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定量核磁共振技术(qNMR)在现代化学、生物医药、食品科学等领域中扮演着至关重要的角色[1, 2]。其原理是基于核磁共振现象,当将含有1H、13C、19F、31P等特定磁性核的样品置于外加磁场中,并施加一定的频率射频脉冲时,特定磁性核会吸收能量并进入激发态。当这些原子核回到基态时,会释放出能量,产生NMR信号[3]。通过分析这些信号的强度,可以定量分析样品中目标分子[4, 5]。与传统的化学分析方法相比,qNMR技术具有准确度高、操作简便、无需标记和高通量检测等优点,已相继被《美国药典》、《欧洲药典》、《中国药典》收录,并广泛应用于多个领域[6]。如用于药物成分的定量分析,检测食品中的添加剂和污染物,监测环境中的有害物质,评估环境污染程度。此外,qNMR还在代谢组学、材料科学等领域中有着重要的应用[7, 8]。
核磁共振磷谱是一种核磁共振分析方法。其用于鉴定含磷化合物的结构,定量分析含磷化合物的含量,或监测含磷化合物动力学及其在生物体中的代谢过程[9]。由于31P原子核天然丰度为100 %,旋磁比较高,化学位移范围宽,使得31P-NMR成为研究含磷化合物极为有效的工具[10, 11]。通过分析31P-NMR谱图,可以获得关于磷化合物的分子结构、化学环境等重要信息[12]。
磷酸氢钙咀嚼片是一种以磷酸氢钙为主要原料,蔗糖、淀粉、硬脂酸镁为辅料制成的补钙类药品,具有参与骨骼形成、骨组织重建、降低毛细血管通透性等药理作用,广泛应用于儿童生长发育的促进和钙质补充。现行中国药典采用乙二胺四醋酸二钠滴定法测定磷酸氢钙片剂中的磷酸氢钙的含量[13, 14],但该法干扰因素多、灵敏度低、操作复杂。本研究建立了一种核磁共振磷谱定量(31P-qNMR)测定磷酸氢钙咀嚼片中磷酸氢钙含量的方法,相比于现有测定方法,操作简单,且专属性强,快速准确。
Determination of phosphorus content in calcium hydrogen phosphate tablets by 31P-qNMR
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摘要:
目的 建立一种高精密度和高专属性的核磁共振磷谱定量方法(31P-qNMR),用于检测磷酸氢钙咀嚼片中含磷药物的含量。 方法 使用六甲基磷酸酰胺作为内标,采集磷酸氢钙咀嚼片剂溶液的磷谱数据。研究磷酸氢钙在浓度范围1.0~9.0 mg/ml内的线性关系,并进行加样回收率和重复性实验。 结果 磷酸氢钙与内标的峰面积比在1.0 ~9.0 mg/ml范围内显示出良好的线性关系,回归方程为A = 0.0043 +0.1836 C。低、中、高3种浓度的加样回收率分别为98.0 %、98.1 %、98.4 %,相对标准偏差(RSD)分别为0.31 %、0.68 %、0.62 %(n = 9)。测定得到的磷酸氢钙咀嚼片中磷酸氢钙的平均含量为98.1 %。结论 成功建立了一种精密度高且专属性强的磷酸氢钙咀嚼片含量检测方法。该方法无需自身对照物,前处理过程简便,检测速度快,为含磷药品质量控制提供了新的技术支撑。 Abstract:Objective To establish a high-precision and high-specificity quantitative nuclear magnetic resonance phosphorus spectrometry method(31P-qNMR)for detecting the phosphorus content in calcium hydrogen phosphate chewable tablets. Methods Hexamethyl phosphoramide was used as an internal standard, phosphorus spectrum data of calcium hydrogen phosphate chewable tablets solution was collected. The linear relationship of calcium hydrogen phosphate within the concentration range of 1.0 to 9.0 mg/ml was studied, and sample recovery rate and repeatability experiments were conducted. Results Calcium hydrogen phosphate and the internal standard demonstrated a good linear relationship within the concentration range of 1.0 to 9.0 mg/ml, with a regression equation A = 0.0043 +0.1836 C. The recovery rates for low, medium, and high concentrations were 98.0%, 98.1%, and 98.4%, respectively, with relative standard deviations(RSD)of 0.31%, 0.68%, and 0.62%, respectively(n= 9). The average content of calcium hydrogen phosphate in the chewable tablets was determined to be 98.1%.Conclusion This study successfully established a high-precision and highly specific method for quantifying the content of calcium hydrogen phosphate in chewable tablets, which did not require a self-reference substance, was simple in sample preparation, and had a fast detection speed. The method could provide a new technical support for the quality control of phosphorus-containing drugs. -
Key words:
- 31P-NMR /
- q-NMR /
- content determination /
- calcium hydrogen phosphate
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[1] CHEN Z, LIAN X F, ZHOU M C, et al. Quantitation of L-cystine in food supplements and additives using 1H qNMR: method development and application[J]. Foods, 2023, 12(12):2421. doi: 10.3390/foods12122421 [2] KIM H C, BAEK K H, LEE Y E, et al. Using 2D qNMR analysis to distinguish between frozen and frozen/thawed chicken meat and evaluate freshness[J]. NPJ Sci Food, 2022, 6(1):44. doi: 10.1038/s41538-022-00159-x [3] MA H, PEDERSEN C M, ZHAO Q, et al. NMR analysis of the Fischer-Tropsch wastewater: Combination of 1D selective gradient TOCSY, 2D DOSY and qNMR[J]. Anal Chim Acta, 2019, 1066:21-27. doi: 10.1016/j.aca.2019.04.007 [4] IWASAKI D, KANAZAWA M, KAWAMOTO F, et al. A new single-reference quantitative method using liquid chromatography with relative molar sensitivity based on 1H-qNMR for khellactone esters from Peucedanum japonicum root extract[J]. Food Chem, 2023, 427:136647. doi: 10.1016/j.foodchem.2023.136647 [5] SCETTRI A, SCHIEVANO E. Quantification of polyols in sugar-free foodstuffs by qNMR[J]. Food Chem, 2022, 390:133125. doi: 10.1016/j.foodchem.2022.133125 [6] WELLS R J, HOOK J M, AL-DEEN T S, et al. Quantitative nuclear magnetic resonance (QNMR) spectroscopy for assessing the purity of technical grade agrochemicals: 2, 4-dichlorophenoxyacetic acid (2, 4-D)and sodium 2, 2-dichloropropionate (Dalapon sodium)[J]. J Agric Food Chem, 2002, 50(12):3366-3374. doi: 10.1021/jf0114379 [7] GIANCASPRO G, ADAMS K M, BHAVARAJU S, et al. The qNMR Summit 5.0: Proceedings and Status of qNMRTechnology[J]. Anal Chem, 2021, 93(36):12162-12169. doi: 10.1021/acs.analchem.1c02056 [8] MUSIO B, RAGONE R, TODISCO S, et al. A community-built calibration system: The case study of quantification of metabolites in grape juice by qNMR spectroscopy[J]. Talanta, 2020, 214:120855. doi: 10.1016/j.talanta.2020.120855 [9] 黄挺, 吴华鑫. 氟磷谱定量核磁共振法进展综述[J]. 计量科学与技术, 2023, 67(5):9-15. doi: 10.12338/j.issn.2096-9015.2023.0095 [10] KOMOROSKI R A, HOLDER J C, PAPPAS A A, et al. 31P NMR of phospholipid metabolites in prostate cancer and benign prostatic hyperplasia[J]. Magn Reson Med, 2011, 65(4):911-913. doi: 10.1002/mrm.22677 [11] DEELCHAND D K, NGUYEN T M, ZHU X H, et al. Quantification of in vivo 31P NMR brain spectra using LCModel[J]. NMR Biomed, 2015, 28(6):633-641. doi: 10.1002/nbm.3291 [12] 黎玲玲, 邹亚娟, 吴节莉, 等. 31P核磁共振内标法测定甘油磷酸胆碱的含量[J]. 药物分析杂志, 2017, 37(11):1933-1936. [13] 国家药典委员会. 中华人民共和国药典: 二部: 2020年版[M]. 北京: 中国医药科技出版社, 2020: 1914. [14] 郭国宁. 磷酸氢钙中钙含量的测定方法[J]. 无机盐工业, 2001, 33(3):41-42,2. doi: 10.3969/j.issn.1006-4990.2001.03.016