19/10/2026
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Nε-Carboxymethyllysine (CML) is one of the most studied advanced glycation end products and is used as glycation marker in foods. To prevent its artifactual formation during sample preparation, CML determination protocols commonly include reduction step prior to acid hydrolysis. However, the effectiveness of this approach may depend on the glycating agents present in the sample. The aim of this study was to evaluate the impact of sodium borohydride reduction on CML determination in pork meat when glyoxylic acid (GA) acts as glycating agent. Raw and cooked pork loin samples were incubated with different amounts of GA and subsequently processed for CML analysis either with or without reduction. CML was quantified by high-performance liquid chromatography-electrospray ionization-ion trap tandem mass spectrometry. In samples not supplemented with GA, NaBH4 treatment increased CML concentrations from 23.2 ± 0.6 to 26.0 ± 1.3 µg g⁻¹ in raw meat and from 24.8 ± 1.1 to 29.7 ± 0.8 µg g⁻¹ in cooked meat. GA supplementation without reduction resulted in modest but significant increases in CML concentration (maximum 50% relative to controls). In contrast, samples subjected to sodium borohydride reduction exhibited a pronounced GA dose-dependent increase in CML levels, reaching up to 200-fold higher values compared to samples without added GA. Mild acidification (pH 2–4) failed to suppress this effect and, under reducing conditions, further promoted CML formation, particularly at pH 2. These findings demonstrate that NaBH4 treatment can convert GA-derived imine intermediate into CML, causing overestimation of native CML concentrations and current reduction-based pretreatment protocols for CML analysis require critical re-evaluation to avoid analytical bias.