High-flux breast milk digestion method

Abstract

The invention provides a high-throughput breast milk digestion method which comprises the following steps: 1, taking a certain amount of breast milk sample, putting the breast milk sample into a container, adding a digestion solvent, and uniformly mixing; 2, placing the container filled with the uniformly mixed sample in a boiling water bath, and digesting; 3, cooling the digested sample to room temperature, and fixing the volume; and 4, directly applying the sample with the constant volume to ICP-MS detection. The breast milk sample obtained through digestion of the method can be used for rapidly detecting the content of inorganic elements and metal elements in the breast milk sample, the method is high in sensitivity, small in needed sampling amount and wide in linear range, hundreds of breast milk samples can be treated, multiple elements can be detected at the same time, and the requirement for large-scale sample detection is well met.

Description

Technical Field

[0001] The present invention relates to the field of breast milk detection, and in particular to a high-throughput breast milk digestion method for detecting inorganic elements and metal elements in breast milk. Background Art

[0002] Breast milk contains a variety of metal elements, such as calcium, iron, zinc and copper, which are essential for the baby's bone development, immunity and brain health. However, contamination by heavy metals such as lead may cause serious harm to the baby's nervous system and cognitive development, and excessive intake of certain metal elements may also pose health risks. The living environment and diet of breastfeeding mothers can affect metal intake. Therefore, it is necessary to monitor the content of the above elements in breast milk to ensure that it is within a safe range, provide a scientific basis for breast milk nutritional analysis, and ensure the healthy growth of infants.

[0003] In the research field of food science, graphite furnace atomic absorption spectrometry (GAAS) is one of the commonly used elemental analysis methods. It is to inject breast milk samples directly into a graphite furnace, use an automatic sampler to inject concentrated nitric acid and hydrogen peroxide as oxidants multiple times, and then use absorption spectroscopy to determine the trace elements in breast milk samples. However, the GAAS method can only detect one element at the same time. When samples of multiple elements need to be analyzed, it takes a long time, is inefficient, and has insufficient detection accuracy for low-concentration samples of certain elements. At the same time, strong oxidants nitric acid and hydrogen peroxide may cause the loss of certain volatile elements under high temperature conditions. In addition, due to the high content of organic matter in breast milk samples, incomplete combustion residues may be produced during the ashing process, affecting the accuracy of the results, so it is necessary to increase the number of treatments with the oxidizing agent. This not only increases the analysis time, but may also introduce contamination.

[0004] Another commonly used method is the combination of microwave digestion and inductively coupled plasma mass spectrometry (ICP-MS). Concentrated nitric acid and hydrogen peroxide are usually used as mixed reagents to digest breast milk samples through closed high-pressure digestion (microwave digestion), and then ICP-MS is used for detection. However, on the one hand, the cost of microwave digestion equipment is relatively high (a microwave digestion instrument costs hundreds of thousands to hundreds of thousands of RMB), and on the other hand, the number of samples for microwave digestion is small and time-consuming. Generally, the number of rotors of microwave digestion instruments is small, and the number of rotors with nominal high throughput is only about dozens, resulting in a small number of samples that can be digested in the same batch. In order to avoid cross-contamination of samples, the digestion tank needs to be cleaned between batches, which greatly increases the sample processing time. In addition, in order to ensure the effectiveness and uniformity of the reaction, microwave digestion requires a large amount of each sample, while breast milk is precious and samples are not easy to obtain. Therefore, the microwave digestion method for breast milk must carefully balance the balance between sample usage and experimental requirements. In addition, microwave digestion is complicated, requiring operators to have certain professional knowledge and experience, and to strictly control digestion conditions, otherwise it may lead to experimental failure, and serious safety accidents may occur. Therefore, microwave digestion has disadvantages such as long time consumption, large amount of reagents and complex process when digesting high-throughput breast milk samples.

[0005] In order to overcome the above problems, the present invention provides a high-throughput breast milk digestion method, which uses a water bath method to digest breast milk samples to detect inorganic elements and metal elements therein. The method of the present invention can obtain accurate data while using a small amount of breast milk sampling, and can process up to hundreds of breast milk samples at the same time, and can detect multiple elements at the same time, thereby well meeting the needs of large-scale sample detection. Summary of the invention

[0006] The object of the present invention is to provide a high-throughput breast milk digestion method, the processing process of which is simple to operate, and the obtained breast milk samples can well meet the ICP-MS detection requirements and can be used to quickly detect the content of inorganic elements and metal elements in breast milk samples. The method has high sensitivity and a wide linear range, and can simultaneously process a large number (for example, up to hundreds) of breast milk samples, and can simultaneously detect multiple elements.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical scheme is adopted:

[0008] A high-throughput breast milk digestion method comprises the following steps:

[0009] 1. Take a certain amount of breast milk sample, put it into a container, add digestion solvent, and mix evenly;

[0010] Among them, the breast milk sampling volume is greater than or equal to 0.05 ml, and the ratio of the breast milk sample to the volume of nitric acid is between 1:5 and 4:5;

[0011] The digestion solvent is concentrated nitric acid, with a concentration of ≥65% and a BV-III grade. The strong oxidizing property of concentrated nitric acid can effectively decompose organic substances in breast milk and release inorganic elements and metal elements therein.

[0012] The sample and the nitric acid solution are mixed evenly to facilitate a full reaction. The mixing can be performed using any suitable means, such as a vortexer.

[0013] 2. Place the container containing the mixed sample in a boiling water bath and heat for 25-30 minutes for digestion; the boiling water bath temperature is maintained at about 100°C;

[0014] In this step, multiple samples can be placed simultaneously according to the size of the water bath and experimental needs;

[0015] The boiling water bath digestion conditions are mild, and the organic components in the breast milk sample are gradually decomposed during the heating process, avoiding the loss of elements or the generation of harmful byproducts that may be caused by high-temperature treatment. In addition, compared with microwave digestion, the water bath digestion method has low sample requirements and can better retain the elements in breast milk, and is particularly suitable for the treatment of small sample quantities.

[0016] 3. Cool the digested sample to room temperature and make it constant to ensure the standardization of the sample volume;

[0017] 4. The sample after volume adjustment is directly used for ICP-MS detection.

[0018] During the ICP-MS detection process, the internal standard method is used for correction to reduce possible signal drift and matrix effect. The internal standard is to add other elements with similar properties to the element to be measured but not contained in the sample to the sample solution. The signal change of the element to be measured is corrected by the stability of the internal standard element signal, thereby improving the accuracy and reproducibility of the test results. ICP-MS has high detection sensitivity and can accurately determine the content of trace inorganic elements and metal elements in breast milk, such as calcium, magnesium, iron, zinc, etc.

[0019] In the method of the present invention, the contents of inorganic elements and metal elements in high-throughput breast milk samples can be accurately measured through water bath digestion pretreatment, ICP-MS high-sensitivity detection and internal standard correction.

[0020] By using the method of the present invention, hundreds of breast milk samples can be processed simultaneously in the same water bath.

[0021] Beneficial Effects

[0022] 1. Compared with traditional microwave digestion or wet digestion, the method of the present invention greatly shortens the sample pretreatment time, and the digestion of breast milk samples can be completed in 30 minutes, which greatly improves the work efficiency;

[0023] 2. The method of the present invention is a high-throughput method, which means that hundreds of breast milk samples can be processed simultaneously in the same water bath, which well meets the needs of large-scale sample testing in the laboratory; and the method of the present invention requires a small amount of breast milk sample, and the minimum sampling amount can be only 0.05 ml.

[0024] 3. The samples digested and treated by the method of the present invention do not need further treatment and can be directly used for ICP-MS analysis, which simplifies the operation steps and can simultaneously detect multiple inorganic elements and metal elements in breast milk samples in one test, thereby improving detection efficiency and data reliability.

[0025] 4. The method of the present invention uses fewer types of solvents and less amount of solvents in the digestion process, thus avoiding the pollution that may be caused by multiple digestion solvents. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The standard curve is obtained by measuring the standard solution with the same acidity as the breast milk digestion solution. The correlation coefficient R 2 All are greater than 0.999, among which Figure 1 a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i and 1j correspond to the elements Mg, Ca, Cr, Mn, Fe, Cu, Zn, Se, Mo and Pb, respectively.

[0027] Figure 2 is the recovery rate of internal standards of different mass numbers in breast milk testing, where Figure 2 a, 2b, 2c, 2d and 2e correspond to the elements Sc, Ge, Y, Rh and Bi, respectively.

[0028] According to the requirements of the national standard determination method, the internal standard recovery rate is between 70% and 130%. It can be seen that Figure 2 The internal standard recoveries in the experiment were all within this range, indicating that the instrument did not drift or cause interference.

[0029] Figure 3 The measurement curves of ICP-MS detection of 10 elements in breast milk samples A, B, and C in Example 1 of the present invention are shown, wherein Figure 3 a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i and 3j correspond to the elements Mg, Ca, Cr, Mn, Fe, Cu, Zn, Se, Mo and Pb, respectively. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments, which are merely illustrative and do not constitute any limitation to the present invention. Based on the teachings of the present invention, all other variations made by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] Take 0.2ml, 0.3ml and 0.4ml of three kinds of breast milk samples respectively, put them into centrifuge tubes, mark them as sample A, sample B and sample C, add concentrated nitric acid with a concentration ≥65% and BV-III grade respectively, and the volumes of concentrated nitric acid are 0.25ml, 0.375ml and 0.5ml respectively; cover the caps of the centrifuge tubes tightly, put them into a vortex instrument, and shake the samples thoroughly; then move the centrifuge tubes containing the samples into a water bath, heat them in a boiling water bath at 100°C for 30 minutes, and then cool them to room temperature to make up the volume to obtain digested breast milk samples.

[0033] Comparative Example 1

[0034] A sample blank was prepared in the same manner as in Example 1.

[0035] Without placing a sample in the centrifuge tube, add 0.5 ml of concentrated nitric acid of ≥65% and grade BV-III, tighten the cap, heat in a boiling water bath at 100°C for 30 minutes, cool to room temperature and set the volume to obtain a sample blank.

[0036] Detection Example

[0037] 1. Preparation of Standards, Quality Controls and Internal Standard Solutions

[0038] 1. Preparation of standard products

[0039] Weigh the single-label standard of the element to be measured, dissolve it in an appropriate amount of 10% nitric acid solution, then transfer it to a volumetric flask, dilute to the calibration volume with 10% nitric acid solution to prepare a stock solution of the standard.

[0040] Select 5-7 different concentration points, dilute the stock solution with 10% nitric acid solution in proportion, and prepare a series of concentrations of standard curve working solutions to cover the expected concentration range of the element to be measured in the sample.

[0041] 2. Preparation of quality control products

[0042] The single-label standard was diluted proportionally to prepare low, medium, and high concentration quality control samples, respectively, to approach the lowest concentration expected in the sample, the concentration in the middle of the standard curve, and the concentration close to the highest concentration expected in the sample.

[0043] 3. Preparation of internal standard solution

[0044] Scandium (Sc), germanium (Ge), indium (In), tellurium (Te), and bismuth (Bi) were used as internal standard elements during detection, and an internal standard solution was prepared using a 2% nitric acid solution.

[0045] 2. ICP-MS Detection

[0046] Instrument conditions: ICP-MS with collision reaction cell and electron dilution function, adjustable collision gas flow, nebulizer temperature 2°C;

[0047] Data processing: With the concentration of the element to be tested as the horizontal axis and the intensity as the vertical axis, a standard curve is made with the horizontal and vertical axes, the correlation coefficient R≥0.999, the readback of the lowest point of the standard curve ≤±20%, the readback of other standard points ≤±15%, and the relative deviation of low, medium and high concentration quality control products ≤±15%.

[0048] Figure 3 The measurement curves of 10 elements in breast milk samples A, B, and C in Example 1 are shown (detected by iQuad 2300 inductively coupled plasma mass spectrometer of Hengsheng Mass Spectrometry (Beijing) Instrument Co., Ltd.). It can be seen that relative to Figure 1 The correlation coefficient R of the standard curve 2 All of them are greater than 0.999, with good linearity, and there is no problem with the acid resistance of the instrument, indicating that the method of the present invention can be well used for the testing of high-throughput breast milk samples.

[0049] 3. Data Processing

[0050] The final content of inorganic elements and metal elements in breast milk was calculated based on the concentration test results of ICP-MS, the sampling amount of breast milk samples, and the constant volume after digestion.

[0051] The final contents of various elements in the obtained breast milk samples are summarized in Table 1 below.

[0052] Table 1: Contents of various elements in breast milk samples

[0053]

[0054] Table 1 shows the content of nutrients and harmful elements in different breast milks. The mother's diet can be adjusted according to the test results to increase the content of nutrients such as calcium, magnesium, zinc, copper, iron and selenium to promote the growth and immune system development of the baby. Excessive content of harmful elements (such as lead (Pb)) may cause learning problems such as delayed intellectual development and learning disabilities in the baby.

[0055] IV. Methodological Validation

[0056] 1. Standard curve requirements

[0057] Standard curve correlation coefficient R 2 ≥0.999( Figure 1 and Figure 3 ), the readback of the lowest point of the standard curve is ≤±20%, the readback of other standard points is ≤±15%, and the relative deviation of the quality control products of low, medium and high concentrations is ≤±15%.

[0058] The standard curve point readback is summarized in Table 2 below.

[0059] Table 2: Standard curve point reading

[0060] time Sample type Label 24Mg[ppm] 44Ca[ppm] 52Cr[ppb] 55Mn[ppb] 57Fe[ppb] 65Cu[ppb] 66Zn[ppb] 78Se[ppb] 98Mo[ppb] 208Pb[ppb] 2024 / 07 / 2512：43：41 BLK -0.0110 -0.2960 0.6350 0.4320 5.1620 -2.02 10.83 0.57 0.192 -0.017 2024-07-25 13：07：12 unknown LQC 0.8353 8.4591 8.4299 8.4060 176.8710 83.3693 89.6976 8.6352 8.6657 8.6641 2024-07-25 13：51：31 unknown MQC 1.7198 16.5927 16.6687 17.4254 356.2640 171.9087 161.1594 15.5690 15.4608 16.0107 2024-07-25 14：30：59 unknown HQC 3.4965 33.7353 33.6174 35.2010 727.0549 353.0385 320.4530 31.8031 31.9953 32.8487 2024-07-25 15：10：35 unknown LQC 0.8495 8.2472 8.4918 8.7614 181.6498 83.7353 85.3134 7.9726 7.9139 7.9450 2024-07-25 15：52：16 unknown MQC 1.7302 17.1113 16.7907 17.5639 358.9059 172.6001 165.2218 16.8324 16.4478 16.4281 2024-07-25 16：31：58 unknown HQC 3.4624 33.3287 32.9503 34.5083 710.6528 346.8303 318.3526 31.4532 31.8548 32.6169 Theoretical value unknown LQC 0.80 8.00 8.00 8.00 160.00 80.00 80.00 8.00 8.00 8.00 Theoretical value unknown MQC 1.60 16.00 16.00 16.00 320.00 160.00 160.00 16.00 16.00 16.00 Theoretical value unknown HQC 3.20 32.00 32.00 32.00 640.00 320.00 320.00 32.00 32.00 32.00 Theoretical value unknown LQC 0.80 8.00 8.00 8.00 160.00 80.00 80.00 8.00 8.00 8.00 Theoretical value unknown MQC 1.60 16.00 16.00 16.00 320.00 160.00 160.00 16.00 16.00 16.00 Theoretical value unknown HQC 3.20 32.00 32.00 32.00 640.00 320.00 320.00 32.00 32.00 32.00 Accuracy unknown LQC 106％ 109％ 97％ 100％ 1076 107％ 99％ 101％ 106％ 109％ Accuracy unknown MQC 108％ 106％ 100％ 106％ 110％ 109％ 94％ 94％ 95％ 100％ Accuracy unknown HQC 110％ 106％ 103％ 109％ 113％ 111％ 97％ 98％ 99％ 103％ Accuracy unknown LQC 108％ 107％ 98％ 104％ 110％ 107％ 93％ 93％ 97％ 100％ Accuracy unknown MQC 109％ 109％ 101％ 107％ 111％ 109％ 96％ 102％ 102％ 103％ Accuracy unknown HQC 109％ 105％ 101％ 106％ 110％ 109％ 96％ 97％ 99％ 102％

[0061] Due to the difference between the matrix of breast milk samples and the matrix of the marking line, when analyzing a large number of breast milk samples, the instrument may drift, resulting in high or low test results. The marking point readback can be corrected and verified in time. As can be seen from Table 2, the relative deviation of the marking point readback is ≤±15%, indicating that the instrument drift is small and the test results are accurate.

[0062] 2. Precision and accuracy

[0063] The quality control products prepared in the above steps were evaluated, and the precision CV% of the target elements was <±15%; the spiked recovery rate was between 85% and 115%, and the precision and accuracy met the methodological requirements. The statistical results of the precision and accuracy of the quality control products are shown in Table 3 below.

[0064] Table 3: Statistical results of precision and accuracy of quality control products

[0065]

[0066] It can be seen from Table 3 that the recoveries of the quality control samples of three different concentrations, low, medium and high, are between 90% and 115%, indicating that the method of the present invention is suitable for digestion testing of breast milk samples.

[0067] 3. Residue

[0068] Since the residual amount is a parameter used to evaluate the residual level of the element to be measured in the ICP-MS after the standard curve test, it is necessary to set 2-3 cleaning needles after the highest point of the mark line. The residual has dropped to within 20% of the lowest point of the standard curve and does not affect the subsequent sample determination.

Claims

1. A high-throughput breast milk digestion method comprising the following steps: (1) Take a certain amount of breast milk sample, put it into a container, add digestion solvent, and mix well; (2) placing the container containing the mixed sample in a boiling water bath and heating for 25-30 minutes for digestion; (3) cooling the digested sample to room temperature and fixing the volume; (4) The sample after volume adjustment is directly used for ICP-MS detection.

2. The method according to claim 1, wherein in step (1), the digestion solvent is concentrated nitric acid, and its concentration is ≥65%.

3. The method according to claim 2, wherein the concentrated nitric acid is of BV-III grade.

4. The method according to claim 1, wherein in step (1), the volume ratio of the breast milk sample to nitric acid is 1:5 to 4:

5.

5. The method according to claim 1, wherein in step (1), the breast milk sampling volume is greater than or equal to 0.05 ml.

6. The method according to claim 1, wherein in step (2), the boiling water bath temperature is maintained at 100°C.

7. The method according to claim 1, wherein in step (2), one or more samples are placed according to the size of the water bath and experimental requirements.

8. The method according to claim 1, wherein in step (4), the ICP-MS detection is calibrated using an internal standard method.

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