Low-field benchtop NMR spectroscopy: status and prospects in natural product analysis †

Introduction: Since a couple of years, low-field (LF) nuclear magnetic resonance (NMR) spectrometers (40 – 100 MHz) have re-entered the market. They are used for various purposes including analyses of natural products. Similar to high-field instruments (300 – 1200 MHz), modern LF instruments can measure multiple nuclei and record two-dimensional (2D) NMR spectra. Objective: To review the commercial availability as well as applications, advantages, limitations, and prospects of LF-NMR spectrometers for the purpose of natural products analysis. Method: Commercial LF instruments were compared. A literature search was performed for articles using and discussing modern LF-NMR. Next, the articles relevant to natural products were read and summarised. Results: Seventy articles were reviewed. Most appeared in 2018 and 2019. Low costs and ease of operation are most often mentioned as reasons for using LF-NMR. Conclusion: As the spectral resolution of LF instruments is limited, they are not used for structure elucidation of new natural products but rather applied for quality control (QC), forensics, food and health research, process control and teaching. Chemometric data handling is valuable. LF-NMR is a rapidly developing niche and new instruments keep being introduced.

atoms. The basics of NMR were developed in the 1950s and the first commercial instruments, like the Varian A60 (60 MHz) appeared in the early 1960s. 1 Synthetic organic chemists and natural product chemists immediately recognised the usefulness of NMR, even if only for proton ( 1 H) at 60 MHz in one dimension.
For example, at that time the group of George Büchi was involved in the structure elucidation of terpenes, including patchoulol ( Figure 1A). In a 1960 article, NMR was not yet used by them but in 1961 NMR first appeared through courtesy of another laboratory. 2 In 1964 their own laboratory had already purchased two Varian 60 MHz machines showing the big demand. 3 In the case of patchoulol, the application of NMR was mostly limited to methyl groups and olefinic protons of dehydration products but even this was highly useful. Only a few years later, NMR played a bigger and more varied role in the group of Koji Nakanishi. Even with NMR, solving the structures of the ginkgolides ( Figure 1B) was a challenging puzzle. 4 Their spectra were more informative than those of patchoulol, which was also due to the fact that they had access to afor that timeadvanced 100 MHz Varian HR-100. Additionally, coupling constants and nuclear Overhauser effect (NOE) were used to determine stereochemistry and double resonance experiments were carried out to determine proton-proton connectivities. 5 This illustrates the rapid developments taking place, which were partially catalysed by the desire to solve complex natural products.
The rest is history, Fourier-transform (FT)-NMR was introduced, which allowed the recording of carbon-13 ( 13 C)-NMR spectra, stronger and stronger superconducting magnets entered the market yielding much more resolution and sensitivity and finally all the 2D NMR techniques were gradually developed leading to the current situation.
However, modern NMR spectrometers are expensive both in terms of initial investment, consumables (liquid helium), maintenance (hardware) and operation (skilled personnel). Thus, they are out of reach for small and medium enterprises (SMEs), governmental quality control (QC) agencies, forensic laboratories and not at all universities students can get hands-on NMR training. This led to the introduction of benchtop NMR spectrometers with permanent magnets . They combine a small footprint, a 5-20× lower price, no consumables, almost zero maintenance and easy operation. The downside is a 5-20× lower resolution and a lower sensitivity. In contrast to low-field (LF)-NMR spectrometers of the 1960s, the new generation is capable of recording 2D spectra.
At first glance modern LF-NMR spectrometers seem to have little merit for natural product or phytochemical analysis as they appear less suitable for structure elucidation and the NMR spectra of even simple natural products exhibit second-order effects. However, natural product analysis is not synonymous with structure elucidation and since 2014 a number of articles have appeared on the application of LF-NMR in natural products analyses, e.g. for QC or forensic purposes. Four reviews on the topic of LF-NMR as a whole have been written by the group of Blümich. Three comprehensively reviewed fundamentals and developments concerning spectroscopy, relaxometry and imaging. [6][7][8] A fourth review focussed specifically on NMR spectroscopy. 9 Rudszuck et al. devoted a review to the QC of crude and edible oils by LF-NMR. 10 Finally Grootveld et al.
summarised applications of LF-NMR in chemical and biochemical analysis. 11 The focus of the current review is on spectroscopic LF-NMR applications involving natural products. Additionally, it reviews available instruments. Its appearance is timely as in 2019 many more LF-NMR articles appeared than ever before and in 2019 there were also many exciting hardware introductions: an autosampler (Magritek), first 100 MHz instrument (Nanalysis), first broadband instrument (Oxford Instruments), < 0.2 Hz line width instrument (Magritek) and a new vendor entering the market (Bruker). Combined, this information might act as an eye-opener for the Phytochemical Analysis readership regarding the application niche of LF-NMR.

| AVAILABLE BENCHTOP NMR SPECTROMETERS
Currently five brands of benchtop NMR instruments are commercially available. The specifications of available instruments are presented in Table 1 based on information available on the web and no responsibility is taken for deviations from these values. Based on Table 1, not one best instrument emerges, all have pros and cons. Prospective buyers should test the instruments, which best meet their needs with their own samples. For instance, if they would like to be able to measure without deuterated solvents, an external lock should be chosen. Overall there is a trend towards higher field strengths and thus heavier instruments. It is debatable whether instruments over 100 kg should still be considered as "movable". For non-research uses, e.g. for QC in SMEs, an autosampler is a necessity. The number of times a particular brand and type was used in the discussed research articles was counted and the following percentages were calculated: Magritek 43  NMR. This is partially caused by the fact that the key players in the LF-NMR field, Bernhard Blümich and Patrick Giraudeau, have been using this instrument.
Two more manufacturers of low-field or cryogen-free instruments exist: Anasazi and HTS-110. However, as their spectrometers cannot be considered as benchtop they are not included in Table 1.

| LF-NMR APPLICATIONS
This review is restricted to articles making use of NMR spectroscopy and commercially available LF-NMR spectrometers. In the following paragraph, all LF-NMR applications involving natural products or plant matrixes have been subdivided in five categories: (1) quality control and adulteration detection, (2) forensic applications, (3) food and health applications, (4) process control, and (5) teaching. In Table S1 there is a listing of relevant papers making use of non-spectroscopic NMR approaches such as relaxation, diffusion and imaging, or home-built equipment.

| LF-NMR in quality control and adulteration detection
This is an important field and the application of NMR in general to detect food fraud was recently separately reviewed. Some attention to LF-NMR was given and according to the authors benchtop NMR is potentially a breakthrough technology in food authentication. 21 Parker et al. in 2014 were the first to apply LF-NMR in phytochemical analysis. 22 They wished to detect adulteration of olive oil with hazelnut oil. This was highly challenging as these two vegetable oils possess an almost identical fatty acid (FA) composition, the only difference being the~6% higher content of double unsaturated fatty acids (UFAs) of hazelnut oil. Thus, the peak area ratio of olefinic peaks (~5.3 ppm, also including H2 of glycerol) versus H1 and H3 of glycerol (~4.2 ppm) was determined for both oils. This ratio was 1.70 and 1.52 for hazelnut and olive oil, respectively, but due to natural variation the most extreme values were almost identical. The final result was that 13% (w/w) hazelnut adulteration could be detected with 95% confidence. Additionally, they used a chemometric approach using the shape of the entire spectrum. This allowed for the detection of 11% adulteration with 95% confidence. Thus, the method is not fool-proof, false negatives and especially false positives will occur occasionally. Compared to an FT-IR method, the 60 MHz NMR method performed better. 22 The earlier-mentioned study was summarised in a subsequent article by this group together with new results. 23 Vegetable oil samples were diluted 1:1 with chloroform (CHCl 3 ) and measured at 60 MHz.
Integration of various triacylglycerol (TAG) peaks (0.9 ppm = ω-3 CH 3 ; 2.7 ppm = bis-allylic CH 2 ; 5.2 ppm = olefinic peak; remaining FAs are saturated) allowed to calculate the percentage of ω-3 FAs, polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFAs) and saturated fatty acids (SFAs), respectively, and an excellent correspondence was found with gas chromatography flame ionisation detector (GC-FID) data. Even for a whole range of complex foods, such as rolls, pies and crisps, a fair correspondence with the labelled SFAs was found.

Through chemometric treatment [principal component analysis (PCA)]
of the LF-NMR data, 10 different vegetable oils could be clearly distinguished. Not surprisingly, as the analytes (TAGs) are the same, the methodology could also distinguish between different types of meats and detect adulteration of sunflower oil with lard. 23 The meat application was also separately published. 24 Riegel investigated adulteration of olive oil with soybean oil. 25  such fraud. 32 However, this proved difficult asespecially in the case of RROthe TAG composition is highly similar leading to LF-NMR spectra, which are difficult to distinguish by a human observer.   The degradation of compounds present in fingerprints (squalene, oleic acid, nonanoic acid) as a function of light exposure was studied to determine the age of human fingerprints. 40 Irradiated pure compounds were dissolved in CDCl 3 and investigated by 60 and 400 MHz NMR. No details on LF-NMR spectra were given.
To deal with more and more seized samples containing psychoactive substances, LF-NMR (60 MHz) was employed to screen samples. 41

| LF-NMR in food and health related applications
LF-NMR was used to distinguish more expensive arabica from cheaper robusta coffees. 43  ies, a precise detection limit could not be given but the adulteration limit was estimated to lie between 10 and 20%. A survey of 27 "100% arabica" coffees in the UK revealed no fraud. 43 In a follow-up article, the sensitivity of the method mentioned earlier was increased by extracting 10 g of coffee with 30 mL of ter methods were more accurate but required a secondary method and are possibly sensitive to process changes. 51 The authors concluded that LF-NMR can be used for on-line monitoring of chemical processes. 51 In their third article, they further investigated the catalyst, mechanism and kinetics. 52 In a biotechnological application, the growth of a yeast and a fungus was followed via on-line LF-NMR (42.5 MHz) measurements of consumed and produced chemicals. 53 The culture broth was pumped

| LF-NMR in teaching
Although NMR can be learned solely by theory, spectra and movies, gaining hands-on experience with preparing an NMR sample, introducing it into a spectrometer, recording your own spectrum in realtime and interpreting it afterwards definitely has added value for students. The field strength of the spectrometer is of lesser importance.  [58][59][60][61][62][63][64][65][66] One of them deals with common OTC (over-the-counter) formulations including the natural products ascorbic acid and caffeine. 61 Another experiment is centred around caffeine. 64 At the university of the author, natural products play a significant role for introducing NMR to students. In the basic course, all students quantitatively analyse the alcohol content of different liquors according to a quantitative protocol published earlier by our group. 67 Further students check the identity and purity of natural products such as piperine, anethol and xanthorrhizol isolated by themselves through interpretation of the spectra. In another course, also 2D spectra are recorded.  30 and detection of an undeclared sugar in fine chemicals. 33 The forensic use is more varied. The non-research real-life application of LF-NMR as a first screening tool of seized drugs with a large library and automated data treatment is the way to go. 41 Food and health applications are varied too, ranging from fraud detection, metabolite screening of food ingredients, 45

| Structure elucidation
The applications confirm that LF-NMR is not that useful for structure elucidation of unknown compounds other than the simplest ones. At best one can get an idea about the identity of a major adulterant or product, which needs to be further proven by HF-NMR, GC-MS or LC-MS. 30

| Sensitivity
In most studies, simply 1D 1 H-NMR spectra are recorded and then sensitivity is not a big issue unless one is dealing with low concentrations. For example, it is less likely that LF-NMR will be applied in the near future for detecting drugs or minor metabolites in biofluids without enrichment or signal enhancement. The recording of 13 C-NMR or 2D spectra is possible on modern LF instruments but can take 24 h or more. However, COSY spectra can be recorded in less than an hour for concentrated samples or even in a few minutes on a modified LF instrument. 27 Research is on-going to increase the sensitivity 10000-fold or more through hyperpolarisation. This would accomplish a 13 C spectrum in one scan, [70][71][72][73][74] or enable metabolite analysis in sec. 75  samples. An alternative approach to increase resolution would be to remove all couplings, like in a projection of a 2D J-resolved spectrum.
Various pulse sequences for this purpose were compared but the results are disappointing in terms of sensitivity (1% of 1D sensitivity) and resolution due to the appearance of artefacts. 79 Also, apart from 2D J-resolved spectra, all other pulse sequences require hardware modifications and simplicity is lost, which again is one of advantages of LF-NMR. Thus, also this approach appears short term doubtful. A simpler approach is the addition of Lanthanide shift reagents to disperse overlapping signals. 80  observed after data treatment. 92 In a follow-up article, they optimised sensitivity and selectivity. 94 96 More applications are bound to follow.
Future publications will mostly originate from academic users but LF-NMR is of particular interest for SMEs and governmental control laboratories, which until now could not afford NMR. Potentially this is a big market. Forensics is another area due to the many natural products (cocaine, morphine, ephedrine, cannabis) or derivatives thereof (heroin, synthetic cannabinoids) used as illegal drugs. A further use is in industrial organic synthesis through flow chemistry where the synthesis can be adapted by means of feedback from the NMR detector.
An example is the synthesis of the natural product carpanone. 85 In all such applications chemometric processing of acquired data will prove essential. It may reveal in an objective manner spectral differences, which are otherwise difficult to discern. In terms of hardware, 80-100 MHz will remain the maximum but through the development of more homogeneous magnetic fields, narrower line widths can be expected, also leading to higher S/N ratios. Finally, it is prudent to realise the main limitation of LF-NMR, i.e. its limited resolution. For instance, in the very first LF-NMR article it was tried to detect the adulteration of olive oil with near-identical hazelnut oil based on TAG profiles. 22 This is almost impossible as the two oils are highly similar.
However, if NMR had not been used as the single analytical technique but rather in combination with refractive index or GC-FID measurements, the combined analytical information might have been convincing. In other words, LF-NMR should in some instances not be used as a stand-alone technique but rather as a complementary technique.