensation technology is matched, so that a honey sample is rapidly and completely digested, the loss of target heavy metal elements is reduced, and the accuracy and precision of the detection method are improved; meanwhile, the threshold of experimental equipment is reduced, safe and controllable operation is guaranteed, and the requirements of various levels of detection laboratories on honey heavy metal trace analysis can be met.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and food testing technology, and in particular to a sample pretreatment method for determining the heavy metal content in honey. Background Technology

[0002] As a natural food, the safety of honey is a major concern. Heavy metal contamination is one of the main risks affecting honey safety. Accurate determination of heavy metal content in honey hinges on sample pretreatment. Currently, the industry commonly uses wet acid digestion to treat complex organic matrix samples like honey, employing strong oxidizing acid systems such as nitric acid-hydrogen peroxide or nitric acid-perchloric acid, combined with high-temperature hot plates or microwave digesters. However, this existing technology has many drawbacks. Not only is the digestion reaction difficult to control and prone to producing large amounts of foam due to honey's rich content of sugars, proteins, and other organic matter, resulting in digestion times exceeding 4 hours and low throughput, but it also requires the addition of excessive acid reagents, increasing costs and introducing laboratory pollution and health risks to operators caused by acid mist and waste liquid. Furthermore, the prolonged high-temperature digestion process easily causes volatile or semi-volatile heavy metals such as cadmium and lead to volatilization and escape, leading to high loss of target elements and decreased accuracy of measurement results. While microwave digestion can improve digestion efficiency, it suffers from high equipment and digestion vessel costs and limited sample processing capacity, making it difficult to popularize in resource-constrained laboratories.

[0003] Therefore, there is an urgent need in the field for a pretreatment method for determining the heavy metal content in honey that is complete in digestion, short in time, requires less acid reagent, and can reduce the loss of target elements. Summary of the Invention

[0004] The purpose of this invention is to provide a sample pretreatment method for determining the heavy metal content in honey. By optimizing the mixed acid system, acid addition method and digestion conditions, and combining it with reflux condensation technology, the method achieves rapid and complete digestion of honey samples, reduces the loss of target heavy metal elements, improves the accuracy and precision of the detection method, lowers the threshold of experimental equipment, and ensures safe and controllable operation. This method can meet the needs of testing laboratories at all levels to conduct trace analysis of heavy metals in honey.

[0005] To achieve the above objectives, the present invention provides a sample pretreatment method for determining the heavy metal content in honey, comprising the following steps: S1. Mix honey with nitric acid in a vortex and pre-oxidize to obtain a premixed solution; S2. Add perchloric acid to the premixed solution obtained in S1, install a reflux condenser, digest, and obtain digestion solution; S3. Cool the digestion solution to room temperature and make up to volume to obtain the sample solution to be tested.

[0006] Preferably, in S1, the mass-to-volume ratio of honey to nitric acid is 0.3-1.0 g: 2.0 mL.

[0007] Preferably, in S1, the mass concentration of nitric acid is 65%-68%.

[0008] Preferably, in S1, the vortex mixing time is 20-40s and the vortex mixing speed is 20-60rpm.

[0009] Preferably, in S1, the pre-oxidation temperature is 18-25℃ and the pre-oxidation time is 10-20min.

[0010] Preferably, in S2, the volume ratio of perchloric acid to nitric acid is 1:1.3-1.5.

[0011] Preferably, in S2, the mass concentration of perchloric acid is 60%-80%, and the addition rate of perchloric acid is 0.1-1.0 mL / min.

[0012] Preferably, in S2, the digestion temperature is 230-250℃ and the digestion time is 2-3h.

[0013] Furthermore, the digestion temperature was 240℃ and the digestion time was 2.75h.

[0014] Therefore, the sample pretreatment method for determining the heavy metal content in honey described above, as used in this invention, has the following beneficial effects: (1) This invention achieves rapid and complete digestion of honey samples by optimizing the mixed acid system, acid addition method and digestion conditions and combining it with reflux condensation technology, thereby reducing the loss of target heavy metal elements, improving the accuracy and precision of the detection method, and lowering the threshold of experimental equipment and ensuring safe and controllable operation. It can meet the needs of testing laboratories at all levels to carry out trace analysis of heavy metals in honey.

[0015] (2) The present invention uses a mixed acid system of nitric acid and perchloric acid in an optimized ratio, which allows nitric acid to fully play its role in matrix decomposition and main oxidation, while controlling the amount of perchloric acid within a reasonable range, so that it only acts as an auxiliary strong oxidant and warming agent to destroy stubborn organic matter, avoiding excessive oxidation potential and temperature due to excessive perchloric acid, effectively inhibiting the generation and escape of volatile heavy metal chlorides, and reducing the loss of target elements; at the same time, the precise ratio of the mixed acid system greatly reduces the total amount of concentrated acid used, reduces reagent costs, and also reduces the generation of acid mist and waste liquid, reducing laboratory environmental pollution and health risks to operators.

[0016] (3) The present invention first adds nitric acid to pre-oxidize the sample, and then slowly adds perchloric acid along the wall in a stepwise acid addition method. This allows the easily oxidized organic matter in the honey to react under mild conditions first, avoiding the violent reaction caused by the direct mixing of the two strong acids and contact with the sample. This effectively alleviates the risk of foam generation and splashing during the digestion process, making the operation safer and more controllable. At the same time, the pre-oxidation allows the sample matrix to be initially decomposed, laying the foundation for the subsequent digestion reaction, improving the overall digestion efficiency, and ensuring that the digestion reaction proceeds smoothly and orderly.

[0017] (4) The present invention precisely sets the digestion temperature and digestion time, and finds the best balance between complete digestion of organic matter and prevention of acid drying out too quickly and loss of target elements. Under the premise of ensuring that the organic matter in the honey sample is completely decomposed and the digestion solution reaches a clear state without organic residue, and meets the requirements of instrument analysis, the digestion time is greatly shortened, and the digestion time of a single sample is controlled within 3 hours, which is much shorter than the 4-6 hours of the traditional method. This significantly improves the throughput of sample analysis in the laboratory and can efficiently complete the detection and processing of large batches of samples.

[0018] (5) The reflux condenser in this invention can, on the one hand, condense the evaporated acid mist and reflux it back into the reaction system, reducing the additional consumption of acid reagents and further reducing reagent costs and acid mist emissions; on the other hand, it can effectively capture and redissolve trace heavy metals that may volatilize with the acid mist, reducing the volatilization loss of target elements from the source, and ensuring the stability of acid concentration during digestion. It can completely destroy difficult-to-digest components such as cellulose and protein, greatly improving the recovery rate of heavy metal elements and ensuring the accuracy of subsequent detection results; at the same time, the design of reflux condenser allows the digestion reaction to be carried out in a closed and stable system, further improving the safety of operation.

[0019] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0020] A sample pretreatment method for determining the heavy metal content in honey includes the following steps: S1. Mix honey with nitric acid in a vortex and pre-oxidize to obtain a premixed solution; S2. Add perchloric acid to the premixed solution obtained in S1, install a reflux condenser, digest, and obtain digestion solution; S3. Cool the digestion solution to room temperature and make up to volume to obtain the sample solution to be tested.

[0021] In this invention, in S1, the mass-to-volume ratio of honey to nitric acid is 0.3-1.0g:2.0mL.

[0022] In this invention, the mass concentration of nitric acid in S1 is 65%-68%.

[0023] In this invention, in S1, the vortex mixing time is 20-40s and the vortex mixing speed is 20-60rpm.

[0024] In this invention, in S1, the pre-oxidation temperature is 18-25℃ and the pre-oxidation time is 10-20min.

[0025] In this invention, honey is initially mixed with nitric acid and pre-oxidized. This step allows easily oxidized organic matter to begin reacting under milder conditions, avoiding subsequent violent reactions.

[0026] In this invention, in S2, the volume ratio of perchloric acid to nitric acid is 1:1.3-1.5.

[0027] In this invention, in S2, the mass concentration of perchloric acid is 60%-80%, and the addition rate of perchloric acid is 0.1-1.0 mL / min.

[0028] In this invention, nitric acid is the main oxidant and matrix decomposing agent, while perchloric acid, as a strong oxidant and warming agent, is controlled at a low level, which is sufficient to completely destroy the residual stubborn organic matter, but not so much as to generate excessively high oxidation potential and temperature due to excessive amount, thereby effectively inhibiting the generation and escape of volatile heavy metal chlorides.

[0029] In this invention, the reflux device can condense the evaporated acid mist and return it to the reaction system. This not only reduces the emission of acid vapor and the additional consumption of reagents, but more importantly, it can effectively capture and redissolve trace heavy metals that may volatilize with the acid mist. This is one of the key technical means to achieve "low loss".

[0030] In this invention, in step S2, the digestion temperature is 230-250℃, preferably 240℃; the digestion time is 2-3h, preferably 2.75h.

[0031] In one embodiment of the present invention, the digestion temperature is 240°C. This temperature is the optimal balance point between ensuring complete digestion of organic matter and preventing the acid from evaporating too quickly and the loss of target elements. Digestion is continued at this temperature for 2.75 hours. During this period, the solution color changes from dark to light, and finally a clear, colorless or pale yellow transparent solution is obtained, indicating that the digestion is complete.

[0032] The technical solution of the present invention will be further described below through embodiments.

[0033] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0034] In this invention, unless otherwise specified, the test materials and instruments are all conventional test materials in the field and can be purchased through commercial channels.

[0035] In an embodiment of the present invention, honey from the Kellem Wollega region of Ethiopia was used as the test sample.

[0036] Example 1 A sample pretreatment method for determining the heavy metal content in honey includes the following steps: S1. Accurately weigh 0.5g of homogenized honey and place it in a 50mL special digestion tube. Add 2.0mL of 65% nitric acid and vortex mix at 40rpm for 30s. Then let it stand at 25℃ for 15min for pre-oxidation to obtain a premixed solution. S2. Add 1.5 mL of 70% perchloric acid along the tube wall to the premixed solution obtained in S1 at a rate of 0.5 mL / min, shake well, connect the digestion tube to the reflux condenser of the Kjeldahl digester (model KDN-20C), set the digestion block temperature to 240℃, and carry out digestion. After 2.75 h, the solution gradually changes from dark brown to clear and transparent, and the digestion solution is obtained. S3. Allow the digestion solution to cool naturally to room temperature, then transfer it all to a 25mL polypropylene volumetric flask. Wash the inner wall of the digestion tube several times with ultrapure water, add the washing solution to the volumetric flask, dilute to the mark, and shake well to obtain the sample solution to be tested.

[0037] Example 2 This embodiment operates the same as in Example 1, except that in S2, the amount of perchloric acid added is 1.35 mL.

[0038] Example 3 This embodiment operates the same as in Example 1, except that in S1, the mass concentration of nitric acid is 68%.

[0039] Comparative Example 1 A sample pretreatment method for determining the heavy metal content in honey includes the following steps: S1. Accurately weigh 0.5g of homogenized honey (same batch as in Example 1) and place it in a 50mL special digestion tube. Add 1.5mL of 70% perchloric acid and vortex mix at 40rpm for 30s. Then let it stand at 25℃ for 15min to obtain a premixed solution. S2. Add 2.0 mL of 65% nitric acid to the premixed solution obtained in S1 in one go, shake well, connect the digestion tube to the ordinary glass bend tube (without reflux condenser) of the Kjeldahl digester, set the digestion block temperature to 240℃, and digest for 2.75 h to obtain the digestion solution. S3. Allow the digestion solution to cool naturally to room temperature, then transfer it all to a 25mL polypropylene volumetric flask. Wash the inner wall of the digestion tube several times with ultrapure water, add the washing solution to the volumetric flask, dilute to the mark, and shake well to obtain the sample solution to be tested.

[0040] Comparative Example 2 A sample pretreatment method for determining the heavy metal content in honey includes the following steps: S1. Accurately weigh 0.5g of homogenized honey (same batch as in Example 1) and place it in a 50mL dedicated digestion tube; S2. Mix 2.0 mL of 65% nitric acid and 1.5 mL of 70% perchloric acid thoroughly before adding them to the digestion tube at once (addition time <10s). Vortex mix at 40 rpm for 30s. Without letting it stand, immediately connect the digestion tube to the reflux condenser of the Kjeldahl digester. Set the digestion block temperature to 240℃ and digest for 2.75h ​​to obtain the digestion solution. S3. Allow the digestion solution to cool naturally to room temperature, then transfer it all to a 25mL polypropylene volumetric flask. Wash the inner wall of the digestion tube several times with ultrapure water, add the washing solution to the volumetric flask, dilute to the mark, and shake well to obtain the sample solution to be tested.

[0041] Observe the phenomena observed during the experiments of Examples 1-3 and Comparative Examples 1-2: Examples 1-3: In S1, after honey and nitric acid are mixed, a small number of bubbles are generated when the mixture is vortexed and allowed to stand. The solution gradually turns dark brown. In S2, when heated and digested, the solution continues to boil and reflux. The dark brown color gradually fades and eventually becomes clear and transparent. In S3, after cooling, the solution is transferred and diluted to a final volume to obtain a colorless and transparent solution.

[0042] Comparative Example 1: In S1, the perchloric acid added directly contacted the honey and reacted violently with the organic matter in the honey, producing a large amount of yellow fumes and posing a risk of boiling over; in S2, after the nitric acid was added at once, the acid mist volatilized severely during the heating and digestion process, the liquid level dropped significantly, and the solution was still light brown after 2.75 hours and was not completely clear.

[0043] Comparative Example 2: The mixing of the two strong acids released heat, causing localized overheating when the sample was added, resulting in a large amount of foam rising and adhering to the inner wall of the digestion tube. Excessive foam occurred in the early stages of digestion; despite the presence of a reflux device, a small amount of sample still overflowed to the lower end of the condenser. After 2.75 hours of digestion, although the solution was basically clear, a ring of carbonized black gelatinous substance adhered to the inner wall of the tube.

[0044] The contents of copper, iron, manganese, nickel, zinc, cadmium, and lead in the sample solution of Example 1 were determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). The optimal instrument operating parameters were: RF power 1500 W, plasma gas flow rate 10.0 L / min, auxiliary gas flow rate 0.3 L / min, nebulizer flow rate 0.7 L / min, and peristaltic pump speed 1.0 mL / min. The preferred analytical wavelengths for the seven key heavy metal elements were as follows: copper (Cu) 327.393 nm, iron (Fe) 238.204 nm, manganese (Mn) 257.610 nm, nickel (Ni) 231.604 nm, zinc (Zn) 206.200 nm, cadmium (Cd) 228.802 nm, and lead (Pb) 220.353 nm.

[0045] Two blank digestion steps were set up for the test sample solution prepared in Example 1. That is, in Example 1, honey was not added in S1, and blank solution was obtained in S3. The remaining operation steps were the same as in Example 1.

[0046] The test sample solution and its blank solution from Example 1 were sequentially injected for analysis. A calibration curve was established using a series of multi-element mixed standard solutions. The instrument software automatically calculated the concentration of heavy metals in the sample based on the calibration curve. The calibration curve showed good linearity, R0. 2 With a value above 0.999, this method meets the requirements for trace analysis for both the limit of detection (MDL) and the limit of quantitation (LOQ) for each element. The results are shown in Table 1.

[0047] Table 1. Comparison of the content of seven heavy metals in Ethiopian honey samples with safety standards.

[0048] As can be seen from Table 1, the method of the present invention successfully determined the heavy metal content in actual honey samples. Honey from the Kellem Wollega region of Ethiopia generally suffers from serious heavy metal contamination, especially lead (Pb) and cadmium (Cd) content, which far exceeds international safety standards and poses a potential threat to public health. This verifies the applicability and detection capability of the method of the present invention to actual samples.

[0049] The recovery rates of seven elements (copper, iron, manganese, nickel, zinc, cadmium, and lead) in the test sample solutions of Example 1 and Comparative Examples 1-2 were tested using the matrix spiked recovery method. The results are shown in Table 2.

[0050] Table 2 Spike recoveries of 7 elements

[0051] As shown in Table 2, the spiked recoveries of seven elements (copper, iron, manganese, nickel, zinc, cadmium, and lead) in the sample solution of Example 1 were significantly higher than those of Comparative Examples 1 and 2. In Comparative Example 1, due to the lack of a reflux device and a different acid addition order, the solution remained light brown after 2.75 hours, indicating incomplete digestion and resulting in lower analytical results. In Comparative Example 2, the lack of a pre-oxidation step and the addition of two acids mixed together led to uncontrolled reaction, resulting in the loss of some elements (such as zinc and cadmium) due to sample adhesion, decreased precision, and an increase in the relative standard deviation (RSD) to 3.85%-5.20%. Furthermore, the digestion tube was difficult to clean, affecting subsequent measurements. These findings fully demonstrate that the digestion method of this invention has excellent accuracy and precision and can be reliably applied to the trace heavy metal analysis of complex honey matrices.

[0052] Therefore, the present invention adopts the above-mentioned sample pretreatment method for determining the heavy metal content in honey. By optimizing the mixed acid system, acid addition method and digestion conditions, and combining it with reflux condensation technology, the honey sample is rapidly and completely digested, reducing the loss of target heavy metal elements, improving the accuracy and precision of the detection method, while lowering the threshold of experimental equipment and ensuring safe and controllable operation. It can meet the needs of testing laboratories at all levels to carry out trace analysis of heavy metals in honey.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A sample pretreatment method for determining the heavy metal content in honey, characterized in that: Includes the following steps: S1. Mix honey with nitric acid in a vortex and pre-oxidize to obtain a premixed solution; S2. Add perchloric acid to the premixed solution obtained in S1, install a reflux condenser, digest, and obtain digestion solution; S3. Cool the digestion solution to room temperature and make up to volume to obtain the sample solution to be tested.

2. The sample pretreatment method for determining the heavy metal content in honey according to claim 1, characterized in that: In S1, the mass-to-volume ratio of honey to nitric acid is 0.3-1.0 g: 2.0 mL.

3. The sample pretreatment method for determining the heavy metal content in honey according to claim 1, characterized in that: In S1, the mass concentration of nitric acid is 65%-68%.

4. The sample pretreatment method for determining the heavy metal content in honey according to claim 1, characterized in that: In S1, the vortex mixing time is 20-40s, and the vortex mixing speed is 20-60rpm.

5. The sample pretreatment method for determining the heavy metal content in honey according to claim 1, characterized in that: In S1, the pre-oxidation temperature is 18-25℃ and the pre-oxidation time is 10-20min.

6. The sample pretreatment method for determining the heavy metal content in honey according to claim 1, characterized in that: In S2, the volume ratio of perchloric acid to nitric acid is 1:1.3-1.

5.

7. The sample pretreatment method for determining the heavy metal content in honey according to claim 1, characterized in that: In S2, the mass concentration of perchloric acid is 60%-80%, and the addition rate of perchloric acid is 0.1-1.0 mL / min.

8. The sample pretreatment method for determining the heavy metal content in honey according to claim 1, characterized in that: In S2, the digestion temperature is 230-250℃ and the digestion time is 2-3 hours.

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