Identification and Quantification of Triacylglycerols in Expired Dairy Products and Seafood Using NARP-HPLC-APCI-MS

Reporter US Volume 33.4 (Food & Beverage Supplement)

Marco Beccaria1 and Luigi Mondello1,2
1Chromaleont s.r.l., c/o “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 Messina, Italy
2“Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 Messina, Italy

Dietary fats, whether derived from dairy, like cheese and yogurt, or marine organisms, like tuna and shrimp, are very complex sources of triacylglycerols (TAGs). Factors such as climate, diet, and stage of lactation influence the TAG profile. Two current realities contribute to the high level of interest in the TAG composition of dietary fats. First, TAGs play important roles in physiology and nutrition. Second, there is a need to reduce, recycle, or repurpose food waste. Many foods that are past their consumer shelf-life may still have important components, such as TAGs, that can be used as dietary supplements or for research purposes.

The extraction of the lipid fraction is a critical step in the analysis of total lipids because contamination or improper extraction may lead to erroneous results. During lipid extraction, samples should be prepared and analyzed carefully to prevent oxidation of lipids and hydrolysis, since artifact production can compromise the identification and quantification of the lipid fraction components. In this report, we describe procedures to extract lipids from different fatty food samples and analyze their TAG profile using LC/MS methodology. This work is an initial step that will ultimately be applied to researching the changes to the TAG profile as the food ages.

Extracting Samples for Lipid Analysis

Exhaustive extraction of the whole lipid content for the dairy samples was achieved according to the Schmid–Bondzynski–Ratzlaff (SBR) extraction method (IDF-International Standard 5B, 1986)1. For the yogurt samples, ISO 14156:2012 method was used to ensure the exhaustive extraction of the whole lipid content. The tuna and shrimp were treated with the Bligh and Dyer method2.

1. Cheese
Various cheeses were studied, including mozzarella, buffalo mozzarella, invernizzina, gorgonzola, stracchino, mascarpone, buffalo ricotta, and robiola. The lipid fraction was extracted by adding 10 g of the cheese sample (accurately weighed), 20 mL of 0.2 N hydrochloric acid, and 20 mL of ethyl alcohol to a volumetric flask. The flask was placed in a 50 °C water bath and gently shaken for 30 minutes with constant magnetic stirring until a homogenous suspension was formed. After cooling the flask to room temperature under running water, 210 mL of n-hexane and diethyl ether (1:2, v/v) were added and the mixture was shaken for an additional 15 minutes. The suspension was left to stand for 10 minutes to allow phase separation. This extraction protocol was repeated three times. The organic extracts were pooled, dried over anhydrous sodium sulfate, filtered, and taken to dryness under vacuum. The final dry residue was stored at - 18 °C until use.

2. Yogurt
The extraction of the yogurt lipid fraction proceeded by adding 10 g of the yogurt sample (accurately weighed), 20 mL of 0.5 N NH3, and 80 mL of ethyl alcohol to a separatory funnel. While swirling the flask, 100 mL of diethyl ether followed by 100 mL of n-pentane were added. The suspension was left to stand for 10 minutes to allow phase separation. The ether phase was recovered and dried over anhydrous sodium sulfate, filtered, and then taken to dryness by a rotary evaporator. The final dry residue was stored at -18 °C until use.

3. Seafood
The lipid fraction of tuna and shrimp was extracted by adding 10 g of the sample (accurately weighed) and 30 mL of chloroform/methanol (1:2, v/v) to a separatory funnel. After stirring, the organic phase was removed and transferred to another separatory funnel along with 10 mL of chloroform and 10 mL of distilled water followed by stirring. The resulting solution was divided between five or six centrifuge tubes and centrifuged for 15 minutes at 3,000 rpm. Two different phases were obtained. The lower phase, which contained the lipid fraction, was collected and reserved. The upper phase was collected and combined with the remaining solid sample in the first separatory funnel and re-extracted two additional times. The three resulting extracts were pooled, sonicated, dried over anhydrous sodium sulphate, filtered, and then taken to dryness under vacuum. The final dry residue was stored at -18 °C until use.

Analysis by NARP-HPLC-APCI-MS

Samples ranging from 15 to 30 mg of each of the dry residue from the final lipid extracts were diluted with 1 mL of acetone and filtered through a 0.45 ìm Acrodisc® nylon membrane filter prior to LC/MS analysis. The TAG profiles of the lipid extracts were analyzed using an optimized NARP (non-aqueous reversed-phase)-HPLC method in combination with positive atmospheric- pressure chemical ionization mass spectrometry (APCI-MS) detection. High-resolution separation was provided by an Ascentis® Express U/HPLC column packed with C18-modified Fused-Core® silica particles.

Results and Discussion

Representative chromatograms of the various dairy products tested are shown in Figures 1 through 3. In Figure 4 the enlargement of two regions of the TIC chromatogram of NARP-HPLC-APCI-MS analysis of invernizzina cheese is shown. In Table 1 the identified TAGs obtained by NARP-HPLC-APCI-MS analysis of the invernizzina cheese sample, which was the most complex one, are reported. Understanding how the TAG profile is modified during storage and after the expiration date is currently under investigation by our laboratory. This data will be correlated with the fatty acid profile obtained by GC-FID/MS experiments. The aim will be to define the nutritional value after the expiration date of different products to evaluate the possibility of utilizing them for other purposes, such as animal feed, or extraction of bioactive compounds for use as nutritional supplements or in biochemical research.

Table 1. TAGs in the Invernizzina Cheese Sample Identified by NARP-HPLC-APCI-MS
Retention time (RT), compound number (N), and area % are reported. Diacylglycerols (DAGs) are identified with [ ] symbol.

 

NARP-HPLC-APCI-MS Analysis of TAGs from Noccioline Mozzarella Cheese

Figure 1. NARP-HPLC-APCI-MS Analysis of TAGs from Noccioline Mozzarella Cheese

Conditions
column:
Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 µm (Product No. 53829-U); mobile phase: [A] acetonitrile; [B] 2-propanol (IPA); gradient: 0 to 70% B in 50 min; held at 70% B for 5 min; to 0% B in 1 min; flow rate: 1 mL/min; detector: MS, APCI(+), mass range 250–1100 m/z; injection: 7 to 20 µL; sample: lipid extract, 15 to 30 mg/mL in acetone
LC/MS CONDITIONS: Instrument: Shimadzu LCMS-2010 APCI(+): range 250-1100 m/z; nebulizing gas: (N2); flow: 2.0 L/min; heat block: 300 °C; CDL temperature: 300 °C; interface temperature: 450 °C; detector voltage: 1.6 kV

 

NARP-HPLC-APCI-MS Analysis of TAGs from Yogurt

Figure 2. NARP-HPLC-APCI-MS Analysis of TAGs from Yogurt
Conditions as in Figure 1.
 

NARP-HPLC-APCI-MS Analysis of TAGs from Invernizzina Cheese

Figure 3. NARP-HPLC-APCI-MS Analysis of TAGs from Invernizzina Cheese
Conditions as in Figure 1. Partition Number (PN): The total number of carbon atoms minus twice the number of double bonds.
 

Enlargements of the TIC Chromatogram of the NARP-HPLC-APCI-MS Analysis of Invernizzina Cheese

Figure 4. Enlargements of the TIC Chromatogram of the NARP-HPLC-APCI-MS Analysis of Invernizzina Cheese

Panel A: 0 - 25.5 minutes
Panel B: 25.5 - 50 minutes

Legal Information

Acrodisc is a registered trademark of Pall Corporation
Ascentis is a registered trademark of Sigma-Aldrich Co. LLC
Fused-Core is a registered trademark of Advanced Materials Technology, Inc.

Materials

     
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