Determination of Oxalic Acid Content in Fruits and Vegetables by High-Performance Liquid Chromatography (HPLC)

Determination of Oxalic Acid Content in Fruits and Vegetables by High-Performance Liquid Chromatography (HPLC)
1. Experimental Principle and Method Design
As a common organic acid in fruits and vegetables, oxalic acid content directly affects the taste and nutritional value of food. This experiment uses reversed-phase high-performance liquid chromatography (RP-HPLC). Under acidic mobile phase conditions, oxalic acid is baseline-separated from interfering substances using a C18 chromatographic column. An ultraviolet detector set at 210 nm is used for quantitative analysis, based on the UV absorption characteristics of the carboxyl groups in oxalic acid molecules.
2. Standard Curve and Sample Preparation
A gradient dilution method is used to prepare standard solutions: accurately weigh 25.0 mg of oxalic acid dihydrate and dilute to 25 mL with ultrapure water to obtain a 1 mg/mL stock solution. This is sequentially diluted into a series of standard solutions at 50, 100, 200, 400, and 800 µg/mL, with each concentration injected in triplicate.
For sample preparation, microwave-assisted extraction is used: weigh 5.00 g of fruit/vegetable homogenate, add 10 mL of 0.1 mol/L hydrochloric acid solution, treat at 60°C with microwave for 10 minutes, filter through a 0.45 µm membrane filter, and collect the filtrate for testing.
3. Optimization of Chromatographic Conditions
The mobile phase is a 0.01 mol/L potassium dihydrogen phosphate buffer (pH 2.5)–acetonitrile (95:5) system, with a flow rate of 0.8 mL/min and column temperature of 35°C. Adjusting the acetonitrile proportion shows that when the organic phase exceeds 10%, the retention time of oxalic acid shortens to less than 3 minutes, but peak tailing occurs. Under the final optimized conditions, the retention time of oxalic acid is 4.2 minutes, achieving complete separation from adjacent citric acid and malic acid (resolution > 1.5).
4. Method Validation
Linear range verification shows a good linear relationship between peak area and concentration in the range of 10–1000 µg/mL (R² = 0.9993). The limit of detection (LOD), determined by the signal-to-noise ratio method, is 0.5 µg/mL. In repeatability tests, the RSD for six replicate determinations of the same spinach sample is 1.8%. Spike recovery experiments at three levels (80%, 100%, and 120%) yield average recoveries of 98.2%, 102.4%, and 97.8%, respectively, meeting the requirements for quantitative analysis.
5. Analysis of Real Samples
Six commercially available fruits and vegetables were tested: spinach (356 ± 12 mg/100 g), celery (215 ± 9 mg/100 g), tomato (18 ± 2 mg/100 g), apple (6 ± 1 mg/100 g), banana (not detected), and broccoli (89 ± 5 mg/100 g). A comparison with the national standard method (GB 5009.277-2016) shows that the relative deviation between the two methods is less than 5%, verifying the reliability of this method. In particular, the HPLC method exhibits higher sensitivity than traditional titration methods when analyzing low-content samples.
6. Key Notes
Strictly control the pH value during sample pretreatment. If the pH of the extract exceeds 3, calcium oxalate precipitation will lead to low test results. The mobile phase should be prepared fresh daily to avoid salt precipitation and column blockage. After every 20 injections, flush the column with acetonitrile–water (30:70) for 30 minutes to prevent column efficiency degradation. For pigment-containing samples (e.g., purple cabbage), it is recommended to add a solid-phase extraction purification step before injection.
7. Application and Expansion Directions
This method can be extended to the detection of oxalic acid in biological samples such as urine and blood. By adjusting the mobile phase composition (e.g., adding tetrabutylammonium hydroxide), oxalic acid and its metabolites can be determined simultaneously. Combined with a mass spectrometry detector, a more precise trace detection method can be established. In the food processing industry, this method can provide important data support for the selection of low-oxalic acid varieties and the optimization of cooking processes.