Allantoin in human urine quantified by ultra-performance liquid chromatography–tandem mass spectrometry

Abstract

Uric acid is a potent antioxidant and scavenger of singlet oxygen and other radicals in humans. Allantoin, the predominant product of free radical-induced oxidation of uric acid is efficiently excreted in the urine and has potential as a biomarker of oxidative stress. We developed a rapid and specific assay for urinary allantoin using ultra performance liquid chromatography-tandem mass spectrometry suitable for high-throughput clinical studies. The method required minimal sample preparation, was accurate (mean error 6%), precise (intra- and inter-day imprecision: <8%) and sensitive (limit of detection: 0.06 pmol). Allantoin levels measured in control samples were comparable to literature values.

Today’s oxidative stress markers

“…There are several reports concerning the determination of allantoin in human urine [10][11][12][13][14]. Allantoin (5-ureidohydantoine) has recently been considered a promising biomarker of prooxidative processes [12,13] specific only for apes and humans [15].…”

“…It requires the use of sensitive and specific analytical techniques [21]. Allantoin was determined by means of capillary zone electrophoresis (CZE) [27], enzymatic assay and enzyme cycling method [28], capillary electrophoresis with UV detection (CE-UV) [29], high performance liquid chromatography -UV detection (HPLC-UV) [9,30], ultra performance liquid chromatography -tandem mass spectrometry (HPLC/UPLC-MS/MS) [10][11][12][13][14]21], and gas chromatography -mass spectrometry (GC-MS) [20,24,31], and allantoin is detected readily in urine, plasma, and synovial fluid [25].…”

“…In the fridge, it remains stable for 12 days, while in the deep frozen (-70°C) urine sample, it is stable at least for 15 weeks [10,12]. Gas chromatography -mass spectrometry is more sensitive than the HPLC method.…”

Detection of allantoin in clinical samples using hydrophilic liquid chromatography with stable isotope dilution negative ion tandem mass spectrometry

“…In addition there is at least a 10-fold improvement in previously reported detection limits [22,29] and accurate quantification is achieved by using a stable isotope of allantoin as an internal standard. Due to the high sensitivity and specificity of this method, it is possible to use as little as 10 L of sample.…”

“…Using HILIC also avoids the 2 h derivatisation step that the newer GC/MS method entails. The throughput of this method has the potential to be increased using ultra high performance liquid chromatography to shorten runs, as has recently been published [29]. The method also has the required sensitivity to detect differences between allantoin levels of health controls and individuals with inflammatory pathologies associated with oxidative stress.…”