Microextraction method traces tea polycyclic aromatic hydrocarbons with green assessment

Tea is the world’s most popular beverage after water, with global consumption estimated at roughly 7.3 billion kilograms in 2023 and projected to climb to 8.3 billion kilograms by 2029. Brewed from the leaves of Camellia sinensis and Camellia assamica, tea is prized for its antioxidant activity, cardiovascular benefits, and potential cognitive enhancement. But a growing body of research has revealed an unwelcome passenger in tea leaves: polycyclic aromatic hydrocarbons, or PAHs, a family of organic contaminants formed whenever organic matter burns incompletely. Some of these compounds are classified as priority pollutants by the United States Environmental Protection Agency, and long-term exposure has been linked to carcinogenicity, genetic damage, and impairment of the immune system.

Now a team of researchers led by Kim Liu Tan and Yong Foo Wong, working with collaborators including Jalal T. Althakafy, Yin-Hui Leong, Yong Shen Chua, and Faiz Bukhari Mohd Suah, has unveiled a new analytical method that can detect eight of the most hazardous PAHs in tea using a fraction of the solvent demanded by conventional techniques. The work, published in Food Chemistry: X, combines dispersive liquid–liquid microextraction, known as DLLME, with ultra-performance liquid chromatography and fluorescence detection, and it delivers detection limits that rival or surpass far more cumbersome methods.

The problem the researchers set out to solve is both chemical and practical. PAHs typically occur in food at trace concentrations, embedded within complex matrices that are rich in pigments, polyphenols, and other interfering compounds. Traditional approaches such as liquid-liquid extraction and QuEChERS require large volumes of sample and organic solvents and involve laborious multi-step procedures. Solid-phase extraction offers an alternative, but conventional cartridges are costly and generally cannot be reused. For food-safety laboratories tasked with screening hundreds of samples, the trade-off between sensitivity, speed, cost, and environmental footprint has long been a source of frustration.

DLLME offers a clever way out of that dilemma. The technique relies on a ternary solvent system: an aqueous sample solution, a few tens of microliters of an extraction solvent in which the PAHs readily dissolve, and a disperser solvent that, when rapidly injected, shatters the extractant into a cloud of fine droplets. Because the collective surface area of these droplets is enormous, analytes migrate from the aqueous phase into the organic droplets within seconds. A brief centrifugation step then settles the droplets into a small, dense sediment that can be collected and injected directly into the chromatograph. The entire extraction consumes only microliters of toxic solvent, which is why DLLME consistently earns superior scores on green-chemistry metrics such as AGREE and AGREEprep compared with classical extraction protocols.

The Malaysian team optimized every parameter of this process with typical rigor. They screened extraction solvents, dispersive solvents, salt content, pH, sample volume, vortex duration, and centrifugation conditions. Among the findings: adding salt, a step often used to boost extraction in other contexts, actually hurt performance, because the target PAHs are strongly non-polar and added salt increased the viscosity of the aqueous phase, slowing the diffusion of analytes toward the organic droplets. Sample pH likewise mattered little, since PAHs are non-ionizable and do not undergo acid–base dissociation; neutral pH simply gave the most consistent recoveries. The optimum recipe settled on 10 milliliters of prepared tea sample, 40 seconds of vortex mixing, and 3 minutes of centrifugation at 3000 times gravity.

Once the extraction chemistry was locked down, the group validated the complete DLLME–UPLC-FLD method against internationally accepted criteria. Matrix-matched calibration curves spanning 75 to 400 nanograms per liter for most analytes, and 200 to 1000 nanograms per liter for indeno(1,2,3-c,d)pyrene, produced correlation coefficients above 0.99. The higher range for the latter compound reflects its intrinsically low fluorescence quantum yield and short fluorescence lifetime, which make it inherently harder to detect optically. Limits of detection ranged from 3.87 to 32.5 nanograms per liter, and limits of quantification from 11.73 to 98.47 nanograms per liter. Those detection limits are lower than those reported for a magnetic solid-phase extraction–GC-MS method applied to soft drinks and non-alcoholic beers, and comparable to a membrane-assisted solvent extraction GC-MS protocol used for river water, apple juice, red wine, and milk.

Precision and accuracy proved equally robust. Intra-day relative standard deviations, measured over nine replicates, were no more than 0.10 percent for retention times and 9.21 percent for peak areas. Across 27 measurements taken over three consecutive days, inter-day precision remained within 1.60 percent for retention times and 9.91 percent for peak areas. Recovery studies, performed by spiking tea samples at three concentration levels corresponding to low, medium, and high points within the calibration range, yielded mean recoveries between 85.13 and 101.71 percent, comfortably within the acceptance windows demanded by regulatory food-safety guidelines.

The eight PAHs targeted by the method are precisely those recommended for monitoring by the European Food Safety Authority: benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(ghi)perylene, chrysene, dibenz(a,h)anthracene, and indeno(1,2,3-c,d)pyrene. These compounds can accumulate in tea leaves from contaminated soil, air, or water during cultivation, and can also form during tea manufacturing, particularly in high-temperature processing steps such as drying and firing. Because tea is infused in hot water before consumption, the fraction of PAHs that actually transfers into the brewed beverage is a critical determinant of dietary exposure, and reliable, sensitive measurement is the essential first step in any risk assessment.

The choice of fluorescence detection paired with ultra-performance liquid chromatography deserves particular attention. Gas chromatography coupled to mass spectrometry remains the regulatory gold standard for PAH analysis, and tandem GC-MS/MS is increasingly favored for trace-level alkylated, nitro-, and oxy-PAHs. But GC-based methods demand derivatization-free volatilization of analytes and expensive instrumentation. HPLC with fluorescence detection offers comparable selectivity and sensitivity for the parent, unsubstituted PAHs, with simpler operation and lower running costs. UPLC’s sub-two-micrometer particles sharpen peaks and shorten run times, which multiplies the number of samples a laboratory can process per day. Coupled with a rapid, solvent-sparing DLLME cleanup, the workflow becomes genuinely practical for routine surveillance rather than a research curiosity.

Beyond the raw analytical numbers, the study explicitly assessed the greenness of the new protocol using modern metric tools, and DLLME scored higher than both liquid-liquid extraction and solid-phase extraction for analyzing complex food matrices. In an era when analytical chemistry laboratories are under mounting pressure to reduce their solvent waste and carbon footprint, this dimension of the work is far from cosmetic. A method that uses microliters of extractant instead of tens of milliliters dramatically cuts both operating costs and hazardous waste disposal burdens, while the absence of ionic liquids or deep eutectic solvents removes additional preparation steps that had limited the practicality of earlier DLLME variants applied to tea.

The implications for public health are direct. With a validated, fast, and green method in hand, regulators and food producers can screen tea batches for PAH contamination more frequently and more cheaply, feeding better data into dietary exposure models that estimate how much of these carcinogens consumers actually ingest. The authors frame their work as method development, validation, greenness assessment, and dietary exposure risk assessment rolled into one, an end-to-end pipeline that takes a contamination problem from bench chemistry to consumer-relevant risk numbers. For the billions of people who begin their day with a cup of tea, that pipeline offers reassurance that the science watching over the supply chain is getting faster, cleaner, and more precise.