HPLC-UV Quantifies Thiols in Berry Superfruits

In an article published in the journal Food Chemistry,¹ researchers from Poland’s University of Lodz presented the first methods for determining total and reduced glutathione (GSH), cysteine (Cys), cysteinyl-glycine (Cys-Gly), and homocysteine (Hcy) in berries using high-performance liquid chromatography with ultraviolet (HPLC-UV) detection.

What are Superfruits, and Why Has Research into Their Beneficial Compounds—Particularly Thiols in Berries—Remained Limited?

"Superfruits" is basically a marketing term for fruits that are natural and unaltered—not genetically modified or heavily processed—but that pack an especially high amount of nutrients known to be good for your health. Even though so-called functional foods are popular for their potential health perks, we still do not have thorough data on exactly what is in them or how much. There is also limited detailed information about which specific beneficial compounds are responsible for these health effects. While superfruits contain a wide variety of compounds, most research so far has focused mainly on vitamins, minerals, and antioxidants—with polyphenols (a group of plant compounds) getting the most attention out of all of them.^2-4

The authors point out in the paper1 that, even though berries are well known for their antioxidant qualities, no one has yet done a thorough study to map out the complete range of thiol compounds (sulfur-containing antioxidants) they contain. “Furthermore,” they continue, “to the best of our knowledge, a critical analytical bottleneck exists, as no validated method is currently available for the accurate quantification of both reduced and total thiol levels in such complex plant tissues.”¹

How Was the Method Developed and Validated?

This method uses N-acetylcysteine as a reference point for measurement. To prepare samples, researchers first blended them in a buffer solution, then used tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to break apart certain bonds within proteins, but only when they wanted to measure total thiol content (a type of sulfur-containing compound). Next, they added a tagging chemical to make the target compounds easier to detect, treated the sample with an acid to stabilize it, and finally filtered everything through a fine membrane to remove any solid particles.¹

To separate and measure the compounds, they ran the samples through a specialized column using a technique that gradually changes the mix of two liquids (trichloroacetic acid and acetonitrile) to pull apart the different substances. The method proved reliable across a range of concentrations—working well for measuring glutathione (GSH) between 0.5 and 50 micromoles per liter, and for cysteine and cysteine-glycine between 0.5 and 20 micromoles per liter.¹

What Did the Method Reveal About Thiol Compound Levels in Different Berries?

When applied to a variety of berries sold in Poland (including strawberries, raspberries, blueberries, blackberries, mixed forest berries, haskap berries, wild strawberries, currants, gooseberries, cranberries, chokeberries, and elderberries) the results showed that glutathione was by far the most abundant of these sulfur-containing compounds. Cysteine showed up in smaller amounts, cysteine-glycine was barely detectable, and homocysteine was not found at all.¹

“From a practical perspective,” write the authors of the paper,¹ “our results offer valuable insights into public health, helping health-conscious individuals make informed dietary choices and purchase decisions. The proposed analytical tools are well-suited for evaluating the impact of food processing on antioxidant levels. A key strength of these methods lies in their simplified sample preparation procedure and excellent analyte stability at all stages. The entire protocol for both reduced and total thiols takes roughly 40 min using no more than 25 mL of inexpensive chemicals. These features make our methods ideally suited for high-throughput food quality control.”

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References

1. Gaweł. M.; Głowacki, R.; Piechocka, J. Analysis of Berry Superfruits for Reduced and Total Thiol Content by LC-UV-based Methods. Food Chem. 2026, 529, 151137. DOI: 10.1016/j.foodchem.2026.151137
2. Chang, S. K.; Alasalvar, C.; Shahidi, F. Superfruits: Phytochemicals, Antioxidant Efficacies, and Health Effects - A Comprehensive Review. Crit Rev Food Sci Nutr. 2019, 59 (10), 1580-1604. DOI: 10.1080/10408398.2017.1422111
3. Liu, J.; Xu, D.; Chen, S. et al. Superfruits in China: Bioactive Phytochemicals and Their Potential Health Benefits - A Review. Food Sci Nutr. 2021, 9 (12), 6892-6902. DOI: 10.1002/fsn3.2614
4. Nagaraja, S. K.; Mir, J. I.; Verma, M. K. et al. A Comprehensive Review on Nutrition, Phytochemistry, Pharmacology and Food Application Perspectives of Bioactive Rich Superfruits. Future Postharvest Food2026, 1-22. DOI: 10.1002/fpf2.70040