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Quantitative NMR (qNMR) has been around for a long time, but also has great potential to solve future problems in any quantitative analysis. As a primary method, it differs fundamentally from chromatographic methods: it is better described as a quantum mechanical balance. Successful implementation of qNMR requires certain attention to detail. 'qNMR - the handbook' is intended to be a guide for analysts to help understand the fundamental principles of NMR and the significant points relating to its implementation for quantitation. Regulatory considerations of qNMR adoption are explained. NMR fundamentals are explained to provide understanding. Together with many useful examples, the book is a compelling addition to the laboratory's reference library, providing all the tools that any practitioner should know to successfully implement qNMR. The authors are qNMR pioneers and come from a variety of backgrounds including business, government, and academia.
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DEDICATION
In memory of
1967 - 2023
He dedicated himself to advancing the science
he loved, and it benefits others.
and is deeply missed.
CONTENTS
1. F
OREWORD
1.1.
Aesthetics, semantics, and holistics
1.2.
Metalanguages
1.3.
Purpose
1.4.
How to use the book
2. I
NTRODUCTION
2.1.
Going quantitative
2.2.
Other Quantitative Techniques
2.3.
The Growing Importance of qNMR
2.4.
Representative Examples of qNMR Applications
2.4.1.
Pharmaceutical drug quality assessment
2.4.2.
Natural products and food chemistry
2.4.3.
Metabolomics
2.4.4.
High-resolution NMR spectroscopy of technical samples and mixtures
2.4.5.
Forensics
2.4.6.
Nuclear surrogacy
2.5.
Pros and cons of qNMR
3. B
ASICS OF
NMR
SPECTROSCOPY
3.1
.
Solution NMR spectroscopy as your method of choice
3.2.
Spectral semantics
3.3.
Chemical Shift and Multiplets
3.4.
J- coupling
3.4.1.
Homonuclear coupling
3.4.2.
Heteronuclear coupling
3.5.
Excitation Profile and Relaxation
3.6.
Processing
3.7.
Line shape
3.8.
Influence of magnet field strength
3.9.
Concentration proportionality
3.10.
Quantification of heteronuclei
4. C
ONCEPTS OF Q
NMR
4.1.
SCSSRS – The six commandments of qNMR
4.1.1.
Selectivity
4.1.2.
Chemical Inertness
4.1.3.
Solubility
4.1.4.
Stability
4.1.5.
Relaxation
4.1.6.
Sufficient resolution
4.2.
Calculating concentration
4.2.1.
Purity
4.2.2.
100% Method
4.2.3.
Relative Concentration
4.2.4.
Absolute Concentration
4.2.5.
Concentration determination without using an internal standard
4.2.6.
ERETIC and HERETIC
4.2.7.
PULCON/Principle of reciprocity
4.2.8.
Standard addition
4.3.
Metrological considerations
4.3.1.
Metrology and traceability
4.3.2.
Uncertainty budget
4.3.3.
Traceability to the SI system
4.4
.
Calibration standards
5. S
AMPLE PREPARATION
5.1.
Before We Start: What do We Actually want to measure by qNMR?
5.2.
Number of replicates
5.3.
How to weigh accurately
5.4.
Environmental factors
5.5.
Operation of the balance
5.6.
Weighing
5.6.1.
Weighing process in pharmacopoeia
5.7.
NMR Sample Tubes
5.8.
Sample homogeneity
5.9.
Ideal solvents
5.9.1.
General
5.9.2.
Solvent physical properties
5.9.3.
Practicalities
5.9.4.
Removing labile Hs – D
2
O wash
5.10.
Solvent effects – examples
5.11.
Calibrants – experimental requirements
5.12.
Ready-to-use solution with calibrants
5.13.
Chemical shift reference material
6. D
ATA ACQUISITION
6.1.
Fundamentals
6.2.
Bulk magnetisation
6.3.
The NMR Spectrometer
6.4.
RF Pulses and the Receiver
6.5.
Pulse flip angle
6.6.
Pulse sequences
6.6.1.
The “pulse-acquire” pulse sequence
6.7.
Excitation band
6.8.
Transmitter offset and sweep width
6.9.
Receiver gain
6.9.1.
Automatic receiver gain optimisation
6.10.
Sample rotation (“spinning”)
6.11
.
Temperature regulation
6.12.
Repetition time
6.13.
Time domain points
6.14.
Number of scans
6.15.
Decoupling
6.15.1.
Broadband
1
H decoupling – q
13
C{
1
H} NMR
6.15.2.
Broadband
13
C decoupling – q
1
H{
13
C} NMR
6.16.
Solvent suppression
6.17.
Determination of T
1
6.18.
Determination of the 90° pulse width
6.19.
Specialist pulse sequences
6.20.
Relaxation reagents
6.21.
qNMR using unusual nuclei
7. D
ATA PROCESSING
7.1.
Acquiring the NMR spectrum using the spectrometer
7.2.
The NMR Signal
7.3.
Poor peak line shape - consequences and causes
7.3.1.
The sample
7.3.2.
2
H lock
7.3.3.
Poor sample shimming
7.4.
Spectrum size (data point density) and acquisition time
7.5.
Spectral artifacts
7.6.
FID truncation
7.7.
Fourier transform (FT)
7.8.
Critical processing steps before FFT
7.8.1.
Apodisation
7.8.2.
Line broadening
7.8.3.
Combined weighting functions
7.8.4.
Apodisation functions that must be strictly avoided for qNMR
7.8.5.
Zero filling
7.9.
Critical processing steps after FFT
7.10.
Phase correction
7.10.1.
Automatic phase correction
7.10.2.
Magnitude calculation and power spectrum
7.11
.
Baseline correction
7.11.1.
Partial baseline correction (region)
8. D
ATA ANALYSIS
8.1.
Standard integration
8.1.1.
Practicalities of sum integration
8.1.2.
Integration: slope and bias
8.2.
Model-based qNMR - general
8.3.
Cases where peak fitting may be advised
8.4.
Presenting deconvolution results
8.5.
Line fitting in the frequency domain
8.5.1.
Line functions
8.5.2.
Fitting: baseline and phase considerations
8.5.3.
High-quality, conventional line fitting
8.5.4.
“Quick” line fitting
8.5.5.
Line fitting optimised for qNMR
8.5.6.
“Edited sum” combination integration method
8.6.
Line fitting in the time domain (CRAFT)
8.7.
Quantum Mechanical fitting approaches
8.8.
Suitability tests
9. A
UTOMATED ANALYSIS OF
NMR
SPECTRA
9.1.
Automated NMR analysis of specific matrices
9.2.
Quantification using a reference database
9.3.
Automated qNMR analysis of unknown mixtures
9.4.
Automated integration of 2D NMR spectra
10. Q
UALITY
A
SSURANCE
& V
ALIDATION
10.1.
Special features of qNMR spectroscopy
10.2.
ISO/IEC 17025 – Worldwide Quality Standard for Testing and Calibration Laboratories
10.2.1.
Calibration and test laboratories
10.2.2.
Relation to other standards and nomenclature
10.2.3.
ISO/IEC 17025 ─ General requirements
10.2.4.
ISO/IEC 17025 – Technical requirements
10.2.5.
ISO/IEC 17025 – Process requirements
10.2.6
.
Accreditation
10.2.7.
GxP Specialties
10.3.
ISO 24583:2022 - General requirements for
1
H NMR internal standard
11. R
EGULATIONS FOR PHARMACEUTICAL DRUG FORMULATIONS
11.1.
Impurities in drugs
11.2.
Assay and composition
11.3.
NMR spectroscopy, an option in a regulatory environment?
11.4.
Drugs and excipients controlled by NMR spectroscopy in the PhEur and USP
11.5.
Natural products and the JP and AHP
11.6.
Industrial standards
11.7.
Outlook and opportunities
12. C
ONCLUSIONS
13. A
PPENDIX
13.1.
Abbreviations
13.2.
My first qNMR Analysis
13.3.
Relevant
1
H Acquisition and Processing Parameters
13.4.
Deuterated (
2
H) solvent NMR spectra
13.4.1.
Acetonitrile-d
3
13.4.2.
Benzene-d
6
13.4.3.
Chloroform-d
13.4.4.
Dichloromethane-d
2
13.4.5.
Dimethyl formamide-d
7
13.4.6.
Dimethyl sulphoxide-d
6
13.4.7.
Formic acid d
2
13.4.8.
Methanol-d
4
13.4.9.
Tetrahydrofuran-d
8
13.4.10.
Trifluoroacetic acid-d
13.4.11.
Water (D
2
O)
13.4.12.
Tables of residual solvent shifts
13.4.13.
Residual
1
H water signals in deuterated solvents
13.5.
Common reference compounds and their properties
13.6.
International qNMR activities and communities
13.6.1
.
Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM)
13.6.2.
ValidNMR group of the
P
ractical
A
pplications of
N
MR in
I
ndustry
C
onference
13.6.3.
qNMR-Summits
13.6.4.
qNMR norming activities in ISO/TC 34 food products
14. R
EFERENCES
1
FOREWORD
When I moved from university to an NMR laboratory in the chemical industry at the end of the 1980s, I wanted to apply my experience with quantitative NMR from my graduate studies to real laboratory operations. For this purpose, I had quantitatively evaluated some of the results of the normal NMR spectra and presented them to colleagues from other areas of analysis, mainly chromatography. A dear colleague replied to me as follows:
"An analyst would rather use his colleague's toothbrush than his method."
For more than 30 years I have been successfully applying quantitative NMR spectroscopy in a professional setting, but still have the feeling that not much has changed regarding the above statement. Understandably, there is still a distrust of qNMR on the part of mainstream methodologists from the HPLC laboratories and specialists in elemental analysis, but surprisingly also from within the ranks of NMR spectroscopy itself. Even the term “qNMR” is a pleonasm because NMR always has the potential to be interpreted quantitatively – only the precision is affected by the details of the experiment and analysis. NMR spectroscopists often take for granted important, distinguishing points about NMR that deserve restating: the method is non-destructive, and no physical separation of chemical species is required.
This book is intended to give everyone the opportunity to make friends with qNMR as a reliable method that offers the solution to a variety of problems in pharmaceutical analysis, food analysis, or even environmental analysis or diagnostics. For some of us – by which I mean pioneers and advocates of qNMR – the method clearly is most discussed in the metrology discipline, alongside primary methods such as weighing or coulometry. Through logical considerations and unambiguous experiments, all quantitative measurements can, in principle, be traced back to a uniform standard, e.g., water.
One does not have to solve all analytical quantitative problems using NMR spectroscopy, but a lot can be achieved. Let's turn this statement around: you don't have to solve everything with HPLC or titration, even if you have always done so. Technical and financial efforts must be considered to decide on the right method. In addition, there is always the need to consider the principle of traceability to SI, or to judge the robustness of the method under GxP.
This book is not a collection of methods, but it is intended to lay the foundations for a general acceptance of NMR in the canon of classical organic and inorganic analysis. It will hopefully be an eye-opener for many readers, as well as a blueprint for successful and confident adoption and use of NMR for myriad quantitation tasks.
In April 2023, my mentor, Prof. Dr. Stefan Berger, passed away. He was not only my teacher but also a role model and friend. His practical application of NMR spectroscopy in daily work is accessible at every NMR workstation, notably through his books "100 and 200 Experiments." In the early 80s, he introduced an automated NMR system with sample changer and multiple access points.
Allow me to share an anecdote from the mid-80s. At that time, a complete research group had not yet formed, as two points can only make a straight line. Nevertheless, there was a desire to give a presentation each week in the weekly seminar, alternating each week per person. Stefan Berger spoke about FT NMR and the Ernst equation. Some things are unforgettable, such as his prophecy regarding Richard Ernst: "He will receive the Nobel Prize for this someday!"
Finally, we discussed an unresolved issue concerning isotope effects, going back to the roots. The problem may now be resolved, perhaps only philosophically, but the commitment to continue pursuing such matters in his spirit remains.
In our last conversation, we discussed an unresolved issue related to isotope effects, going back to the roots. The problem may be merely philosophical and now resolved, but the commitment to pursue such matters in his spirit remains his legacy.
Prof. Dr. Bernd Diehl, Spectral Service, July 2023
1.1.AESTHETICS, SEMANTICS, AND HOLISTICS
To establish and apply an analytical method based on aesthetic criteria is not a scientific argument. In the case of NMR spectroscopy, however, we can point out such aspects; this could certainly have the effect that both already active NMR analysts and sceptics from the chromatography guild think outside the box. First, a few philosophical words about semiotics and the linguistic origins of the term “spectroscopy”.
The human cognitive apparatus is limited to our five senses. In fact, these senses are our analytical tools, both from a qualitative and quantitative perspective. Because of the biological and physical limitations of these, our primary analytical instruments, we have discovered or invented tools that extend our limited near vision to everything from the cosmic level to the atomic and below.
The space of the molecules or even of the atoms resist direct observation, and quantum mechanical phenomena are somewhat ‘ghostly’ to us. The term spectroscopy can be split into two parts, the Latin “spectrum” and the Greek “σκοπεῖν” (skopein). Spectrum means “image”, whilst skopein means “to look at”. Spectroscopy is, therefore, “image viewing”. In a more modern sense, the term spectrum is closely related to a rainbow, if not to sociological terms. A more detailed consideration brings us a little closer to the original because spectrum also means “appearance” or “spirit”. In English, “spectral” is still completely connected to this linguistic origin.
Spectroscopy enables our human senses to observe the ghostly world of atoms and molecules that is otherwise hidden from us. Thus, NMR spectroscopy in particular - as the name suggests - penetrates the (atomic) core of matter.
Quantum mechanical phenomena are very closely related to symmetry and asymmetry, and indeed this is directly reflected in NMR spectra. For some, an NMR spectrum is a scraggy mountain range, whilst for others it is a structure of the highest aesthetic.
1.2.METALANGUAGES
The specialist language, and steps taken with qNMR are:
In the first step of an NMR investigation, an order amongst disordered states is created by the applied magnetic field. The second step is to perturb this order by exciting it with electromagnetic energy. It is not this perturbation that is measured in an NMR experiment, but the FID, the energy emitted when the system returns to the undisturbed, resting state. It is somewhat analogous to ringing a bell; the measurement is made by the acoustically decaying noise that we can directly receive with our sense of hearing and that our mind interprets (even without Fourier transformation).
Firstly, the FID provides a holistic picture of what is being inspected. However, it is presented in a form that is not directly readable by humans - a foreign language, so to speak, that requires translation. This translation is a mathematical operation, the Fourier transform, which turns the time domain spectrum into the familiar, human-readable, frequency domain spectrum. If you look closely, you can still see a ghostly spark in the mathematical solution of a Fourier transformation in the real and imaginary spectra.
For non-specialists, an NMR spectrum is also like a foreign language. A further translation step is required to convert this spectrum, a seemingly random mixture of seemingly random lines, into one or more molecular formulas. With these molecular formulas, trained chemists can at least use their language and converse in it. For a person who is not trained in science, of course, chemistry must be translated into words again, even into different real languages. The path of knowledge thus leads one through a multitude of necessary translations from one metalanguage to a higher and different one. Finally, the information reaches the questioner at some point, namely “What is this?” and “How much is it?”
Along the translation chain, some of the original information is inevitably lost at each step. A critical loss of information occurs when the spectrum is translated into a molecular formula. Even die-hard NMR spectroscopists sometimes forget to pass on the quantitative information of an NMR spectrum or even, indeed, that it exists.
More information may be lost in this cascade of metalanguages that were originally part of a holistic NMR experiment. For example, information about molecular dynamics and other interactions of matter may not be described in a non-destructive, contactfree space-time experiment. An NMR spectroscopist observes nature by making it vibrate like a bell without striking it. He does not break matter down into its constituent parts to reconstruct the amount and type of matter from the fragments of a mass spectrum, or separate and isolate individual substances from complex mixtures of such via chromatography.
These fundamental differences must be known and understood because they are the cause of many of the misunderstandings between users of NMR spectroscopy and chromatography.
Quantitative NMR must therefore be validated according to its own unique principles and not simply by adopting standard chromatographic procedures. The canon of necessary validation steps between NMR spectroscopy and chromatography is crucially different. Conversely, one should not demand experiments that are nonsensical for NMR spectroscopy, either out of lack of understanding or pure opportunism.
1.3.PURPOSE
From the start, we wish to define the content of this book and somewhat limit the scope of quantitative NMR described herein. As a primary relative method, virtually any NMR spectrum can also be considered and evaluated from a quantitative point of view - and the steps required that describe a correct and reliable way to perform this is the unswerving focus. Areas of diagnostics, food screeners, metabolomics and similar analytics, partly based on statistical methods such as principal component analyses (PCA), should be mentioned but these will not be discussed here in detail. Likewise, this book does not deal with the rather less successful and infrequently applied coupling of HPLC and NMR in high-throughput quantitative analyses.
In this book we focus on targeted analyses analogous to the classical content determinations of organic or inorganic defined molecules. The qNMR method is to be presented here as a powerful alternative to classical chromatography or titration, the advantages and disadvantages of which can be weighed from the following discussions. We would also like to show in some chapters that the application of qNMR is not limited to small molecules and protons but can, with few restrictions, be extended to all NMR active nuclei and molecules. The only requirement is solubility in a solvent, normally perdeuterated. These are fundamental tenets that apply to all extensions of the method.
The book is titled “qNMR – The Handbook” because we do not seek only to review relevant publications; neither is it a detailed instruction or walkthrough for special analytical procedures in the manner of a cookbook. Rather, it is intended as a guide for analysts to help understand the related, fundamental principles of NMR and the important points relating to its implementation for quantitation.
Any new process must be learned, and we therefore start each chapter with the key learnings for review.
We have drawn on our many years of experience with the qNMR method: NMR fundamentals are combined with practical aspects, and we discuss the regulatory side of the various guidelines, including ICH, ISO, Pharmacopoeias.
1.4.HOW TO USE THE BOOK
People reading this book might come from different scientific backgrounds. Some might be new to the field of NMR spectroscopy, in which case the “Basics” (chapter 3, page →) is a perfect point of reference. Readers who have already had some degree of training in the basic theory and have used NMR spectra for structural elucidation can most likely skip this chapter and continue directly with chapter 4 (p →), which deals with the core concepts of quantification. The same philosophy holds true for other chapters, as we present topics at both basic and detailed levels. The reader can choose the chapters they are interested in. Thus, there is a certain degree of repetition in the introductory part of any given chapter, and we have added cross-references to other chapters.
For people who are interested in a more in-depth discussion of certain aspects, we have added the information about selected publications - either papers or books - where more detailed information can be found. However, the literature is not exhaustively reviewed, and the references should be considered entry points to further, detailed reading.
Since the title “qNMR - the handbook” could equally be “qNMR for Beginners”, we also have added some “key learnings” - a higher-level overview at the beginning of each of the chapters. You should consider these learnings to determine whether you have understood and assimilated the knowledge for qNMR provided in the chapter. If not, you might consider going back to corresponding paragraphs and try to do the experiment again. We have collected all learnings at the end of the book.
Chapters 6-8 describe quite general NMR fundamentals that will interest anyone new to the topic. They stand on their own as an accessible description of key NMR elements. So as not to overburden the reader, a strong reliance is put on simple figures and examples to graphically illustrate key points, rather than long, wordy descriptions. Fuller explanations are always available in books and review articles.
The workflow of a typical qNMR measurement is described in the appendix. If you are beginner to qNMR, it might be good to start with a simple example, such as the quantification of one compound, before continuing with the assessment of a mixture of components and even more complex investigations. We also include data for reference, such as spectra of common deuterated solvents.
We have focused on methodologies that are in common use at the time of writing. We plan to revise the book and will reassess the weight given to these topics and add more as becomes appropriate.
2
INTRODUCTION
history of NMR in general
history of qNMR
most important milestones
sharp rise in the number of publications
qNMR in comparison to alternative techniques
applications in various work areas
After the introduction of quantum mechanics and the experimental proof of electron spin in the Stern-Gerlach experiment, the interest in physics research in the 1930s and 1940s turned to the experimental proof of the nuclear magnetic moment (nuclear spin). These experiments took place against the background of the still underdeveloped high-frequency superconducting magnet technology, using comparatively weak permanent magnets. Early experimental approaches for calorimetric detection of nuclear magnetic resonance were unsuccessful [1]. For the detection of the nuclear magnetic effect, Purcell performed experiments on solid paraffin [2], and Bloch experimented with water [3]. A good overview of the first developments in this field can be found in a review article by Wertz [4].
Instrumental technology was repeatedly revolutionised in leaps and bounds as a result of important technical developments [5]. In 1948, the company Varian was founded [6]. The first commercial NMR devices were built in 1952 [7], and initially exploited the newly introduced sweep technique in which radio frequency was kept constant, and the spectrum was recorded during the continuous change of the magnetic field.
Advancing computer technology and the introduction of the Fast Fourier Transform (FFT) algorithm [8–10] made it possible to introduce the Pulse Fourier Transform technique [11, 12] to record NMR spectra, which is still used almost exclusively today [13]. The method allows repetitive signal acquisitions to be co-added, and the often weak signal-to-noise to be improved. Huge advances followed key technical advancements such as the introduction of high-field cryo magnets, and pulse-field gradient units. Since the mid-1960s, the Overhauser effect [14] has been used as one of the most important experimental phenomena in NMR spectroscopy in the determination of the spatial arrangement of nuclei in a molecule. Before then, the effect was mainly used to increase the sensitivity of weak NMR nuclei in heteronuclear experiments. NMR has an important role to play in the understanding of molecular motion and interactions. In all cases, this understanding is at the atomic level; that is, which atoms of a molecule are involved, rather than a general picture that follows a bulk measurement.
Figure 2-1. A. 90 MHz high-field NMR spectrometer (1976). Note the use of an electromagnet. B. A modern benchtop (compact magnet) NMR Spectrometer (80 MHz)
The use of superconducting cryo magnets began in the 1970s and led to ever higher field strengths and improved magnetic field homogeneities in NMR instruments. This dramatically increased the sensitivity and resolution of NMR devices. Varian introduced the first commercial superconducting spectrometer, the HR-220, in 1962–1964, and the 300 MHz SC-300 followed in 1967. In 1969, the German company Bruker GmbH entered the market with a 270 MHz device. High-field NMR instruments based on superconducting magnets have also been offered by the Japanese manufacturer JEOL since 1973. The field strengths of the NMR magnets currently supplied commercially allow 1H NMR experiments at up to 1.2 GHz (Figure 2-2B), which are mainly used to elucidate protein structures. Such high field strengths are not necessary for quantification purposes. In fact, most NMR instruments that are routinely used for quantitation operate at 300–500 MHz.
Figure 2-2. A. A typical modern high-field NMR instrument installation based on a cryo magnet (500 MHz). B. A state-of-the-art very high-field NMR spectrometer (1.2 GHz). These high-performance systems are very expensive to buy and maintain, rare, and reserved for specialist applications such as protein and DNA/RNA studies. Source: Bruker BioSpin
The notion of co-adding the spectra of a single sample was introduced with the Computer of Average Transients, or CAT. The biggest step forward took place in 1966 when Ernst and Anderson demonstrated the considerable sensitivity advantage enjoyed by acquiring spectra in pulse Fourier transform (FT) mode. This approach persists even today.
Since the 1970s, the software has also seen continuous improvement. First suggested by J. Jeener (1971) [15], two- and multidimensional multi-pulse techniques were implemented for the first time in 1974 by the late Prof. R. R. Ernst [16]. These techniques enable the targeted extraction of information from complex NMR spectra and are in widespread use today. Due to the simultaneous increase in sensitivity, the investigation of less sensitive nuclei also becomes routine. Ernst also developed a technique for broadband decoupling of 13C NMR spectra [17], and received the Nobel Prize in Chemistry in 1991 for this and his work on multidimensional NMR spectroscopy [18]. In the 1980s, the development of the automation of NMR experiments also started.
In the 1990s, many pulse sequences were greatly improved and shortened by the incorporation of short, pulsed field gradient (PFG) elements in pulse sequences [19]. These techniques enable the targeted and precise extraction of the desired information from the spectra in a unique way and have, in turn, contributed to a significant improvement in the quality of spectra and, indeed, the NMR technique in general. Thus, their use is widespread. The coupling of NMR spectroscopy with chromatographic techniques (LC-NMR, SFC-NMR) [20–23] became commercially available in the mid-1990s and broadened the spectrum of analytical methodology in many fields, especially in pharmaceutical research where high throughput is a factor. The introduction of micro-and nano-sample heads (≤ 3mm OD tubes), which provide high-resolution NMR spectra for the smallest sample quantities and with very high signal-to-noise sensitivity, has also contributed greatly to this field of application.
Diagnostic Magnetic Resonance Imaging (MRI) relies on NMR, but has very different apparatus, experiments, and outcomes compared with its “first cousin”, high-resolution NMR. The first magnetic resonance imaging (MRI) examination on a live human patient was performed on July 3, 1977. MRI represented a huge advance in medical diagnostics.
The many pioneers of NMR spectroscopy have greatly advanced the technology with their imaginative ideas [15], often in the face of the scepticism of experts. Today, the development of new NMR hardware continues to be rapid. In addition to continuous improvements in the acquisition and processing software, the signal-to-noise sensitivity of modern probe heads is drastically increased (by a factor of three) by cooling the electronics considerably using liquid nitrogen or helium. This is associated with a reduction in the measurement time by a factor of 12, which makes completely new experiments possible. Flow-through probe heads allow for increased sample throughput in high-throughput NMR for routine structure elucidation in industrial research. All these developments in NMR spectroscopy are reflected in the award of various Nobel prizes over the last 100 years (see Table 2-1).
Table 2-1. Nobel prizes in physics, chemistry, and medicine for NMR spectroscopy. (https://en.wikipedia.org/wiki/List_of_Nobel_laureates)
Year
Laureate(s)
Explanatory statement and discipline of awards
1902
Hendrik Antoon Lorenz (1853–1928, NL)
The investigation of the influence of magnetism upon radiation phenomena (Splitting of spectral lines) (Physics)
1943
Otto Stern (1888–1969, D, USA)
The development of the molecular ray method and the discovery of the magnetic moment of the proton (Physics)
1944
Isidor Isaac Rabi (1898–1988, P, USA)
The resonance method for recording the magnetic properties of atomic nuclei (Physics)
1952
Edward Mills Purcell (1912–1997, USA) & Felix Bloch (1905–1983, CH, USA)
The development of new methods for nuclear magnetic precision measurements and related (application) discoveries; Nuclear resonance spectroscopy (Physics)
1972
John Bardeen (1908–1991, USA) & Leon Neil Cooper (1930, USA)
The development of the Bardeen-Cooper-Schrieffer (BCS) theory of superconductivity (Physics)
1991
Richard Ernst (1933–2021, CH)
The development of the Fourier-Transformation technique for high-resolution NMR spectroscopy (Chemistry)
2002
Kurt Wüthrich (1938, CH)
Method for the elucidation of the 3D structure of biological macromolecules in solution (Chemistry)
2003
Paul Lauterbur (1929–2007, USA) & Sir Peter Mansfield (1933–2017, UK)
Development of magnetic resonance imaging (tomography) (Medicine)
2.1.GOING QUANTITATIVE
As discussed, NMR spectroscopy has mostly been used for chemical structure confirmation and structural elucidation of newly synthesised compounds and structurally complex natural compounds isolated from plants or animals. Using high-resolution NMR instruments, the use of two-dimensional (2D) experiments such as COSY, NOESY, ROESY, HMBC, HSQC, etc., has pushed the boundaries of structural elucidation via NMR spectroscopy. Simultaneously, the quantitative application of NMR spectroscopy (qNMR) has equally been reported from the earliest days of solution NMR and developed into a routine quantitation method, despite this application receiving less attention.
As early as 1963, the first quantitative application of NMR spectroscopy was reported by Jungnickel and Forbes [24]. They stated that “the accuracy of integrated intensities for determining hydrogen types has been demonstrated for a wide variety of chemically different types of hydrogens...”. In the same year, Hollis performed a quantitative analysis of a mixture of aspirin (acetylsalicylic acid), phenacetin, and caffeine by means of 1H NMR spectroscopy [25], extracting mol% values for the components from a single spectrum. Hence, two simple but fundamental quantitative application examples were described in the earliest days of the technique. The observation is not surprising because NMR spectroscopy is, per se, a primary method of measurement. Its operation can be completely described and understood; an uncertainty statement can be written in terms of the SI system [26]. This is based on the fundamental principle that the intensity of an NMR signal, given by the area under the signal, is directly proportional to the number of resonant nuclei that contribute to this signal.
However, “real world”, reproducible quantitative results require detailed protocols to be followed regarding the data acquisition, processing, and evaluation of the spectra. The aim of this book is to present and explain the prerequisites and method protocols for applications that demonstrate the convenience, simplicity, and particular potential of qNMR. Whilst these protocols may seem cumbersome at first, they are quickly adopted as a matter of habit and good practice. qNMR then becomes a very reliable method of first choice.
2.2.OTHER QUANTITATIVE TECHNIQUES
The quantification of a mixture of diverse components of equal or very different content - such as a drug and its impurities - can be easily determined using high performance liquid chromatography (HPLC), and indeed this method is very often considered the gold standard for such. UV detection in HPLC provides very limited structural information but is well suited for quantitation even in the nanomolar range, if necessary. Single wavelength or diode array detectors (DADs) may be used. UV detectors are very common and relatively cheap. For quantitation, conditions that allow the Beer-Lambert Law must be followed, making the detector response linear with concentration. However, the detector response as a function of concentration - molar absorptivity (ε) - is specific to every given species and must be determined using authentic samples. This is the key difference with an NMR-based analysis. With classic NMR, every species in a sample measurement has the same, linear correlation between signal response and concentration - which has huge benefit.
Mass spectrometers (MS) are commonly used as HPLC detectors. These provide useful mass-related information based on the analyte, and possibly that of fragments. There are many ionisation methods and MS instrument types, though they will not be reviewed here. MS detectors have the advantage of being highly sensitive but are far more costly than UV or DAD detectors. An MS detector response that is linear with sample concentration is not necessarily expected. Whilst MS is far from an ideal method for quantitation, it still is in common usage [27]. Steps can be taken to minimise sample- and instrument-related factors that impact on quantitation performance. For example, electrospray ionisation (ESI) is commonly used to improve the performance of MS for quantitation. In the following chapter, the advantages and the field of applications of qNMR are explored in more detail.
2.3.THE GROWING IMPORTANCE OF QNMR
qNMR is now an important pillar in quantification analysis, as attested by the increasing number of publications revealed by Google Scholar when setting the keyword “qNMR” (see
Figure 2-3). While the number of publications for NMR in general has seen steady decline since 2008, qNMR publications have shown ever larger increases.
Figure 2-3. Comparison of the number of publications listed annually in Google Scholar for the keywords "NMR" and "qNMR”
2.4.REPRESENTATIVE EXAMPLES OF QNMR APPLICATIONS
2.4.1.Pharmaceutical drug quality assessment
Synthetic chemists not only look at the NMR spectrum of their new compounds from the structural elucidation point of view, but they also inspect the baseline for impurities and attempt to quantify them, though mostly without following any standard quantification protocol. Consequently, many chemistry and medicinal chemistry journals attempt to determine the purity of new compounds via qNMR (see, e.g., [28, 29]). Along these lines, the content of pharmaceutical drugs and quantification of their impurities is often reported in the literature. A nice example was recently published by Franco et al. for a new antifungal drug candidate [29] whose content was quantified via the ERETIC technique (see section 4.2.6) as a standard. In addition, the method was validated. Even though hundreds of similar examples have been reported and are used in new drug applications to drugs agencies, the quality assessment of drugs makes surprisingly infrequent use of qNMR (see chapter 10). The same is seen for the identification of counterfeit drugs, which may contain an API of low quality, containing many and potentially unknown impurities, or which may even be a different drug to the one purported. In the latter case in particular, NMR spectroscopy outpaces HPLC methods by far because it delivers structural- and quantitative information in a single spectrum, even for mixtures. In addition to the recognition of counterfeit drugs, qNMR can be used for quantitation for the following purposes:
Identification and content determination of drugs and excipient, and characterisation of the composition of polymeric excipients (e.g., amyl nitrate, buserelin, enoxaparin, oxytocin)
Evaluation of the impurities present in pharmaceutical drugs and excipients (e.g., heparin, medronic acid, poloxamer)
Determination of an isomeric composition: ratio of diastereomers and assessment of the enantiomeric excess by means of a chiral additive
Average chain length of fatty alcohols (e.g., Lauromacrogol)
Monitoring the decomposition of a drug
Evaluation of residual solvents present in a drug
The same holds true for synthesised organic compounds. Of note, it goes without saying that NMR spectroscopy is blind to all nuclei which are not measured, e.g., with 1H NMR spectroscopy no inorganic ions can be directly observed. Hence, inorganic counterions, e.g., of pharmaceutical drugs, cannot be seen, but might be assessed using an additional NMR experiment with the corresponding probe, and setting the spectrometer to observe the NMR signal at the correct frequency.
2.4.2.Natural products and food chemistry
The adulteration of food is a long-standing issue that is driven by profit and greed. It is a problem that undoubtedly dates to the times of early human trading, but robust analytical assessment methods have only become available in quite recent times. Amongst the top 10 adulterated foods are organic foods, honey, coffee, olive oil, and fish. Regional authorities are often responsible for food quality and mainly rely on both HPLC/MS and NMR spectrometries to determine important properties such as provenance and content/adulteration. As we show with the representative examples below, qNMR has also become a routine analytical method for food quality assessment.
A detailed analysis of isotope ratios measured from NMR spectra is also in common use in the food industry. Therefore, SNIF-NMR closely examines 13C and 2H NMR spectra for this purpose. A recent review [31] considers its application to wine authenticity testing.
Scientists interested in natural products that originate from the plant or animal kingdom have long made extensive use of simple 1D NMR and 2D NMR spectroscopies because of the structural information afforded. Here, qNMR was and is often used to characterise a complex mixture of a simple plant extract, or related fractions. Prof. G. F. Pauli and his group at the University of Illinois at Chicago (UIC), in particular, have focused on the development of quantitative methods in the last two decades in addition to isolation strategies (for reviews see [32–34]). Those methods are often complementary to applied LC/MS/MS methods.
An extract of a plant, bacterium, or animal can also be characterised without detailed component characterisation using chemometric techniques, such as regression analysis, principal component analysis, or partial least square discriminant analysis, to name just a few. Again, NMR spectra are a perfect data provider. This so-called metabolomics approach - either targeted or non-targeted - makes the interpretation of the data much easier (for a number of examples, see [35, 36] and section 2.4.3). This can even be achieved using both a low-field benchtop instrument and a high-field spectrometer [37].
Numerous applications of qNMR have been described in food chemistry, where the content can also indicate authenticity and quality. 1H NMR spectroscopy of edible oils such as extra-virgin olive oil (Figure 2-4) shows signals that can be attributed to key components and fatty acid types; for example, the percentage of polyunsaturated fatty acids can be easily determined in a single measurement.
Aloe vera extracts (Figure 2-5) are good examples of low-sugar foods. A single 1H NMR spectrum of a sample can afford important information of the natural substance’s makeup and quality. A partial list of these is shown in Table 2-2, below. See sections 4.2.6 and 11.5 for further discussion of A. vera and NMR.
Figure 2-4. 1H NMR spectrum (600 MHz) of extra virgin olive oil mixed with CDCl3. The diagnostic regions used for targeted evaluation of, e.g., polyunsaturated fats are indicated.
Table 2-2. A vera QC attributes and relevant compounds that can be quantified by 1H NMR
QC attribute
Compound(s) that can be quantified
Whole Leaf Markers (WLM)
Iso-citric acid, iso-citric lactone
Preservatives & additives
Citric acid, benzoic acid, sorbic acid, propylene glycol, glycerol, glycine, sucrose
Bacterial degradation indicators
Lactic acid, ethanol, succinic acid, acetic acid, pyruvic acid, fumaric acid, formic acid
Adulterant
Maltodextrin
Freshness indicator/storage stability
Acetylated polymannose
Processing compounds
Maltodextrin
Divalent M
2+
Ca
2+
, Mg
2+
(observed as EDTA complex)
Figure 2-5. 1H NMR spectrum (300 MHz) of an Aloe vera sample. Both endogenous species (black) and added compounds (red) are labelled. Lactic acid content is a marker of freshness, whilst nicotinamide is used as a reference standard (blue)
Other analytical methods rely on a library of authentic samples to build something like a statistical picture of spectral features that indicate food attributes. The so-called Wine-Screener™ [38, 39] can easily detect counterfeit and incorrect processing of wine using non-targeted analysis and has a long-standing tradition in foodstuff examination [40]. The same holds true for the analogous method for fruit juices. More recently, the same approach was applied in honey authentication (FoodScreener™ [38]).
Honey, a high-sugar food, can be directly analysed by 1H NMR for sugars (arabinose, fructose, glucose, etc.), organic acids (formic acid, malic acid, etc.), amino acids (alanine, valine, etc.), and quality parameters (ethanol, 5-hydroxymethylfurfural). Statistical classification methods can be used to determine botanical origin. And finally, NMR is excellent at exposing the widespread practice of honey adulteration.
Regulatory authorities make use of 1H NMR spectroscopy to reveal counterfeits such as the adulteration of honey with corn syrup, and multivariate data analysis methods lead to further detection of botanical origin.
The spectra in Figure 2-6 were obtained from a honey sample that was spiked with minor sugars that are useful to detect product adulteration: isomaltose, palatinose, and raffinose. From the 1D 1H NMR spectra we see that the spectrum is dominated by signals from sugars in the 3ppm to 5 ppm central region – mainly mono- and disaccharides. When the vertical expansion is increased then signals from many more species are revealed. The 2D J-resolved spectrum helps with spectral assignment, and peak deconvolution (Figure 2-6D) allows the peak areas of the important contaminants, and thus their concentration to be determined.
Figure 2-6. 1H NMR spectra of honey (D2O, 400 MHz). A. the dominant peaks in the carbohydrate spectral region. B. Vertical expansion, showing signals from non-carbohydrate species. C. 2D J-resolved spectrum assists with assignments. D. Peak deconvolution of low-concentration sugar peaks. [Spectrum courtesy of Mr J Teipel, CVUA, Germany.]
Coffee can be made from Coffea arabica beans, being the more expensive species, but also having a more delicate flavour, and/or from Coffea canephora (Robusta), which has a stronger taste. Hence, desirable Coffea arabica is often adulterated by the cheaper Coffea robusta. Since Robusta coffee contains 16-O-methylcafestrol (16-OMC), which is not present in Coffea arabica, NMR spectroscopy can be used to easily identify robusta fraud (Figure 2-7). The Lachenmeier group has recently developed and validated a qNMR method using the O-methyl signals of the methylcafestol and other components to check the quality and authenticity of coffee [41]; this approach can be used as a coffee screener. A targeted approach may be used to identify the coffee type and detect possible adulteration, but chemometric approaches have also been shown to be useful.
Figure 2-7. 1H NMR spectra (600 MHz) of an organic extract of roasted coffee beans. Signals attributed to compounds that are unique to each type are highlighted.
The combination of 1H NMR spectroscopy and chemometric methods represents an ideal tool with which to profile foodstuffs, e.g., the differentiation between conventionally and organically grown tomatoes can be achieved using the 1H NMR spectra of hundreds of tomato samples and principal component - as well as linear discriminant - analysis [42
