This work was supported by the Foods for Health Discovery Theme at The Ohio State University and the Department of Food Science and Technology at The Ohio State University. The authors would like to thank the NMR facility at The Ohio State University and the NMR facility at Penn State University.

<ol>
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Am Oil Chem Soc. 70, 225-228 (1993).</li><li>Igarashi, T., Aursand, M., Hirata, Y., Gribbestad, I. S., Wada, S., Nonaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondestructive+quantitative+acid+and+n-3+fatty+acids+in+fish+oils+by+high-resolution+1H+nuclear+magnetic+resonance+spectroscopy.">Nondestructive quantitative acid and n-3 fatty acids in fish oils by high-resolution 1H nuclear magnetic resonance spectroscopy.</a> J. Am. Oil Chem. Soc. 77, 737-748 (2000).</li><li>Plans, M., Wenstrup, M., Saona, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Infrared+Spectroscopy+for+Characterization+Dietary+Omega-3+Oil+Supplements.">Application of Infrared Spectroscopy for Characterization Dietary Omega-3 Oil Supplements.</a> J. Am. Oil Chem. Soc. 92, 957-966 (2015).</li><li>Jian-hua, C. I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+Spectrum+Detection+of+Fish+Oil+DHA+Content+Based+on+Empirical+Mode+Decomposition+and+Independent+Component+Analysis.">Near-infrared Spectrum Detection of Fish Oil DHA Content Based on Empirical Mode Decomposition and Independent Component Analysis.</a> J Food Nutr Res. 2 (2), 62-68 (2014).</li><li>Millen, A. E., Dodd, K. W., Subar, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+vitamin,+mineral,+nonvitamin,+and+nonmineral+supplements+in+the+United+States:+The+1987,+1992,+and+2000+National+Health+Interview+Survey+results.">Use of vitamin, mineral, nonvitamin, and nonmineral supplements in the United States: The 1987, 1992, and 2000 National Health Interview Survey results.</a> J. of Am. Diet Assoc. 104 (6), 942-950 (2004).</li><li>Dwyer, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+developing+analytical+and+label-based+dietary+supplement+databases+at+the+NIH+office+of+dietary+supplements.">Progress in developing analytical and label-based dietary supplement databases at the NIH office of dietary supplements.</a> J. Food Compos. Anal. 21, S83-S93 (2008).</li><li>Monakhova, Y. B., Ruge, I., Kuballa, T., Lerch, C., Lachenmeier, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+coenzyme+Q10+in+food+supplements+using+1H+NMR+spectroscopy.">Rapid determination of coenzyme Q10 in food supplements using 1H NMR spectroscopy.</a> Int. J. Vitam. Nutr. Res. 83 (1), 67-72 (2013).</li><li>Monakhova, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardless+1H+NMR+determination+of+pharmacologically+active+substances+in+dietary+supplements+and+medicines+that+have+been+illegally+traded+over+the+internet.">Standardless 1H NMR determination of pharmacologically active substances in dietary supplements and medicines that have been illegally traded over the internet.</a> Drug Test. Anal. 5 (6), 400-411 (2013).</li><li>Berger, S., Braun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 200 and more NMR experiments: a practical course. , (2004).</li><li>Knothe, G., Kenar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+fatty+acid+profile+by+1H-NMRspectroscopy.">Determination of the fatty acid profile by 1H-NMRspectroscopy.</a> Eur. J. Lipid Sci. Technol. 106, 88-96 (2004).</li><li>Sacchi, R., Medina, J. I., Aubourg, S. P., Paolillo, I. G. L., Addeo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+High-Resolution+13C+NMR+Analysis+of+Lipids+Extracted+from+the+White+Muscle+of+Atlantic+Tuna+(Thunnus+alalunga).">Quantitative High-Resolution 13C NMR Analysis of Lipids Extracted from the White Muscle of Atlantic Tuna (Thunnus alalunga).</a> J. Agric. Food Chem. 41 (8), 1247-1253 (1993).</li><li>Dais, P., Misiak, M., Hatzakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+marine+dietary+supplements+using+NMR+spectroscopy.">Analysis of marine dietary supplements using NMR spectroscopy.</a> Anal. Methods. 7 (12), 5226-5238 (2015).</li><li>Pickova, J., Dutta, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+Oxidation+in+Some+Processed+Fish+Products.">Cholesterol Oxidation in Some Processed Fish Products.</a> J. Anal. Oil Chem. Soc. 80 (10), 993-996 (2003).</li><li>Siddiqui, N., Sim, J., Silwood, C. J. L., Toms, H., Iles, R. A., Grootveld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+analysis+of+encapsulated+marine+oil+supplements+using+high-resolution+1H+and+13C+NMR+techniques.">Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C NMR techniques.</a> J. of Lipid Rsrch. 44 (12), 2406-2427 (2003).</li><li>Sua&#180;rez, E. R., Mugford, P. F., Rolle, A. J., Burton, I. W., Walter, J. A., Kralovec, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C-NMR+Regioisomeric+Analysis+of+EPA+and+DHA+in+Fish+Oil+Derived+Triacylglycerol+Concentrates.">13C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates.</a> J. Am. Oil Chem. Soc. 87, 1425-1433 (2010).</li><li>Youlin, X. A., Moran, S., Nikonowiczband, E. P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Z-restored+spin-echo+13C+1D+spectrum+of+straight+baseline+free+of+hump,+dip+and+roll.">Z-restored spin-echo 13C 1D spectrum of straight baseline free of hump, dip and roll.</a> Magn. Reson. Chem. 46, 432-435 (2008).</li><li>Tengku-Rozaina, T. M., Birch, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+distribution+of+fatty+acids+on+hoki+and+tuna+oil+triglycerides+by+pancreatic+lipase+and+13C+NMR+analysis.">Positional distribution of fatty acids on hoki and tuna oil triglycerides by pancreatic lipase and 13C NMR analysis.</a> Eur. J. Lipid Sci. Technol. 116 (3), 272-281 (2014).</li><li>Berry, S. E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triacylglycerol+structure+and+interesterification+of+palmitic+and+stearic+acid-rich+fats:An+overview+and+implications+for+cardiovascular+disease.">Triacylglycerol structure and interesterification of palmitic and stearic acid-rich fats:An overview and implications for cardiovascular disease.</a> Nutr. Res. Rev. 22 (1), 3-17 (2009).</li><li>Hunter, J. 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The authors have nothing to disclose.

Modifications and Strategies for Troubleshooting
Spectral quality. The linewidth of the NMR signal and thus the resolution of the NMR spectrum is highly dependent on shimming, which is a process for the optimization of the homogeneity of the magnetic field. For routine analysis, 1D shimming is adequate and a 3D shimming is not required, given that it is performed by NMR personnel on a regular basis. If this is not the case, a 3D shimming must be performed prior to analysis using a sample containing 0.6 mL of H2O:D2O (90:10). To achieve a better and faster shimming, the sample needs to be centered in the excitation/detection region of the radio frequency (RF) coil, using the graded depth gauge, before it is placed in the magnet bore. Another factor that affects shimming is spinning the sample at a spin rate of 10-20 Hz. Although spinning the sample at this spin rate improves the radial shims (X, Y, XY, XZ, YZ, X2-Y2, etc.), it is generally not recommended in order to avoid the appearance of spinning side bands of the first or higher order. However, when working on instruments that operate in Larmor frequencies lower than 400 MHz, spinning is recommended for 1D NMR experiments.
Resolution, as well as sensitivity, are affected by the receiver gain (rg) value. Low values of the receiver gain reduce the sensitivity, whereas values higher than appropriate cause overflow of the analog-to-digital converter (ADC). ADC overflow results in nonsymmetrical line-shapes and the signals cannot be used for quantitative purposes because the first points of the free induction decay (FID) may be lost. In most cases, the command &#34;rga&#34; calculates an appropriate rg value. However, in some cases, the rg value calculated by the software is higher than the ideal value and there is a distortion in the Lorentzian shape of the NMR signal. In such a case, the user should manually input a smaller rg value by typing &#34;rg (value)&#34; in the command line. A typical RG value for the samples that analyzed with this protocol is 8.
Often, when using cryogenically cooled NMR probes with a high Quality-factor (Q-factor), a large delay (dead time) &#62;200us between the last pulse and the detection period is required to avoid artifacts such as a hump around the transmitter&#39;s frequency and a rolling in the spectrum&#39;s baseline. However, such a long delay causes a large negative first-order phase error, which can also introduce a baseline rolling and large dips around the base of the strong signals. In these cases, a z-restored spin-echo pulse sequence can be used to produce NMR spectra with significantly improved baselines, although a small sensitivity reduction may occur23.
Phospholipids. In addition to the analysis of fish oil samples rich in triglycerides and ethyl esters, NMR can be used for the analysis of fish oil samples rich in phospholipids (PLs). However, special care is required for such samples because PLs form aggregates, which can cause a significant reduction in the spectral resolution and sensitivity. For the analysis of these samples, a solvent mixture of deuterated chloroform:methanol (CDCl3:CD3OD) in a ratio of 70:30 is required for obtaining spectra of high quality.
Internal standard. BHT was selected as an internal standard in this study because it is a highly symmetric molecule with simple 1H and 13C NMR spectra and none of its peaks overlap with those of fish oil constituents. BHT has a signal in the 1H NMR spectrum, which appears as a singlet at &#948; 6.97 and belongs to the two equivalent aromatic protons (para- position in respect to the OH group) and a signal at &#948; 151.45 in the 13C NMR spectrum which belongs to the aromatic quaternary carbon bearing the -OH group. Both of these signals have no overlap with any of the constituents in fish oil, and thus can be used for quantification purposes. Other compounds such as 1,2,4,5-tetrachloro-3-nitrobenzene (TCNB) or ethylene chloride can also be used as alternative internal standards, however, they are characterized by longer T1 values.
Limitations of the Technique
The quantification of various fatty acids and lipids in fish oil supplements is achieved through integration of the appropriate diagnostic NMR signals in the 1D spectra. Such signals should belong only to a specific sample component and must have no interference with signals from other compounds. This may be an issue for 1H NMR analysis since the 1H NMR spectrum is characterized by low resolution due to the short range of chemical shifts. In addition, the presence of scalar coupling (J) produces multiplets and makes the analysis more complicated. For example, ethyl esters (EE) can be quantified using 1H NMR by the characteristic triplet (J = 7.20 Hz) of the methyl group at &#948; 1.25 and the multiplet at &#948; 4.12, which belongs to the methylene protons of the ester group. However, when using NMR instruments operating in Larmor frequencies lower than 850 MHz, the analysis of EE using 1H NMR should be avoided because of the partial overlapping of the peak at &#948; 4.12 with the peak at &#948; 4.14 of TGs, and the overlapping of the signal at &#948; 1.25 with the broad signal of the aliphatic methylene protons at &#948; 1.23-1.35. Large deviations were also observed between the 1H and 13C analysis of EPA in some samples, 13C NMR was closer to the labeled composition provided by the manufacturer. This is probably due to the overlapping of the signal at &#948; 1.69, which is used for EPA analysis, with signals of other compounds that appear in some types of fish oil supplements. Additional errors in quantifications can arise when using an internal standard due to the uncertain purity of the internal standard and from errors in weighing.
The compositional analysis can be expressed in relative molar concentrations without the use of an internal standard. If results need to be expressed in absolute concentrations, for example as milligram of fatty acid per gram of oil (mg/g), the use of an internal standard is required. However, in cases where the NMR signal of interest belongs to multiple compounds with different molecular weights, the results cannot be expressed as mg/g even when using an internal standard. In addition, the use of internal standard usually increases the length of the analysis because the most common internal standards, such as BHT, are small molecules with high molecular symmetry, which results in long relaxation times. Since the repetition time (delay between pulses + acquisition time) is set according to the longest relaxation time T1 in the sample, the use of an internal standard will increase the duration of the experiments as longer delays between pulses are required. This is an especially important factor for 13C NMR analysis because of the exceptionally long T1 relaxation time of carbon nuclei. The addition of a paramagnetic compound such as Cr(acac)3 can efficiently reduce the T1 relaxation time. The recommended concentration of Cr(acac)3 is 0.75 mg/mL of solution. Higher concentrations of Cr(acac)3 may be considered for further reduction of T1, however, caution is required in order to avoid decreases in the S/N due to the line broadening.
Although the 13C NMR is characterized by a much higher spectral resolution compared to 1H, the sensitivity of the 13C NMR experiment is significantly lower because of the low natural abundance (1.1%) and the low gyromagnetic ratio (67.26 106 rad s-1 T-1) of 13C nuclei. In addition, the long T1 relaxation times of 13C increase the length of the analysis. This may be an issue when the available oil for analysis is limited, because an increased number of scans should be used to achieve a reasonable signal to noise ratio.
Limitations in the sensitivity and the resolution of the NMR spectra prevent the analysis of many minor compounds in fish oil that can be analyzed with other techniques such as GC. For example, 1&#919; NMR is unable to separate individual sterols or fatty acids (e.g. palmitic and stearic) whereas 13C is not able to determine compounds that appear in very low concentration in fish oil such as dodecanoic and myristic acid, which overlap with the signals of all saturated fatty acids at &#948; 173.24 and &#948; 172.82. Although increasing the amount of sample that is analyzed makes the analysis of some minor compounds feasible, caution is required for very concentrated samples, because of their increased viscosity. Very viscous solutions containing more than 150 mg of oil should be avoided because there is a decrease in S/N due to the line broadening caused by the reduced spin-spin T2 relaxation times. In addition, longer delays between pulses are required because of the longer T1 and there are several issues in shimming and thus in resolution.
All the compounds analyzed in fish oil with NMR can be quantified simultaneously in one snapshot without using any separation or purification steps. The NMR analysis is rapid as the 1H spectrum can be recorded in less than one minute, whereas the 13C NMR acquisition lasts 10 min. It should be noted, however, that there are a few factors that affect the data acquisition time. Specifically, for 13C NMR, the 10 min run time can only be achieved without the use of internal standards, and with the use of cryogenically cooled probes, in which the RF coil and the preamplifier are cooled and thus the thermal noise is minimized. A 10-15 fold increase in the experimental time should be expected for 13C NMR analysis when room temperature (conventional) probes are used.
Significance with Respect to Existing Methods
NMR spectroscopy proved to be a powerful tool for qualitative and quantitative determination of the composition of fish oil supplements, and because of its rapidness it has the potential to be applied for the high throughput screening of a vast number of fish oil samples. NMR spectroscopy is by definition a quantitative methodology since the signal area is directly proportional to the number of nuclei that cause the signal. While acutely toxic chemicals are required to prepare NMR samples, this method is environmentally friendly because such small amounts of this chemicals (e.g. CDCl3) are used as opposed to other methods that require large amounts of solvent to elute samples. In addition, NMR has several advantages compared to other analytical methods. No calibration with standards is required prior the analysis, and a minimal sample preparation without any separation and purification steps is usually adopted, which renders NMR a very fast analytical tool. Additionally, 13C NMR is the best available methodology for determining the positional distribution of various fatty acids on the glycerol skeleton. While the enzymatic hydrolysis has been used as an alternative is not always reliable24. This is of specific importance because there is a significant interest in studying the regiospecificity of various fatty acids in foods, as it has been found that this affects their function in human diet25,26.
Future Applications
Despite the agreement between NMR analysis and the products&#39; label, as well as the fact that there are some studies showing agreement between GC and NMR, we believe that more rigorous and comprehensive intra-laboratory studies are required to examine the agreement between NMR and traditional methodologies for the analysis of fish oil constituents using a larger number of samples, fish oil products of different origins, and certified standard solutions.
Another important future application of NMR in fish oil analysis will be the determination of the oxidation products. In addition to the determination of the major compounds in fish oil, several primary and secondary oxidation products in fish oil, such as aldehydes and peroxides, are present. 1H NMR can be potentially applied for the evaluation of the oxidation status in fish oil supplements, under different oxidation conditions, as shown in Figure 3. The biggest challenge in this analysis will be the NMR assignment and the identification of individual oxidation products. Advances in sensitivity of the NMR hardware will also allow the identification of individual sterols using 13C NMR. NMR spectroscopy can also be applied for the analysis of fish tissue as a whole even without any extraction by using High Resolution Magic Angle Spinning (HR-MAS) NMR.
Critical Steps within the Protocol
Two of the most critical steps that affect the accuracy of quantitative NMR spectra involve the selection of a 90&#176; pulse and the use of a delay between pulses &#8805; 5&#215;T1. The pulse angle is proportional to pulse width which is a calibrated NMR parameter that depends on the instrumentation and the sample. A 90&#176; pulse is essential for the complete conversion of longitudinal (z) magnetization to the observable transverse (xy) magnetization. It is important to note that before pulse calibration, the NMR probed needs to be well tuned and matched. This will optimize the transfer of the RF power to the sample and thus maximize S/N and ensure effective decoupling. The probe tuning is mostly affected by the dielectric constant of the sample, so if there are differences in the concentration between samples, repeat the tuning process for each one. The 1D 13C NMR experiment involves both 13C and 1H channels so automatic tuning and matching is necessary for both nuclei.
A delay between pulses longer than 5&#215;T1 ensures the complete recovery of the net magnetization to its initial value. If all the resonances in the spectrum have not completely relaxed before each pulse, the signal is partially suppressed and this leads to inaccuracies in the integration. T1 value is a critical factor that affects the length of the experiment and it depends on the magnetic field strength as well as the viscosity of the sample. Given that the viscosity between samples is similar, T1 relaxation times should be determined for each instrument only in the beginning of the analysis session.
Another important feature of the fish oil analysis with 13C NMR is the selection of the appropriate pulse sequence. The most reliable method for quantitative 13C analysis is the inverse gated decoupling experiment, where broadband proton decoupling is applied only during the acquisition period and thus there is no polarization transfer from 1H to 13C via the nuclear Overhauser effect (NOE). However, while the fully decoupled NMR experiment can be used for quantitative purposes, caution is required when using this experiment because there are different NOE factors among carbons with different multiplicities and therefore integral comparison between methyl, methylene, methane and carbonyl carbons must be avoided. Despite this, when only carbons of similar multiplicity and chemical environment are considered in the analysis, the fully decoupled method is reliable. One example of this is carbonyl carbons of fatty acids which have been found to have no significant differences in the NOE factors after decoupling27. In addition, for carbons bearing protons, the fully decoupled experiment provides higher sensitivity due to the NOE contributions on the NMR signal intensity. A comparison between spectra acquired with the two pulse sequences is shown in Figure S1.

The consumption of n-3 fatty acids in the diet has proven to be beneficial against several conditions such as heart disorders1,2,3, inflammatory diseases4 and diabetes5. The Western diet is considered poor in n-3 fatty acids and thus the consumption of fish oil supplements is recommended to improve the n-6/n-3 balance in consumer&#39;s nutrition1. Despite the recent increase in fish oil supplement consumption, questions remain about the safety, authenticity, and quality of some of these products. The rapid and accurate compositional analysis of fish oil supplements is essential to properly evaluate the quality of these commercial products and ensure consumer safety.
The most common methodologies for the assessment of fish oil supplements are gas chromatography (GC) and Infrared Spectroscopy (IR). While these are highly sensitive methods, they suffer from several drawbacks6. GC analysis is time consuming (4-8 h) because separation and derivatization of individual compounds is required7 and lipid oxidation may occur during the analysis8,9. While IR spectroscopy can be quantitative, a prediction model is required to be constructed using partial least squares regression (PLSR), although there are exceptions in which IR bands can be attributed to a single compound10. PLSR requires the analysis of a large number of samples, which increases the time of the analysis11. For this reason, there is an increasing interest in the development of new analytical methodologies that allow accurate and fast analysis of a large number of fish oil samples. Organizations such as the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH) and the Food and Drug Administration (FDA) have collaborated with the Association of Official Analytical Chemists (AOAC) to develop these new methods12,13.
One of the most promising analytical methods for the screening and the evaluation of multi-component matrices, such as dietary supplements, is Nuclear Magnetic Resonance (NMR) spectroscopy14,15. NMR spectroscopy has several advantages: it is a non-destructive and quantitative technique, it requires minimal to no sample preparation, and it is characterized by excellent accuracy and reproducibility. In addition, NMR spectroscopy is an environmentally friendly methodology because it utilizes only small amounts of solvents. The main drawback of NMR spectroscopy is its relatively low sensitivity compared to other analytical methods, however, recent technological advances in instrumentation such as stronger magnetic fields, cryogenic probes of various diameters, advanced data processing, and versatile pulse sequences and techniques have increased the sensitivity up to the nM range. While NMR instrumentation is high cost, the long-life of NMR spectrometers and the many applications of NMR lower the cost of the analysis in the long run. This detailed video protocol is intended to help new practitioners in the field avoid pitfalls associated with 1H and 13C NMR spectroscopic analysis of fish oil supplements.

<table><tbody><tr><td>Avance III 850 NMR instrument</td><td>Bruker</td><td></td><td></td></tr><tr><td>Avance III 500 NMR instrument</td><td>Bruker</td><td></td><td></td></tr><tr><td>TCI 5 mm probe</td><td>Bruker</td><td></td><td>Helium cooled inverse (proton deetected) NMR probe featuring three independent channels (&lt;sup&gt;1&lt;/sup&gt;H, &lt;sup&gt;13&lt;/sup&gt;C, &lt;sup&gt;15&lt;/sup&gt;N)</td></tr><tr><td>BBO prodigy 5 mm probe</td><td>Bruker</td><td></td><td>Nitrogen cooled observe (X-nuclei detected) probe, featuring two channels; one for &lt;sup&gt;1&lt;/sup&gt;H and &lt;sup&gt;19&lt;/sup&gt;F detectionand one for X-nuclei (covering from &lt;sup&gt;15&lt;/sup&gt;N to &lt;sup&gt;31&lt;/sup&gt;P)</td></tr><tr><td>Spinner turbin</td><td>Bruker</td><td></td><td>NMR spinners are made by polymer materials and they have a rubber o-ring to hold the NMR tube securely in place</td></tr><tr><td>Topspin 3.5</td><td>Bruker</td><td></td><td></td></tr><tr><td>deuterated chloroform</td><td>Sigma-Aldrich</td><td>&amp;nbsp;865-49-6</td><td>99.8 atom % D, contains 0.03 TMS</td></tr><tr><td>2,6-Di-&lt;em&gt;tert&lt;/em&gt;-butyl-4-methylphenol</td><td>Sigma-Aldrich</td><td>&amp;nbsp;128-37-0</td><td>purity &gt;99%</td></tr><tr><td>Fish oil samples</td><td></td><td></td><td></td></tr><tr><td>NMR tubes</td><td>New Era</td><td>NE-RG5-7</td><td>5mm OD Routine &amp;ldquo;R&amp;rdquo; Series NMR Sample Tube</td></tr><tr><td>BSMS</td><td>Bruker</td><td></td><td>Bruker Systems Management System; control system device</td></tr></tbody></table>

nmr spectroscopy as a robust tool for the rapid evaluation of the lipid profile of fish oil supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Watch this Scientific Journal Video about NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements at JoVE.com

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

nmr-spectroscopy-as-robust-tool-for-rapid-evaluation-lipid-profile

Nanyang Technological University

Research

JoVE Journal

Chemistry

25.9K Views.  The Ohio State University. Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

Video: NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Environment

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Biochemistry

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Determination of Total Lipid and Lipid Classes in Marine Samples

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix.
If seawater has been sampled, the first step usually involves separation into operationally defined &#39;dissolved&#39; and &#39;particulate&#39; factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement.
Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).

This protocol is for the determination of lipids in seawater and biological specimens. Lipids in filtrates are extracted with chloroform or mixtures of chloroform and methanol in the case of solids. Lipid classes are measured by rod thin-layer chromatography with flame ionization detection and their sum gives the total lipid content.

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biology

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Neuroscience

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...