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

Summary

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.

Abstract

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.

Introduction

The consumption of n-3 fatty acids in the diet has proven to be beneficial against several conditions such as heart disorders^1,2,3, 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'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 analysis^8,9. 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 methods^12,13.

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) spectroscopy^14,15. 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.

Protocol

1. NMR Sample Preparation

Note: Caution, please consult all relevant material safety data sheets (MSDS) before use. Deuterated chloroform (CDCl₃) used in sample preparation is toxic. Please use all the appropriate safety practices when performing sample preparation including the use of a fume hood and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).

1. Preparation of ¹H and ¹³C samples
   1. Extract 120 µL (~ 110 mg) of fish oil from a dietary capsule using a syringe and place it in a 4 mL glass vial. Record the weight of the fish oil.
   2. Sample dissolution
      1. Dissolve approximately 120 µL of fish oil in 500 µL of CDCl₃ containing 0.01% of tetramethylsilane (TMS) which is used as a reference for the ¹H and ¹³C chemical shifts.
         NOTE: TMS is used only for chemical shift calibration (see step numbers 2.2.1.2.7 and 2.2.2.2.7), not for quantification (see step numbers 2.2.1.3 and 2.2.2.3) purposes.
      2. Prepare a 2,6-di-tert-butyl-4-methylphenol (BHT) stock solution, if quantification expressed in mg/g is desired, by dissolving approximately 220 mg of BHT and 15 mg of Chromium(III) acetylacetonate (Cr(acac)₃) in 20 mL of CDCl₃ containing 0.01% of TMS. Use 500 µL of the stock solution to dissolve 100 mg (± 10 mg) of fish oil.
   3. After dissolving the oil (this takes a few seconds), transfer all of the solution directly into a high quality 5-mm NMR tube and attach a cap. Analyze the samples within 24 h after preparing the samples.

2. NMR Instrument preparation

Note: Caution, beware that the presence of strong magnetic fields produced by NMR instruments can affect medical devices and implants such as pacemakers and surgical prostheses, as well as electronic items such as credit cards, watches, etc. Additional caution is required when the analysis is performed using non-shielding magnets. Two NMR instruments were used for the acquisition of ¹H and ¹³C NMR spectra; a spectrometer operating at 850.23 MHz and 213.81 MHz for ¹H and ¹³C nuclei, respectively, equipped with a triple resonance helium-cooled inverse (TCI) 5 mm probe and a spectrometer operating at 500.20 MHz and 125.77 MHz for ¹H and ¹³C nuclei, respectively, equipped with a broad band observed (BBO) nitrogen-cooled 5 mm probe. All experiments were performed at 25 ± 0.1 ºC and the spectra were processed by a standard NMR data analysis acquisition and processing software package (see Materials List).

1. Preparation for acquiring the NMR spectra
   Note: ¹H and ¹³C NMR spectra can be acquired consequently without removing the sample from the instrument.
   1. Insert the NMR tube into a spinner turbine (see Materials List).
   2. Place the spinner and the tube on the top of a graded depth gauge and gently push the top of the tube until its bottom part touches the bottom of the gauge.
   3. Place NMR sample in an open spot of the SampleCase. Note the slot number the sample is placed in.
   4. To load the sample in the NMR, return to the control computer and type 'sx #', where # is the slot in the SampleCase holding your sample.
   5. Wait for the deuterium signal of CDCl₃ to appear on the lock window screen. If it does not automatically appear, type "lockdisp". As soon as the deuterium signal is visible, type "lock" on the command line and select "CDCl₃" from the solvent's list in order to lock the sample using the CDCl₃ deuterium resonance.
      NOTE: Deuterium signal may not appear if previous user used a different solvent. User should wait for the indicator that the sample is down, then lock.
   6. Type "bsmsdisp" in the command line to ensure spinning is not active. If the "SPIN" button is green, click it to deactivate spinning.
   7. Type the "new" command to create a new data set. Enter a name for the data set in the "NAME" tab and the experiment number in the "EXPNO" tab. Use number "1" in the "PROCNO" tab. In the "Experiment" tab, hit "Select" and choose the "PROTON" parameter file. Write the title of the experiment in the "TITLE" tab. Click "OK."
   8. Type "getprosol" in the command line to obtain the standard parameters for the current NMR probe and solvent.
   9. Repeat step 2.1.7 for ¹³C, selecting the "C13IG" pulse sequence in the "Experiment" tab for the 1D ¹³C inverse gated decoupled experiment.
   10. Type "getprosol" in the command line to obtain the standard parameters for the current NMR probe and solvent.
   11. Type the command "atma" to perform automatic tuning and matching of the probe for both carbon and proton nuclei.
   12. Perform one-dimensional gradient shimming to achieve a highly homogeneous magnetic field, and thus optimum line shape for the NMR signals.
       1. Use the standard automatic procedure for 1D shimming, simply by sequentially executing the commands "qu topshim 1dfast ss", "qu topshim tuneb ss," and "qu topshim report" on the command line.
2. Parameter optimization
   1. 90° pulse calibration
      1. Create a new data set for ¹H (see steps 2.1.7 and 2.1.8).
      2. Type the command "paropt" on the command line to start the automation program for calibrating the 90° pulse. Select pulse duration, p1, as the parameter to be modified.
      3. Start with "2" µs as the initial value of p1, enter "2" µs increments and perform "16" experiments.
      4. Create a new data set for ¹³C (see step 2.1.9) and repeat the process for ¹³C nuclei (see steps 2.2.1.2 and 2.2.1.3).
   2. T₁ measurement measured by the null method¹⁶ for ¹H
      NOTE: The null method uses the inversion recovery pulse sequence, consisting of a 180° pulse follow by a delay (tau), to allow relaxation along the z axis and a final 90° pulse which creates the observable transverse magnetization.
      1. Create a new data set for ¹H (see steps 2.1.7 and 2.1.8).
      2. Type "pulprog t1ir1d" to change the pulse sequence to the inversion-recovery experiment.
      3. Type the following commands on the command line to set up the spectral width in ppm, the center of the RF transmitter, the number of scans the number of dummy scans and the number of data points "sw 8", "o1p 3.8", "ns 2", "ds 2" and "td 64K".
      4. Type "p1 (value)" and enter the duration values for 90° pulse as determined by the pulse calibration (see step 2.2.1) and type "p2 (value)" for the 180° pulse (the duration value for the 180° pulse is the 90° pulse duration multiplied by two).
      5. Set the recycle delay to a very large value, such as 10 s by typing "d1 10".
      6. Set tau to a short value, such as 10 ms, by typing "d7 10ms" in the command line.
      7. Set the receiver gain (RG) to an appropriate value using the command "rga" for automatic calculation of RG.
      8. Run a spectrum by typing the command "zg".
      9. Execute Fourier-transformation by typing "efp" in the command line.
      10. Perform automatic phase correction by typing the command "apk" in the command line. If additional phase adjustments are required to further improve the spectrum, click on the "Process tab," then click on the "Adjust Phase" icon to enter the phase correction mode.
          1. Use the zero-order (0) and first-order (1) phase correction icons by dragging the mouse until all the signals are in negative absorption mode. Apply and save the phase correction values by clicking the "Return and Save" button to exit the phase correction mode.
      11. Increase the tau until all peaks are either positive or nulled by repeating steps 2.2.2.6-2.2.2.9. To determine the T₁ value, simply divide the tau value where the peak is nulled with ln2.
   3. T₁ measurement measured by the null method¹⁶ for ¹³C
      1. Create a new data set for ¹³C (see step 2.1.9)
      2. Type "pulprog t1irpg" to change the pulse sequence to the inversion-recovery experiment for carbon nuclei.
      3. Type the following commands on the command line to set up the spectral width in ppm, the center of the RF transmitter, the number of scans, the number of dummy scans and the number of data points: "sw 200", "o1p 98", "ns 8", "ds 2"and "td 64K".
      4. Type "p1 (value)" and enter the duration values for 90° pulse as determined by the pulse calibration (see step 2.2.1) and type "p2 (value)" for the 180° pulse (the duration value is the 90° pulse duration multiplied by two).
      5. Set the recycle delay to a very large value, such as 100 s by typing "d1 100".
      6. Set tau to a short value, such as 100 ms by typing "d7 100ms" in the command line.
      7. Set the receiver gain (RG) to an appropriate value using the command "rga" for automatic calculation of RG.
      8. Run a spectrum by typing the command "zg".
      9. Execute Fourier-transformation by typing "efp" in the command line.
      10. Perform Automatic phase correction by typing the command "apk" in the command line. If additional phase adjustments are required to further improve the spectrum, click on the "Adjust Phase" icon and the phase correction icons for zero-order (0) and first-order phase (1) correction.
          1. While clicking on the zero-order and first-order phase correction icons, drag the mouse until all the signals are in negative absorption mode. Apply and save the phase correction values by clicking the "Return and Save" button to exit the phase correction mode.
      11. Increase the tau until all peaks are either positive or nulled by repeating steps 2.2.3.6-2.2.3.9. To determine the T₁ value, simply divide the tau value where the peak is nulled with ln2.
3. One-dimensional (1D) NMR Spectra
   1. ¹H-NMR spectra
      1. Acquisition of the NMR data
         1. Go to the ¹H data set created in step 2.1.7 and use the standard "pulse-acquire" pulse sequence, "zg", by typing "pulprog zg" in the command line.
         2. Type the following commands on the command line to set up the spectral width in ppm, the center of the RF transmitter, the number of scans, the number of dummy scans, the number of data points and the pulse duration for a 90° pulse angle: "sw 8", "o1p 3.8", "ns 2", "ds 2", "td 64K" and "p1 (as determined by pulse calibration)" (see step 2.2.1).
            NOTE: 32K data points can be used for the 500 MHz instrument.
         3. Set a relaxation delay of 7 s for the 500 MHz instrument or 9 s for the 850 MHz instrument by typing "d1 7s" or "d1 9s", respectively, in the command line.
         4. Set the receiver gain (RG) to an appropriate value using the command "rga" for automatic calculation of RG.
         5. Type "digmod baseopt" to acquire a spectrum with improved baseline.
         6. Start the acquisition by typing the pulse-acquire command "zg" in the command line.
      2. Processing of the NMR data
         1. Type "si 64K" in the command line to apply zero-filling and set the size of the real spectrum to 64K.
         2. Set the line broadening parameter to 0.3 Hz by typing "lb 0.3" in the command line to apply a weighting function (exponential decay) with a line broadening factor of 0.3 Hz prior to Fourier transform.
         3. Execute Fourier-transformation by typing "efp" in the command line.
         4. Perform Automatic phase correction by typing the command "apk" in the command line. If additional phase adjustments are required to further improve the spectrum, click on the "Process tab," then click on the "Adjust Phase" icon and the phase correction icons for zero-order (0) and first-order (1) phase correction.
            1. While clicking on the zero-order and first-order phase correction icons, drag the mouse until all of the signals are in positive absorption mode. Apply and save the phase correction values by clicking the "Return and Save" button to exit the phase correction mode.
         5. Apply a polynomial fourth-order function for base-line correction upon integration by typing the command "abs n".
            NOTE: This ensures a flat spectral baseline with a minimum intensity.
         6. Report chemical shifts in ppm from TMS (δ = 0). Click on the calibration ("Calib. Axis") icon, and place the cursor with the red line on top of the TMS NMR signal (peak closest to 0). Left click and type in "0".
      3. NMR data analysis
         1. Integrate the spectral region from δ 1.1 to δ 0.6 as well as the peaks at δ 4.98, δ 5.05 and δ 5.81 using the "Integrate" icon (under the "Process" tab) and the highlight ("Define new Region") icon. Left click and drag through the integrals.
            NOTE: If there is need to focus on a region, click on the highlight icon to deactivate and left click and drag the mouse to zoom in on the region. To adjust the threshold intensity, use the middle mouse button if needed. Click on the highlight icon again to make the integration function active, then move to the next peak.
            1. Normalize the sum of the above integrals to 100 by right clicking on the integral value that appears under the signal and select "Normalize sum of integrals". Input the value "100" in the box and click the "Return and Save" to exit the integration mode.
         2. When using BHT as an internal standard, integrate the peak at δ 6.98 and set the integral equal to the millimoles of BHT per 0.5 mL of the stock solution.
         3. Integrate the peaks of interest (see step 2.3.1.3.1) extending 10 Hz from each side of the peak, when possible.
         4. Proceed to perform ¹³C-NMR spectra acquisition and processing in a similar manner.
   2. ¹³C-NMR spectra
      1. Acquisition of the NMR data
         1. Go to the ¹³C data set and use the inverse gated decoupled pulse sequence, "zgig" by typing "pulprog zgig" in the command line.
            NOTE: To run a carbon experiment with the standard broadband decoupled pulse sequence, type "pulprog zgpg" in the command line.
         2. Type the following commands on the command line to set up the spectral width in ppm, the center of the RF transmitter, the number of scans, the number of dummy scans, the number of data points and the pulse duration for a 90° pulse angle: "sw 200", "o1p 95", "ns 16" "ds 2", "td 64K" and "p1 (as determined by pulse calibration)" (see step 2.2.1.4).
         3. Set a relaxation delay of 35 s for the 500 MHz instrument or 45 s for the 850 MHz instrument by typing "d1 35s" or "d1 45s", respectively, in the command line. When using BHT, relaxation delay should be 50 s in the 500 MHz instrument and 60 s in the 850 MHz instrument.
         4. Set the receiver gain (RG) to an appropriate value using the command "rga" for automatic calculation of RG.
         5. Type "digmod baseopt" in the command line to acquire a spectrum with improved baseline.
         6. Start the acquisition by typing the pulse-acquire command "zg" in the command line.
      2. Processing of the NMR data
         1. Type "si 64K" in the command line to apply zero-filling and set the size of the real spectrum to 64K.
         2. Set the line broadening parameter to 1.0 Hz by typing "lb 1.0" in the command line to apply a weighting function (exponential decay) with a line broadening factor of 1.0 Hz prior to Fourier transform.
         3. Execute Fourier-transformation by typing "efp" in the command line.
         4. Perform Automatic phase correction by typing the command "apk" in the command line. If additional phase adjustments are required to further improve the spectrum, click on the "Process tab," then click on the "Adjust Phase" icon and the phase correction icons for zero-order (0) and first-order phase (1) correction.
            1. While clicking on the zero-order and first-order phase correction icons, drag the mouse until all of the signals are in positive absorption mode. Apply and save the phase correction values by clicking the "Return and Save" button to exit the phase correction mode.
               NOTE: For carbon spectra recorded on Larmor frequency of 214 MHz (the 850 MHz instrument) the correction of the frequency dependent errors (first-order) may be challenging and time consuming for less experienced users because of the large off-resonance effects of the 90° pulse.
         5. Apply a polynomial fourth-order function for base-line correction upon integration by typing the command "abs n" in the command line.
         6. Report chemical shifts in ppm from TMS (δ = 0). Click on the calibration ("Calib. Axis") icon, and place the cursor with the red line on top of the NMR signal to be referenced. Left click and type in "0".
      3.  NMR data analysis
         1. Integrate the spectral region from δ 175 to δ 171 using the "Integrate" icon (under the "Process" tab) and the highlight ("Define new Region") icon. Left click and drag through the integrals.
            NOTE: If there is need to focus on a region, click on the highlight icon to deactivate and left click and drag the mouse to zoom in on the region. Click on the highlight icon again to make the integration function active, then move to the next peak.
            1. Set the integral to 100 by doing a right click on the integral value that appears under the signal and select "Calibrate Current Integral". Input the value "100" in the box and click the "Return and save" to exit the integration mode.
         2. When using BHT as an internal standard, integrate the peak at δ 151.45 and set the integral equal to the millimoles of BHT per 0.5 mL of the stock solution.
         3. Integrate the peaks of interest extending 5 Hz from each side of the peak (see step 2.3.2.3.1).

Results

¹H and ¹³C NMR spectra were collected for commercially available fish oil supplements using two NMR instruments; an 850 MHz and a 500 MHz spectrometer. These spectra can be used for the quantitative determination of components of fish oil, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), as well other compounds such as n-1 acyl chains and nutritionally important index such as the n-6/n-3 ratio. The quantification can be p...

Discussion

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 ...

Materials

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