NARP-HPLC-APCI-MS Analysis of TAGs from Noccioline Mozzarella Cheese on Ascentis^® Express C18

Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 μm particles (53829-U)

[A] acetonitrile; [B] 2-propanol (IPA)

0 to 70% B in 50 min; held at 70% B for 5 min; to 0% B in 1 min

1 mL/min

MS, APCI(+), mass range 250–1100 m/z

7-20 μL

lipid extract, 15 to 30 mg/mL in acetone

Shown here is a procedure to extract lipids from cheese and analyse the TAG profile using LC/MS methodology.

Sample preparation: 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. 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 μm Acrodisc nylon membrane filter prior to LC/MS analysis.

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

Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany