药学学报

Object	Objective	Instrument	Pulse sequence	Publishing time
Orlistat in tablets^[3]	Study a qNMR method to quantify orlistat in tablets	500 MHz, CryoProbe	¹H NMR	2017
Dipotassium glycyrrhizinat^[4]	Establish a quantitative method based on ¹H qNMR and develop for assessing the purity of dipotassium glycyrrhizinate	500 MHz, CryoProbe	¹H NMR	2021
Organic calibration standards^[5]	Perform qNMR analysis in conjunction with the mass balance to afford greater confidence in the purity assessment of organic calibration standards	400 or 600 MHz, BBFO probes	¹H NMR	2015
PE, PET, and PS^[6]	Find solvents suitable for quantitative analysis of MP particles and to validate the calibration curve method for the quantitative analysis of MP particles by qNMR	500 MHz, TH ATM probe	¹H NMR	2019
DNT^[7]	Quantify DNT belonging to the third-generation neonicotinoid pesticides, which are among the most common residuals in a variety of food commodities	800 MHz, CPQCI CryoProbe	¹H NMR	2023
Danshen injection^[8]	Establish a comprehensive method for quantitative determination of complex ingredients in TCM injections	600 MHz	¹H NMR	2022
Futicasone propionate and azelastine hydro-chloride in nasal spray formulation^[9]	Conduct quality control on the dosage forms	400 MHz	¹H NMR	2021
Pomegranate seed oil^[10]	Assess the concentration of conjugated fatty acids for the identification of pomegranate seed oil	600 MHz, ONE NMR probe	¹H NMR	2022
Cannabinoids^[11]	Characterize and determine the main non-psychoactive cannabinoids in eight different hemp varieties	600 MHz	¹³C NMR	2019
Choline^[14]	Determine choline in commercial matrices and additives	500 MHz, TBO-LF probe	¹⁴N NMR	2021
Difluprednate^[15]	Reveal drug multiphase distribution of oil-in-water nanoemulsion, and provide reference for drug development and quality monitoring	600 MHz, liquid nitrogen-cooled prodigy TCI-F probe	¹⁹F NMR	2022
Fluorinated NPS^[16]	Establish NMR methods to quantify 11 types of fluorinated NPS	400 MHz	¹⁹F NMR	2022
Cyclophosphamide hydrate^[18]	Determine the purity of cyclophosphamide hydrate	600, 500, and 400 MHz	¹H NMR, ³¹P NMR	2021
Organophosphorus compound, sofosbuvir^[19]	Quantitative analysis of organic compounds containing ³¹P	600 MHz, CryoProbe; 500 and 400 MHz, normal probes	³¹P NMR	2022
Phytocannabinoids^[20]	Develop a quantitation method for cannabinoids	400 MHz	¹H NMR, COSY	2022
11-α-Hydroxymo-grosides^[21]	Achieve quality control of luo han guo fruits and extracts	400 MHz, PABBO broad-band probe	COSY, Bs-HSQC	2021
Heparin^[24]	Profile the substitution patterns of K5-PS derivatives	500 MHz, TXI probe	HSQC	2005
Epoxide formation in oil and mayonnaise^[26]	Assess the formation of hydroperoxides, aldehydes, and epoxides under accelerated shelf-life conditions	600 MHz, CryoProbe	HSQC	2022
Diester-type C19-diterpenoid alkaloids^[27]	Establish a fast 2D HSQC qNMR method with high efficiency and accuracy	600 MHz, CP21 BBO 600S3 BB-H & F-D-05 Z XT CryoProbe	HSQC	2023
Levofloxacin^[36]	Obtain the relative and absolute content of enantiomers in levofloxacin cream	400 MHz, BBFO probe	J-Compensated Q-HSQC	2020
Saccharides^[40]	Assess pulmonary deposition in impaction experiments of saccharides employed as carriers in dry powder inhaler formulations to select the appropriate carriers	600 MHz, normal probe	¹H NMR	2019
Diterpene acids^[41]	Quantify diterpenoid acids to address the issue of inaccuracy in the quantification of diterpenoid acids with weak UV absorption based on chromatography with UV detector	300 MHz, PABBO broad band probe	HSQC	2018
Anthraquinones^[42]	Select duroquinone and rutin with similar molecular characteristics to the target compounds as alternative standards for quantification, to address the dependence of chromatographic techniques on standards	400 MHz, PABBO broad band probe	HSQC	2019
Alkaloids^[43]	Explore the complemen-tarity of qNMR methods by combining ¹H NMR and 2D Q-QUIPU HSQC	700 MHz, normal probe	¹H NMR, Q-QUIPU HSQC	2019
Cycloartane triterpenes^[44]	Solve the adulteration problem of Actaea racemosad in the market	600 MHz, TXI CryoProbe	¹H NMR	2020
Minor components in mango juice^[45]	Explore the quantitative characteristics of band-selective excitation	500 MHz	Band-selective excitation ¹H NMR	2017
6 primary metabolites in pomegranate juice^[46]	Evaluate the quality problems such as adulteration	700 MHz, TXO CryoProbe	CPMG, ZG, QEC-HSQC	2020
Isomaltulose^[47]	Establish a NMR method to quantify isomaltulose, other monosaccharides and disaccharides in food within a short time	500 MHz, Prodigy CryoProbe	HSQC combined with 50% NUS	2022
Juice, wine, honey, and olive oil	Utilize complex statistical models to detect origin authenticity, production process control, false labeling, sample similarity, and species purity	400 MHz	Completely automated Bruker FoodScreener™
Metabolic and lipo-protein of COVID-19 patients^[48]	Analyze the metabolic status of COVID-19 patients	600 MHz, TXI probe	Bruker IVDr	2021
Bovine liver extract^[34]	Quantify metabolites in bovine liver extract	700 MHz, QCI probe	Constant-time gsHSQC0	2011
Thiocoraline in an extract from Verrucosispora sp.^[49]	Quantify micromolar natural product in complex extracts	700 MHz, QCI probe	Phase-cycled HSQC0, non-constant-time gsHSQC0	2011
Structural units of lignin^{[32, 50, 51]}	Analyze the degree of polymerization and branching of lignin, and evaluate its structure	600 MHz, triple resonance z-gradient probe^[32], CryoProbe^{[52, 53]}	Q HSQC, QQ HSQC, ³¹P NMR	2003, 2011, 2011.
Vitamin D2 and D3^[52]	Analyze the micellization degree of vitamin D in cream to optimize the formula	600 MHz FT-NMR	¹H NMR	2020
GlcNAc and (GlcNAc)₂^[53]	Analyze the decomposition activity of chitinase and screen the best conditions for enzyme activity	500 MHz	¹H NMR	2011

Object	Objective	Instrument	Pulse sequence	Publishing time
Orlistat in tablets^[3]	Study a qNMR method to quantify orlistat in tablets	500 MHz, CryoProbe	¹H NMR	2017
Dipotassium glycyrrhizinat^[4]	Establish a quantitative method based on ¹H qNMR and develop for assessing the purity of dipotassium glycyrrhizinate	500 MHz, CryoProbe	¹H NMR	2021
Organic calibration standards^[5]	Perform qNMR analysis in conjunction with the mass balance to afford greater confidence in the purity assessment of organic calibration standards	400 or 600 MHz, BBFO probes	¹H NMR	2015
PE, PET, and PS^[6]	Find solvents suitable for quantitative analysis of MP particles and to validate the calibration curve method for the quantitative analysis of MP particles by qNMR	500 MHz, TH ATM probe	¹H NMR	2019
DNT^[7]	Quantify DNT belonging to the third-generation neonicotinoid pesticides, which are among the most common residuals in a variety of food commodities	800 MHz, CPQCI CryoProbe	¹H NMR	2023
Danshen injection^[8]	Establish a comprehensive method for quantitative determination of complex ingredients in TCM injections	600 MHz	¹H NMR	2022
Futicasone propionate and azelastine hydro-chloride in nasal spray formulation^[9]	Conduct quality control on the dosage forms	400 MHz	¹H NMR	2021
Pomegranate seed oil^[10]	Assess the concentration of conjugated fatty acids for the identification of pomegranate seed oil	600 MHz, ONE NMR probe	¹H NMR	2022
Cannabinoids^[11]	Characterize and determine the main non-psychoactive cannabinoids in eight different hemp varieties	600 MHz	¹³C NMR	2019
Choline^[14]	Determine choline in commercial matrices and additives	500 MHz, TBO-LF probe	¹⁴N NMR	2021
Difluprednate^[15]	Reveal drug multiphase distribution of oil-in-water nanoemulsion, and provide reference for drug development and quality monitoring	600 MHz, liquid nitrogen-cooled prodigy TCI-F probe	¹⁹F NMR	2022
Fluorinated NPS^[16]	Establish NMR methods to quantify 11 types of fluorinated NPS	400 MHz	¹⁹F NMR	2022
Cyclophosphamide hydrate^[18]	Determine the purity of cyclophosphamide hydrate	600, 500, and 400 MHz	¹H NMR, ³¹P NMR	2021
Organophosphorus compound, sofosbuvir^[19]	Quantitative analysis of organic compounds containing ³¹P	600 MHz, CryoProbe; 500 and 400 MHz, normal probes	³¹P NMR	2022
Phytocannabinoids^[20]	Develop a quantitation method for cannabinoids	400 MHz	¹H NMR, COSY	2022
11-α-Hydroxymo-grosides^[21]	Achieve quality control of luo han guo fruits and extracts	400 MHz, PABBO broad-band probe	COSY, Bs-HSQC	2021
Heparin^[24]	Profile the substitution patterns of K5-PS derivatives	500 MHz, TXI probe	HSQC	2005
Epoxide formation in oil and mayonnaise^[26]	Assess the formation of hydroperoxides, aldehydes, and epoxides under accelerated shelf-life conditions	600 MHz, CryoProbe	HSQC	2022
Diester-type C19-diterpenoid alkaloids^[27]	Establish a fast 2D HSQC qNMR method with high efficiency and accuracy	600 MHz, CP21 BBO 600S3 BB-H & F-D-05 Z XT CryoProbe	HSQC	2023
Levofloxacin^[36]	Obtain the relative and absolute content of enantiomers in levofloxacin cream	400 MHz, BBFO probe	J-Compensated Q-HSQC	2020
Saccharides^[40]	Assess pulmonary deposition in impaction experiments of saccharides employed as carriers in dry powder inhaler formulations to select the appropriate carriers	600 MHz, normal probe	¹H NMR	2019
Diterpene acids^[41]	Quantify diterpenoid acids to address the issue of inaccuracy in the quantification of diterpenoid acids with weak UV absorption based on chromatography with UV detector	300 MHz, PABBO broad band probe	HSQC	2018
Anthraquinones^[42]	Select duroquinone and rutin with similar molecular characteristics to the target compounds as alternative standards for quantification, to address the dependence of chromatographic techniques on standards	400 MHz, PABBO broad band probe	HSQC	2019
Alkaloids^[43]	Explore the complemen-tarity of qNMR methods by combining ¹H NMR and 2D Q-QUIPU HSQC	700 MHz, normal probe	¹H NMR, Q-QUIPU HSQC	2019
Cycloartane triterpenes^[44]	Solve the adulteration problem of Actaea racemosad in the market	600 MHz, TXI CryoProbe	¹H NMR	2020
Minor components in mango juice^[45]	Explore the quantitative characteristics of band-selective excitation	500 MHz	Band-selective excitation ¹H NMR	2017
6 primary metabolites in pomegranate juice^[46]	Evaluate the quality problems such as adulteration	700 MHz, TXO CryoProbe	CPMG, ZG, QEC-HSQC	2020
Isomaltulose^[47]	Establish a NMR method to quantify isomaltulose, other monosaccharides and disaccharides in food within a short time	500 MHz, Prodigy CryoProbe	HSQC combined with 50% NUS	2022
Juice, wine, honey, and olive oil	Utilize complex statistical models to detect origin authenticity, production process control, false labeling, sample similarity, and species purity	400 MHz	Completely automated Bruker FoodScreener™
Metabolic and lipo-protein of COVID-19 patients^[48]	Analyze the metabolic status of COVID-19 patients	600 MHz, TXI probe	Bruker IVDr	2021
Bovine liver extract^[34]	Quantify metabolites in bovine liver extract	700 MHz, QCI probe	Constant-time gsHSQC0	2011
Thiocoraline in an extract from Verrucosispora sp.^[49]	Quantify micromolar natural product in complex extracts	700 MHz, QCI probe	Phase-cycled HSQC0, non-constant-time gsHSQC0	2011
Structural units of lignin^{[32, 50, 51]}	Analyze the degree of polymerization and branching of lignin, and evaluate its structure	600 MHz, triple resonance z-gradient probe^[32], CryoProbe^{[52, 53]}	Q HSQC, QQ HSQC, ³¹P NMR	2003, 2011, 2011.
Vitamin D2 and D3^[52]	Analyze the micellization degree of vitamin D in cream to optimize the formula	600 MHz FT-NMR	¹H NMR	2020
GlcNAc and (GlcNAc)₂^[53]	Analyze the decomposition activity of chitinase and screen the best conditions for enzyme activity	500 MHz	¹H NMR	2011

定量核磁共振技术应用及发展

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郭聪聪 , 王映红 ^*

药学学报 | 综述 2023,58(12): 3599-3607

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药学学报 | 综述 2023, 58(12): 3599-3607

定量核磁共振技术应用及发展

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郭聪聪, 王映红^*

作者信息

中国医学科学院、北京协和医学院药物研究所, 北京 100050

通讯作者:

*王映红, Tel: 86-10-63165217, E-mail: wyh@imm.ac.cn

Application and development of quantitative nuclear magnetic resonance technology

Cong-cong GUO, Ying-hong WANG^*

Affiliations

Institute of Meteria Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2023-12-12 doi: 10.16438/j.0513-4870.2023-0511

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摘要

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核磁共振定量(quantitative nuclear magnetic resonance, qNMR) 技术样品处理简单、重复性强, 在化合物定量方面具有明显优势。近年来逐渐得到应用的二维qNMR分析, 能较好解决复杂体系中不同成分的量化问题, 在医药、食品、化工等领域逐渐得到应用。本文将从qNMR分析方法、影响因素、实验优化、应用领域等多方面对其进行综述, 以促进qNMR技术的广泛有效应用。

关键词

核磁共振 / 定量 / 影响因素 / 实验优化 / 应用

Abstract

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Quantitative nuclear magnetic resonance (qNMR) technology has significant advantages in quantification due to its simple sample processing and high reproducibility. Two-dimensional qNMR analysis, which can solve the quantification problem of different components in complex systems, has gradually been applied in medicine, food, metabonomics, chemical engineering, and other fields. This paper reviews the analysis methods, influencing factors, experimental optimization, application fields, and other aspects of qNMR to promote its wide and effective application.

Key words

nuclear magnetic resonance / quantitation / influencing factor / experimental optimization / application

引用本文

郭聪聪, 王映红. 定量核磁共振技术应用及发展. 药学学报, 2023 , 58 (12) : 3599 -3607 . DOI: 10.16438/j.0513-4870.2023-0511

Cong-cong GUO, Ying-hong WANG. Application and development of quantitative nuclear magnetic resonance technology[J]. Acta Pharmaceutica Sinica, 2023 , 58 (12) : 3599 -3607 . DOI: 10.16438/j.0513-4870.2023-0511

正文

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核磁共振(nuclear magnetic resonance, NMR) 可以提供多种谱图, 对包括大分子化合物在内的物质进行结构确证。Hollis^[1]于1963年首次利用¹H NMR对阿司匹林、非那西汀和咖啡因的混合物进行定量, 将NMR引入定量领域。NMR作为一种定量技术, 具有对标准品依赖程度低, 测试后的样品可回收及可同时分析多个化合物等优势^[2]。定量NMR (quantitative NMR, qNMR) 包括一维(one-dimensional, 1D) 和二维(two-dimensional, 2D) 技术, 1D qNMR操作简便, 应用较早, 也最为普遍。常用的1D qNMR包括¹H^[3-10]、¹³C^{[11, 12]}、¹⁵N^{[13, 14]}、¹⁹F^{[15, 16]}、³¹P^[17-19]等核磁技术。¹H NMR具有高灵敏度, 且氢原子核在化合物中分布广泛, 应用普遍; ¹⁹F、³¹P天然丰度均为100%, 灵敏度高, 可用于分析含有相应原子的化合物; ¹³C和¹⁵N的天然丰度较低, 分别为1.1%和0.37%, 灵敏度低, 多用于分析浓度较大或经¹³C、¹⁵N标记的溶液体系。2D NMR交叉峰由两个原子核的振动频率共同决定, 使重叠信号得以分开, 更有利于复杂混合体系的定量分析。常用于定量的2D NMR包括COSY (homonuclear correlation spectroscopy)^{[20, 21]}、TOCSY (total correlation spectroscopy)^{[22, 23]}、HSQC (heteronuclear single quantum coherence)^[24-27]。其中HSQC为异核单量子相干, 检测核为氢核, 间接维为具有更大谱宽的碳谱, 同时具备¹H NMR的高灵敏度和¹³C NMR的高分辨率, 应用相对较多。NMR技术具有高重复性、样品处理简便的显著优势, 随着高磁场谱仪、超低温探头等核磁技术的发展, NMR的灵敏度得到提高, 在定量领域具有更加可观的应用前景, 因此本文将对1D qNMR与qHSQC的影响因素、应用及研究进展进行论述。

1 qNMR的理论基础

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NMR的研究对象是具有磁矩的原子核, 在静磁场中, 具有磁矩的原子核产生能级裂分, 此时, 对样品施加一个特定频率的电磁波, 原子核发生能级跃迁, 即产生核磁共振。核磁共振信号强度与相应原子核数量成正比, 这形成了核磁共振定量的基础。

qNMR可以使用外标法和内标法进行定量, 其中内标法更为常用, 其计算方法遵循以下公式(1):

(1)$ {W}_{\mathrm{R}}=\frac{\left(\frac{{I}_{\mathrm{R}}}{{n}_{\mathrm{R}}}\right){M}_{\mathrm{R}}}{\left(\frac{{I}_{\mathrm{S}}}{{n}_{\mathrm{S}}}\right){M}_{\mathrm{S}}}{W}_{\mathrm{S}}{m}_{\mathrm{S}}\mathrm{\%} $

其中, R为目标分析物, S为内标, W_R为目标分析物的实际测定值, W_S为内标实际称样量, I为目标信号积分值, M为相对分子质量, m_s%为内标纯度。

2 qNMR实验优化

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基于1D qNMR分析重点在于保障积分准确性, 必须准确考虑影响信号强度的因素, 包括信号峰纯度、采样参数和处理参数, 并对实验条件进行优化。除以上影响1D qNMR信号强度的因素外, 2D qNMR峰体积会受到更多采样参数的影响, 如影响qHSQC信号强度的因素还包括直接碳氢耦合(direct carbon-hydrogen coupling constants, ¹J_CH)、同核耦合(homonuclear coupling constants, J_HH) 及横向弛豫时间(transversal relaxation time, T₂), 同时, 优化qHSQC脉冲序列可在一定程度上补偿以上参数的影响, 提高定量分析的精确度。本文将对1D qNMR和2D qHSQC涉及的参数进行讨论, 对于其他2D qNMR读者可根据自身需求关注相关文献。

2.1 峰纯度

基于NMR的定量分析需保证选择的定量峰不与其他共振信号重叠, 否则会引起定量结果偏大, 可利用2D NMR对峰纯度进行验证, 当出现重叠无法准确定量时, 可通过以下方式进行解决。

2.1.1 更换氘代试剂

溶剂会引起化学位移改变^[28], 因此当定量峰与其他信号重叠时, 可以更换氘代试剂观察信号峰的分离情况, 但必须同时保证样品的溶解性, 必要时可使用以使定量峰和其他信号峰分离的溶剂为主的混合氘代试剂进行溶解。

2.1.2 选择合适的样品浓度

氢和碳的化学位移会随浓度发生变化, 因此可适当调整分析样品的浓度获得高纯度的定量峰。在此过程中, 灵敏度是不可忽视的因素, 为保证定量方法精密度的相对标准偏差(relative standard deviation, RSD) 在1%之内, 对于¹H NMR要求信噪比 > 250, ¹⁹F NMR > 300, ³¹P NMR > 600^[29]。

2.1.3 添加辅助试剂

使用的辅助试剂包括有机手性试剂环糊精及镧系位移试剂铕、镨和镱, 会使化学位移出现更大的变化, 其中镧系试剂会引起信号展宽, 其展宽程度与场强呈现正相关, 因此镧系试剂适于基于低磁场仪器的定量分析^[30]。

2.1.4 选择1D异核NMR或多维NMR

异核1D NMR包括¹³C、¹⁵N、³¹P和¹⁹F, 其中¹³C、¹⁵N的天然丰度较低, 灵敏度低, 适于分析含量较多的样品, 否则需要较长的采样时间, 实验效率较低。与此相比, 2D NMR可在另一维度将重叠信号展开, 且灵敏度高于¹³C和¹⁵N NMR, 在近十年被开发用于定量领域^[31]。

2.2 采样参数

影响核磁信号强度的因素除原子核数量外, 还存在诸多其他因素, 包括弛豫时间、耦合作用、采样时间、脉冲激发位置等, 这些均是实验结果的误差来源。为提高定量准确性, 必须了解各参数对信号强度的影响及其优化方法。

2.2.1 纵向弛豫时间(longitudinal relaxation time, T₁)

qNMR要求一个脉冲激发完成后, 给予足够的弛豫延迟, 保证纵向磁化矢量恢复完全。采用90°激发, 脉冲激发间隔为5T₁时, 纵向磁化矢量可恢复99.3%, 满足qNMR要求。然而, T₁受到很多因素影响, 如: pH值、溶液浓度等, 因此应准确测定不同条件下目标峰的T₁, 保证弛豫延迟≥ 5T_max。此外, 为减少实验时间, 可采用30°激发, 此时弛豫延迟≥ 4T_max, 纵向磁化矢量即可恢复99.5%。

2.2.2 采样时间(acquisition time, AQ)

采样时间要足够长, 保证自由衰减信号(free induction delay, FID) 不被截断, 否则会使信号强度降低。若采样时间结束前, FID未衰减结束, 获得的核磁信号会被扭曲, 强信号根部会出现对称的振荡信号。

2.2.3 偏共振效应

偏共振效应是指所有范围的基团无法被脉冲均匀激发, 因此为减少偏共振效应对信号强度的影响, 采集qNMR时, 应将激发频率设定为化合物的定量峰与内标峰之间的中心频率。

2.2.4 直接碳氢耦合(¹J_CH)

直接碳氢耦合是影响HSQC信号强度的主要因素之一, 二者之间的关系遵从公式(2)。

(2)$ I\propto \mathrm{s}\mathrm{i}{\mathrm{n}}^{2}\left(\pi \mathrm{*}{J}_{\mathrm{t}\mathrm{r}\mathrm{u}\mathrm{e}}\right)/2{J}_{\mathrm{t}\mathrm{u}\mathrm{n}\mathrm{e}} $

其中, I为信号强度, J_true为目标信号峰实际的¹J_CH, J_tune为调制的碳氢耦合常数(在Bruker软件中为采样参数CNST2)。常规HSQC实验中, J_tune通常为145 Hz, 足以使不同基团产生信号, 不出现信号丢失。然而, 利用HSQC进行定量时, 为使内标和定量峰响应一致, 当分析样品为单一物质时, 将J_tune设置为内标峰和定量峰¹J_CH的均值即可, 若期望同时定量分析多个化合物, 则各化合物选择的定量峰应具有相近的¹J_CH, 并将J_tune设置为内标峰和定量峰¹J_CH的均值。

2.2.5 同核耦合(J_HH)

同核耦合会引起信号强度降低, 二者之间的关系遵循公式(3)^[32]。

(3)$ I\propto \mathrm{c}\mathrm{o}{\mathrm{s}}^{2}\left(\pi \Delta {J}_{\mathrm{H}\mathrm{H}\mathrm{i}}\right) $

其中, I为定量峰积分值, Δ = 1/(2¹J_CHtune), J_tune为调制的耦合常数(在Bruker软件中为采样参数CNST2), J_HH为同核耦合常数, 因此定量峰应尽量选择单峰或耦合较少的信号峰。

2.2.6 横向弛豫时间(T₂)

横向弛豫时间与峰强度的关系为I ∝ exp(-2Δ/T₂)^[32]。T₂较大时, 其对信号强度的影响可忽略不计, 因此当目标化合物为小分子时, T₂可以不进行测定。对大分子进行定量, 若定量基团T₂为同一数量级, 其对定量峰产生的影响一致, 此时横向弛豫的影响可不纳入考虑范围; T₂相差较大时, 横向弛豫对信号强度的影响不可忽视, 此时可对T₂进行准确评估, 校准信号强度, 或选择短时脉冲序列, 减少横向磁化矢量的丢失。

2.2.7 改善脉冲序列

与1D qNMR相比, ¹J_CH是影响HSQC信号强度的特异性因素。J_true与J_tune偏差越大, 信号损失越大, 当定量化合物较多, 且定量峰的¹J_CH相差较大时, 基于标准程序的HSQC定量不再适用。此外, HSQC的间接维为具有较大谱宽的碳谱, 偏共振效应对信号强度的影响更加明显, 当目标化合物的定量信号峰分布较分散时, 仅通过设定激发频率的位置无法补偿偏共振效应的影响。因此为提高HSQC定量实用性, 可通过改进脉冲序列补偿¹J_CH和偏共振效应对信号强度的影响。

Hu等^[33]提出相循环HSQC0 (time-zero HSQC), 即: 将基础的HSQC序列模块进行重复, 对不同重复次数(i) 获得的HSQC谱进行积分(A_i), 以重复次数为横坐标, ln (A_i) 为纵坐标, 进行线性回归, 截距即为ln (A₀), 以上方式获得的A₀与化合物浓度线性相关, 可直接用于定量分析。该研究利用此方式对丙氨酸/蛋氨酸/3-羟基丁酸钠混合溶液进行定量, 验证了该方法的准确性。Hu等^[34]进一步提出了gsHSQC0 (gradient-selective HSQC0), 包括恒定时间gsHSQC0和可变gsHSQC0, 其中可变gsHSQC0信号衰减程度相对较低, 更适于测定浓度较低的化合物。然而, 随着重复次数增加, HSQC信号强度会衰减, 当化合物含量较少或仪器设备场强较低时, HSQC3, 甚至HSQC2无法达到定量信噪比。Sette等^[35]利用相循环HSQC0, 并分别利用600 MHz和400 MHz谱仪对HSQC0对场强的耐用性进行探讨, 结果表明场强为400 MHz时, HSQC2和HSQC3信噪比较低, 定量无法实现, 当增加扫描次数, 并与非均匀采样(non-uniform sampling, NUS) 合用时, 定量准确性依旧无法得到保证。因此, 该序列的使用应基于高场强的核磁共振仪。

Wang等^[36]提出J-compensated Q-HSQC, 保证 ¹J_CH = 120~170 Hz具有一致的信号响应。该序列在CPMG INEPT (Carr-Purcell-Meiboom-Gill insensitive nuclei enhanced by polarization transfer) 中使用复合90°射频脉冲, 同时使用XY-16进行相位循环, 对J_CH和J_HH进行调制, 改善脉冲激发的不均匀性; 碳通道INEPT利用TanhTan脉冲取代硬180°反转脉冲, 演化期用Crp60com.4取代硬180°重聚焦脉冲, 减少射频场不均一性和非共振效应; 在演化期引入固定时间法, 对较宽范围的碳碳耦合进行补偿^[37], 减少间接维的线形展宽(相位畸变) 及伪影, 提高谱图分辨率。

上述脉冲序列中, 影响信号强度的因素获得了不同程度的补偿。然而与碳相连的氢的个数对信号强度的影响均存在, 在定量时仍需对交叉峰进行准确归属。Makela等^[38]提出QEC-HSQC (quantitative, equal carbon response HSQC)。此脉冲序列在第一次INEPT后引入重聚焦周期, 在演化期后不引入相应的去聚焦周期, 实现不同类型的碳信号响应一致。另外, 该脉冲序列在INEPT中用180° Shaka-6组合脉冲补偿偏共振效应^[39]; 在梯度场引入短时G2和G7脉冲以清除180°脉冲的缺陷, 引入较长时间G3脉冲以抑制t1噪音。用二甲马钱子碱和金鸡纳啶对脉冲序列进行评估, 结果发现不同类型碳积分值的RSD分别为5.96%和7.08%, 证明了该序列的可行性; 同时利用含有薄荷醇和没食子酸丙酯的混合样品探讨了该序列定量的准确性。

2.3 处理参数

谱图的处理方式也会影响积分强度, 包括傅里叶变换前后的处理。对于傅里叶变换之前窗函数的选择, 较大的LB (line broadening for em) 会增加信噪比, 但同时会引起信号展宽, 因此适于信号分辨率较高的谱图。其次, 充零会在一定程度上增加分辨率, 但充零点数(size of real spectrum, SI) 不能超过采样点数(time domain size, TD) 的两倍。最重要的是积分范围要求是半峰宽的64倍, 保证积分99%的信号强度。

3 qNMR的应用

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qNMR对所有基团均可检测, 可在无对照品的情况下, 同时实现定性和定量分析, 且样品处理简单、可回收, 实验重复性强, 弥补了其他分析技术的不足, 因此在各领域的应用逐渐增加(表 1^{[3-11, 14-16, 18-21, 24, 26, 27, 32, 34, 36, 40-53]})。

3.1 在药物分析方面的应用

美国药典29-NF24中已经公布了几种通过¹H NMR测定的药物, qNMR已成为目前测定原料药及片剂中主药含量的新工具。

Sun等^[3]利用¹H NMR对奥利司他片剂进行含量测定, 该方法定量限为0.014 mg·mL^-1, 平均回收率达99.45%, 测定结果与高效液相色谱(HPLC) 一致。Zhang等^[4]选择富马酸和邻苯二甲酸氢钾作为内标, 利用¹H NMR对自行合成的甘草酸二钾进行纯度鉴定, 所建立方法呈现出良好的准确性、精密度和耐用性, 基于两种内标的平均回收率均大于99%, 分析结果与HPLC-UV一致。Babenko等^[40]分析喷雾制剂中多糖辅料的体外肺部沉积, 以苯甲酸钠为内标, 利用¹H NMR定量分析糖类含量, 对载体颗粒进行筛选, 以选择合适的药物载体, 保证药物输送效率, 为吸入制剂的优化提供参考。

手性化合物分析传统上使用HPLC进行, 其手性柱昂贵, 样品前处理复杂耗时, 相比之下, qNMR具有显著优势, Wang等^[36]利用qHSQC, 分析左氧氟沙星乳膏中对映体含量, 前处理仅需半小时即可完成, 与HPLC相比缩短了6倍; 在乳膏基质¹H NMR中手性中心周围的信号被药物辅料重叠, 因此本研究利用J-compensated Q-HSQC, 使用(R)-1, 1′-联萘-2, 2′-二基磷酸盐与氧氟沙星相互作用, 使H-1a信号分离; 参比信号为与定量峰T₂近似的马钱子碱C-12/H-12交叉峰, 获得乳膏中左氧氟沙星相对含量为60.2%, 绝对定量值为182 mg·g^-1。

分析聚合物中某成分的含量较困难, 通常需采用高尖端且昂贵的技术, 如: X射线光电子光谱、二级离子串联飞行时间质谱、等温滴定量热法等。与此相比, qNMR操作简单、价格适中、可直接检测成分含量。Guerrini等^[24]利用HSQC对硫酸化肝素进行分析, 通过与HPLC结果比较, 证明了HSQC定量肝素的准确性, 进而对硫酸盐K5-PS衍生物进行了充分表征。

色谱技术为中药活性成分传统的分析方法, 然而, 中药材成分复杂, 各化合物具有不同的理化性质, 难以实现多组分同时定量。相比之下qNMR操作简便, 且可以同时对多化合物进行定性定量分析, 成为中药材物质含量测定的有效技术。

Cicek等^[41]利用HSQC对古巴油中8种二萜酸进行定量, 总采样时间为3 h。此外, 将HSQC与75% NUS联合应用^[42], 采样时间缩短, 实现了欧鼠李中蒽醌类化合物的定量分析。Le等^[43]对白毛莨中生物碱进行定量, 利用快速的¹H qNMR定量分析稳定性低的小檗碱, 利用Q QUIPU (quick quantitative perfected and pure shifted) HSQC对氢化小檗碱和黄连碱进行准确定量, 且定量结果与UHPLC-MS和UHPLC-UV一致, 证明了核磁共振联合应用在复杂基质中的定量潜能。

Imai等^[44]利用¹H NMR对Actaea racemosa (AR) 及其近缘物种A. podocarpa (AP)、A. cordifolia (AC) 根状茎和地上部分的70%甲醇提取物中总芳烷三萜含量进行测定。利用COSY确证了δ 0.20~0.45与0.46~0.62信号峰纯度, 考虑到信号分离程度, 选定δ 0.20~0.45外旋H-19信号进行定量, 校准信号为残余DMSO-d₅, 最终获得AR的根状茎和地上部分中总芳烷三萜含量分别为7.2%~19.3%和3.8%~20.8%, AC为13.9%~28.5%和7.5%~8.7%, AP仅含有1.1%~4.0%和2.1%~3.3%。基于此, 有助于解决市场中AR掺假问题。

3.2 在食品分析方面的应用

为扩展qNMR定量动态范围, 实现浓度分布广泛代谢物的准确定量, Ryu等^[45]将选择性激发氢谱和传统氢谱联合应用, 以HMF和TSP-d₄分别作为低场区和高场区定量峰参照物, 对芒果汁中次生物质进行了准确定量。本文利用丙氨酸标准溶液验证了选择性激发氢谱定量准确度, 定量方法选定标准曲线法; 同时代谢物莽草酸在低场区和高场区均有可用于定量的信号, 因此利用两种方法分别对两个区域的信号进行分析, 二者定量结果一致, 进一步证实了选择性激发氢谱标准曲线法定量的准确性。Tang等^[46]对石榴汁中6种初级代谢物进行靶向分析, 以邻苯二甲酸氢钾和二甲基丙二酸为内标, 氯化锰为弛豫试剂, 选择CPMG、ZG和QEC-HSQC对市售石榴汁进行定量分析, 从而对石榴汁掺假等质量问题进行评估。Fels等^[47]利用HSQC标准程序与50% NUS联合应用对食品中糖进行定量分析, 将分析结果与酶法测定、高效离子交换色谱(HPAEC) 及气相色谱(GC) 的分析结果进行对比, 验证所建立方法的准确性, 与传统糖的分析时间高于60 min相比, 本研究所建立的方法仅需27 min即可实现定量。

Bruker的FoodScreener^TM是一个完全自动化的NMR食品分析平台(https://www.bruker.com/en/products-and-solutions/mr/nmr-food-solutions/food-screener.html)。目前含有果汁、葡萄酒、蜂蜜和橄榄油4个模块, 可以提供基于400 MHz的NMR分析和筛选报告。该平台仅需要简单的样品制备即可识别并完全量化化合物, 通过复杂的统计模型可实现产地真实性、生产过程控制、虚假标签、样品相似性和物种纯度的检测, 研究人员仅需将谱图结果传输至Bruker与参考数据库进行比较, 之后便会收到一份易于解释的报告。该平台的使用是全自动化, 且不需要研究人员具备核磁专业知识。

3.3 在代谢组学分析中的应用

生物体液中代谢物的精确定量, 可通过标准代谢物图谱库, 自动对基于标准化程序获得的代谢谱进行精确定量。利用三维互相关(three-dimensional cross correlation, 3DCC) 和反卷积的方式对谱图进行处理^[54], 即: 将液相或质谱获得的信息与NMR信息结合, 从复杂的代谢物核磁谱图中提取出纯化合物的NMR谱图, 使用数学方式进行处理, 获得纯化学位移谱的信号强度。除此之外, 利用某些软件或程序(如Chenomx NMR Suite、COLMARm、Bruker IVDr等) 对谱图进行自动反褶积处理, 即: 将峰值位置和峰强度与谱库中纯化合物的谱图进行匹配, 实现代谢物的定量分析^[55]。其中IVDr为布鲁克生物样本库工具, 还可自动对标准化程序获得的脂蛋白谱进行精确定量, Schmelter等^[48]利用Bruker IVDr工具对新型冠状病毒感染(COVID-19) 患者的代谢和脂蛋白特征进行分析, 将分析结果与健康患者及COVID-19阴性的心源性患者进行比较, 发现与健康患者相比, COVID-19患者表现出严重血脂异常和代谢状态的深度改变。

Hu等^[34]利用常时gsHSQC0对牛肝提取物进行定量, 总采样时间为2 h。在样品中添加DSS和2-吗啉乙磺酸分别作为低浓度和高浓度代谢物定量的内标, 测量浓度范围可达0.1~120 mmol·L^-1, 成功定量了牛肝脏提取物中的23种代谢物。Hu等^[49]还利用相循环HSQC0和非常时gsHSQC0脉冲对Verrucosispora sp.的提取物噻可拉林进行选择性定量, 测定时加入弛豫试剂乙酰丙酮铬, 获得噻可拉林的平均含量为1.0% (w/w), 实现了复杂提取物中微摩尔级天然产物的定量分析, 总采样时间为4 h 15 min。

3.4 在化工类分析中的应用

Heikkinen等^[32]利用Q HSQC对云杉木中磨木木质素结构单元进行绝对定量, 以DMSO为溶剂, 4-(1-羟乙基)-2-甲氧基苯酚为内标, 对β-O-4、β-5、dibenzodioxocin和β-β4种结构单体进行准确定量。Crestini等^[50]采用QQ (quick quantitative) HSQC、³¹P NMR对C-9及不同结构单体进行定量, 评价软木和硬木磨木木质素样品的聚合程度及分支程度, 并对木质素结构进行评估^[51]。

护肤霜基质复杂, 维生素D2、D3的甲基信号峰(δ 0.5) 重叠严重, 因此Robertson等^[52]以δ 6处双峰为定量峰, TSP为内标, 利用¹H NMR分别以MeOD和D₂O为溶剂, 对护肤霜中游离维生素D2、D3进行定量, 分析乳霜中维生素D胶束化程度, 进而对乳膏配方进行优化。

3.5 其他

qNMR在对照品纯度标定中表现出显著优势, 其可以直接对目标化合物进行分析, 特异性较强, 实验过程简便。Davies等^[5]利用¹H NMR对21种标准物质进行分析, 并将结果与质量平衡法进行比较, 多数物质通过两种方法获得的纯度值等价; 而硫酸睾酮含有一种与主成分吸收波长不一致的杂质, 导致其产生不同的响应因子, 利用HPLC-UV进行纯度测定时, 定量结果偏大, 而NMR可以在谱图中区分该成分, 显示出qNMR在该物质纯度鉴定中的优势。

Liu等^[53]利用¹H NMR, N-乙酰基作为靶信号, 应用反褶积技术确定N-乙酰D-葡萄糖胺(GlcNAc) 和N, N′-二乙酰壳二糖(GlcNAc)₂的含量, 同时利用H-α/H-β信号对α/β GlcNAc和β-α/β-β (GlcNAc)₂的相对含量进行确定, 实现了几丁质水解产物实时分析, 基于此可以分析几丁质酶的分解活性, 筛选酶活性的最佳条件, 实现α/β GlcNAc和β-α/β-β (GlcNAc)₂的有效制备。

Peez等^[6]利用¹H NMR定量测定聚乙烯(polyethy-lene, PE)、聚酯(polyethylene terephthalate, PET) 和聚苯乙烯(polystyrene, PS)。以氘代试剂残余质子信号为内标, PE以C₇D₈为溶剂, 以H-1和H-2整体积分进行定量; PET以CDCl₃/TFA 4∶1为溶剂, 定量信号为H-1; PS以CDCl₃为溶剂, 定量峰为芳香环信号; 利用标准曲线法对样品进行定量。该方法的定量限低于环境相关浓度的下限, 精密度均大于99%; 准确度测定值均在相应置信区间, 线性相关系数在0.90~0.99之间, 证明了该方法分析微塑料含量的实用性。与传统分析方法相比, qNMR获得数值为质量浓度, 丰度单位一致, 使研究结果更具有可比性。

4 总结与展望

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本文对qNMR的应用、实验准确性的影响因素及实验方式进行简要综述。与其他定量分析技术相比, qNMR具有对标准品依赖程度低、样品处理简单、重复性强等显著优势。特别是近年来逐渐得到应用的2D qNMR分析, 能较好解决复杂体系中不同成分的量化问题, 在医药、食品、化工等领域应用逐渐增多。当然, qNMR仍然存在不足, 与其他定量方法相比, 其灵敏度相对较低, 但是随着近年来超低温探头^[56]、更高磁场仪器、定量溶解动态核极化^[57]、激光和低温探头辅助核磁共振^[58]等技术的发展, NMR灵敏度和分辨率获得提高。利用HSQC定量分析时, 为达到足够的信噪比和分辨率, 采样时间通常较长, 因此还需将NUS^[59]、VRT (variation of the repetition time)^[60]、光谱折叠^[61]等加速实验采集的技术与之有机结合; 并且, 自动化分析COLMARq^[62]的发展会使其操作可行性增加。

此外, 改进的HSQC脉冲序列并未收录于商业软件中, 因此要求实验人员具备专业的脉冲方面知识, 这大大限制了它的一般应用。尽管如此, 随着NMR技术的不断发展, qNMR的应用前景依然可期。

作者贡献: 郭聪聪负责文献检索、论文撰写及修改; 王映红负责论文的专业性和规范性审阅。

利益冲突: 所有作者声明不存在利益冲突。

基金

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国家科技重大专项(2018ZX09711001-002-004)

参考文献

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文献

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参考文献引证文献

排序方式：

[1]

Hollis DP. Quantitative analysis of aspirin, phenacetin, and caffeine mixtures by nuclear magnetic resonance spectrometry[J]. Anal Chem, 1963, 35: 1682-1684.

https://doi.org/10.1021/ac60204a043

[2]

Singh S, Roy R. The application of absolute quantitative ¹H NMR spectroscopy in drug discovery and development[J]. Expert Opin Drug Discov, 2016, 11: 695-706.

https://doi.org/10.1080/17460441.2016.1189899

[3]

Sun SS, Jin MX, Zhou X, et al. The application of quantitative ¹H-NMR for the determination of orlistat in tablets[J]. Molecules, 2017, 22: 1517.

https://doi.org/10.3390/molecules22091517

[4]

Zhang YY, Zhang J, Zhang WX, et al. Quantitative ¹H nuclear magnetic resonance method for assessing the purity of dipotassium glycyrrhizinate[J]. Molecules, 2021, 26: 3549.

https://doi.org/10.3390/molecules26123549

[5]

Davies SR, Jones K, Goldys A, et al. Purity assessment of organic calibration standards using a combination of quantitative NMR and mass balance[J]. Anal Bioanal Chem, 2015, 407: 3103-3113.

https://doi.org/10.1007/s00216-014-7893-6

[6]

Peez N, Janiska MC, Imhof W. The first application of quantitative ¹H NMR spectroscopy as a simple and fast method of identification and quantification of microplastic particles (PE, PET, and PS)[J]. Anal Bioanal Chem, 2019, 411: 823-833.

https://doi.org/10.1007/s00216-018-1510-z

[7]

Li X, Zhang W, Li X, et al. Purity assessment of dinotefuran using mass balance and quantitative nuclear magnetic resonance[J]. Molecules, 2023, 28: 3884.

https://doi.org/10.3390/molecules28093884

[8]

Li W, Zhao F, Yang J, et al. Development of a comprehensive method based on quantitative ¹H NMR for quality evaluation of traditional Chinese medicine injection: a case study of danshen injection[J]. J Pharm Pharmacol, 2022, 74: 1006-1016.

https://doi.org/10.1093/jpp/rgac034

[9]

El-Masry AA, El-Wasseef DR, Eid M, et al. Quantitative proton nuclear magnetic resonance method for simultaneous analysis of fluticasone propionate and azelastine hydrochloride in nasal spray formulation[J]. R Soc Open Sci, 2021, 8: 210483.

https://doi.org/10.1098/rsos.210483

[10]

Ün İ, Ün ŞŞ, Tanrıkulu N, et al. Assessing the concentration of conjugated fatty acids within pomegranate seed oil using quantitative nuclear magnetic resonance (qNMR)[J]. Phytochem Anal, 2022, 33: 452-459.

https://doi.org/10.1002/pca.3101

[11]

Marchetti L, Brighenti V, Rossi MC, et al. Use of ¹³C-qNMR spectroscopy for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (Hemp)[J]. Molecules, 2019, 24: 1138.

https://doi.org/10.3390/molecules24061138

[12]

Zheng L, Yu P, Zhang Y, et al. Evaluating the bio-application of biomacromolecule of lignin-carbohydrate complexes (LCC) from wheat straw in bone metabolism via ROS scavenging[J]. Int J Biol Macromol, 2021, 176: 13-25.

https://doi.org/10.1016/j.ijbiomac.2021.01.103

[13]

Joubert V, Silvestre V, Lelievre M, et al. Position-specific ¹⁵N isotope analysis in organic molecules: a high-precision ¹⁵N NMR method to determine the intramolecular ¹⁵N isotope composition and fractionation at natural abundance[J]. Magn Reson Chem, 2019, 57: 1136-1142.

https://doi.org/10.1002/mrc.4903

[14]

Ruiz-muelle AB, Moreno PG, Fernandez I. Quantitative quadrupolar NMR (qQNMR) using nitrogen-14 for the determination of choline in complex matrixes[J]. Talanta, 2021, 230: 122344.

https://doi.org/10.1016/j.talanta.2021.122344

[15]

Wang D, Park JH, Zheng J, et al. Multiphase drug distribution and exchange in oil-in-water nanoemulsion revealed by high-resolution ¹⁹F qNMR[J]. Mol Pharm, 2022, 19: 2142-2150.

https://doi.org/10.1021/acs.molpharmaceut.2c00025

[16]

Liu CM, Song CH, Jia W, et al. The application of ¹⁹F NMR spectroscopy for the analysis of fluorinated new psychoactive substances (NPS)[J]. Forensic Sci Int, 2022, 340: 111450.

https://doi.org/10.1016/j.forsciint.2022.111450

[17]

Giummarella N, Balakshin M, Koutaniemi S, et al. Nativity of lignin carbohydrate bonds substantiated by biomimetic synthesis[J]. J Exp Bot, 2019, 70: 5591-5601.

https://doi.org/10.1093/jxb/erz324

[18]

Uchiyama N, Hosoe J, Sugimoto N, et al. Purity determination of cyclophosphamide hydrate by quantitative ³¹P-NMR and method validation[J]. Chem Pharm Bull, 2021, 69: 630-638.

https://doi.org/10.1248/cpb.c21-00109

[19]

Uchiyama N, Kiyota K, Hosoe J, et al. Quantitative ³¹P-NMR for purity determination of sofosbuvir and method validation[J]. Chem Pharm Bull, 2022, 70: 892-900.

https://doi.org/10.1248/cpb.c22-00639

[20]

Dadiotis E, Mitsis V, Melliou E, et al. Direct quantitation of phytocannabinoids by one-dimensional ¹H qNMR and two-dimensional ¹H-¹H COSY qNMR in complex natural mixtures[J]. Molecules, 2022, 27: 2965.

https://doi.org/10.3390/molecules27092965

[21]

Cicek SS, Esposito T, Girreser U. Prediction of the sweetening effect of Siraitia grosvenorii (luo han guo) fruits by two-dimensional quantitative NMR[J]. Food Chem, 2021, 335: 127622.

https://doi.org/10.1016/j.foodchem.2020.127622

[22]

Bingol K, Zhang F, Bruschweiler-Li L, et al. Quantitative analysis of metabolic mixtures by two-dimensional ¹³C constant-time TOCSY NMR spectroscopy[J]. Anal Chem, 2013, 85: 6414-6420.

https://doi.org/10.1021/ac400913m

[23]

Schlippenbach TV, Oefner PJ, Gronwald W. Systematic evaluation of non-uniform sampling parameters in the targeted analysis of urine metabolites by ¹H, ¹H 2D NMR spectroscopy[J]. Sci Rep, 2018, 8: 4249.

https://doi.org/10.1038/s41598-018-22541-0

[24]

Guerrini M, Naggi A, Guglieri S, et al. Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy[J]. Anal Biochem, 2005, 337: 35-47.

https://doi.org/10.1016/j.ab.2004.10.012

[25]

Zhang L, Gellerstedt G. Quantitative 2D HSQC NMR determination of polymer structures by selecting suitable internal standard references[J]. Magn Reson Chem, 2007, 45: 37-45.

https://doi.org/10.1002/mrc.1914

[26]

Boerkamp VJP, Merkx DWH, Wang J, et al. Quantitative assessment of epoxide formation in oil and mayonnaise by ¹H-¹³C HSQC NMR spectroscopy[J]. Food Chem, 2022, 390: 133145.

https://doi.org/10.1016/j.foodchem.2022.133145

[27]

Lin Q, Meng C, Liu J, et al. An optimized two-dimensional quantitative nuclear magnetic resonance strategy for the rapid quantitation of diester-type C19-diterpenoid alkaloids from Aconitum carmichaelii[J]. Anal Chem, 2023, 95: 8452-8460.

https://doi.org/10.1021/acs.analchem.2c05109

[28]

Pauli GF, Kuczkowiak U, Nahrstedt A. Solvent effects in the structure dereplication of caffeoyl quinic acids[J]. Magn Reson Chem, 1999, 37: 827-836.

https://doi.org/10.1002/(SICI)1097-458X(199911)37:11<827::AID-MRC568>3.0.CO;2-W

[29]

Holzgrabe U. Quantitative NMR spectroscopy in pharmaceutical applications[J]. Prog Nucl Magn Reson Spectrosc, 2010, 57: 229-240.

https://doi.org/10.1016/j.pnmrs.2010.05.001

[30]

Casy AF. Chiral discrimination by NMR spectroscopy[J]. Trac-Trend Anal Chem, 1993, 12: 185-189.

https://doi.org/10.1016/0165-9936(93)87021-O

[31]

Giraudeau P. Challenges and perspectives in quantitative NMR[J]. Magn Reson Chem, 2017, 55: 61-69.

https://doi.org/10.1002/mrc.4475

[32]

Heikkinen S, Toikka MM, Karhunen PT, et al. Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: application to wood lignin[J]. J Am Chem Soc, 2003, 125: 4362-4367.

https://doi.org/10.1021/ja029035k

[33]

Hu K, Westler WM, Markley JL. Simultaneous quantification and identification of individual chemicals in metabolite mixtures by two-dimensional extrapolated time-zero ¹H-¹³C HSQC (HSQC(0))[J]. J Am Chem Soc, 2011, 133: 1662-1665.

https://doi.org/10.1021/ja1095304

[34]

Hu K, Ellinger JJ, Chylla RA, et al. Measurement of absolute concentrations of individual compounds in metabolite mixtures by gradient-selective time-zero ¹H-¹³C HSQC with two concentration references and fast maximum likelihood reconstruction analysis[J]. Anal Chem, 2011, 83: 9352-9360.

https://doi.org/10.1021/ac201948f

[35]

Sette M, Lange H, Crestini C. Quantitative HSQC analyses of lignin: a practical comparison[J]. Comput Struct Biotechnol J, 2013, 6: e201303016.

https://doi.org/10.5936/csbj.201303016

[36]

Wang T, Liu Q, Wang M, et al. Quantitative measurement of a chiral drug in a complex matrix: a J-compensated quantitative HSQC NMR method[J]. Anal Chem, 2020, 92: 3636-3642.

https://doi.org/10.1021/acs.analchem.9b04591

[37]

Hallenga K, Lippens GM. A constant-time ¹³C-¹H HSQC with uniform excitation over the complete ¹³C chemical shift range[J]. J Biomol NMR, 1995, 5: 59-66.

https://doi.org/10.1007/BF00227470

[38]

Makela V, Helminen J, Kilpelainen I, et al. Quantitative, equal carbon response HSQC experiment, QEC-HSQC[J]. J Magn Reson, 2016, 271: 34-39.

https://doi.org/10.1016/j.jmr.2016.08.003

[39]

Shaka AJ. Composite pulses for ultra-broadband spin inversion, chemical[J]. Chem Phys Lett, 1985, 120: 201-205.

https://doi.org/10.1016/0009-2614(85)87040-8

[40]

Babenko M, Peron JR, Kaialy W, et al. ¹H NMR quantification of spray dried and spray freeze-dried saccharide carriers in dry powder inhaler formulations[J]. Int J Pharm, 2019, 564: 318-328.

https://doi.org/10.1016/j.ijpharm.2019.03.030

[41]

Cicek SS, Pfeifer barbosa AL, Girreser U. Quantification of diterpene acids in Copaiba oleoresin by UHPLC-ELSD and heteronuclear two-dimensional qNMR[J]. J Pharm Biomed Anal, 2018, 160: 126-134.

https://doi.org/10.1016/j.jpba.2018.07.034

[42]

Cicek SS, Ugolini T, Girreser U. Two-dimensional qNMR of anthraquinones in Frangula alnus (Rhamnus frangula) using surrogate standards and delay time adaption[J]. Anal Chim Acta, 2019, 1081: 131-137.

https://doi.org/10.1016/j.aca.2019.06.046

[43]

Le PM, Milande C, Martineau E, et al. Quantification of natural products in herbal supplements: a combined NMR approach applied on goldenseal[J]. J Pharm Biomed Anal, 2019, 165: 155-161.

https://doi.org/10.1016/j.jpba.2018.11.062

[44]

Imai A, Lankin DC, Godecke T, et al. NMR based quantitation of cycloartane triterpenes in black cohosh extracts[J]. Fitoterapia, 2020, 141: 104467.

https://doi.org/10.1016/j.fitote.2019.104467

[45]

Ryu S, Koda M, Miyakawa T, et al. Quantitation of minor components in mango juice with band-selective excitation NMR spectroscopy[J]. J Agric Food Chem, 2017, 65: 9547-9552.

https://doi.org/10.1021/acs.jafc.7b03336

[46]

Tang F, Hatzakis E. NMR-based analysis of pomegranate juice using untargeted metabolomics coupled with nested and quantitative approaches[J]. Anal Chem, 2020, 92: 11177-11185.

https://doi.org/10.1021/acs.analchem.0c01553

[47]

Fels L, Ruf F, Bunzel M. Quantification of isomaltulose in food products by using heteronuclear single quantum coherence NMR-experiments[J]. Front Nutr, 2022, 9: 928102.

https://doi.org/10.3389/fnut.2022.928102

[48]

Schmelter F, Föh B, Mallagaray A, et al. Metabolic and lipidomic markers differentiate COVID-19 from non-hospitalized and other intensive care patients[J]. Front Mol Biosci, 2021, 8: 737039.

https://doi.org/10.3389/fmolb.2021.737039

[49]

Hu K, Wyche TP, Bugni TS, et al. Selective quantification by 2D HSQC0 spectroscopy of thiocoraline in an extract from a sponge-derived Verrucosispora sp[J]. J Nat Prod, 2011, 74: 2295-2298.

https://doi.org/10.1021/np200503c

[50]

Crestini C, Melone F, Sette M, et al. Milled wood lignin: a linear oligomer[J]. Biomacromolecules, 2011, 12: 3928-3935.

https://doi.org/10.1021/bm200948r

[51]

Sette M, Wechselberger R, Crestini C. Elucidation of lignin structure by quantitative 2D NMR[J]. Chemistry, 2011, 17: 9529-9535.

https://doi.org/10.1002/chem.201003045

[52]

Robertson C, Lucas RA, Le Gresley A. Scope and limitations of nuclear magnetic resonance techniques for characterisation and quantitation of vitamin D in complex mixtures[J]. Skin Res Technol, 2020, 26: 112-120.

https://doi.org/10.1111/srt.12773

[53]

Liu FC, Su CR, Wu TY, et al. Efficient H-NMR quantitation and investigation of N-acetyl-D-glucosamine (GlcNAc) and N, N'- diacetylchitobiose (GlcNAc)(2) from chitin[J]. Int J Mol Sci, 2011, 12: 5828-5843.

https://doi.org/10.3390/ijms12095828

[54]

Wiegandt A, Meyer B. Unambiguous characterization of N-glycans of monoclonal antibody cetuximab by integration of LC-MS/MS and ¹H NMR spectroscopy[J]. Anal Chem, 2014, 86: 4807-4014.

https://doi.org/10.1021/ac404043g

[55]

Lipfert M, Rout MK, Berjanskii M, et al. Automated tools for the analysis of 1D-NMR and 2D-NMR spectra[J]. Methods Mol Biol, 2019, 2037: 429-449.

[56]

Hassan A, Quinn CM, Struppe J, et al. Sensitivity boosts by the CPMAS CryoProbe for challenging biological assemblies[J]. J Magn Reson, 2020, 311: 106680.

https://doi.org/10.1016/j.jmr.2019.106680

[57]

Lerche MH, Karlsson M, Ardenkjaer-larsen JH, et al. Targeted metabolomics with quantitative dissolution dynamic nuclear polarization[J]. Methods Mol Biol, 2019, 2037: 385-393.

[58]

Okuno Y, Mecha MF, Yang H, et al. Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration[J]. Proc Natl Acad Sci U S A, 2019, 116: 11602-11611.

https://doi.org/10.1073/pnas.1820573116

[59]

Zambrello MA, Schuyler AD, Maciejewski MW, et al. Nonuniform sampling in multidimensional NMR for improving spectral sensitivity[J]. Methods, 2018, 138-139: 62-68.

https://doi.org/10.1016/j.ymeth.2018.03.001

[60]

Macura. S. Accelerated multidimensional NMR data acquisition by varying the pulse sequence repetition time[J]. J Am Chem Soc, 2009, 131: 9606-9607.

https://doi.org/10.1021/ja9011063

[61]

Merchak N, Silvestre V, Rouger L, et al. Precise and rapid isotopomic analysis by ¹H-¹³C 2D NMR: application to triacylglycerol matrices[J]. Talanta, 2016, 156-157: 239-244.

https://doi.org/10.1016/j.talanta.2016.05.031

[62]

Li DW, Leggett A, Bruschweiler-Li L, et al. COLMARq: a web server for 2D NMR peak picking and quantitative comparative analysis of cohorts of metabolomics samples[J]. Anal Chem, 2022, 94: 8674-8682.

https://doi.org/10.1021/acs.analchem.2c00891

2023年第58卷第12期

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doi: 10.16438/j.0513-4870.2023-0511

接收时间：2023-04-26
首发时间：2025-11-21
出版时间：2023-12-12

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收稿日期：2023-04-26
修回日期：2023-08-03

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国家科技重大专项(2018ZX09711001-002-004)

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中国医学科学院、北京协和医学院药物研究所, 北京 100050

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*王映红, Tel: 86-10-63165217, E-mail: wyh@imm.ac.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Object	Objective	Instrument	Pulse sequence	Publishing time
Orlistat in tablets^[3]	Study a qNMR method to quantify orlistat in tablets	500 MHz, CryoProbe	¹H NMR	2017
Dipotassium glycyrrhizinat^[4]	Establish a quantitative method based on ¹H qNMR and develop for assessing the purity of dipotassium glycyrrhizinate	500 MHz, CryoProbe	¹H NMR	2021
Organic calibration standards^[5]	Perform qNMR analysis in conjunction with the mass balance to afford greater confidence in the purity assessment of organic calibration standards	400 or 600 MHz, BBFO probes	¹H NMR	2015
PE, PET, and PS^[6]	Find solvents suitable for quantitative analysis of MP particles and to validate the calibration curve method for the quantitative analysis of MP particles by qNMR	500 MHz, TH ATM probe	¹H NMR	2019
DNT^[7]	Quantify DNT belonging to the third-generation neonicotinoid pesticides, which are among the most common residuals in a variety of food commodities	800 MHz, CPQCI CryoProbe	¹H NMR	2023
Danshen injection^[8]	Establish a comprehensive method for quantitative determination of complex ingredients in TCM injections	600 MHz	¹H NMR	2022
Futicasone propionate and azelastine hydro-chloride in nasal spray formulation^[9]	Conduct quality control on the dosage forms	400 MHz	¹H NMR	2021
Pomegranate seed oil^[10]	Assess the concentration of conjugated fatty acids for the identification of pomegranate seed oil	600 MHz, ONE NMR probe	¹H NMR	2022
Cannabinoids^[11]	Characterize and determine the main non-psychoactive cannabinoids in eight different hemp varieties	600 MHz	¹³C NMR	2019
Choline^[14]	Determine choline in commercial matrices and additives	500 MHz, TBO-LF probe	¹⁴N NMR	2021
Difluprednate^[15]	Reveal drug multiphase distribution of oil-in-water nanoemulsion, and provide reference for drug development and quality monitoring	600 MHz, liquid nitrogen-cooled prodigy TCI-F probe	¹⁹F NMR	2022
Fluorinated NPS^[16]	Establish NMR methods to quantify 11 types of fluorinated NPS	400 MHz	¹⁹F NMR	2022
Cyclophosphamide hydrate^[18]	Determine the purity of cyclophosphamide hydrate	600, 500, and 400 MHz	¹H NMR, ³¹P NMR	2021
Organophosphorus compound, sofosbuvir^[19]	Quantitative analysis of organic compounds containing ³¹P	600 MHz, CryoProbe; 500 and 400 MHz, normal probes	³¹P NMR	2022
Phytocannabinoids^[20]	Develop a quantitation method for cannabinoids	400 MHz	¹H NMR, COSY	2022
11-α-Hydroxymo-grosides^[21]	Achieve quality control of luo han guo fruits and extracts	400 MHz, PABBO broad-band probe	COSY, Bs-HSQC	2021
Heparin^[24]	Profile the substitution patterns of K5-PS derivatives	500 MHz, TXI probe	HSQC	2005
Epoxide formation in oil and mayonnaise^[26]	Assess the formation of hydroperoxides, aldehydes, and epoxides under accelerated shelf-life conditions	600 MHz, CryoProbe	HSQC	2022
Diester-type C19-diterpenoid alkaloids^[27]	Establish a fast 2D HSQC qNMR method with high efficiency and accuracy	600 MHz, CP21 BBO 600S3 BB-H & F-D-05 Z XT CryoProbe	HSQC	2023
Levofloxacin^[36]	Obtain the relative and absolute content of enantiomers in levofloxacin cream	400 MHz, BBFO probe	J-Compensated Q-HSQC	2020
Saccharides^[40]	Assess pulmonary deposition in impaction experiments of saccharides employed as carriers in dry powder inhaler formulations to select the appropriate carriers	600 MHz, normal probe	¹H NMR	2019
Diterpene acids^[41]	Quantify diterpenoid acids to address the issue of inaccuracy in the quantification of diterpenoid acids with weak UV absorption based on chromatography with UV detector	300 MHz, PABBO broad band probe	HSQC	2018
Anthraquinones^[42]	Select duroquinone and rutin with similar molecular characteristics to the target compounds as alternative standards for quantification, to address the dependence of chromatographic techniques on standards	400 MHz, PABBO broad band probe	HSQC	2019
Alkaloids^[43]	Explore the complemen-tarity of qNMR methods by combining ¹H NMR and 2D Q-QUIPU HSQC	700 MHz, normal probe	¹H NMR, Q-QUIPU HSQC	2019
Cycloartane triterpenes^[44]	Solve the adulteration problem of Actaea racemosad in the market	600 MHz, TXI CryoProbe	¹H NMR	2020
Minor components in mango juice^[45]	Explore the quantitative characteristics of band-selective excitation	500 MHz	Band-selective excitation ¹H NMR	2017
6 primary metabolites in pomegranate juice^[46]	Evaluate the quality problems such as adulteration	700 MHz, TXO CryoProbe	CPMG, ZG, QEC-HSQC	2020
Isomaltulose^[47]	Establish a NMR method to quantify isomaltulose, other monosaccharides and disaccharides in food within a short time	500 MHz, Prodigy CryoProbe	HSQC combined with 50% NUS	2022
Juice, wine, honey, and olive oil	Utilize complex statistical models to detect origin authenticity, production process control, false labeling, sample similarity, and species purity	400 MHz	Completely automated Bruker FoodScreener™
Metabolic and lipo-protein of COVID-19 patients^[48]	Analyze the metabolic status of COVID-19 patients	600 MHz, TXI probe	Bruker IVDr	2021
Bovine liver extract^[34]	Quantify metabolites in bovine liver extract	700 MHz, QCI probe	Constant-time gsHSQC0	2011
Thiocoraline in an extract from Verrucosispora sp.^[49]	Quantify micromolar natural product in complex extracts	700 MHz, QCI probe	Phase-cycled HSQC0, non-constant-time gsHSQC0	2011
Structural units of lignin^{[32, 50, 51]}	Analyze the degree of polymerization and branching of lignin, and evaluate its structure	600 MHz, triple resonance z-gradient probe^[32], CryoProbe^{[52, 53]}	Q HSQC, QQ HSQC, ³¹P NMR	2003, 2011, 2011.
Vitamin D2 and D3^[52]	Analyze the micellization degree of vitamin D in cream to optimize the formula	600 MHz FT-NMR	¹H NMR	2020
GlcNAc and (GlcNAc)₂^[53]	Analyze the decomposition activity of chitinase and screen the best conditions for enzyme activity	500 MHz	¹H NMR	2011

Object

Objective

Instrument

Pulse sequence

Publishing time

Orlistat in tablets^[3]

Study a qNMR method to quantify orlistat in tablets

500 MHz, CryoProbe

¹H NMR

2017

Dipotassium glycyrrhizinat^[4]

Establish a quantitative method based on ¹H qNMR and develop for assessing the purity of dipotassium glycyrrhizinate

500 MHz, CryoProbe

¹H NMR

2021

Organic calibration standards^[5]

Perform qNMR analysis in conjunction with the mass balance to afford greater confidence in the purity assessment of organic calibration standards

400 or 600 MHz, BBFO probes

¹H NMR

2015

PE, PET, and PS^[6]

Find solvents suitable for quantitative analysis of MP particles and to validate the calibration curve method for the quantitative analysis of MP particles by qNMR

500 MHz, TH ATM probe

¹H NMR

2019

DNT^[7]

Quantify DNT belonging to the third-generation neonicotinoid pesticides, which are among the most common residuals in a variety of food commodities

800 MHz, CPQCI CryoProbe

¹H NMR

2023

Danshen injection^[8]

Establish a comprehensive method for quantitative determination of complex ingredients in TCM injections

600 MHz

¹H NMR

2022

Futicasone propionate and azelastine hydro-chloride in nasal spray formulation^[9]

Conduct quality control on the dosage forms

400 MHz

¹H NMR

2021

Pomegranate seed oil^[10]

Assess the concentration of conjugated fatty acids for the identification of pomegranate seed oil

600 MHz, ONE NMR probe

¹H NMR

2022

Cannabinoids^[11]

Characterize and determine the main non-psychoactive cannabinoids in eight different hemp varieties

600 MHz

¹³C NMR

2019

Choline^[14]

Determine choline in commercial matrices and additives

500 MHz, TBO-LF probe

¹⁴N NMR

2021

Difluprednate^[15]

Reveal drug multiphase distribution of oil-in-water nanoemulsion, and provide reference for drug development and quality monitoring

600 MHz, liquid nitrogen-cooled prodigy TCI-F probe

¹⁹F NMR

2022

Fluorinated NPS^[16]

Establish NMR methods to quantify 11 types of fluorinated NPS

400 MHz

¹⁹F NMR

2022

Cyclophosphamide hydrate^[18]

Determine the purity of cyclophosphamide hydrate

600, 500, and 400 MHz

¹H NMR,
³¹P NMR

2021

Organophosphorus compound, sofosbuvir^[19]

Quantitative analysis of organic compounds containing ³¹P

600 MHz, CryoProbe; 500 and 400 MHz, normal probes

³¹P NMR

2022

Phytocannabinoids^[20]

Develop a quantitation method for cannabinoids

400 MHz

¹H NMR, COSY

2022

11-α-Hydroxymo-grosides^[21]

Achieve quality control of luo han guo fruits and extracts

400 MHz, PABBO broad-band probe

COSY,
Bs-HSQC

2021

Heparin^[24]

Profile the substitution patterns of K5-PS derivatives

500 MHz, TXI probe

HSQC

2005

Epoxide formation in oil and mayonnaise^[26]

Assess the formation of hydroperoxides, aldehydes, and epoxides under accelerated shelf-life conditions

600 MHz, CryoProbe

HSQC

2022

Diester-type C19-diterpenoid alkaloids^[27]

Establish a fast 2D HSQC qNMR method with high efficiency and accuracy

600 MHz, CP21 BBO 600S3 BB-H & F-D-05 Z XT CryoProbe

HSQC

2023

Levofloxacin^[36]

Obtain the relative and absolute content of enantiomers in levofloxacin cream

400 MHz, BBFO probe

J-Compensated Q-HSQC

2020

Saccharides^[40]

Assess pulmonary deposition in impaction experiments of saccharides employed as carriers in dry powder inhaler formulations to select the appropriate carriers

600 MHz, normal probe

¹H NMR

2019

Diterpene acids^[41]

Quantify diterpenoid acids to address the issue of inaccuracy in the quantification of diterpenoid acids with weak UV absorption based on chromatography with UV detector

300 MHz, PABBO broad band probe

HSQC

2018

Anthraquinones^[42]

Select duroquinone and rutin with similar molecular characteristics to the target compounds as alternative standards for quantification, to address the dependence of chromatographic techniques on standards

400 MHz,
PABBO broad band probe

HSQC

2019

Alkaloids^[43]

Explore the complemen-tarity of qNMR methods by combining ¹H NMR and 2D Q-QUIPU HSQC

700 MHz, normal probe

¹H NMR,
Q-QUIPU HSQC

2019

Cycloartane triterpenes^[44]

Solve the adulteration problem of Actaea racemosad in the market

600 MHz, TXI CryoProbe

¹H NMR

2020

Minor components in mango juice^[45]

Explore the quantitative characteristics of band-selective excitation

500 MHz

Band-selective excitation ¹H NMR

2017

6 primary metabolites in pomegranate juice^[46]

Evaluate the quality problems such as adulteration

700 MHz, TXO CryoProbe

CPMG,
ZG,
QEC-HSQC

2020

Isomaltulose^[47]

Establish a NMR method to quantify isomaltulose, other monosaccharides and disaccharides in food within a short time

500 MHz, Prodigy CryoProbe

HSQC combined with 50% NUS

2022

Juice, wine, honey, and olive oil

Utilize complex statistical models to detect origin authenticity, production process control, false labeling, sample similarity, and species purity

400 MHz

Completely automated Bruker FoodScreener™

Metabolic and lipo-protein of COVID-19 patients^[48]

Analyze the metabolic status of COVID-19 patients

600 MHz, TXI probe

Bruker IVDr

2021

Bovine liver extract^[34]

Quantify metabolites in bovine liver extract

700 MHz, QCI probe

Constant-time gsHSQC0

2011

Thiocoraline in an extract from Verrucosispora sp.^[49]

Quantify micromolar natural product in complex extracts

700 MHz, QCI probe

Phase-cycled HSQC0,
non-constant-time gsHSQC0

2011

Structural units of lignin^{[32, 50, 51]}

Analyze the degree of polymerization and branching of lignin, and evaluate its structure

600 MHz, triple resonance z-gradient probe^[32], CryoProbe^{[52, 53]}

Q HSQC,
QQ HSQC,
³¹P NMR

2003,
2011,
2011.

Vitamin D2 and D3^[52]

Analyze the micellization degree of vitamin D in cream to optimize the formula

600 MHz FT-NMR

¹H NMR

2020

GlcNAc and (GlcNAc)₂^[53]

Analyze the decomposition activity of chitinase and screen the best conditions for enzyme activity

500 MHz

¹H NMR

2011