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....

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....

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

List of materials used in this article

Name	Company	Catalog Number	Comments
Avance III 850 NMR instrument	Bruker
Avance III 500 NMR instrument	Bruker
TCI 5 mm probe	Bruker		Helium cooled inverse (proton deetected) NMR probe featuring three independent channels (1H, 13C, 15N)
BBO prodigy 5 mm 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