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利伐沙班纯品(≥99%,CAS:366789-02-8,Molecular Weight:435.88)和高密度聚乙烯(颗粒直径:40~48 µm,CAS:9002-88-4)均购自上海阿拉丁生化科技股份有限公司。
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首先确定目标浓度范围。因为高密度聚乙烯在远红外波段透明[17],文中将利伐沙班与150 mg高密度聚乙烯混合、研磨并通过液压机压制成直径为13 mm、表面光滑的压片。这些压片中的利伐沙班浓度不同,用以模拟不同浓度的利伐沙班样本,具体的利伐沙班浓度(C, mol/L)计算公式如下:
$$ {{C}}=w/MV $$ (1) $$ {{V}}={\rm{\pi }}{{r}}^{2}{{d}} $$ (2) 式中:w和M分别是利伐沙班的质量和分子量(435.88);d和r分别是利伐沙班/聚乙烯混合压片的厚度和半径(13 mm)。所有片剂均控制在150 mg,质量损失控制在1%以下。根据公式(1)和(2)换算得利伐沙班浓度分别为0 mmol/L,43 mmol/L,86 mmol/L,129 mmol/L,172 mmol/L,215 mmol/L和430 mmol/L。测试时,纯PE压片用作参考,其测试光谱作为其余样品测试的参考光谱。
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实验中采用传统的傅里叶变换红外光谱仪来检测利伐沙班样品。所用设备FTIR光谱仪购自布鲁克公司,型号为VERTEX 80v,信噪比优于55000∶1,检测中设置扫描分辨率为4 cm−1,扫描次数为64次,样品仓在室温(~22 ℃) 下,真空环境中,以减少水蒸气对实验的影响。
Raman光谱检测:将利伐沙班溶解在二甲基亚砜(DMSO)中,配制成不同浓度的利伐沙班溶液。取液体样品2 mL装入比色皿进行宏观大尺寸液体样品的拉曼检测。所用设备为自制拉曼光谱仪,激发波长532 nm,激光功率为500 mW,光栅为600 gr/mm。
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文中采用傅里叶变换红外光谱仪对制备的利伐沙班压片进行了检测。得到浓度为430 mmol/L利伐沙班的远红外吸收谱,如图1所示。由图1可知,430 mmol/L利伐沙班36.6 cm−1、49.3 cm−1、80.6 cm−1、和96.2 cm−1处有明显的特征吸收峰,在73.3 cm−1附近存在一个不明显的肩峰。
图 1 430 mmol/L利伐沙班的远红外谱图表征
Figure 1. Far-IR characteristic absorption spectrum of 430 mmol/L rivaroxaban detected by far-IR spectroscopy
在明确利伐沙班的远红外特征吸收峰后,接着采用傅里叶变换红外光谱仪检测了不同浓度同利伐沙班压片的远红外吸收光谱,用于研究利伐沙班浓度与其远红外吸收光谱的变化规律关系。具体实验中,将不同质量的利伐沙班与PE混合,得到浓度分别43 mmol/L,86 mmol/L,129 mmol/L,172 mmol/L,215 mmol/L和430 mmol/L的样品,测得这些样品的吸收光谱如图2所示。图中清楚地显示,随着利伐沙班的浓度增大,其四个远红外特征吸收峰的幅值呈现有规律地上升趋势。
图 2 (a) 不同浓度利伐沙班的远红外吸收光谱图;(b) 不同浓度利伐沙班样品在96.2 cm−1处吸收度与其浓度的线性关系
Figure 2. (a) Far-IR absorbance spectra of rivaroxaban with different concentration; (b) Linear relationship between absorbance and concentration of rivaroxaban with different concentrations at 96.2 cm−1
为准确地定量分析利伐沙班,分别提取不同浓度利伐沙班样品在其最强的远红外特征吸收峰(即96.2 cm−1吸收峰)的吸光度(A),分析吸光度与利伐沙班浓度(C)之间的变化关系。根据朗伯-比尔定律,对数据进行线性拟合,拟合结果如图2(b)所示,相应的线性拟合函数表达式为:
$$ {{A}}=1.933\;63{{C}}+0.037\;83 $$ (3) 拟合函数的R2为0.9914,标准差SD为0.00155,表明实验结果与线性拟合函数高度吻合。随着利伐沙班的浓度增大,其远红外吸收光谱上对应的特征吸收峰的吸收系数也呈线性递增,符合与朗伯-比尔定律,且误差范围小。对于后期利伐沙班的定量检测,只需得到待测样品的利伐沙班远红外特征吸收峰位置处的吸收系数值,结合线性拟合函数,就可以反推得出待测物质中利伐沙班的浓度。
Quantitative detection of rivaroxaban based on far-IR absorbance spectroscopy and Raman spectroscopy
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摘要: 利伐沙班是一种新型口服抗凝药,它具有疗效确切、安全性好、使用方便等优点,所以经常用于静脉血栓栓塞性疾病的预防与治疗,以及非瓣膜性房颤的卒中预防。由于利伐沙班在患者体内的浓度会影响其对凝血因子Xa的抑制作用,这导致患者的临床反应有个体差异,影响最终治疗效果。为了更加合理地使用利伐沙班,临床上需要监测人体血液或尿液中利伐沙班的浓度。针对该临床需求,文中基于远红外指纹谱和拉曼特征谱在物质有效识别和定量分析的优势,采用傅里叶变换红外光谱仪和激光共聚焦拉曼光谱系统,针对液体状态下利伐沙班进行识别并定量检测。文中先通过傅里叶变换红外光谱仪检测利伐沙班的远红外吸收谱随其浓度发生的变化,再通过激光共聚焦拉曼光谱系统检测了利伐沙班的拉曼光谱随其浓度发生的变化,最后比较了远红外光谱法与拉曼法的准确率。经过比较,远红外检测的精度比拉曼光谱检测的精度提升2倍。这些结果对临床医学中利伐沙班的使用具有重要意义。Abstract: Rivaroxaban is a new type of oral anticoagulant, which has the advantages of definite curative effect, good safety and convenient use, so it is often used in the prevention and treatment of venous thromboembolic diseases and stroke prevention of non valvular atrial fibrillation. Due to the concentration of rivaroxaban in patients, it will affect the inhibition of coagulation factor Xa, which leads to individual differences in the clinical response of patients and affects the final treatment effect. In order to use rivaroxaban more reasonably, it is necessary to monitor the concentration of rivaroxaban in human blood or urine. For the clinical needs, based on the advantages of far-infrared fingerprint spectrum and Raman characteristic spectrum in effective identification and quantitative analysis of substances, Fourier transform infrared spectrometer and laser confocal Raman spectroscopy system were used to identify and quantitatively detect rivaroxaban in liquid state. In this paper, the change of far-infrared absorption spectrum of rivaroxaban with its concentration was detected by Fourier transform infrared spectrometer, and then the change of Raman spectrum of rivaroxaban with its concentration was detected by laser confocal Raman spectroscopy system. Finally, the accuracy of far-infrared spectroscopy method and Raman method was compared. After comparison, it is proved that the accuracy of far-infrared detection is 2 times higher than that of Raman spectrum detection. These results are of great significance for the use of rivaroxaban in clinical medicine.
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