Fluorine (19F) Nuclear Magnetic Resonance Spectroscopy For Real Time Maraviroc Analysis From Microparticulate Systems

Real time analysis of pharmaceuticals in controlled release nano and microsystems remains a challenge. It is hypothesized that fluorine 19 nuclear magnetic resonance (¹⁹F qNMR) can be used for real time quantification and in vitro release of maraviroc (MVC). The release of maraviroc was analyzed in simulated body fluids from spray dried sodium alginate microspheres (MS) using the ¹⁹F qNMR method. Calibration produced a linearity curve in concentration range (0.42 mg/ml – 15 mg/ml), and the limits of detection and quantification values were 0.97 mg/ml and 2.93 mg/ml, respectively. The method was confirmed to be specific, accurate, precise, and robust (%RSE > 2%). MVC was successfully microencapsulated (18% w/w) as evidenced by the FT-IR spectra and SEM images. The MS had an average diameter of 2.522 ± 0.15 μm, with a zeta potential of – 61.31 ± 2.1 mV. Overall, the ¹⁹F qNMR method enabled a direct and real time quantification of MVC for an efficient drug release kinetics. This approach could be potentially used to quantify fluorinated drug potency, purity, and stability, and evaluate in vitro release kinetic from different formulations.     

定量核磁共振技术及其在药学领域中的应用进展

综述定量核磁共振技术的基本原理以及在新药研发中的应用进展。定量核磁共振作为一种定量分析方法,专属性强、准确快捷,可与其他定量方法相互补充,用于新药研发的诸多环节,如药品质量控制、候选药物纯度测定及药物代谢研究等,在药学领域有很好的应用前景。     

Quantitative Analysis of Hydroxyurea and Urea by Proton Nuclear Magnetic Resonance (NMR) Spectroscopy

Quantitative ¹H-nuclear magnetic resonance (NMR) procedures are described for measuring hydroxyurea and urea. Dimethylsulfoxide-d₆ is used as the solvent for both assays; the internal standards employed are urea and p-dichlorobenzene for hydroxyurea and urea, respectively. The analysis for hydroxyurea is based on the comparison of the area of the – NH₂ peak of hydroxyurea with the area of the urea – NH₂ peak area. The analysis of urea is based on the comparison of the – NH₂ peak of urea with the area of the p-dichlorobenzene singlet. The ¹H-NMR assays yield results that are precise and agree well with results of the more cumbersome and less specific USP procedures.     

The Place of Nuclear Magnetic Resonance Spectroscopy in Drug Metabolism Studies

Abstract

1. An elementary introduction is provided to the theory of n.m.r. spectroscopy.

2. The different modes of operation are described with particular attention to the preparation and examination of small samples.

3. Examples are given of applications of n.m.r. spectroscopy to structure elucidation problems in drug metabolism studies. The particular value of n.m.r. in locating the positions of new hydroxyl groups is emphasized.     

Solid-State Nuclear Magnetic Resonance Spectroscopy: Theory and Pharmaceutical Applications

The theory of solid-state nuclear magnetic resonance (NMR) spectroscopy is reviewed, with specific discussions of magnetic interactions in the solid state. Each magnetic interaction (Zeeman, dipole-dipole, chemical-shift, spin–spin, and quadrupolar) is addressed and manifestations of these interactions in the solid state NMR spectrum are explained. The techniques of high-power decoupling, magic-angle spinning, and cross-polarization, used to acquire highly resolved solid-state NMR spectra, are also illustrated. Application of solid-state NMR to pharmaceutical problem solving and methods development is then briefly reviewed.     

核磁共振法定量影响因素探析

定量核磁共振技术作为一种定量分析方法,具有操作容易,样品制备简单,非破坏性,分析时间短,专属性强,准确度高等优点,可以作为质量平衡定值方法的一种有力补充。定量核磁共振方法有很多种,例如^1H-qNMR,^13C-qNMR,^19F-qNMR和^31P-qNMR等,^1H-qNMR是最广泛使用的。核磁定量结果的影响因素有很多,最主要的干扰因素为定量样品制备,核磁共振实验参数设定,数据处理参数设定等,本文将详细讨论影响核磁定量结果的因素。     

基于核磁共振对盐酸阿比多尔片的定性和定量研究

目的 基于核磁共振对盐酸阿比多尔片进行定性和定量研究。方法 采用盐酸阿比多尔化学位移δ:7.88 ppm处质子峰为定量峰,马来酸化学位移δ:6.33 ppm处为内标峰,在温度为298 K,弛豫延迟时间10 s,采样时间为4.01 s,扫描次数为32次的条件下采集混合物的核磁共振图谱,对定量峰和内标峰积分后计算其含量。比较核磁共振定量法与HPLC的测定结果。结果 盐酸阿比多尔线性范围是10.53~28.09 mg·mL^-1,回收率在99.76%~101.01%,精密度和稳定性良好,方法学专属性较高。核磁共振定量法与HPLC的测定结果基本一致,而核磁共振定量法具有快速、准确的优势。结论 建立的核磁共振定量法可用于盐酸阿比多尔片定性和定量研究,提供了一种新的质量控制方法。     

Assessment of Higher Order Structure Comparability in Therapeutic Proteins Using Nuclear Magnetic Resonance Spectroscopy

In this work, we applied nuclear magnetic resonance (NMR) spectroscopy to rapidly assess higher order structure (HOS) comparability in protein samples. Using a variation of the NMR fingerprinting approach described by Panjwani et al. [2010. J Pharm Sci 99(8):3334–3342], three nonglycosylated proteins spanning a molecular weight range of 6.5–67 kDa were analyzed. A simple statistical method termed easy comparability of HOS by NMR (ECHOS-NMR) was developed. In this method, HOS similarity between two samples is measured via the correlation coefficient derived from linear regression analysis of binned NMR spectra. Applications of this method include HOS comparability assessment during new product development, manufacturing process changes, supplier changes, next-generation products, and the development of biosimilars to name just a few. We foresee ECHOS-NMR becoming a routine technique applied to comparability exercises used to complement data from other analytical techniques.     

1H和19F核磁共振定量法测定塞来昔布含量

目的 分别采用¹H核磁共振定量法和¹⁹F核磁共振定量法测定塞来昔布绝对含量,并将2种方法的测定结果与质量平衡法测定结果进行比较。方法 ¹H核磁共振定量法以CD₃OD为溶剂,塞来昔布δ 6.92处质子峰作为定量峰,马来酸δ 6.33处为内标峰。在弛豫延迟时间10 s,采样时间4.01 s,扫描次数为32条件下采集混合物的氢谱。¹⁹F核磁共振定量法以CD₃OD为溶剂,4-溴-2-氟-乙酰苯胺为内标,在谱宽240 ppm,中心频率-94.26 ppm,样品扫描次数为16次条件下采集氟谱,计算¹H和¹⁹F核磁共振定量法的测试结果。结果 ¹H核磁共振定量法和¹⁹F核磁共振定量法测定结果相近,且与质量平衡法测定结果基本一致。结论 核磁共振定量法可以用来测定塞来昔布含量,具有准确、高效的优势。     

脑内肿瘤诊断中核磁共振波谱的应用价值分析

目的:探究核磁共振波谱在脑内肿瘤诊断中的应用价值。方法:选取某院2016年3月~2018年3月期间收治的74例疑似为脑内肿瘤患者作为对象,分别予以正电子发射型计算机断层显像(PET)-CT诊断及核磁共振波谱诊断,以患者的病理学诊断结果作为"金标准",比较不同检查方法的准确率、敏感度及特意度。结果:经过病理学诊断74例疑似脑内肿瘤患者确诊共52例,占比例为70.27%,应用PET诊断的准确率63.46%低于应用核磁共振波谱诊断准确率96.15%,有统计学差异性(χ~2=17.244,P<0.05);应用核磁共振波谱诊断的敏感度59.62%、特异度86.36%均高于PET诊断38.46%、59.09%,有统计学差异(P<0.05)。结论:脑内肿瘤诊断中应用核磁共振波谱,准确率高,敏感性、特异性好,可以广泛应用。     

1H核磁共振定量法测定氢溴酸右美沙芬片含量

目的采用1H核磁共振定量法(quantitative proton nuclear magnetic resonance,qHNMR)测定氢溴酸右美沙芬片中氢溴酸右美沙芬含量。方法 qHNMR以氢溴酸右美沙芬化学位移δ7.05~7.15 ppm处质子峰为定量峰,苯甲酸苄酯δ5.38ppm处为内标峰,在温度为300 K,采样时间为4.01 s,弛豫延迟时间10 s,扫描次数为32次条件下采集混合物qHNMR图谱。比较qHNMR与HPLC的含量测定结果。结果 qHNMR专属性较高,线性范围5.88~23.08mg·mL^-1,回收率为98.4%~99.7%,定量限为0.45 mg·mL^-1,检测限为0.10 mg·mL^-1。qHNMR含量测定结果与HPLC的测定结果基本一致。结论 qHNMR可用于氢溴酸右美沙芬片中氢溴酸右美沙芬的含量测定。     

核磁共振内标法测定维生素C片中维生素C的含量

目的:建立核磁共振测定维生素C片中维生素C含量的方法。方法:采用Varian unity Inova600MHz核磁共振仪,溶剂为氘代二甲亚砜,内标为苯,测定温度为21℃,观察频率为599.951MHz,谱宽为9599.2Hz,脉冲宽度为11.65μs,采样时间为2s,延迟时间为5s,采样次数为32次,在此条件下采集样品1H-NMR谱。结果:供试片中维生素C含量为40.1%,RSD=1.38%,平均回收率为100.9%。结论:核磁共振内标法测定维生素C片中维生素C的含量,方法简便、准确,可用于维生素C的定量分析。     

Characterization of Aptamer BC 007 Substance and Product Using Circular Dichroism and Nuclear Magnetic Resonance Spectroscopy

Possible unwanted folding of biopharmaceuticals during manufacturing and storage has resulted in analysis schemes compared to small molecules that include bioanalytical characterization besides chemical characterization. Whether bioanalytical characterization is required for nucleotide-based drugs, may be decided on a case-by-case basis. Nucleotide-based pharmaceuticals, if chemically synthesized, occupy an intermediate position between small-molecule drugs and biologics. Here, we tested whether a physicochemical characterization of a nucleotide-based drug substance, BC 007, was adequate, using circular dichroism (CD) spectroscopy. Nuclear magnetic resonance confirmed CD data in one experimental setup. BC 007 forms a quadruplex structure under specific external conditions, which was characterized for its stability and structural appearance also after denaturation using CD and nuclear magnetic resonance. The amount of the free energy (ΔG⁰) involved in quadruplex formation of BC 007 was estimated at +8.7 kJ/mol when dissolved in water and +1.4 kJ/mol in 154 mM NaCl, indicating structural instability under these conditions. However, dissolution of the substance in 5 mM of KCl reduced the ΔG⁰ to ?5.6 kJ/mol due to the stabilizing effect of cations. These results show that positive ΔG⁰ of quadruplex structure formation in water and aqueous NaCl prevents BC 007 from preforming stable 3-dimensional structures, which could potentially affect drug function.     

经阴道彩超联合核磁共振成像在凶险型前置胎盘的应用

目的:探讨经阴道彩超联合核磁共振成像在凶险型前置胎盘植入临床诊断方面的临床价值。方法:选取2014年1月~2016年1月某院收治的疑似凶险型前置胎盘合并胎盘植入患者98例,在妊娠32~34周分别进行经阴道彩超和核磁共振成像检查,比较单纯经阴道彩超、核磁共振成像和两种方法联合诊断的准确性情况。结果:联合诊断在凶险性前置胎盘植入诊断符合率、特异性、漏诊率、误诊率方面较经阴道彩超、核磁共振成像有显著性差异,P<0.05。结论:联合诊断可以有效提高凶险型前置胎盘植入临床诊断的符合率、敏感性、特异性,对于漏诊率、误诊率具有重要临床价值。     

帕金森病患者壳核质子磁共振波谱研究

目的利用质子磁共振波谱(1H MRS)技术研究帕金森病(PD)患者壳核的代谢变化及意义.方法PD患者46例和正常对照30例行1H MRS检查,测定双侧壳核的N-乙酰天门冬氨酸/肌酸(NAA/Cr)、N-乙酰天门冬氨酸/胆碱复合物(NAA/Cho)和胆碱复合物/肌酸(Cho/Cr)比值.结果早期PD患者有症状侧肢体和中晚期PD患者症状严重侧肢体的对侧壳核NAA/Cr比值低于对照组(P＜0.05),未予多巴胺制剂治疗的PD患者症状严重侧肢体的对侧壳核NAA/Cr比值低于对照组(P＜0.05).结论1H MRS检测到壳核的NAA/Cr比值降低,有助于PD以及早期PD的诊断;壳核NAA/Cr比值受多巴胺制剂治疗的影响,NAA/Cr可作为纹状体神经元功能异常的可逆性标志.     

Advances in the Specificity of Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Based Structural Characterisation Methods for Synthetic Oligonucleotides

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (¹H) and carbon (¹³C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences.     

Characterization of Cromolyn Sodium Hydrates and Its Formulation by 23Na-Multiquantum and Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy

We characterized cromolyn sodium (CS) hydrates and evaluated their molecular states in low-dose formulations using Na-multiquantum magic-angle spinning (MQMAS) nuclear magnetic resonance (NMR) analysis. Two CS hydrates, low-water-content hydrated form and high-water-content hydrated form containing 2–3 and 5–6 hydrates, respectively, were prepared by humidification. Single-crystal X-ray diffraction and powder X-ray diffraction analysis revealed that these CS hydrates contained sodium channel structures and that water molecules were adsorbed on the sodium nucleus. ¹³C-cross-polarization/MAS NMR spectra of these hydrates revealed similar results, confirming that the water molecules were adsorbed not on the cromolyn skeletons but mainly on the sodium nucleus. In contrast, ²³Na-MQMAS NMR analysis allowed us to clearly distinguish these hydrates without discernible effects from quadrupolar interaction. Thus, MQMAS NMR analysis is a valuable tool for evaluating salt drugs and their formulations. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2738–2747, 2013     

Evaluation of Liver Function by Co-administration Methodology Using 13C-Labelled Benzoic Acid and Hippuric Acid Coupled with Nuclear Magnetic Resonance Spectroscopy

The amount of hippuric acid synthesized and excreted in the urine after benzoic acid loading (hippuric acid test) is a useful index of liver function. However, the hippuric acid test gives erroneous results in the event of failure of renal excretory function. A new stable isotope co-administration methodology using nuclear magnetic resonance (NMR) spectroscopy has been developed to overcome this defect. [7-¹³C]Benzoic acid and [glycine carbonyl-¹³C]hippuric acid ([gly-¹³C]hippuric acid), each 0.4–0.6 mmol kg^?1 were simultaneously administered intravenously as probes to normal or liver-injured rats and the urine was analysed by 100 MHz ¹³C NMR spectroscopy. Consequently, urinary excretion of [7-¹³C]hippuric acid formed from [7-¹³C]benzoic acid and [gly-¹³C]hippuric acid was successfully traced with very simple and convenient procedures. The urinary excretion of [7-¹³C]hippuric acid indicated the combined functions of hippuric acid synthesis and renal excretion, whereas that of [gly-¹³C]hippuric acid was indicative of renal excretion of hippuric acid only. The heights of resonances for C7 of [7-¹³C]hippuric acid and the glycine carbonyl carbon of [gly-¹³C]hippuric acid were used to calculate the concentrations of labelled hippuric acids. [7-¹³C]Hippuric acid was excreted more slowly than [gly-¹³C]hippuric acid by both normal and liver-injured rats. The liver-injured rats excreted the labelled hippuric acids more slowly than the normal rats. The kinetic parameters were computed for the individual rats on the basis of Michaelis-Menten elimination for benzoic acid and first-order elimination for hippuric acid. The maximum rates of metabolism (V_max) (4.8–5.8 μmol min^?1 kg^?1) and the renal elimination rate constants of hippuric acid (K_re) (0.010–0.021 min^?1) in the liver-injured rats were lower than those (V_max 6.7–11.8 μmol min^?1 kg^?1; K_re 0.026–0.045 min^?1) in the normal rats. These results have demonstrated that liver function can be evaluated from the V_max value even though the renal function of hippuric acid excretion (K_re) is impaired. Thus the co-administration methodology is feasible and can remove the defect of the previous hippuric acid test. These results could form the basis for a more convenient and reliable hippuric acid test in man.     

Evaluation of Lipopeptide (Daptomycin) Aggregation Using Fluorescence,Light Scattering,and Nuclear Magnetic Resonance Spectroscopy

The aggregation behavior and critical aggregation concentration (CAC) values of daptomycin in aqueous solutions were evaluated under the external factors of pH, temperature, daptomycin concentration, and calcium ions concentration by using the complementary characterization techniques, fluorescence, dynamic and static light scattering, and nuclear magnetic resonance (NMR) spectroscopy. On the basis of the intrinsic fluorescence resonance energy transfer of daptomycin, the CAC values were identified by an upward inflection of the fluorescence emission from Kyn-13 at 460 nm. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM at pH 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy.     

Insights into Nano- and Micron-Scale Phase Separation in Amorphous Solid Dispersions Using Fluorescence-Based Techniques in Combination with Solid State Nuclear Magnetic Resonance Spectroscopy

Purpose

Miscibility between the drug and the polymer in an amorphous solid dispersion (ASD) is considered to be one of the most important factors impacting the solid state stability and dissolution performance of the active pharmaceutical ingredient (API). The research described herein utilizes emerging fluorescence-based methodologies to probe (im)miscibility of itraconazole (ITZ)-hydroxypropyl methylcellulose (HPMC) ASDs.

Methods

The ASDs were prepared by solvent evaporation with varying evaporation rates and were characterized by steady-state fluorescence spectroscopy, confocal imaging, differential scanning calorimetry (DSC), and solid state nuclear magnetic resonance (ssNMR) spectroscopy.

Results

The size of the phase separated domains for the ITZ-HPMC ASDs was affected by the solvent evaporation rate. Smaller domains (<10 nm) were observed in spray-dried ASDs, whereas larger domains (>30 nm) were found in ASDs prepared using slower evaporation rates. Confocal imaging provided visual confirmation of phase separation along with chemical specificity, achieved by selectively staining drug-rich and polymer-rich phases. ssNMR confirmed the results of fluorescence-based techniques and provided information on the size of phase separated domains.

Conclusions

The fluorescence-based methodologies proved to be sensitive and rapid in detecting phase separation, even at the nanoscale, in the ITZ-HPMC ASDs. Fluorescence-based methods thus show promise for miscibility evaluation of spray-dried ASDs.