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5-oxoprolinemia is underdiagnosed cause of metabolic acidosis in cases of paracetamol overdose.Capillary electrophoresis as method for simultaneous quantification of paracetamol and 5-oxoproline in serum.Monitoring of the incidence of 5-oxoprolinemia by the determination of both analyte levels in patient samples.High anion gap metabolic acidosis frequently complicates acute paracetamol overdose and is generally attributed to lactic acidosis or compromised hepatic function. However, metabolic acidosis can also be caused by organic acid 5-oxoproline (pyroglutamic acid). Paracetamol’s toxic intermediate, N-acetyl-p-benzoquinoneimine irreversibly binds to glutathione and its depletion leads to subsequent disruption of the gamma glutamyl cycle and an excessive 5-oxoproline generation. This is undoubtedly an underdiagnosed condition because measurement of serum 5-oxoproline level is not readily available. A simple, cost effective, and fast capillary electrophoresis method with diode array detection (DAD) for simultaneous measurement of both paracetamol (acetaminophen) and 5-oxoproline in serum was developed and validated. This method is highly suitable for clinical toxicology laboratory diagnostic, allowing rapid quantification of acidosis inducing organic acid 5-oxoproline present in cases of paracetamol overdose. The calibration dependence of the method was proved to be linear in the range of 1.3–250 μg mL−1, with adequate accuracy (96.4–107.8%) and precision (12.3%). LOQ equaled 1.3 μg mL−1 for paracetamol and 4.9 μg mL−1 for 5-oxoproline.