The new approach, described in Frontiers in Analytical Science by Rong Fu, Diane J. Sutcliffe, Ashok R. Dinasarapu, Ellen J. Hess and H. A. Jinnah, relies on ultra performance liquid chromatography coupled with photodiode array ultraviolet detection, commonly abbreviated UPLC-PDA. Unlike mass spectrometry-based metabolomics, which can be expensive and technically demanding, PDA detection exploits the strong ultraviolet absorbance of purines at 254 nanometers, allowing straightforward identification by retention time and spectral profile. The system consists of a Waters ACQUITY H-Class plus core with a quaternary solvent manager and an eLambda PDA detector, operating on a 2.1 by 100 millimeter reverse-phase C18 column packed with 1.8 micrometer particles at 40 degrees Celsius.

Separation chemistry is the crux of the method. Purines carry ionizable groups whose charge states shift dramatically with pH, so the Emory team designed a stepped gradient between two carefully formulated buffers. Buffer A contains 10 millimolar ammonium acetate and 1.5 millimolar tetrabutylammonium phosphate at pH 5.0, while buffer B contains 10 millimolar ammonium phosphate with the same ion-pairing reagent and 25 percent acetonitrile at pH 7.0. The tetrabutylammonium phosphate acts as an ion-pairing agent, taming the highly polar nucleotides so they interact with the reverse-phase column and elute as discrete peaks rather than co-eluting in the void volume. The gradient holds 100 percent buffer A for 11 minutes, ramps to 75 percent buffer B, then climbs to 100 percent buffer B before the column re-equilibrates for 9.5 minutes ahead of the next injection.

With this configuration, the method resolves all 15 target metabolites: ATP, ADP, AMP, adenosine, adenine, GTP, GDP, GMP, IMP, ZMP, guanosine, hypoxanthine, inosine, xanthine and uric acid. Only 5 microliters of sample are injected per run. Calibration with purified standards confirmed linear detection across a remarkable four-order-of-magnitude range from 0.1 to 100 micromolar, with all metabolites detectable down to approximately 100 nanomolar. Reproducibility proved equally impressive. When the same sample was analyzed five times within a single batch and across three batches on different days, the within-run coefficient of variance averaged 0.8 percent and the between-run variance averaged 1 percent for most analytes, comfortably under the 2 percent threshold the authors cite as evidence of excellent reliability.

The improvements over the team’s earlier HPLC-PDA method are substantial. The original technique required 65 minutes per sample; the new UPLC version cuts that to 33 minutes. Sensitivity is higher with half the sample volume, a critical advantage when working with scarce clinical specimens or small cell cultures. Perhaps most importantly, the new method cleanly discriminates GMP from IMP, two metabolites that co-eluted and could not be separately quantified with the older protocol. Because GMP and IMP occupy distinct positions in purine interconversion pathways, their separate measurement opens new analytical possibilities for studies of guanine nucleotide synthesis and purine salvage.

To demonstrate performance in genuine biological material, the researchers applied the method to induced pluripotent stem cells derived from three publicly available lines: KOLF2.0, NCRM1 and PGP-1. Each line was studied in matched pairs, with and without a c.508C>T stop codon mutation in the HPRT1 gene. This mutation abolishes hypoxanthine-guanine phosphoribosyltransferase, the salvage enzyme whose complete loss causes Lesch-Nyhan disease, a rare X-linked disorder marked by self-injurious behavior, dystonia and severe motor impairment. Cells were grown in six-well plates to roughly one million cells per well, with culture medium freshly replaced 24 hours before collection.

Sample preparation demanded meticulous attention to chemistry. For intracellular purines, cells were released by trypsinization, pelleted, and extracted in 0.2 molar perchloric acid, which instantly denatures the enzymes that would otherwise degrade labile nucleotides. Sonication at 4 degrees Celsius liberated the contents, and after centrifugation the acidic supernatant was neutralized with potassium carbonate, precipitating potassium perchlorate before final filtration through PVDF microcentrifuge tubes. For extracellular purines, 450 microliters of conditioned medium were acidified with perchloric acid to 0.1 molar and processed identically. Intracellular results were normalized to total cellular protein measured with a BCA assay, while extracellular results were corrected for culture volume and duration.

The biological findings were striking. In keeping with decades of prior work, the adenine derivatives dominated the intracellular purine pool, with ATP present in the greatest amounts, roughly 33 to 46 nanomoles per milligram protein across the lines, followed by GTP at 11 to 16 nanomoles per milligram. The adenylate energy charge, calculated as ([ATP] + 0.5 × [ADP]) / ([ATP] + [ADP] + [AMP]), ranged from 0.81 to 0.88, confirming that all cultures retained a healthy nucleotide energy state above the 0.80 benchmark typical of viable cells. Multivariate analysis of variance revealed significant main effects for both mutation status and cell type, along with a significant interaction between the two variables.

Most intracellular purines showed small or no changes in the HPRT1-mutant cells, but the extracellular compartment told a very different story. Conditioned medium contained no detectable purines except hypoxanthine, which was elevated by an average of 30-fold in the mutant lines, with individual increases ranging from 12-fold to as much as 225-fold depending on the genetic background. The explanation is elegant: without functional HPRT, the cells cannot recycle hypoxanthine back into nucleotides, so they simply dump it into the extracellular space. This finding underscores why measuring extracellular purines is essential, since changes outside the cell were far more obvious than those within it.

The authors acknowledge limitations. The method focuses on free purines and does not capture purines incorporated into DNA, RNA, NAD(H), NADP(H), FAD(H) or other complex molecules, though it can be adapted for some of these derivatives. Cyclic nucleotides and deoxynucleotides occur at levels too low for direct detection without prior sample concentration. The team also emphasizes the extraordinary speed of purine degradation: within seconds, ATP is dephosphorylated to ADP and then AMP, and within minutes AMP is further catabolized to adenosine, inosine, hypoxanthine and uric acid. Tissue measurements demand freeze-clamping in situ or focused microwave irradiation to halt degradation. Finally, the authors caution that isolated ATP measurements can be misleading, since they cannot distinguish energy impairment from wholesale purine depletion; only the simultaneous measurement of ATP, ADP and AMP, captured by the adenylate energy charge, provides a faithful picture of cellular energy state. With its speed, sensitivity and comprehensive coverage, the new UPLC-PDA method appears well suited to simultaneous analysis of the most abundant biologically relevant purines across blood, urine, cerebrospinal fluid and cultured cells, offering a powerful new lens on disorders of purine metabolism.

Subject of Research: Biomedical Analysis and Diagnostics

Article Title: Comprehensive measurement of purines in biological samples

Article References: Fu, R., Sutcliffe, D. J., Dinasarapu, A. R., Hess, E. J., & Jinnah, H. A. (2025). Comprehensive measurement of purines in biological samples. Frontiers in Analytical Science, 5, Article 1600781. https://doi.org/10.3389/frans.2025.1600781

Image Credits: AI Generated

DOI: 10.3389/frans.2025.1600781

Keywords: purine metabolism, UPLC-PDA, Lesch-Nyhan disease, hypoxanthine, adenylate energy charge, metabolomics, HPRT1, analytical chemistry, Comprehensive, measurement, purines, biological

The research, published in Food Chemistry: X, arrives against a sobering epidemiological backdrop. An estimated 55.8 million people were living with gout in 2020, a 150.6 percent increase in total cases since 1990, with the steepest growth concentrated in high-income regions such as North America. Men are affected roughly three times as often as women, a gap attributed largely to estrogen’s role in promoting renal excretion of urate. While aging populations contribute to the trend, the disproportionate rise in wealthy nations points toward modifiable lifestyle factors, including obesity and the consumption of purine-rich foods and drinks. Beer, brewed from nucleic-acid-rich barley malt, consistently tops the purine league table among alcoholic beverages, and epidemiological studies have linked its consumption to roughly a 1.5-fold higher gout risk compared with nondrinkers, versus a more modest 1.15-fold increase for spirits.

To measure purines across a sprawling beverage landscape, the team first had to solve an analytical problem. Traditional HPLC-UV methods for purine quantification demand harsh acid hydrolysis and multi-step enzymatic treatments that can stretch sample preparation across several days. Tandem mass spectrometry offers superior sensitivity but relies on expensive triple-quadrupole instruments that many laboratories cannot justify for routine screening. The researchers instead developed a targeted method using a single-quadrupole QDA detector operated in selected ion recording mode, coupled to an HPLC system with an ion-pairing gradient on a T3 column. By monitoring the precise mass-to-charge ratios of eight compounds, adenine, guanine, hypoxanthine, xanthine, adenosine, guanosine, cytidine and uridine, the method achieved mass-selective specificity without hydrolysis, enzymatic derivatization, or a triple-quadrupole price tag, making it well suited to quality control and large comparative studies.

With the method in hand, the team analyzed 43 commercially available and experimentally produced beverages, spanning twelve full-strength beers, four commercial non-alcoholic beers, ten experimental non-alcoholic beers brewed in-house, six sake, wine, cider, and a range of distilled and blended drinks. The results traced a clear hierarchy. Full-strength beers showed both the highest concentrations and the widest spread of total purines, from about 45.6 milligrams per liter in a light rice-adjunct lager to 223.4 milligrams per liter in a robust stout. Dark, malt-forward styles such as porters, stouts and barrel-aged rye ales clustered at the top, while lighter lagers brewed with substantial corn or rice adjuncts sat at the bottom. Guanosine dominated the purine profile of every beer, contributing roughly 41 to 68 percent of the total.

The numbers carry real dietary weight. A single 12-ounce bottle of the highest-purine stout would deliver approximately 79 milligrams of purines, nearly 20 percent of the 400 milligram daily intake limit recommended in Japan for managing hyperuricemia and gout. The researchers attribute beer’s purine richness to barley itself, whose proteins are abundant in glutamine and glycine, the amino acid precursors of the purine ring, and whose nucleic acids are liberated during malting, when germination activates nucleases that chop grain DNA and RNA into soluble nucleotides. Yeast, meanwhile, appears to do little to clean up the mess. The brewer’s yeast salvage pathways primarily assimilate purine bases rather than nucleosides, so guanosine and adenosine survive fermentation largely intact and accumulate in the finished beer.

The study’s most striking finding concerns non-alcoholic beer, a market valued at roughly 22 to 24 billion dollars and projected to nearly double within a decade. Because non-alcoholic designation is defined purely by ethanol concentration, typically below 0.5 percent by volume, it places no constraint on the non-volatile compounds that remain. Physical dealcoholization methods such as membrane filtration selectively strip ethanol while leaving dissolved solutes untouched, and the data bore this out dramatically. One membrane-processed non-alcoholic porter contained 168.5 milligrams of purines per liter, statistically indistinguishable from full-strength beers overall and comparable to its own full-strength counterpart at 182.9 milligrams per liter. Statistically, the membrane-filtered non-alcoholic beers as a group could not be separated from full-strength beers by their purine content.

Not every alcohol-removal strategy behaved the same way, however. A commercially distilled non-alcoholic lager contained just 15.3 milligrams per liter, roughly seven times lower than its full-strength counterpart, and the experimental beers brewed with a maltose-negative yeast strain, which produce minimal ethanol without any downstream alcohol removal, averaged below 50 milligrams per liter. But the researchers caution against crediting the technology alone. Because purines are non-volatile, distillation should not remove them, and the team hypothesizes that the low-purine distilled product reflects a lighter base formulation rather than the vacuum still itself. The experimental beers told the same story: when the grist was reformulated with reduced barley malt, purine levels fell regardless of the fermentation strategy used.

To probe the role of raw materials directly, the team brewed ten pilot-scale non-alcoholic beers at the university’s Center for Beverage Innovation, substituting 50 percent of the barley malt with rice, corn, cassava, millet, sorghum, wheat or unmalted barley under otherwise identical conditions. The results were consistent and quantifiable. Replacing half the malt with low-nucleic-acid adjuncts cut total purine concentrations by approximately 25 to 51 percent relative to the all-malt control, depending on the adjunct source. The most dramatic case was a beer brewed entirely from malted rice, which contained a mere 0.71 milligrams of purines per liter. Correlation analysis reinforced the mechanism: total purines tracked strongly with original extract, free amino nitrogen, and the proportion of barley malt in the grist, while the share of alternative starches correlated negatively, confirming that purine load rises and falls with malt-derived nucleic acid input.

Other beverage categories fell neatly into place. Sake, brewed from polished rice, contained between 8.5 and 40.2 milligrams per liter and displayed a strikingly different fingerprint, dominated by xanthine at 37 to 94 percent of the total rather than guanosine, a divergence the authors attribute to rice-based raw materials and the distinct fermentation biology of sake production. Red wine registered 29.7 milligrams per liter while white wine showed essentially none. Cider, malt beverages and vodka-based tea hovered near or below 2 milligrams per liter, and distilled spirits, including whiskey, tequila and soju, along with sugar-based hard seltzers, contained no detectable purines at all, since the compounds are excluded during distillation and absent from sugar washes.

The authors are careful to frame these findings as measures of dietary purine exposure rather than direct predictions of gout risk, noting that ethanol itself independently influences uric acid metabolism and that individual susceptibility, diet and clinical factors all contribute. The commercial samples represented single products rather than production batches, each experimental beer was brewed once, and the method quantified only free purine bases and nucleosides, not purines locked in nucleotides or intact nucleic acids. Still, the practical implications are clear. For brewers seeking to formulate genuinely low-purine products, the most direct lever is the grist: swap barley malt for low-protein adjuncts, or explore yeast strains and enzymes that enhance nucleoside assimilation. For consumers managing gout or hyperuricemia, the message is subtler but arguably more important: the non-alcoholic label says nothing about purines, and a dark, malt-heavy non-alcoholic stout may deliver nearly the same purine punch as the regular version on the shelf beside it.

Subject of Research: Quantification of purine derivatives in beer and fermented beverages and the effect of raw materials and dealcoholization on purine content

Article Title: Targeted analysis of purine derivatives in beer and fermented beverages using HPLC-QDA

Article References: Sen, R., Schubert, C., Rani, H., & Lafontaine, S. (2026). Targeted analysis of purine derivatives in beer and fermented beverages using HPLC-QDA. Food Chemistry: X, Article 104565. https://doi.org/10.1016/j.fochx.2026.104565

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104565

Keywords: purines, beer, non-alcoholic beer, gout, hyperuricemia, uric acid, barley malt, HPLC, mass spectrometry, sake, wine, food chemistry