High Resolution UHPLC Analysis of Naringin in Grapefruit Juice

John W. Henderson Jr, Judy Berry and Maureen Joseph, Agilent Technologies Inc., Wilmington, Delaware, USA.

Grapefruit juice was analysed by gradient UHPLC for naringin — a major flavanoid in grapefruit juice that gives it its bitter taste. A ZORBAX Rapid resolution HD (RRHD) StableBond-C18 2.1 × 150 mm, 1.8 μm column was able to resolve over 60 peaks, including naringin in 7 minutes, and was re-equilibrated in just three minutes by operating at a high flow rate (0.8 mL/min). The high pressure generated (965 bar) is well within the 1200 bar operating range of the RRHD column and 1290 Infinity LC.

Introduction

Gradient reversed-phase HPLC with long (150 mm) 1.8 μm columns are an option for analysing complex matrices with numerous analytes, such as grapefruit juice. Sufficiently flushing the column with strong solvent and re-equilibrating with weaker starting conditions is necessary but can be time consuming. Rapid resolution high definition (RRHD) 1.8 μm, columns can withstand pressures of up to 1200 bar, so doubling or quadrupling the flow rate is reasonable and proportionally reduces the total analysis time including re-equilibration.

Experimental

See figures for method parameters. A grapefruit was hand squeezed and its juice filtered through a 0.22 μm RC syringe filter directly into an autosampler vial for analysis.

Results

Figure 1 shows the analysis of filtered grapefruit juice using a mobile phase gradient with an initial isocratic hold, a linear ramp in organic solvent to elute peaks of interest, a high organic flush and a re-equilibration to starting conditions. The 2.1 × 150 mm, 1.8 μm column offers high efficiency even at a high 0.8 mL/min flow rate and a system pressure range of 480 to 965 bar, well within the 1200 bar limit of the RRHD column and Agilent 1290 Infinity LC. More than 60 peaks in grapefruit juice were resolved in 7 min, including the main peak of interest, naringin. The amount of naringin was 0.12 mg/mL juice.

Figure 1: High resolution gradient analysis of grapefruit juice.

Figure 2 superimposes chromatograms of six injections. Retention time RSD for naringin is 0.03% and RSD for peak area is 0.83, indicating that 3 minutes is sufficient for both flushing with strong solvent and re-equilibration to initial gradient conditions. The benefit of an RRHD 2.1 × 150 mm column UHPLC is clearly demonstrated: a re-equilibration time of just 3 min versus 10 min — the time needed to flush and re-equilibrate (5 column volumes each) a 2.1 × 150 mm column with 3.5 or 5 μm particles operated at 0.2 mL/min.

Figure 2: Six chromatograms superimposed of a gradient UHPLC analysis for naringin in grapefruit juice.

Conclusion

The high efficiency and resolving power of sub-2 μm RRHD columns and the ability to operate at high flow rates under UHPLC conditions, make these columns a valuable tool for rapid gradient analysis of complex samples.

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