Rapid LC–MS-MS Analysis of Heterocyclic Amines in Salmon

Heterocyclic amines (HCAs) are a class of compounds that are formed when amino acids from meat proteins react with glucose and creatine under high-temperatures, as is associated with frying or grilling over an open flame (1–3). Studies have correlated the consumption of high levels of HCAs with an increased risk of some forms of cancer. Though this has only been demonstrated in animal models, investigations into the possible correlation between cooked meat consumption and elevated cancer risks will continue in both Europe and the United States (1).

Analysis of HCAs in food products can be challenging due to a number of factors including: (1) their relatively low level in food products, necessitating the use of highly sensitive techniques such as LC–MS-MS; (2) the complex nature of the sample matrix, which can compromise HPLC column lifetime and confound analysis; and (3) the basic nature of these analytes, which can lead to peak tailing issues. Herein, we present a sensitive method for the analysis of HCAs which utilizes solid-phase extraction (SPE) for cleanup and a sub-2 µm core-shell UHPLC column for the subsequent LC–MS-MS analysis.

Figure 1: Representative chromatogram of 12 heterocyclic amines analyzed using a Kinetex 1.7 µm C18 core-shell UHPLC column.

Experimental Conditions

Sample Preparation and Solid-Phase Extraction:

For detailed sample preparation and SPE conditions, please contact Phenomenex. In brief, salmon tissue meat purchased from a grocery store was pulverized and then spiked with a mixture of HCAs and appropriate internal standards at a final concentration of 25 ng/g of tissue. This mixture was then subject to liquid-liquid extraction using hexane and acetonitrile. The aqueous acetonitrile layer was recovered for subsequent solid phase extraction using a polymer-based strong cation-exchange cartridge (Strata™ -X-C 60 mg/3 mL).

Figure 2: A representative extracted ion chromatogram for 7,8- and 4,8-dimethyl IQx, isomeric HCAs that must be baseline-resolved to enable accurate quantitation using LC–MS-MS.

LC/MS/MS Conditions:

Column: Kinetex® 1.7 µm C18 with SecurityGuard™ ULTRA C18

Dimensions: 50 × 3.0 mm

Mobile Phase: A: 30 mM Ammonium formate; B: Acetonitrile

Gradient: (80:20) A/B to (40:60) A/B in 3 min, then step to 100% B and hold for 1 min, then re-equilibrate at (80:20) A/B for 1 min

Flow Rate: 600 µL/min

Column Temperature: Ambient

Detection: MS using API 4000™ detector

Injection Concentration: 25 ng/mL

Sample:

References

(1) http://www.cancer.gov/cancertopics/factsheet/Risk/cooked-meats.

(2 )P. Arvidsson et al., J. of Food Sci. 64(2), 216–221 (1999).

(3) T. Sugimura et al., Cancer Sci. 95(4), 290–299 (2004).

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