Analysis of Limonin in Citrus Juice Using QuEChERS and LC–MS/MS

This application note outlines a simple, fast, and cost-effective QuEChERS-based method for the determination of limonin in citrus juice. Limonin is extracted from a variety of juice samples using acetonitrile and citrate-buffered salts. The sample extract undergoes cleanup by dispersive-SPE (dSPE) using primary-secondary amine (PSA), C18, and graphitized carbon black (GCB) to remove unwanted matrix components, including sugars, acids, and pigments, and yield a clear sample extract. Analysis is performed by LC–MS/MS using a Selectra® C18 HPLC column (although HPLC-UV can also be used).

Procedure

1. Sample Extraction:

a) Allow citrus juice to come to room temperature and thoroughly mix the sample before analysis.

b) Transfer 10 mL of juice into a 50 mL polypropylene centrifuge tube.

c) Add 10 mL of acetonitrile.

d) Add the contents of the ECQUEU7-MP Mylar pouch and vortex or shake (manually or mechanically) for 5 min.

• For this work a SPEX® SamplePrep® 2010 Geno/Grinder® was used (1500 rpm).

e) Centrifuge the samples at ≥3000 rcf for 5 min.

2. Sample Clean-up of Extract

a) Transfer 1 ml of supernatant to a CUMPSC1875CB2CT 2 mL dSPE tube.

b) Vortex the samples for 30 s.

c) Centrifuge the samples at ≥3000 rcf for 5 min.

d) Transfer 500-600 µL of purified supernatant into an autosampler vial for analysis.

Instrumental

Results

Samples were fortified with limonin at 10 µg/mL (50 µL of a 2 mg/mL acetonitrile standard). A six-point matrix-matched calibration curves were prepared at 1, 2, 5, 10, 15, and 20 µg/mL. Non-fortified juice samples were extracted according to the QuEChERS procedure described above and 1 mL of purified extracts were fortified with 5, 10, 25, 50, 75, and 100 µL of a 200 µg/mL acetonitrile standard.

* One outlier was omitted from the final results.

Conclusion

This application note outlines a simple, fast, and cost-effective QuEChERS-based method for the determination of limonin in citrus juice. Citrus juice samples are extracted with citrate-buffered QuEChERS salts followed by dSPE cleanup of the supernatant using PSA, C18, and GCB, yielding a clear extract. Analysis is performed by LC–MS/MS using a Selectra® C18 HPLC column. Various citrus juices (lemon, lime, orange, and grapefruit) were evaluated for recovery and repeatability using fortified samples. Excellent recovery (88 to 97%) and repeatability (≤ 7.5%) were obtained using the outlined method.

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