Preparation method of bovine whole blood freeze-dried quality control product for metal element detection

Abstract

The application relates to a preparation method of a bovine whole blood lyophilized quality control product for metal element detection. First, plasma and blood cells are separated; then a uniform blood cell solution without precipitation is obtained from the obtained blood cells; then the base values of the plasma and the blood cell solution are detected respectively; then a standard solution is prepared, the volume of the standard solution required by each metal element is calculated, the required standard solutions are mixed together to obtain a mixed standard solution; the mixed standard solution is diluted with purified water, and alkali is added to adjust the pH; then the mixed standard solution is uniformly mixed with the plasma, the blood cell solution is added, and is uniformly mixed to obtain a pre-lyophilization solution; finally, the pre-lyophilization solution is divided and pre-frozen, and vacuum freeze-drying is carried out to obtain a bovine whole blood quality control product lyophilized powder. The preparation process is simple and easy to operate, not only can the production process standardization be easily realized, but also the uniformity and stability of the quality control product can be more effectively improved.

Description

Technical Field

[0001] This invention relates to a method for preparing whole blood quality control products, specifically a method for preparing freeze-dried bovine whole blood quality control products for metal element detection. Background Technology

[0002] With the increasing application of inductively coupled plasma mass spectrometry (ICP-MS) in detecting multiple metal elements in human whole blood samples in recent years, quality control products have played a crucial role in ensuring quality during the detection of various elements. However, existing whole blood quality control product preparation methods are mostly solution-based. Although the preparation process is simple, the solution state can only be stored at -20°C, which is not conducive to long-distance transportation and long-term storage.

[0003] Patent 104965092A discloses a method for preparing a whole blood quality control sample containing metal elements. The main preparation method is as follows: A) collecting bovine whole blood; B) determining the concentration of metal elements in the bovine whole blood; C) adding metal elements to adjust the concentration of metal elements in the bovine whole blood to the target concentration; D) centrifuging or filtering to remove fiber precipitates; E) adding preservatives and gently mixing. This method seems simple, but the added metal elements may combine with proteins in the bovine whole blood to form insoluble metal salt precipitates. Therefore, it is necessary to remove the precipitates and continue adding metal elements in small amounts multiple times based on the test results. The entire process requires repeated testing, which may consume a large amount of protein in the bovine whole blood or cause significant red blood cell agglutination. Furthermore, the required amount of added elements will vary depending on the baseline value of each batch of bovine whole blood, potentially leading to different protein consumption between batches and batch-to-batch variations.

[0004] Patent 106370872A discloses a method for adding a high concentration of metal elements to bovine whole blood and a quality control product of bovine whole blood, including the following steps: A) collecting bovine whole blood and separating plasma from blood cells by centrifugation; B) adding a high concentration of metal elements to the plasma; C) mixing the spiked plasma with blood cells to obtain bovine whole blood with added high concentration of metal elements. This patent uses a 0.05mm diameter nebulizer to nebulize the standard solution into the plasma. Although this can reduce protein precipitation, the nebulization speed is extremely slow. Without additional power such as a peristaltic pump, the nebulization speed of the standard solution may be less than 100μL / min, and nebulizing 30mL of solution may take 300min. Furthermore, the nebulizer is a precision instrument component, requiring precise control of the argon gas flow rate and pressure, which places high demands on the production equipment and is not conducive to large-scale production.

[0005] The concentration distribution of metallic elements in the human body is quite wide. Some elements, such as magnesium, calcium, iron, copper, and zinc, are mostly distributed in the microgram range, while others, such as cobalt, manganese, selenium, and lead, are mostly distributed in the nanogram range. To prepare quality control products with appropriate concentrations, high-purity single-element standards are required. Commercially available single-element standard solutions are prepared in highly concentrated strong acid solutions. However, whole blood has a complex matrix with a high protein content, making it prone to precipitation upon contact with acids and metal salt ions. Furthermore, if there is a significant amount of acid in the matrix, during freeze-drying, as the moisture content decreases, the acid accumulates, creating a strongly acidic environment that causes protein precipitation, coagulation, and blackening, affecting the homogeneity of the reconstituted solution. Therefore, this invention provides a method for preparing metallic element quality control products. To improve the stability of the quality control products, the whole blood quality control products are prepared as freeze-dried powders with uniform concentration and good stability. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method for preparing freeze-dried bovine whole blood quality control products for metal element detection. This invention not only facilitates the standardization of production processes but also more effectively improves the uniformity and stability of the quality control products.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A method for preparing a lyophilized quality control sample of bovine whole blood for metal element detection includes the following steps: (1) Collect whole bovine blood and separate plasma and blood cells; (2) After mixing the obtained blood cells and removing the supernatant solution, freeze them; take out the frozen blood cells, thaw them naturally, centrifuge them, take the supernatant solution and vacuum filter them to obtain a uniform blood cell solution without precipitation; (3) Detect the base values ​​of plasma and blood cell solution respectively; (4) Prepare standard solutions; calculate the required volume of standard solution for each metal element based on the baseline value, target concentration and planned volume, mix the required standard solutions together to obtain a mixed standard solution; dilute the mixed standard solution with purified water, and then add alkali to adjust the pH; (5) Mix the mixed standard solution with plasma, then add blood cell solution and mix well to obtain the solution before lyophilization; (6) Dispense the solution before freeze-drying into clean containers, pre-freeze, and then freeze-dry under vacuum to obtain the freeze-dried powder of bovine whole blood quality control product.

[0008] This invention reduces protein precipitation in the matrix through three methods: First, high-concentration standard solutions are prepared by dissolving metal salts in purified water, while other single-element standard solutions are diluted with purified water to reduce acid introduction. Second, the mixed standard solution is first diluted with purified water to obtain a lower concentration of salt and acid before mixing with plasma to reduce precipitation. Third, after dilution with purified water, the mixed standard solution is not directly mixed with plasma; instead, an alkaline solution is first added to neutralize the strong acid, resulting in a weakly acidic mixed standard solution before mixing with plasma. Because the standard solution, plasma, and red blood cell solution are mixed in a stepwise, large-volume manner, this invention can obtain a solution with uniform concentration in a shorter time.

[0009] Preferably, in step (1), the separation is centrifugal separation.

[0010] Preferably, in step (2), the freezing temperature is below -20°C, and the filter membrane used for vacuum filtration is a polypropylene fiber filter cloth with pore sizes of 0.5μm, 1μm, 5μm, and 10μm. More preferably, the pore size is 0.5μm, resulting in a homogeneous solution without large particles.

[0011] After freeze-thaw centrifugation, there is no clear boundary between the upper solution and the lower solid tissue in the blood cell solution, and it may contain some floating matter that cannot settle. Therefore, further filtration is required to remove large particles and obtain a homogeneous solution. Because the blood cell solution has high viscosity, conventional filter membranes are difficult to use. This invention selects a polypropylene fiber filter cloth suitable for filtering red blood cell solutions, which will not introduce interfering elements.

[0012] Preferably, in step (4), the metallic elements include magnesium, calcium, manganese, iron, cobalt, copper, zinc, selenium, and lead.

[0013] Preferably, the standard solution includes magnesium solution, calcium solution, iron solution, zinc solution, copper standard solution, lead solution, manganese solution, cobalt solution, and selenium solution; the raw materials for the standard solution include magnesium chloride hexahydrate, calcium chloride dihydrate, zinc chloride, ferric chloride hexahydrate, manganese, copper, cobalt, lead, and selenium.

[0014] Preferably, the concentrations of the magnesium solution are 10–50 mg / mL, the calcium solution is 10–50 mg / mL, the iron solution is 10–50 mg / mL, the zinc solution is 2–10 mg / mL, the copper solution is 200–1000 μg / mL, the lead solution is 20–100 μg / mL, the manganese solution is 4–20 μg / mL, the cobalt solution is 2–10 μg / mL, and the selenium solution is 20–100 μg / mL.

[0015] The human body has a wide distribution of elemental concentrations and requires high purity of raw material standards. This invention selects suitable raw materials to prepare standard solutions of appropriate concentrations, thereby reducing interference introduced by the standards.

[0016] Preferably, in step (4), the alkali is an aqueous solution of tetramethylammonium hydroxide with a concentration of 10-25 wt.%; the amount added is 1-1 / 20 of the volume of the mixed standard solution.

[0017] Preferably, the clean container is a polypropylene sample bottle (wash-free PP bottle); the expansion coefficient of the reconstituted lyophilized bovine whole blood quality control sample is 1 to 1.1.

[0018] Because conventional borosilicate glass vials require prolonged soaking and cleaning, and cross-contamination is easily caused without automatic cleaning equipment, this invention uses self-cleaning plastic bottles that can be used directly and are convenient for large-scale production.

[0019] Preferably, the freezing temperature is below -20℃ and the pre-freezing time is more than 12 hours.

[0020] Preferably, the preparation method of the lyophilized bovine whole blood quality control sample for metal element detection includes the following steps: (1) Collect bovine whole blood and centrifuge at 4000 rpm for 8 min to separate plasma and blood cells; (2) After centrifuging the collected blood cells, remove the supernatant solution and freeze at -80℃ for 17h. Thaw naturally at room temperature. After the blood cell solution is completely thawed, centrifuge at 4000rpm for 8min and take the supernatant solution for vacuum filtration to obtain a uniform blood cell solution without precipitate. The filter membrane used is a polypropylene fiber filter cloth with a pore size of 0.5μm. (3) Detect the baseline values ​​of plasma and blood cell solutions respectively; (4) Preparation of standard solution: Weigh 677.67 mg of magnesium chloride hexahydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a magnesium elemental solution of 20 mg / mL; Weigh 588.04 mg of calcium chloride dihydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a calcium elemental solution of 40 mg / mL; Weigh 33.55 mg of zinc chloride into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a 4 mg / mL zinc elemental solution; Weigh 386.19 mg of ferric chloride hexahydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a 20 mg / mL iron solution; Add 40 μL of 1 mg / mL manganese single element solution to a 5 mL centrifuge tube, then add 1960 μL of purified water, vortex mix for 5 min to obtain a 20 μg / mL manganese element solution; Add 30 μL of 1 mg / mL cobalt single element solution to a 5 mL centrifuge tube, then add 2970 μL of purified water, vortex mix for 5 min to obtain a 10 μg / mL cobalt element solution; Add 200 μL of 1 mg / mL lead single element solution to a 5 mL centrifuge tube, then add 1800 μL of purified water, vortex mix for 5 min to obtain a 100 μg / mL lead element solution; Add 200 μL of 1 mg / mL selenium single element solution to a 5 mL centrifuge tube, then add 1800 μL of purified water, vortex mix for 5 min to obtain a 100 μg / mL selenium element solution. Based on the baseline value, target concentration, and planned preparation volume, calculate the required volume of standard solution for each metal element, mix the required standard solutions together to obtain a mixed standard solution; dilute the mixed standard solution with purified water, and then add 25 wt.% tetramethylammonium hydroxide aqueous solution to adjust the pH. The volume of the 25 wt.% tetramethylammonium hydroxide aqueous solution is 1 / 10 of the volume of the mixed standard solution. (5) Take 1 volume of mixed standard solution, add 1 volume of plasma, and mix by inverting the container; then add 1 volume of blood cell solution, and mix by inverting the container to obtain the solution before lyophilization. (6) Dispense the solution before freeze-drying into polypropylene sample bottles, pre-freeze at -20℃ for more than 12 hours, and then freeze-dry under vacuum to obtain the freeze-dried powder of bovine whole blood quality control product.

[0021] The beneficial effects of this invention are as follows: 1. The preparation process of this invention is simple and easy to operate, which not only makes it easy to standardize the production process, but also more effectively improves the uniformity and stability of the quality control products. 2. This invention successfully filters and obtains a homogeneous blood cell solution without introducing interfering elements; this invention also prepares standard solutions of appropriate concentrations, reducing interference introduced by the standards. 3. This invention can not only effectively reduce the generation of protein precipitation in the matrix, but also obtain a solution with uniform concentration in a short time. 4. This invention takes into account the effect of volume expansion after freeze-drying, so that the element concentrations are almost unchanged before and after freeze-drying. Attached Figure Description

[0022] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below through embodiments. These embodiments are for illustrative purposes only and are not intended to limit the present invention. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0025] Reference Figure 1 A method for preparing lyophilized bovine whole blood quality control material for metal element detection includes the following steps: (1) Collect bovine whole blood and centrifuge at 4000 rpm for 8 min to separate plasma and blood cells; (2) After centrifuging the collected blood cells, remove the supernatant solution and freeze at -80℃ for 17h. Thaw naturally at room temperature. After the blood cell solution is completely thawed, centrifuge at 4000rpm for 8min and take the supernatant solution for vacuum filtration to obtain a uniform blood cell solution without precipitate. The filter membrane used is a polypropylene fiber filter cloth with a pore size of 0.5μm. (3) Detect the baseline values ​​of plasma and blood cell solutions respectively; (4) Prepare standard solutions; calculate the required volume of standard solution for each metal element based on the baseline value, target concentration and planned volume, mix the required standard solutions together to obtain a mixed standard solution; dilute the mixed standard solution with purified water, and then add 25 wt.% tetramethylammonium hydroxide aqueous solution to adjust the pH. The volume of the 25 wt.% tetramethylammonium hydroxide aqueous solution is 1 / 10 of the volume of the mixed standard solution. (5) Take 1 volume of mixed standard solution, add 1 volume of plasma, and mix by inverting the container; then add 1 volume of blood cell solution, and mix by inverting the container to obtain the solution before lyophilization. (6) Dispense the solution before freeze-drying into polypropylene sample bottles, pre-freeze at -20℃ for more than 12 hours, and then freeze-dry under vacuum to obtain the freeze-dried powder of bovine whole blood quality control product.

[0026] Different filtration membranes for filtering blood cell solutions Whole bovine blood meeting biosafety regulations was collected and centrifuged at 4000 rpm for 8 min to separate plasma and blood cells. The collected blood cells were mixed and centrifuged again, the supernatant was removed, and the mixture was frozen at -80℃ for 17 h, then thawed naturally at room temperature. After complete thawing, the blood cell solution was centrifuged at 4000 rpm for 8 min, and the supernatant was vacuum filtered. The filter membranes used included ordinary filter paper, mixed cellulose (MCE) membrane, polytetrafluoroethylene (PTFE) membrane, polyethersulfone (PES) membrane, and polypropylene fiber filter cloth. Testing revealed that only polypropylene fiber filter cloth and filter paper could effectively filter the solution; other membranes had difficulty filtering. Filter paper could not be used for long periods and quickly broke, leading to filtration failure. Therefore, polypropylene fiber filter cloth was ultimately selected. Further optimization of the pore size of the polypropylene fiber filter cloth (0.5 μm, 1 μm, 5 μm, 10 μm) was performed, and a 0.5 μm pore size was ultimately chosen to obtain a homogeneous solution without large particles.

[0027] Preparation of standard solutions In the human body, the normal range for iron is typically 200-500 μg / mL, while the range for manganese and cobalt is usually below 30 ng / mL, a difference of approximately 10,000 times. Iron standard materials often contain trace amounts of impurities such as manganese and cobalt, which significantly impacts the preparation of quality control materials containing these elements, making it difficult to obtain quality control materials with a reasonable distribution range. This study compares iron standards from different sources and measures the manganese and cobalt content in a 100 μg / mL iron solution to select a suitable iron standard solution.

[0028] A 20 mg / mL iron solution was prepared using anhydrous ferric chloride powder, and diluted 200 times to obtain a 100 μg / mL solution. A 20 mg / mL iron solution was also prepared using ferric chloride hexahydrate (ferric chloride hexahydrate, ferric chloride hexahydrate) powder, and diluted 200 times to obtain a 100 μg / mL solution. A 1 mg / mL iron single-element solution standard (GBW(E)080123) was diluted 10 times to obtain a 100 μg / mL solution. Inductively coupled plasma mass spectrometry (ICP-MS) was used for detection, and the results are shown in Table 1. Table 1 The test results in Table 1 show that the iron element solution prepared with anhydrous ferric chloride contains more than 400 ng / mL of manganese and 4.63 ng / mL of cobalt per 100 μg / mL. Using this solution to prepare quality control products containing both manganese and cobalt would severely affect the target values. The national standard for single-element iron solution contains 5% HCl at a concentration of only 1 mg / mL. The concentration in most human populations is between 200-500 μg / mL. If used as a quality control solution, the concentration is too low, requiring a large volume, and other elements cannot be diluted to achieve the target concentration. Therefore, ferric chloride hexahydrate is preferred as the raw material for the iron element solution.

[0029] The preparation methods for the standard solutions of each element are as follows: Weigh 677.67 mg of magnesium chloride hexahydrate (magnesium chloride hexahydrate) into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex for 5 min to obtain a magnesium solution of 20 mg / mL. Weigh 588.04 mg of calcium chloride dihydrate (calcium chloride dihydrate) into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex for 5 min to obtain a calcium solution of 40 mg / mL. Weigh 33.55 mg of zinc chloride into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex for 5 min to obtain a zinc solution of 4 mg / mL. Weigh 386.19 mg of ferric chloride hexahydrate (ferric chloride hexahydrate) into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex for 5 min to obtain an iron solution of 20 mg / mL.

[0030] Add 40 μL of a 1 mg / mL manganese single-element solution to a 5 mL centrifuge tube, then add 1960 μL of purified water. Vortex for 5 min to obtain a 20 μg / mL manganese solution. Add 30 μL of a 1 mg / mL cobalt single-element solution to a 5 mL centrifuge tube, then add 2970 μL of purified water. Vortex for 5 min to obtain a 10 μg / mL cobalt solution. Add 200 μL of a 1 mg / mL lead single-element solution to a 5 mL centrifuge tube, then add 1800 μL of purified water. Vortex for 5 min to obtain a 100 μg / mL lead solution. Add 200 μL of a 1 mg / mL selenium single-element solution to a 5 mL centrifuge tube, then add 1800 μL of purified water. Vortex for 5 min to obtain a 100 μg / mL selenium solution. Copper was prepared directly using a 1 mg / mL copper single-element solution.

[0031] pH adjustment The plasma and blood cell solutions obtained above were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) to determine the baseline values ​​of each element. Based on the target concentration and preparation volume, and according to the concentrations obtained in the preparation of the standard solutions, the required volumes of each solution to be added were calculated. The expected concentrations for high and low quality control, determined according to the distribution range of each element in the human body, are shown in Table 2. Table 2 Based on a preparation volume of 100 mL, mixing 1 volume of standard solution, 1 volume of plasma, and 1 volume of erythrocyte solution yields 33.3 mL of each. The required volumes of each element solution are shown in Table 3. Table 3 Because the single-element metal solutions are all stored in strong acid solutions such as nitric acid and hydrochloric acid, although they are diluted with pure water, the acidity of the mixed solution is still relatively strong. Direct addition to the blood matrix can easily lead to precipitation. Therefore, this study selected tetramethylammonium hydroxide, an organic strong base with high purity and extremely low metal element content, to neutralize the strong acid solution. The solutions of each element in the high and low quality control samples were mixed according to the volumes in Table 3 above, and then purified water was added to 33.3 mL. Five aliquots were prepared in the same way for each sample. 1 / 2 (Group A), 1 / 5 (Group B), 1 / 10 (Group C), and 1 / 20 (Group D) of the mixed standard volume of alkaline solution (25 wt.% tetramethylammonium hydroxide aqueous solution) (Group AD) and one sample without alkaline solution (E) were added. After mixing, it was found that Group A showed element precipitation after standing for a period of time, while the others did not show obvious precipitation. At the same time, as the amount of alkali added increased, the solution gradually changed from colorless and transparent to yellow and the color deepened. Adding 33.3 mL of plasma to each of the remaining BE groups revealed no significant precipitation in groups B and C, while group E, which did not contain alkaline solution, showed the most precipitation. Centrifuging solutions B and C and comparing their concentrations with theoretical concentrations revealed that the iron, lead, and selenium concentrations in group B were 5–10% lower than in group C. This indicates that although both groups appeared to be clear solutions, the excessive addition of alkali could still affect the element concentrations. Therefore, it is preferable to add 25 wt.% tetramethylammonium hydroxide aqueous solution at a volume of 1 / 10 of the mixed standard volume.

[0032] Volume expansion coefficient of freeze-dried quality control products Add 33.3 mL of erythrocyte solution to the above group C solution, mix by inverting for 5 min, and then dispense the mixture into 0.5 mL, 0.525 mL, and 0.55 mL portions. Freeze at -80℃ for at least 17 hours, then freeze-dry for 24 hours using a vacuum freeze dryer. After removal, reconstitute each sample with 500 μL of purified water. Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect the concentration of each element in the quality control sample. The results are shown in Table 4, comparing the freeze-dried and unfreezed results with those before freeze-drying, using high-quality control as an example.

[0033] Table 4 As shown in Table 4, the results of reconstitution of the 0.55 mL lyophilized powder were closest to those of the solution before lyophilization, indicating that reconstitution with the same volume would increase the volume of the reconstituted solution by about 10%. To prevent significant changes in the concentration of the solution before and after lyophilization, it is preferable to use 0.55 mL for dispensing and 0.5 mL for reconstitution.

[0034] Preparation of lyophilized quality control products Prepare 600 mL high- and low-quality control solutions separately. Take the plasma and blood cell solutions obtained above and use inductively coupled plasma mass spectrometry to test the baseline values ​​of each element. Based on the target concentration and preparation volume, calculate the required volume of each solution to be added according to the concentration of the solutions prepared above. Mix the solutions of each element in the high- and low-quality control solutions, and then add purified water to 200 mL. Vortex mix for 5 min, then add 1 / 10 of the mixed standard volume of 25 wt.% tetramethylammonium hydroxide aqueous solution, and vortex mix again for 5 min. Add 200 mL of plasma and mix upside down for 5 min. Finally, add 200 mL of red blood cell solution and mix upside down for 5 min. Aliquot each solution into 1.8 mL wash-free PP bottles at 0.55 mL. Aliquot 500 bottles each for high- and low-quality control solutions, freeze at -80℃ for at least 17 hours, then freeze-dry using a vacuum freeze dryer for 24 hours. After removal, cap and store at 2-8℃.

[0035] Uniformity and stability of freeze-dried quality control products Uniformity Ten bottles each of the above-mentioned lyophilized quality control samples (high and low) were randomly selected and reconstituted with 0.5 mL of purified water. Each sample was tested three times, and the coefficient of variation (CV) was calculated. The results are shown in Table 5. The intra- and inter-sample CVs for each element were all <5%, indicating that the preparation process was reliable and the intra- and inter-sample homogeneity of each element was good. However, if a conventional process is used, where standards are directly added to whole blood samples and then lyophilized, insoluble matter or black clumps will appear in the lyophilized powder, making reconstitution difficult and resulting in poor homogeneity.

[0036] Table 5 Stability (long-term stability) The prepared high and low quality control samples were stored at 2-8℃. Ten bottles were randomly selected at 0 days, 3 months, 6 months, 12 months, and 18 months for testing, and the test results were compared with those at 0 days. The test results are shown in Tables 6 and 7.

[0037] Table 6. Low-quality control stability results Table 7 High-quality control stability results The results showed that after 18 months, the mean values ​​of all elements were within 10% of the values ​​at day 0, indicating that the freeze-dried whole blood quality control product had good stability when stored at 2-8℃. The concentrations obtained from the high and low quality control preparations were mostly within 10% of the expected target concentrations, indicating that the effects of protein precipitation, alkali addition, etc., on each element during preparation were minimal. Furthermore, the raw materials used were of high purity, and the preparation process was simple and easy to standardize, allowing for the production of quality control products with lower concentrations for elements such as manganese and lead that are susceptible to environmental and material consumable effects.

[0038] Stability (accelerated stability at 37℃) The prepared high and low quality control samples were stored at 37°C and tested at 0, 3, 7, and 14 days. The test results were compared with those at 0 days. The test results are shown in Tables 8 and 9.

[0039] Table 8. Low-quality control stability results Table 9. High-quality control stability results The results showed that the mean values ​​of all elements detected after 14 days of acceleration were within 10% of the values ​​detected at day 0, indicating that the freeze-dried whole blood quality control product had good stability after 14 days of acceleration at 37℃. It can maintain stable performance during long-distance transportation or use between laboratories without consuming much energy.

[0040] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. A method for preparing lyophilized bovine whole blood quality control material for metal element detection, characterized in that, Includes the following steps: (1) Collect whole bovine blood and separate plasma and blood cells; (2) After mixing the obtained blood cells and removing the supernatant solution, freeze them; take out the frozen blood cells, thaw them naturally, centrifuge them, and then vacuum filter the supernatant solution to obtain a uniform blood cell solution without precipitate; in step (2), the freezing temperature is below -20℃, and the filter membrane used for vacuum filtration is a polypropylene fiber filter cloth with pore sizes of 0.5μm, 1μm, 5μm, and 10μm; (3) Detect the baseline values ​​of plasma and blood cell solutions respectively; (4) Prepare standard solutions; calculate the required volume of standard solution for each metal element based on the baseline value, target concentration and planned volume, and mix the required standard solutions together to obtain a mixed standard solution; dilute the mixed standard solution with purified water, and then add alkali to adjust the pH to weakly acidic; the metal elements include magnesium, calcium, manganese, iron, cobalt, copper, zinc, selenium and lead, and the alkali is tetramethylammonium hydroxide aqueous solution with a concentration of 10-25 wt.%, and the amount added is 1-1 / 20 of the volume of the mixed standard solution; (5) Mix the mixed standard solution with plasma, then add blood cell solution and mix well to obtain the solution before lyophilization; (6) Dispense the solution before freeze-drying into clean containers, pre-freeze, and then freeze-dry under vacuum to obtain freeze-dried bovine whole blood quality control powder.

2. The method for preparing lyophilized bovine whole blood quality control material for metal element detection according to claim 1, characterized in that, In step (1), the separation is centrifugal separation.

3. The method for preparing lyophilized bovine whole blood quality control material for metal element detection according to claim 1, characterized in that, The standard solutions include magnesium solution, calcium solution, iron solution, zinc solution, copper standard solution, lead solution, manganese solution, cobalt solution, and selenium solution; the raw materials for the standard solutions include magnesium chloride hexahydrate, calcium chloride dihydrate, zinc chloride, ferric chloride hexahydrate, manganese, copper, cobalt, lead, and selenium.

4. The method for preparing the lyophilized bovine whole blood quality control sample for metal element detection according to claim 3, characterized in that, The concentrations of magnesium, calcium, iron, zinc, copper, lead, manganese, cobalt, and selenium solutions are 10–50 mg / mL, 10–50 mg / mL, 2–10 mg / mL, 200–1000 μg / mL, 20–100 μg / mL, 4–20 μg / mL, 2–10 μg / mL, and 20–100 μg / mL, respectively.

5. The method for preparing lyophilized bovine whole blood quality control material for metal element detection according to claim 1, characterized in that, The clean container is a polypropylene sample bottle; the expansion coefficient of the reconstituted lyophilized bovine whole blood quality control sample is 1 to 1.

1.

6. The method for preparing lyophilized bovine whole blood quality control material for metal element detection according to claim 1, characterized in that, The pre-freezing temperature is below -20℃, and the pre-freezing time is more than 12 hours.

7. The method for preparing lyophilized bovine whole blood quality control material for metal element detection according to claim 1, characterized in that, The specific steps are as follows: (1) Collect bovine whole blood and centrifuge at 4000 rpm for 8 min to separate plasma and blood cells; (2) After centrifuging the collected blood cells, remove the supernatant solution and freeze at -80℃ for 17h. Thaw naturally at room temperature. After the blood cell solution is completely thawed, centrifuge at 4000rpm for 8min and take the supernatant solution for vacuum filtration to obtain a uniform blood cell solution without precipitate. The filter membrane used is a polypropylene fiber filter cloth with a pore size of 0.5μm. (3) Detect the baseline values ​​of plasma and blood cell solutions respectively; (4) Preparation of standard solutions: Weigh 677.67 mg of magnesium chloride hexahydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a magnesium elemental solution of 20 mg / mL; Weigh 588.04 mg of calcium chloride dihydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a calcium elemental solution of 40 mg / mL; Weigh 33.55 mg of zinc chloride into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a zinc elemental solution of 4 mg / mL; Weigh 386.19 mg of ferric chloride hexahydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve it, vortex mix for 5 min to obtain a 20 mg / mL iron solution; Add 40 μL of 1 mg / mL manganese single element solution to a 5 mL centrifuge tube, then add 1960 μL of purified water, vortex mix for 5 min to obtain a 20 μg / mL manganese element solution; Add 30 μL of 1 mg / mL cobalt single element solution to a 5 mL centrifuge tube, then add 2970 μL of purified water, vortex mix for 5 min to obtain a 10 μg / mL cobalt element solution; Add 200 μL of 1 mg / mL lead single element solution to a 5 mL centrifuge tube, then add 1800 μL of purified water, vortex mix for 5 min to obtain a 100 μg / mL lead element solution; Add 200 μL of 1 mg / mL selenium single element solution to a 5 mL centrifuge tube, then add 1800 μL of purified water, vortex mix for 5 min to obtain a 100 μg / mL selenium element solution. Based on the baseline value, target concentration, and planned preparation volume, calculate the required volume of standard solution for each metal element, mix the required standard solutions together to obtain a mixed standard solution; dilute the mixed standard solution with purified water, and then add 25 wt.% tetramethylammonium hydroxide aqueous solution to adjust the pH. The volume of the 25 wt.% tetramethylammonium hydroxide aqueous solution is 1 / 10 of the volume of the mixed standard solution. (5) Take 1 volume of mixed standard solution, add 1 volume of plasma, and mix by inverting the container; then add 1 volume of blood cell solution, and mix by inverting the container to obtain the solution before lyophilization. (6) Dispense the solution before freeze-drying into polypropylene sample bottles, pre-freeze at -20℃ for more than 12 hours, and then freeze-dry under vacuum to obtain the freeze-dried powder of bovine whole blood quality control product.

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