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

Abstract

The invention relates to a preparation method of a bovine whole blood freeze-dried quality control product for metal element detection. The preparation method comprises the following steps: firstly, separating to obtain plasma and blood cells; then obtaining a uniform blood cell solution without precipitation from the obtained blood cells; respectively detecting the substrate values of the plasma solution and the blood cell solution; then preparing standard substance solutions, calculating the volume of the standard substance solutions needing to be added into each metal element, and mixing the required standard substance solutions together to obtain a mixed standard substance solution; adding purified water to dilute the mixed standard substance solution, and adding alkali to adjust pH; uniformly mixing the mixed standard substance solution with plasma, adding the blood cell solution, and uniformly mixing to obtain a solution before freeze-drying; and finally, sub-packaging and pre-freezing the solution before freeze-drying, and performing vacuum freeze-drying to obtain the bovine whole blood quality control freeze-dried powder. The preparation process is simple and easy to operate, the standardization of the production process is easy to realize, and the uniformity and stability of the quality control product are more effectively improved.

Description

Technical Field

[0001] The present invention relates to a method for preparing a whole blood quality control product, in particular to a method for preparing a bovine whole blood freeze-dried quality control product for metal element detection. Background Art

[0002] With the increasing application of inductively coupled plasma mass spectrometry (ICP-MS) in the detection of various metal elements in human whole blood samples in recent years, quality control products have played a vital role in ensuring the quality of these elements. However, existing whole blood quality control product preparation methods mostly use solution-based quality control products. While the preparation process is simple, these solutions can only be stored in a frozen environment at -20°C, making them difficult to transport over long distances and store for extended periods.

[0003] Patent 104965092A discloses a method for preparing a metal element whole blood quality control product. The main preparation method is: A) collecting bovine whole blood; B) determining the concentration of metal elements in the bovine whole blood; C) adding metal element substances to adjust the concentration of metal elements in the bovine whole blood to the target concentration; D) centrifuging or filtering to remove fibrous precipitates; E) adding preservatives and gently mixing. This method seems relatively simple, but the added metal elements may combine with the proteins in the bovine whole blood to form insoluble metal salt precipitates. Therefore, it is necessary to remove the precipitate and continue to add metal element substances in small amounts and multiple times according to the test results. The whole process requires repeated testing, which may consume a large amount of protein in the bovine whole blood or cause more red blood cell agglutination. In addition, due to the different base values ​​of each batch of bovine whole blood, the mass of elements that need to be added will also be different, which may cause different protein consumption in different batches, resulting in differences between batches.

[0004] Patent 106370872A discloses a method for adding high-concentration metal elements to bovine whole blood and a bovine whole blood quality control product, comprising the following steps: A) collecting bovine whole blood and separating the plasma from blood cells by centrifugation; B) adding a high-concentration metal element substance to the plasma; and C) mixing the spiked plasma with the blood cells to obtain bovine whole blood with high-concentration metal elements. This patent uses a 0.05mm diameter nebulizer to atomize a standard solution into the plasma. While this can reduce protein precipitation, the atomization speed is extremely slow. Without additional power, such as a peristaltic pump, the atomization speed of the standard solution may be less than 100μL / min, and atomizing 30mL of solution may take 300 minutes. Furthermore, the nebulizer is a precision instrument component that requires precise control of the argon gas flow rate and pressure, placing high demands on production equipment and hindering large-scale production.

[0005] The concentration distribution of metal elements in the human body is relatively wide. The distribution range of some elements such as magnesium, calcium, iron, copper, zinc, etc. is mostly in the microgram level, and the distribution range of some elements such as cobalt, manganese, selenium, lead, etc. is mostly in the nanogram level. If you need to prepare a quality control product with a reasonable concentration, you need to use a single element standard with higher purity. Commercially available single element standard solutions are all prepared in a strong acid solution with a relatively high concentration, and the whole blood matrix has a high complex protein content, which is very easy to precipitate when in contact with acid and metal salt ions. In addition, if there is more acid in the matrix, as the water content decreases during freeze drying, the acid aggregates to form a strong acid environment, causing the protein precipitate to coagulate and turn black, affecting the uniformity of the reconstituted solution. Therefore, the present invention provides a method for preparing a metal element quality control product. At the same time, in order to improve the stability of the quality control product, the whole blood quality control product is prepared into a freeze-dried powder with uniform concentration and good stability. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, the present invention provides a method for preparing a freeze-dried bovine whole blood quality control product for metal element detection. The present invention not only easily realizes the standardization of the production process, but also more effectively improves the uniformity and stability of the quality control product.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: A method for preparing a freeze-dried bovine whole blood quality control product for metal element detection comprises the following steps: (1) Collect whole blood from cattle and separate it into plasma and blood cells; (2) Mix the obtained blood cells, remove the upper solution, and freeze them; take out the frozen blood cells, thaw them naturally, centrifuge them, and vacuum filter the upper solution to obtain a uniform blood cell solution without precipitation; (3) Detect the base value of plasma and blood cell solution respectively; (4) Prepare standard solutions; calculate the volume of standard solution required for each metal element based on the base value, target concentration, and planned preparation 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 the blood cell solution and mix well to obtain the pre-lyophilization solution; (6) The pre-lyophilized solution was divided into clean containers, pre-frozen, and then vacuum-freeze-dried to obtain the bovine whole blood quality control freeze-dried powder.

[0008] The present invention reduces protein precipitation in the matrix through three methods: first, using purified water to dissolve metal salts to prepare a high-concentration standard solution, and then diluting other single-element standard solutions with purified water to reduce the introduction of acid; second, the mixed standard solution is first diluted with purified water to obtain lower concentrations of salt and acid, and then mixed with plasma to reduce precipitation; third, after the mixed standard solution is diluted with purified water, it is not directly mixed with plasma. Instead, an alkaline solution is first added to neutralize the strong acid to obtain a weakly acidic mixed standard solution, which is then mixed with plasma. Because the standard solution, plasma, and red blood cell solution are mixed in large volumes in a step-by-step manner, the present invention can produce a solution of uniform concentration in a relatively short 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 0.5 μm, 1 μm, 5 μm, or 10 μm. More preferably, the pore size is 0.5 μm, and a uniform solution free of large particles is obtained by filtration.

[0011] After freeze-thaw centrifugation, there is no clear boundary between the upper solution and the lower solid tissue layer of the blood cell solution, and some floating objects that cannot be settled may be contained. Therefore, further filtration is required to remove large particles to obtain a uniform solution. Due to the high viscosity of the blood cell solution, conventional filter membranes are difficult to filter. The present invention screens out a polypropylene fiber filter cloth suitable for filtering red blood cell solutions without introducing interfering elements.

[0012] Preferably, in step (4), the metal 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 of the standard solution include magnesium chloride hexahydrate, calcium chloride dihydrate, zinc chloride, iron chloride hexahydrate, manganese single element, copper single element, cobalt single element, lead single element and selenium single element.

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

[0015] The concentration distribution of human elements is relatively wide, and the purity of the raw material standard is relatively high. The present invention selects suitable raw materials to prepare standard solutions with appropriate concentrations, thereby reducing the interference introduced by the standard.

[0016] Preferably, in step (4), the base is an aqueous solution of tetramethylammonium hydroxide with a concentration of 10 to 25 wt. %; and the amount added is 1 to 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 bovine whole blood freeze-dried quality control product after reconstitution is 1 to 1.1.

[0018] Because conventional borosilicate glass vials require long-term soaking and cleaning, and are very likely to cause cross contamination if there is no automatic cleaning equipment, the present invention uses a no-cleaning plastic bottle that can be used directly, facilitating large-scale production.

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

[0020] Preferably, the method for preparing the freeze-dried bovine whole blood quality control product for metal element detection comprises the following steps: (1) Collect whole blood from cattle and centrifuge it at 4000 rpm for 8 min to separate plasma and blood cells; (2) The collected blood cells were mixed and centrifuged, and the upper layer of solution was removed. The mixture was placed in a -80°C environment and frozen for 17 hours, and then thawed naturally at room temperature. After the blood cell solution was completely thawed, it was centrifuged at 4000 rpm for 8 minutes, and the upper layer of solution was vacuum filtered to obtain a uniform blood cell solution without precipitation. The filter membrane used was a polypropylene fiber filter cloth with a pore size of 0.5 μm. (3) Detect the baseline values ​​of plasma and blood cell solution 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, and vortex mix for 5 minutes to obtain a 20 mg / mL magnesium solution; Weigh 588.04 mg of calcium chloride dihydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 40 mg / mL calcium solution; Weigh 33.55 mg of zinc chloride into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 4 mg / mL zinc solution. Weigh 386.19 mg of ferric chloride hexahydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 20 mg / mL iron solution; Take 40 μL of 1 mg / mL manganese solution and add it to a 5 mL centrifuge tube. Then add 1960 μL of purified water and vortex mix for 5 minutes to obtain a 20 μg / mL manganese solution. Take 30 μL of 1 mg / mL cobalt solution and add it to a 5 mL centrifuge tube. Then add 2970 μL of purified water and vortex mix for 5 minutes to obtain a 10 μg / mL cobalt solution. Take 200 μL of 1 mg / mL lead solution and add it to a 5 mL centrifuge tube. Then add 1800 μL of purified water and vortex mix for 5 minutes to obtain a 100 μg / mL lead solution. Take 200 μL of 1 mg / mL selenium solution and add it to a 5 mL centrifuge tube. Then add 1800 μL of purified water and vortex mix for 5 minutes to obtain a 100 μg / mL selenium solution. Calculate the required volume of standard solution for each metal element based on the baseline value, target concentration, and planned preparation volume. Mix the required standard solutions to create a mixed standard solution. Dilute the mixed standard solution with purified water, and then adjust the pH by adding 25 wt.% tetramethylammonium hydroxide aqueous solution (1 / 10 the volume of the mixed standard solution). (5) Take 1 volume of mixed standard solution, add 1 volume of plasma, and mix by inverting; then add 1 volume of blood cell solution, and mix by inverting to obtain the pre-lyophilized solution; (6) The pre-lyophilized solution was divided into polypropylene sample bottles, pre-frozen at -20°C for more than 12 hours, and vacuum freeze-dried to obtain the bovine whole blood quality control freeze-dried powder.

[0021] The beneficial effects of the present invention are: 1. The preparation process of the present invention is simple and easy to operate, which not only facilitates the standardization of the production process, but also more effectively improves the uniformity and stability of the quality control product; 2. The present invention successfully filters to obtain a uniform blood cell solution without introducing interfering elements; the present invention prepares a standard solution with an appropriate concentration to reduce the interference introduced by the standard; 3. The present invention can not only effectively reduce the generation of protein precipitation in the matrix, but also obtain a solution with uniform concentration in a shorter time; 4. The present invention takes into account the effect of volume expansion after freeze-drying, so that the element concentration before and after freeze-drying remains almost unchanged. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flowchart of the present invention. DETAILED DESCRIPTION

[0023] The technical solutions of the present invention are further specifically described below through examples. These examples are provided for the purpose of illustrating the present invention and are not intended to limit the present invention. All other examples obtained by persons of ordinary skill in the art based on the examples in this application without creative work are intended to fall within the scope of protection of this application.

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

[0025] Reference Figure 1 A method for preparing a freeze-dried bovine whole blood quality control product for metal element detection comprises the following steps: (1) Collect whole blood from cattle and centrifuge it at 4000 rpm for 8 min to separate plasma and blood cells; (2) The collected blood cells were mixed and centrifuged, and the upper layer of solution was removed. The mixture was placed in a -80°C environment and frozen for 17 hours, and then thawed naturally at room temperature. After the blood cell solution was completely thawed, it was centrifuged at 4000 rpm for 8 minutes, and the upper layer of solution was vacuum filtered to obtain a uniform blood cell solution without precipitation. The filter membrane used was a polypropylene fiber filter cloth with a pore size of 0.5 μm. (3) Detect the baseline values ​​of plasma and blood cell solution respectively; (4) Prepare standard solutions; calculate the volume of standard solution required for each metal element based on the base value, target concentration, and planned preparation 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; then add 1 volume of blood cell solution, and mix by inverting to obtain the pre-lyophilized solution; (6) The pre-lyophilized solution was divided into polypropylene sample bottles, pre-frozen at -20°C for more than 12 hours, and vacuum freeze-dried to obtain the bovine whole blood quality control freeze-dried powder.

[0026] Filtration of blood cell solution using different filter membranes Bovine whole blood, meeting biosafety regulations, was collected and centrifuged at 4000 rpm for 8 minutes to separate plasma and blood cells. The collected blood cells were mixed and centrifuged, the supernatant removed, and then frozen at -80°C for 17 hours before thawing naturally at room temperature. After the blood cell solution was completely thawed, it was centrifuged at 4000 rpm for 8 minutes, 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 effectively filtered the solution, while other filter membranes struggled. Filter paper, on the other hand, could not be used for long periods and quickly broke, leading to filtration failure. Therefore, polypropylene fiber filter cloth was ultimately chosen. The pore sizes of the polypropylene fiber filter cloth were further optimized to 0.5μm, 1μm, 5μm, and 10μm, ultimately selecting a 0.5μm filter to produce a uniform solution free of large particles.

[0027] Preparation of standard solution The normal range of iron in the human body is typically 200-500 μg / mL, while manganese and cobalt typically range from 30 ng / mL, representing a concentration distribution difference of approximately 10,000-fold. Iron standards contain small amounts of impurities such as manganese and cobalt, which significantly impact the preparation of quality control products containing these three elements simultaneously, making it difficult to obtain quality control products with a reasonable distribution range. By comparing iron standards from different sources and measuring the manganese and cobalt content in 100 μg / mL iron solutions, we can select an appropriate iron standard solution.

[0028] Anhydrous ferric chloride powder was used to prepare a 20 mg / mL iron solution, which was diluted 200-fold to obtain a 100 μg / mL solution. Ferric chloride hexahydrate (ferric chloride hexahydrate, ferric chloride hexahydrate) powder was used to prepare a 20 mg / mL iron solution, which was diluted 200-fold to obtain a 100 μg / mL solution. A 1 mg / mL iron single element solution standard substance (GBW(E)080123) was diluted 10-fold to obtain a 100 μg / mL solution. The results were detected by inductively coupled plasma mass spectrometry, as shown in Table 1: Table 1 The test results in Table 1 indicate that anhydrous ferric chloride contains over 400 ng / mL of manganese and 4.63 ng / mL of cobalt per 100 μg / mL of iron solution. Using this solution to prepare quality control products containing both manganese and cobalt would significantly impact the target values. The national standard for single-element iron solution contains 5% HCl and a concentration of only 1 mg / mL. The human body has a concentration range of 200-500 μg / mL for most people. If used as a quality control solution, this concentration is too low, requiring a large volume and preventing dilution of other elements to the target concentration. Therefore, ferric chloride hexahydrate is the preferred raw material for the iron solution.

[0029] The preparation scheme of each element standard solution is as follows: Weigh 677.67 mg of magnesium chloride hexahydrate (MgCl2·6H2O) into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 20 mg / mL magnesium solution. Weigh 588.04 mg of calcium chloride dihydrate (CaCl2·6H2O) into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 40 mg / mL calcium solution. Weigh 33.55 mg of zinc chloride into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 4 mg / mL zinc solution. Weigh 386.19 mg of ferric chloride hexahydrate (FeCl2·6H2O) into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 20 mg / mL iron solution.

[0030] Add 40 μL of a 1 mg / mL manganese solution to a 5 mL centrifuge tube, then add 1960 μL of purified water and vortex mix for 5 minutes to obtain a 20 μg / mL manganese solution. Add 30 μL of a 1 mg / mL cobalt solution to a 5 mL centrifuge tube, then add 2970 μL of purified water and vortex mix for 5 minutes to obtain a 10 μg / mL cobalt solution. Add 200 μL of a 1 mg / mL lead solution to a 5 mL centrifuge tube, then add 1800 μL of purified water and vortex mix for 5 minutes to obtain a 100 μg / mL lead solution. Add 200 μL of a 1 mg / mL selenium solution to a 5 mL centrifuge tube, then add 1800 μL of purified water and vortex mix for 5 minutes to obtain a 100 μg / mL selenium solution. Copper can be prepared directly using a 1 mg / mL copper solution.

[0031] pH adjustment The plasma and blood cell solutions obtained above were taken and the baseline values ​​of each element were tested using inductively coupled plasma mass spectrometry. Based on the target concentration and preparation volume, the volume of each solution required was calculated according to the concentration of the solution prepared in the preparation of the standard solution. The expected concentrations of high and low quality controls were determined based on the distribution range of each element in the human body, as shown in Table 2: Table 2 Based on a 100 mL preparation volume, 1 volume of standard solution, 1 volume of plasma, and 1 volume of red blood cell solution are mixed, i.e., 33.3 mL each. The volumes of each element solution that needs to be added are shown in Table 3: Table 3 Because single-element metal solutions are stored in strong acid solutions such as nitric acid and hydrochloric acid, even though diluted with pure water, the mixed solutions are still highly acidic, easily forming precipitation when added directly to the blood matrix. Therefore, in this study, tetramethylammonium hydroxide, a high-purity organic base with extremely low metal content, was used to neutralize the strong acid solutions. The high and low quality control solutions were mixed according to the volumes shown in Table 3 above, and purified water was added to 33.3 mL. Five aliquots were prepared using the same method, each containing 1 / 2 (Group A), 1 / 5 (Group B), 1 / 10 (Group C), and 1 / 20 (Group D) of the mixed standard volume (Groups AD), along with one aliquot without alkaline solution (Group E). After mixing, elemental precipitation occurred in Group A after standing for a period of time, while no significant precipitation was observed in the other aliquots. Furthermore, as the amount of alkaline solution added increased, the solution gradually turned from colorless and transparent to yellow and darkened in color. 33.3 mL of plasma was then added to each of the remaining BE groups. No significant precipitation was observed in Groups B and C, while Group E, which did not contain alkaline solution, produced the most precipitation. After centrifugation of Solutions B and C, the differences between the values ​​of the various elements in the solutions and the theoretical concentrations were measured. The values ​​of elements such as iron, lead, and selenium in Group B were found to be 5-10% lower than those in Group C. This indicates that although Groups B and C appear to be clear solutions, the addition of excess alkali can still affect the concentrations of these elements. Therefore, it is recommended that the volume of the 25 wt.% tetramethylammonium hydroxide aqueous solution be 1 / 10 of the volume of the mixed standard.

[0032] Volume expansion coefficient of freeze-dried quality control products The Group C solution was added to 33.3 mL of the red blood cell solution and mixed by inversion for 5 minutes. The mixed solution was then aliquoted into 0.5 mL, 0.525 mL, and 0.55 mL portions, respectively. The aliquots were frozen at -80°C for at least 17 hours and then freeze-dried in a vacuum freeze dryer for 24 hours. Each aliquot was then reconstituted with 500 μL of purified water. The concentrations of various elements in the quality control samples were determined by inductively coupled plasma-mass spectrometry. The test results, using the high-quality control as an example, were compared after lyophilization with those before lyophilization. The results are shown in Table 4.

[0033] Table 4 As shown in Table 4, the results after reconstitution of 0.55 mL of lyophilized powder are closest to those of the pre-lyophilized solution, indicating that reconstitution with the same volume will increase the volume of the reconstituted solution by approximately 10%. To minimize significant changes in solution concentration before and after lyophilization, it is preferred to use 0.55 mL for aliquots and 0.5 mL for reconstitution.

[0034] Preparation of freeze-dried quality control products Prepare 600 mL of high and low quality control solutions, respectively. Take the plasma and blood cell solutions obtained above and measure the baseline values ​​of each element using inductively coupled plasma-mass spectrometry. Calculate the required volume of each solution based on the target concentration and the prepared volume, according to the solution concentrations obtained above. Mix the high and low quality control solutions for each element and add purified water to 200 mL. After vortexing for 5 minutes, add 1 / 10 the volume of the mixed standard solution in a 25 wt.% aqueous solution of tetramethylammonium hydroxide and vortex again for 5 minutes. Add 200 mL of plasma and mix by inversion for 5 minutes. Finally, add 200 mL of the red blood cell solution and mix by inversion for 5 minutes. Aliquot 0.55 mL of each vial into 1.8 mL disposable PP bottles. Dispense 500 vials of each high and low quality control solution. Freeze at -80°C for at least 17 hours, then freeze-dry in a vacuum freeze dryer for 24 hours. Remove, cap, and store at 2-8°C.

[0035] Uniformity and stability of freeze-dried quality control products Uniformity Ten bottles of each high and low quality control sample were randomly sampled and reconstituted with 0.5 mL of purified water. Each bottle of quality control sample was tested three times, and the coefficient of variation (CV) of the test results was calculated. The results are shown in Table 5. The intra- and inter-bottle coefficients of variation for each element were <5%, indicating that this preparation process is reliable and that the homogeneity of each element is good both within and between bottles. However, if the conventional process is used to directly add the standard to the whole blood sample and then freeze-dry, insoluble matter or black clots will appear in the lyophilized powder, making it difficult to dissolve during reconstitution 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°C. Ten bottles were randomly sampled for testing at 0 days, 3 months, 6 months, 12 months, and 18 months, 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 tested were within 10% of the 0-day values, demonstrating the excellent storage stability of this freeze-dried whole blood quality control product at 2-8°C. The deviations between the concentrations obtained from both the high and low quality control formulations and the expected target concentrations were mostly within 10%, indicating that the elements were minimally affected by factors such as protein precipitation and alkali addition during the formulation process. Furthermore, the high-purity raw materials used and the simplified formulation process facilitated standardization, enabling the production of lower concentrations of quality control products for elements susceptible to environmental influences such as manganese and lead.

[0038] Stability (37°C accelerated stability) The prepared high and low quality control samples were stored at 37°C and tested on day 0, 3 days, 7 days, and 14 days, and the test results were compared with those on day 0. 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 relative deviations of the mean test results of all elements after 14 days of acceleration compared with those at 0 days were all within 10%, indicating that the freeze-dried whole blood quality control product had good stability after 14 days of acceleration at 37°C, and could 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 specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a freeze-dried bovine whole blood quality control product for metal element detection, characterized in that: The steps include: (1) Collect whole blood from cattle and separate it into plasma and blood cells; (2) Mix the obtained blood cells, remove the upper solution, and freeze them; take out the frozen blood cells, thaw them naturally, centrifuge them, and vacuum filter the upper solution to obtain a uniform blood cell solution without precipitation; (3) Detect the baseline values ​​of plasma and blood cell solution respectively; (4) Prepare standard solutions; calculate the volume of standard solution required for each metal element based on the base value, target concentration, and planned preparation 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 the blood cell solution and mix well to obtain the pre-lyophilization solution; (6) The pre-lyophilized solution was divided into clean containers, pre-frozen, and then vacuum-freeze-dried to obtain the bovine whole blood quality control freeze-dried powder.

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

3. The method for preparing the freeze-dried bovine whole blood quality control product for metal element detection according to claim 1, characterized in that: 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 0.5 μm, 1 μm, 5 μm, and 10 μm.

4. The method for preparing the freeze-dried bovine whole blood quality control product for metal element detection according to claim 1, characterized in that: In step (4), the metal elements include magnesium, calcium, manganese, iron, cobalt, copper, zinc, selenium, and lead.

5. The method for preparing the freeze-dried bovine whole blood quality control product for metal element detection according to claim 4, characterized in that: 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 of the standard solution include magnesium chloride hexahydrate, calcium chloride dihydrate, zinc chloride, iron chloride hexahydrate, manganese single element, copper single element, cobalt single element, lead single element and selenium single element.

6. The method for preparing the freeze-dried bovine whole blood quality control product for metal element detection according to claim 5, characterized in that: The concentration of the magnesium element solution is 10-50 mg / mL, the concentration of the calcium element solution is 10-50 mg / mL, the concentration of the iron element solution is 10-50 mg / mL, the concentration of the zinc element solution is 2-10 mg / mL, the concentration of the copper element solution is 200-1000 μg / mL, the concentration of the lead element solution is 20-100 μg / mL, the concentration of the manganese element solution is 4-20 μg / mL, the concentration of the cobalt element solution is 2-10 μg / mL, and the concentration of the selenium element solution is 20-100 μg / mL.

7. The method for preparing the freeze-dried bovine whole blood quality control product for metal element detection according to claim 1, characterized in that: In step (4), the base is an aqueous solution of tetramethylammonium hydroxide with a concentration of 10 to 25 wt. %; the amount added is 1 to 1 / 20 of the volume of the mixed standard solution.

8. The method for preparing the freeze-dried bovine whole blood quality control product for metal element detection according to claim 1, characterized in that: The clean container is a polypropylene sample bottle; the expansion coefficient of the bovine whole blood freeze-dried quality control product after reconstitution is 1 to 1.

1.

9. The method for preparing the freeze-dried bovine whole blood quality control product 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.

10. The method for preparing the freeze-dried bovine whole blood quality control product for metal element detection according to claim 1, characterized in that: The specific steps are: (1) Collect whole blood from cattle and centrifuge it at 4000 rpm for 8 min to separate plasma and blood cells; (2) The collected blood cells were mixed and centrifuged, and the upper layer of solution was removed. The mixture was placed in a -80°C environment and frozen for 17 hours, and then thawed naturally at room temperature. After the blood cell solution was completely thawed, it was centrifuged at 4000 rpm for 8 minutes, and the upper layer of solution was vacuum filtered to obtain a uniform blood cell solution without precipitation. The filter membrane used was a polypropylene fiber filter cloth with a pore size of 0.5 μm. (3) Detect the baseline values ​​of plasma and blood cell solution 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, and vortex mix for 5 minutes to obtain a 20 mg / mL magnesium solution; Weigh 588.04 mg of calcium chloride dihydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 40 mg / mL calcium solution; Weigh 33.55 mg of zinc chloride into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 4 mg / mL zinc solution. Weigh 386.19 mg of ferric chloride hexahydrate into a 5 mL centrifuge tube, add 4 mL of purified water to dissolve, and vortex mix for 5 minutes to obtain a 20 mg / mL iron solution; Take 40 μL of 1 mg / mL manganese solution and add it to a 5 mL centrifuge tube. Then add 1960 μL of purified water and vortex mix for 5 minutes to obtain a 20 μg / mL manganese solution. Take 30 μL of 1 mg / mL cobalt solution and add it to a 5 mL centrifuge tube. Then add 2970 μL of purified water and vortex mix for 5 minutes to obtain a 10 μg / mL cobalt solution. Take 200 μL of 1 mg / mL lead solution and add it to a 5 mL centrifuge tube. Then add 1800 μL of purified water and vortex mix for 5 minutes to obtain a 100 μg / mL lead solution. Take 200 μL of 1 mg / mL selenium solution and add it to a 5 mL centrifuge tube. Then add 1800 μL of purified water and vortex mix for 5 minutes to obtain a 100 μg / mL selenium solution. Calculate the required volume of standard solution for each metal element based on the baseline value, target concentration, and planned preparation volume. Mix the required standard solutions to create a mixed standard solution. Dilute the mixed standard solution with purified water, and then adjust the pH by adding 25 wt.% tetramethylammonium hydroxide aqueous solution, where 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; then add 1 volume of blood cell solution, and mix by inverting to obtain the pre-lyophilized solution; (6) The pre-lyophilized solution was divided into polypropylene sample bottles, pre-frozen at -20°C for more than 12 hours, and vacuum freeze-dried to obtain the bovine whole blood quality control freeze-dried powder.

Images