Method for in vitro detection of the content of exchangeable magnesium ions in bone mineral

Abstract

The application discloses a method for detecting the content of exchangeable magnesium ions in bone mineral, and comprises the following steps: contacting powder bone mineral with a proper amount of alendronate in water to promote the exchangeable magnesium ions in the bone mineral to be basically completely released into the aqueous solution; and then determining the content of magnesium ions in the aqueous solution to calculate the content of the exchangeable magnesium ions in the bone mineral. The method provides a chemical detection means for the determination of the content of the exchangeable magnesium ions in the bone mineral.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry and bone material detection technology, specifically relating to an in vitro detection method for the content of easily exchangeable magnesium ions in bone minerals. Background Technology

[0002] Magnesium is an essential metallic element for the human body, playing a crucial role in ATP synthesis, DNA and RNA structural stability, enzymatic reactions, maintenance of normal cardiac function, and blood pressure stability. Therefore, accurately assessing the body's magnesium status has significant physiological and clinical implications.

[0003] The total magnesium content in the human body, by mass, is approximately 25 ± 3 g, of which about 60% is stored in bones, accounting for about 0.5% of total bone mineral content. The remaining approximately 40% of magnesium is found in soft tissues and extracellular fluid. Extracellular fluid magnesium accounts for only about 1% of the total magnesium in the human body, of which about 5%–10% is easily exchangeable magnesium bound to anions (such as phosphate, citrate, and carbonate ions) [AR Morrison. Magnesium homeostasis: Lessons from humangenetics. Clin. J. Am. Soc. Nephrol. 18, 969-978 (2023)]. Serum magnesium ions (Mg 2+ The steady-state regulation of Mg concentration depends on the synergistic effects of the intestine, bones, and kidneys. 2+ When there is a temporary deficiency, the body mainly relies on the release of easily exchangeable magnesium ions from bones to maintain the normal range of serum magnesium concentration in the range of 0.70 to 1.25 mmol / L [J. Vormann. Magnesium: nutrition and homoeostasis. AIMS Public Health 3, 329-340 (2016)].

[0004] Magnesium ions have a variety of physiological functions. They are cofactors for more than 300 enzymes in the body and participate in more than 600 enzymatic reactions [JHFD Baaij, JGJ Hoenderop, RJM Bindels. Magnesium in man: implications for health and disease. Physiol. Rev. 95, 1-46 (2015); FH Nielsen. Dietary magnesium and chronic disease. Adv. Chronic Kidney D.25, 230-235 (2018)], playing a key role in ATP synthesis.Magnesium stabilizes the structure of nucleic acids (DNA and RNA) [K. Nam, ARA Thodika, S. Tischlik, C. Phoeurk, TM Nagy, L. Schierholz, J. Ådén, P. Rogne, M. Drescher, AE Sauer-Eriksson, M. Wolf-Watz. Magnesium induced structural reorganization in the active site of adenylate kinase. Sci. Adv. 10, eado5504 (2024)], and plays an important role in muscle contraction, regulation of nerve signals, and regulation of tissue fibrosis [R. Yamagami, JL Bingaman, EA Frankel, PCBevilacqua. Cellular conditions of weakly chelated magnesium ions strongly promote RNA stability and catalysis. Nat. Commun. 9, 2149 (2018); C. Xu, Y. Luo, Z. Cai, J. Ji, Z. Guo, Z. Cai, C. You, Y. Zhou, Z. Chen, W. Zhang, N.Gong, J. Wang. Magnesium ions attenuate tendon graft fibrosis during its ligamentization after ACL reconstruction through modulation of fibroblast tomyofibroblast trans-differentiation by promoting PGE2secretion. Bioact.Mater. 52, 474-491 (2025)].Magnesium is essential for maintaining bone mineral density and preventing osteoporosis [M.Farsinejad-Marj, P. Saneei, A. Esmaillzadeh. Dietary magnesium intake, bonemineral density and risk of fracture: a systematic review and meta-analysis. Osteoporos Int. 27, 1389-1399 (2016)], and participates in insulin secretion and signal transduction [SR deSousa Melo, LR dos Santos, T. da Cunha Soares, B. Cardoso, T. Dias, J. Morais, M. Sousa, T. Sousa, N. Silva, L. Silva, K. Cruz, D. Marreiro. Participation of magnesium in the secretion and signaling pathways of insulin: an updated review. Biol. Trace Elem. Res. 200, 3545-3553 (2022)].Furthermore, magnesium deficiency is associated with increased anxiety behavior [SB Sartori, N. Whittle, A. Hetzenauer, N. Singewald. Magnesium deficiency induces anxiety and HPA axis dysregulation:modulation by therapeutic drug treatment. Neuropharmacology 62, 304-312(2012)]. Magnesium plays an important role in maintaining normal heart rhythm and blood pressure regulation [M. Houston. The role of magnesium in hypertension and cardiovascular disease. J. Clin. Hypertens(Greenwich) 13, 843-847 (2011)]. Magnesium supplementation can also alleviate oxidative stress by increasing the activity of antioxidant enzymes [C. Orhan, B. Er, PBD Deeh, AA Bilgic, SP Ojalvo, JR Komorowski, K. Sahin. Different sources of dietary magnesium supplementation reduces oxidative stress by regulation Nrf2 and NF-κB signaling pathways in high-fatdiet rats. Biol. Trace Elem. Res.]. 199, 4162-4170 (2021)].

[0005] Given the aforementioned wide range of physiological functions, the body relies on the rapid release of easily exchangeable magnesium ions from bones to maintain physiological homeostasis when magnesium is deficient. 25 Mg and 26Studies on magnesium metabolism kinetics using Mg isotopes have shown that approximately 24% of the magnesium in the body of healthy men is rapidly exchanged, suggesting that readily exchangeable magnesium ions in bone constitute a magnesium ion pool that can closely buffer the magnesium concentration in extracellular fluid [M. Sabatier, F. Pont, MJ Arnaud, JR Turnlund. Acompartmental model of magnesium metabolism in healthy men based on twostable isotope tracers. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R656-663 (2003)]. Research indicates that this buffering capacity based on readily exchangeable magnesium ions in bones declines with age throughout a person's life, and eventually, nearly half of the readily exchangeable magnesium ion potential in bones may be lost. This magnesium loss may manifest in two ways: firstly, a decrease in the content of readily exchangeable magnesium ions; and secondly, external magnesium... 2+ It is difficult to enter the existing magnesium ion pool, resulting in a decrease in magnesium exchange capacity [AIMS Public Health 3, 329-340 (2016)].

[0006] Currently, the main medical method for directly detecting magnesium ion content in bone minerals is bone biopsy. Indirect methods include dual-energy X-ray absorptiometry, ultrasound bone mineral density testing, MRI and other imaging techniques, as well as biochemical indicators such as serum magnesium levels and 24-hour urinary magnesium excretion. However, changes in bone mineral density cannot directly indicate bone magnesium status [RK Rude, HEGruber. Magnesium deficiency and osteoporosis: animal and human observations. J. Nutr. Biochem. 15, 710-716 (2004)]. A method that can identify and quantify magnesium ions in bone is needed. 2+ Horizontal imaging technology is still in the early stages of research and development [AC Seifert, AC Wright, SL Wehrli, HH Ong, C.Li, FW Wehrli]. 31P NMR relaxation of cortical bone mineral at multiplemagnetic field strengths and levels of demineralization. NMR Biomed. 6, 1158-1166 (2013); M. Liu, X. Yu, M. Li, N. Liao, A. Bi, Y. Jiang, S. Liu, Z. Gong,W. Zeng. Fluorescent probes for the detection of magnesium ions (Mg 2+ ): fromdesign to application. RSC Adv. 8, 12573-12587 (2018); B. Pinto-Pacheco, Q.Lin, CW Yan, S. de Melo Silva, D. Buccella. Lanthanide-based luminescentprobes for biological magnesium: accessing polyphosphate-bound Mg 2+ . Chem. Commun. 59, 3586-3589 (2023)]. 25 Mg and 26 Although stable Mg isotopes have been used to determine the content of exchangeable magnesium ions in bone minerals [Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R656-663(2003)], due to their high natural abundance, it is difficult to effectively indicate the state of magnesium using only a small number of isotopes. 28 The half-life of Mg does not match the half-life of bone magnesium, resulting in a large testing error [Brit. J. Nutr. 99 (S3), S24-S36 (2008)].

[0007] In summary, bone biopsy is currently the primary clinical method for directly detecting magnesium ion content in bone minerals. However, existing technologies lack in vitro chemical detection methods for the content of easily exchangeable magnesium ions in bone minerals. Developing such a method would help maximize the detection potential of bone biopsy samples, providing chemical data on the content of easily exchangeable magnesium ions in bone while obtaining information on bone morphology and microstructure evolution. The need for early warning of osteoporosis and its complications continues to grow due to multiple factors, including declining levels of easily absorbed magnesium in food, population aging, and unhealthy lifestyles such as sedentary lifestyles and lack of exercise. The data on easily exchangeable magnesium ion content in bone provided by this method is expected to offer a new biochemical indicator for assessing bone magnesium nutritional status and early screening research for bone metabolism-related diseases. Summary of the Invention

[0008] To overcome the above problems, this invention provides a method for detecting the content of easily exchangeable magnesium ions in bone minerals based on the interaction between alendronic acid and bone minerals. By contacting an appropriate amount of alendronic acid with bone minerals in an aqueous solution and allowing them to interact, the easily exchangeable magnesium ions in the bone minerals are almost completely released into the aqueous solution. Subsequently, the concentration of magnesium ions in the aqueous solution is measured, and the mass of the easily exchangeable magnesium ions and the percentage of this mass relative to the total mass of magnesium ions in the bone minerals are calculated, i.e., the percentage content of easily exchangeable magnesium ions in the bone minerals. This invention provides a chemical means for determining the content of easily exchangeable magnesium ions in bone minerals.

[0009] Specifically, the present invention provides an in vitro detection method for the content of readily exchangeable magnesium ions in bone minerals, comprising the following steps: reacting bone minerals with alendronic acid in water to obtain a reaction mixture; determining the magnesium ion concentration in the aqueous solution of the reaction mixture and calculating the total mass M1 of magnesium ions in the aqueous solution; and calculating the percentage content of readily exchangeable magnesium ions in the bone minerals according to the formula (M1 / M0)×100%, where M0 is the mass of total magnesium ions in the bone minerals.

[0010] In this article, the percentage of exchangeable magnesium ions in bone minerals refers to the percentage of the mass of exchangeable magnesium ions in bone minerals relative to the total mass of magnesium ions.

[0011] In some embodiments, bone minerals are reacted with alendronic acid in water to obtain a reaction mixture; after the reaction is complete, the reaction mixture is subjected to solid-liquid separation to obtain a filtrate or supernatant; the pH of the filtrate or supernatant is adjusted to 2.5-3.0, the concentration of magnesium ions in the solution is determined, and the mass M1 of magnesium ions in the aqueous solution is calculated.

[0012] In some embodiments, the weight ratio of alendronic acid to bone mineral is (4.0–15.0):100.

[0013] In some embodiments, the weight ratio of alendronic acid to bone minerals is (4.0 to 10.0):100.

[0014] In some embodiments, the weight ratio of alendronic acid to bone minerals is (5.0–8.0):100.

[0015] In some embodiments, the weight ratio of alendronic acid to the bone mineral is 4.0:100, 5.0:100, 6.0:100, 7.0:100, 8.0:100, 9.0:100, 10.0:100, 11.0:100, 12.0:100, 13.0:100, 14.0:100, or 15.0:100.

[0016] In some embodiments, the weight ratio of the bone minerals to the water is (1-2):100, and the water is deionized water.

[0017] In some embodiments, the temperature of the contact reaction is 20°C to 50°C. In some embodiments, the temperature of the contact reaction is 20°C to 40°C. In some embodiments, the temperature of the contact reaction is 25°C to 36°C.

[0018] In some embodiments, the contact reaction time is 72–120 h. In some embodiments, the contact reaction time is 96–120 h.

[0019] In some embodiments, the contact reaction is carried out under stirring conditions at a stirring rate of 60–300 rpm.

[0020] In some embodiments, the bone minerals are in powder form.

[0021] In some embodiments, the method for determining the total magnesium ion mass M0 in the bone minerals includes the following steps: weighing a bone mineral sample of mass M, adding an appropriate amount of concentrated hydrochloric acid or concentrated nitric acid as a solvent, and optionally performing ultrasonic-assisted dissolution to completely dissolve the sample (the solution is clear, and no visible insoluble particles are observed with the naked eye), to obtain a sample solution; transferring the sample solution and making up to volume with water, adjusting the pH value of the solution to 2.0-3.5; determining the magnesium ion concentration in the obtained solution, and calculating the total magnesium ion mass M0 according to the formula M0= C0× V, where V is the volume of the solution.

[0022] In some implementations, the method includes the following steps: Powdered bone minerals were prepared according to the method described herein; The powdered bone minerals are reacted with alendronic acid in water to obtain a reaction mixture; the magnesium ion concentration in the aqueous solution of the reaction mixture is determined, and the mass M1 of magnesium ions in the aqueous solution is calculated; the percentage content of easily exchangeable magnesium ions in the bone minerals is calculated according to the formula (M1 / M0)×100%, wherein M0 is determined according to the method described herein.

[0023] In one specific embodiment, the method includes: reacting powdered bone minerals with alendrolic acid in water for 96 h; after the reaction is complete, separating the reaction mixture into solid and liquid phases by filtration or centrifugation; adding hydrochloric acid or nitric acid to the resulting filtrate or supernatant to adjust the pH to approximately 2.8; and making up to volume with water; determining the magnesium ion concentration in the resulting solution; calculating the magnesium ion mass M1 in the solution; and calculating the content of easily exchangeable magnesium ions in the bone minerals based on the total magnesium ion mass M0 in the bone minerals using the formula (M1 / M0)×100%.

[0024] In one specific implementation, the method for preparing the bone minerals includes: adding a bone sample and water into a hydrothermal reactor made of polytetrafluoroethylene and reacting it hydrothermally at 190 °C for 3 h; after cooling, removing the bone sample and grinding it into powder in an agate mortar; immersing the obtained powdered sample in 30% hydrogen peroxide at room temperature for 1–2 h, optionally with ultrasonic assistance, followed by solid-liquid separation by vacuum filtration; further placing the obtained solid sample into an ethanol-water solution (volume ratio approximately 1:1), stirring at 85 °C for 4 h, and again performing solid-liquid separation by vacuum filtration; drying the obtained solid sample using a rotary evaporator under reduced pressure at a drying temperature of 60 °C–80 °C, a vacuum degree below 5 kPa, a rotation speed of approximately 100 rpm, and a drying time of 1–2 h to obtain powdered bone minerals.

[0025] In one specific implementation, the method for determining the total magnesium ion mass M0 in the bone minerals includes: weighing a bone mineral sample of mass M and placing it in a quartz bottle, adding an appropriate amount of concentrated hydrochloric acid or concentrated nitric acid to completely dissolve the sample (the solution is clear, and no visible insoluble particles are observed); transferring the dissolved sample solution to a volumetric flask, rinsing the quartz bottle with water, and transferring the washings to the volumetric flask as well; adding water to near the mark, adjusting the pH of the solution to approximately 2.8 with hydrochloric acid or nitric acid, and making up to volume; determining the concentration C0 of magnesium ions in the resulting solution, and calculating the total magnesium ion mass M0 according to the formula M0 = C0 × V, where V is the volume of the final volume.

[0026] In some specific implementations, the bone sample is a mammalian bone sample.

[0027] The method provided by this invention is simple to operate, and all required tests can be performed using conventional analytical testing equipment in the field, exhibiting good operability and scalability. This method can be used to determine the content of easily exchangeable magnesium ions in bone minerals, thereby supporting research on the relationship between easily exchangeable magnesium ions in bone and related biochemical indicators. This method is also applicable to biomimetic bone materials containing easily exchangeable magnesium ions. Based on the basic principles of this method, it is expected to be further expanded to fields involving the detection of easily exchangeable magnesium ions, such as the chemical evaluation of artificial bone repair materials, quality control of bone organoids, elemental analysis of bone fossils, and detection of forensic bone samples. The easily exchangeable magnesium ion content data obtained by this method can be used for research on bone metabolism mechanisms and scientific assessments related to bone health. Detailed Implementation

[0028] The invention will be further explained below with reference to specific examples.

[0029] Unless otherwise specified, all reagents mentioned in the following examples are of analytical grade or higher, all water is deionized water, and all glass containers are made of high borosilicate glass.

[0030] Example Preparation of powdered bone minerals A suitable amount of bone sample and water were added to a hydrothermal reactor made of polytetrafluoroethylene (PTFE) and reacted at 190 °C for 3 hours. After cooling, the softened bone sample was removed and ground into powder in an agate mortar. The powdered sample was then immersed in 30% hydrogen peroxide at room temperature for 1–2 hours, optionally with ultrasonic-assisted treatment, followed by solid-liquid separation by vacuum filtration. The resulting solid was further placed in an ethanol-water solution (volume ratio approximately 1:1) and stirred at 85 °C for 4 hours, followed by solid-liquid separation by vacuum filtration again. The resulting solid was further dried using a rotary evaporator under reduced pressure at a temperature of 60 °C–80 °C, a vacuum degree below 5 kPa, a rotation speed of approximately 100 rpm, and a drying time of 1–2 hours. The resulting dried powder sample is the powdered bone mineral.

[0031] Unless otherwise specified, the powdered bone minerals used in the following examples are all derived from the same batch of bovine femurs and prepared according to the method described above. It is understood that the present invention is not limited to this specific source, and bone minerals from other mammalian bone sources can achieve similar technical effects.

[0032] It should be noted that methods for treating natural bones to obtain bone mineral powder using steps such as hydrothermal treatment, hydrogen peroxide impregnation, and heating and stirring with an ethanol-water solution are already known to those skilled in the art. This invention is not limited to the specific preparation methods described above; any known method capable of obtaining bone mineral powder can be used in this invention. Currently, there are no reports on using the reaction of bone minerals with alendrolic acid in water for the detection of readily exchangeable magnesium ions. Based on this discovery, this invention provides an in vitro method for detecting the content of readily exchangeable magnesium ions in bone minerals.

[0033] Determination of total magnesium ion mass and percentage content in bone minerals The total magnesium ion content in bone minerals was determined using acid digestion-inductively coupled plasma optical emission spectrometry (ICP-OES). The specific procedure is as follows: 100.0 mg of bone minerals was weighed into a quartz bottle, and concentrated hydrochloric acid or concentrated nitric acid (1–3 mL) was added as a solvent to completely dissolve the sample (the solution was clear, and no visible insoluble particles were observed). Sonication was optionally used for this purpose. The dissolved sample solution was transferred to a 100 mL volumetric flask. The quartz bottle was rinsed with water (10 mL × 3), and the rinsing solution was transferred to the volumetric flask as well. Water was added to near the mark, and the pH of the solution was adjusted to approximately 2.8 with hydrochloric acid or nitric acid. The volume was then brought to 100 mL. The magnesium ion concentration C0 (mg / L) in the solution was determined by ICP-OES, and the result was 5.31 ± 0.16 mg / L. The total magnesium ion content M0 of the bone minerals was obtained by multiplying the measured magnesium ion concentration C0 by the final volume (0.1 L), which was 0.531 ± 0.016 mg. According to the formula (M0 / M)×100%, where M is the mass of the weighed bone minerals, which is taken as 100.0 mg in this embodiment, the mass percentage of magnesium ions in the bone mineral sample is calculated to be 0.53±0.02%.

[0034] Unless otherwise stated, the bone minerals used in the following examples were all prepared by this method, and their magnesium ion mass percentage content was 0.53±0.02%.

[0035] Example 1 (1) In a 250 mL flask, add water (100 mL) and bone minerals (1.5000 g), then add alendronic acid (75.0 mg), and stir the resulting reaction mixture in a sealed container at 20 ℃~36 ℃ for 96 h at a stirring speed of 60~300 rpm; (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.8. (3) Transfer the filtrate or supernatant obtained in step (2) to a 100 mL volumetric flask. Wash the solid in the filtration funnel with a small amount of water 2-3 times, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration of magnesium ions (C1) in the sample solution by ICP-OES. The concentration is 20.32±0.12 mg / L. Then calculate the mass of magnesium ions in the solution (M1) as 2.03±0.01 mg. (5) Divide the M1 (2.03±0.01 mg) obtained in step (4) by the total magnesium mass M0 (7.95±0.30 mg) of the bone minerals, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the bone minerals as (25.6±1.0)%.

[0036] Example 2 (1) In a 250 mL flask, add water (100 mL) and bone minerals (1.5000 g), then add alendronic acid (90.0 mg). Stir the resulting reaction mixture in a sealed container at 20 ℃~36 ℃ for 96 h at a stirring speed of 60~300 rpm. (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.8. (3) Transfer the filtrate or supernatant obtained in step (2) to a 100 mL volumetric flask. Wash the solid in the filtration funnel with a small amount of water 2-3 times, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration of magnesium ions (C1) in the sample solution by ICP-OES. The concentration is 20.11±0.08 mg / L. Then calculate the mass of magnesium ions (M1) in the solution as (2.01±0.01) mg. (5) Divide the M1 (2.01±0.01 mg) obtained in step (4) by the total magnesium mass M0 (7.95±0.30 mg) of the bone minerals, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the bone minerals as (25.3±1.0)%.

[0037] Example 3 (1) In a 250 mL flask, add water (100 mL) and bone minerals (1.5000 g), then add alendronic acid (100.0 mg). The resulting reaction mixture is stirred in a sealed container at 20 ℃~36 ℃ for 96 h at a stirring speed of 60~300 rpm. (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.8. (3) Transfer the filtrate or supernatant obtained in step (2) to a 100 mL volumetric flask. Wash the solid in the filtration funnel with a small amount of water 2-3 times, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration of magnesium ions (C1) in the sample solution by ICP-OES. The concentration is 20.55±0.06 mg / L. Then calculate the mass of magnesium ions in the solution (M1) as 2.05±0.01 mg. (5) Divide the M1 (2.05±0.01 mg) obtained in step (4) by the total magnesium mass M0 (7.95±0.30 mg) of the bone minerals, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the bone minerals as (25.9±1.0)%.

[0038] Example 4 (1) In a 1000 mL flask, add water (600 mL) and bone minerals (6.0000 g), then add alendronic acid (300.0 mg), and stir the resulting reaction mixture in a sealed container at 20 ℃~36 ℃ for 120 h at a stirring speed of 60~300 rpm; (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.7. (3) Transfer the obtained filtrate or supernatant to a 1000 mL volumetric flask. Wash the solid in the filtration funnel 2-3 times with an appropriate amount of water, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask 2-3 times with an appropriate amount of water, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube 2-3 times with an appropriate amount of water, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration of magnesium ions (C1) in the sample solution by ICP-OES. The concentration is 8.05±0.30 mg / L. Then calculate the mass of magnesium ions in the solution (M1) as 8.05±0.30 mg. (5) Divide the M1 (8.05±0.30 mg) obtained in step (4) by the total magnesium mass M0 (31.80±1.20 mg) of the bone minerals, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the bone minerals as (25.3±1.3)%.

[0039] Example 5 (1) In a 250 mL flask, add water (100 mL) and bone minerals (1.5000 g), then add alendronic acid (60.0 mg), and stir the resulting reaction mixture in a sealed container at 20 ℃~36 ℃ for 96 h at a stirring speed of 60~300 rpm; (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.8. (3) Transfer the filtrate or supernatant obtained in step (2) to a 100 mL volumetric flask. Wash the solid in the filtration funnel with a small amount of water 2-3 times, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration of magnesium ions (C1) in the sample solution by ICP-OES. The concentration is 17.83±0.09 mg / L. Then calculate the mass of magnesium ions in the solution (M1) as 1.78±0.01 mg. (5) Divide the M1 (1.78±0.01 mg) obtained in step (4) by the total magnesium mass M0 (7.95±0.30 mg, calculated in the same way as above) of the bone minerals, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the bone minerals as (22.4±0.9)%.

[0040] Example 6 (1) In a 250 mL flask, add water (100 mL) and bone minerals (1.5000 g), then add alendronic acid (75.0 mg), and stir the resulting reaction mixture in a sealed container at 20 ℃~36 ℃ for 120 h at a stirring speed of 60~300 rpm; (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.8. (3) Transfer the filtrate or supernatant obtained in step (2) to a 100 mL volumetric flask. Wash the solid in the filtration funnel with a small amount of water 2-3 times, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration of magnesium ions (C1) in the sample solution by ICP-OES. The concentration is 19.93±0.11 mg / L. Then calculate the mass of magnesium ions in the solution (M1) as 1.99±0.01 mg. (5) Divide the M1 (1.99±0.01 mg) obtained in step (4) by the total magnesium mass M0 (7.95±0.30 mg) of the bone minerals, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the bone minerals as (25.1±1.0)%.

[0041] Example 7 (1) In a 250 mL flask, add water (100 mL) and bone minerals (1.5000 g), then add alendronic acid (200.0 mg). The resulting reaction mixture is stirred in a sealed container at 20 ℃~36 ℃ for 72 h at a stirring speed of 60~300 rpm. (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.8. (3) Transfer the filtrate or supernatant obtained in step (2) to a 100 mL volumetric flask. Wash the solid in the filtration funnel with a small amount of water 2-3 times, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration of magnesium ions (C1) in the sample solution by ICP-OES. The concentration is 21.87±0.08 mg / L. Then calculate the mass of magnesium ions in the solution (M1) as 2.19±0.01 mg. (5) Divide the M1 (2.19±0.01 mg) obtained in step (4) by the total magnesium mass M0 (7.95±0.30 mg) of the bone minerals, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the bone minerals as (27.5±1.0)%.

[0042] Example 8 This embodiment is used to verify the applicability of the method of the present invention in magnesium-doped calcium hydroxyphosphate materials containing citrate.

[0043] (1) In a 250 mL flask, add water (100 mL) and calcium hydroxyphosphate (1.0000 g, of which the citrate doping amount is 0.5 wt.%, using sodium citrate as raw material; the magnesium doping amount is 0.2 wt.%, using magnesium chloride as raw material), and then add alendronic acid (50.0 mg). The resulting reaction mixture is stirred in a closed container at 20 ℃~36 ℃ for 96 h at a stirring speed of 60~300 rpm. (2) After the reaction is complete, the mixture obtained in step (1) is separated into solid and liquid by filtration or centrifugation. Hydrochloric acid or nitric acid is added dropwise to the obtained filtrate or supernatant to adjust the pH value to about 2.8. (3) Transfer the filtrate or supernatant obtained in step (2) to a 100 mL volumetric flask. Wash the solid in the filtration funnel with a small amount of water 2-3 times, and add the washing liquid to the filtration flask and transfer it to the volumetric flask; then wash the filtration flask with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. If centrifugation is used in step (2), wash the centrifuge tube with a small amount of water 2-3 times, and add the washing liquid to the volumetric flask. Finally, add water to make up to the mark and shake well; (4) Take a sample from the solution obtained in step (3) and determine the concentration (C1) of magnesium ions in the sample solution by ICP-OES. The concentration is 3.61±0.09 mg / L. Then calculate the mass of magnesium ions in the solution M1=0.36±0.01 mg. (5) Divide M1 obtained in step (4) by the total magnesium mass M0 (2.05±0.05 mg) of the calcium hydroxyphosphate co-doped with citrate and magnesium, i.e. (M1 / M0)×100%, to obtain the content of easily exchangeable magnesium ions in the material as (17.6±0.7)%.

[0044] The above results demonstrate that the method of this invention can be applied to the chemical evaluation of readily exchangeable magnesium ions in magnesium-doped calcium hydroxyphosphate biomimetic bone materials containing citrate, providing a feasible analytical tool for their quality control and performance studies. Based on this, this method is expected to be further extended to the chemical evaluation of readily exchangeable magnesium ions in biomimetic bone materials with other compositions (such as different doping elements or doping ratios).

[0045] The results of all the above embodiments show that reacting alendronate with bone minerals in water utilizes its exchange / release effect on magnesium ions on the surface of bone minerals, allowing for the near-complete release of readily exchangeable magnesium ions from the bone minerals into the aqueous solution. This enables accurate quantitative detection of the readily exchangeable magnesium ion content in bone minerals by measuring the magnesium ion concentration in the aqueous solution. Currently, there are no reports on using the reaction of bone minerals with alendronate in water to detect the content of readily exchangeable magnesium ions. This is fundamentally different from the clinical application and technical purpose of alendronate sodium as a bone resorption inhibitor. This invention provides a reliable chemical analytical method for determining the content of readily exchangeable magnesium ions in bone minerals.

[0046] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An in vitro method for detecting the content of easily exchangeable magnesium ions in bone minerals, characterized in that, Includes the following steps: (a) A suitable amount of bone minerals is reacted with alendronic acid in water to obtain a reaction mixture; (b) Determine the concentration of magnesium ions in the aqueous solution of the reaction mixture obtained in step (a), and calculate the mass M1 of magnesium ions in the aqueous solution; (c) The percentage content of easily exchangeable magnesium ions in the bone mineral is calculated according to the formula (M1 / M0)×100%, where M0 represents the mass of total magnesium ions in the bone mineral.

2. The method according to claim 1, characterized in that, Steps (a) and (b) are as follows: (a) Bone minerals are reacted with alendronic acid in water to obtain a reaction mixture; after the reaction is complete, the reaction mixture is subjected to solid-liquid separation to obtain a filtrate or a supernatant; (b) Adjust the pH of the filtrate or supernatant to 2.5 to 3.0, determine the concentration of magnesium ions in the solution, and calculate the mass of magnesium ions M1.

3. The method according to claim 1, characterized in that, In step (a), the weight ratio of alendronic acid to bone mineral is (4.0 to 15.0):

100.

4. The method according to claim 3, characterized in that, The weight ratio of alendronic acid to the bone mineral is (5.0–8.0):

100.

5. The method according to claim 1, characterized in that, In step (a), the weight ratio of the bone minerals to the water is (1-2):100, and the water is deionized water.

6. The method according to claim 1, characterized in that, In step (a), the temperature at which the bone minerals react with alendronic acid in water is 25 ℃ to 36 ℃.

7. The method according to claim 1, characterized in that, In step (a), the bone minerals and alendronic acid are reacted in water for 72 to 120 hours.

8. The method according to claim 1, characterized in that, In step (a), the contact reaction between the bone minerals and alendronic acid in water is carried out under stirring conditions at a stirring rate of 60–300 rpm.

9. The method according to claim 1, characterized in that, The method for determining the total magnesium ion mass M0 in bone minerals includes the following steps: (a) Provide a bone mineral sample of mass M; (b) The bone mineral sample is mixed with concentrated hydrochloric acid or concentrated nitric acid, and optionally sonicated to completely dissolve the sample to obtain a sample solution; (c) Transfer the sample solution and dilute it to volume with water, adjusting the pH of the solution to 2.0–3.5; (d) Determine the magnesium ion concentration (C0) in the solution obtained in step (c), and calculate the total magnesium ion mass M0 according to the formula M0 = C0 × V, where V is the constant volume.

10. The method according to any one of claims 1 to 9, characterized in that, It also includes a method for preparing the bone minerals, comprising the following steps: (a) Take a bone sample and place it in a hydrothermal reactor. Add water and heat to react. After cooling, take out the bone sample and grind it into powder. (b) The powdered sample obtained in step (a) is immersed in hydrogen peroxide and then subjected to solid-liquid separation; (c) The solid sample separated in step (b) is placed in an ethanol-water solution and heated and stirred to perform solid-liquid separation again; (d) Dry the solid sample obtained in step (c) to obtain the bone mineral.