Analysis of ascorbic acid, citric acid and benzoic acid in orange juice

Importance of the Topic

Quality assessment of antioxidants and preservatives in fruit juices is essential for food safety, regulatory compliance and nutritional labeling. Ascorbic acid (vitamin C) acts both as an antioxidant and nutritional marker, while citric and benzoic acids serve as acidity regulators and microbial growth inhibitors. Reliable quantification of these compounds supports industry quality control, shelf-life studies, and consumer protection.

Study Objectives and Overview

This application note describes the development, partial validation, and transfer of a reversed-phase HPLC method to a faster UHPLC protocol for simultaneous determination of ascorbic acid, citric acid, and benzoic acid in orange juice. Key aims included:

• Establishing linearity, precision, detection limits and recovery in aqueous and matrix samples
• Evaluating robustness of critical chromatographic parameters
• Quantifying analytes in commercial orange juice brands
• Transferring the method to a short UHPLC run to reduce analysis time and solvent usage

Instrumentation

• HPLC: Agilent 1260 Infinity Binary Pump, Autosampler, Thermostat, Column Compartment, Diode Array Detector (DAD)
• UHPLC: Agilent 1290 Infinity Binary Pump, Autosampler, Thermostat, Column Compartment, DAD
• Column: Agilent Poroshell 120 EC-C18, 4.6×100 mm, 2.7 µm (HPLC) and 3.0×75 mm, 2.7 µm (UHPLC)
• Detector wavelengths: 210 nm (citric acid), 230 nm (benzoic acid), 243.5 nm (ascorbic acid)

Methodology

Standards of ascorbic, citric and benzoic acids were prepared in phosphate buffer (pH 2.5) and diluted to cover expected juice concentrations. Orange juice samples were acidified to pH 2.5, centrifuged and filtered prior to injection.
Chromatographic conditions:

• Mobile phase A: 20 mM KH2PO4, pH 2.5; B: 60% methanol + 40% acetonitrile
• Gradient elution: 5% B hold, ramp to 25% B, then to 90% B for HPLC (25 min), or to 70% B for UHPLC (5 min)
• Flow: 1.0 mL/min (HPLC), 1.5 mL/min (UHPLC); column temperature 20 °C; sample tray at 4 °C
• Injection: 5 µL (HPLC), 4 µL (UHPLC)

Main Results and Discussion

Linearity was demonstrated over 10–225 µg/mL for ascorbic acid (R2 = 0.9998), 5,500–9,000 µg/mL for citric acid (R2 = 0.9995) and 0.2–50 µg/mL for benzoic acid (R2 ≈ 1.0). LOD/LOQ for benzoic acid were 0.05/0.2 µg/mL (S/N = 3/16). Precision (RSD) of peak area was below 1.9% and retention time below 0.13%. Robustness testing (±2% flow, ±5% temperature, ±5% injection volume, ±3 nm wavelength) confirmed method stability, though ascorbic acid area was sensitive to parameters, highlighting the need for tight control of temperature and flow.
Recovery studies in spiked juice achieved >90% for all analytes. Analysis of three commercial orange juices showed variable native and added levels: ascorbic acid from 35 to 145 µg/mL, citric acid from 3,160 to 8,900 µg/mL, and trace benzoic acid (0–2.25 µg/mL), sometimes undiclosed on labels.

The UHPLC transfer shortened run time five-fold to 5 minutes while maintaining the same sensitivity and linearity. Solvent consumption decreased by 68%, and potential losses of labile ascorbic acid during autosampler storage were minimized.

Benefits and Practical Applications

• High throughput quality control of citrus juices and other beverages for antioxidants and preservatives
• Compliance with food additive regulations and nutritional labeling
• Reduced analysis time and solvent use by UHPLC improves laboratory efficiency
• Reversible gradient and DAD detection enable flexible method adaptation to related matrices

Future Trends and Opportunities

Advances in column technologies and UHPLC–MS coupling may allow further reduction in analysis time and enhanced selectivity. Miniaturized systems and green solvents will align with sustainability goals. Integration of online sample preparation and chemometric approaches can streamline multi-analyte monitoring in complex food matrices.

Conclusion

A robust HPLC–DAD method was developed and partially validated for simultaneous quantification of ascorbic, citric and benzoic acids in orange juice. The method transferred successfully to a rapid UHPLC format, preserving analytical performance while boosting throughput and reducing solvent use. Both approaches provide reliable tools for food quality control.

References

1. INCHEM. Chemical Safety Information on Benzoic Acid and Derivatives. URL accessed 2011.
2. US FDA. Benzene in Soft Drinks and Other Beverages data through May 16, 2007.
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5. Novakova L, Solich P, Solichova D. HPLC Methods for Determination of Ascorbic and Dehydroascorbic Acids. Trends Anal Chem. 2008;27:942–958.
6. Margolis SA, Park E. Stability of Ascorbic Acid in Solutions Stored in Autosampler Vials. Clin Chem. 2001;47:1463–1464.