Quantification of allantoin and other metabolites of the purine degradation pathway in human plasma samples using a newly developed HILIC-LC-MS/MS method.

The development of a simple HILIC-LC-MS/MS method to quantify the plasma levels of allantoin, inosine, hypoxanthine, and adenosine, using stripped plasma for the bioanalytical method validation, was the purpose of this study. Chromatographic separation conducted using an XBridge BEH Amide column (2.1 × 150 mm, 3.5 µm) was achieved under gradient elution with two mobile phases: 0.1% formic acid-ACN (5:95) and 0.1% formic acid-ACN (50:50). Multiple reaction monitoring MS detection was performed using a triple quadrupole. The method validation experiments were performed according to the European Medicines Agency and the U.S. Food and Drug Administration guidelines. The lower LOQ was 50 nM, 5 nM, 20 nM, and 2 nM for allantoin, inosine, hypoxanthine, and adenosine, respectively. The recovery was repeatable and stable. The intraday precision ranged from 1.6% to 6.5%, while the interday precision ranged from 3.4% to 58.7%. Therefore, it is necessary to make a matrix-matched calibration curve each day to overcome this issue. Since the quality control samples' stability did not always comply with the guidelines, the samples need to be analyzed soon after collection.

Recent progress in the LC-MS/MS analysis of oxidative stress biomarkers.

The presence of a dynamic and balanced equilibrium between the production of reactive oxygen (ROS) and nitrogen (RNS) species and the in-house antioxidant defense mechanisms is characteristic for a healthy body. During oxidative stress (OS), this balance is switched to increased production of ROS and RNS, exceeding the capacity of physiological antioxidant systems. This can cause damage to biological molecules, leading to loss of function and even cell death. Nowadays, there is increasing scientific and clinical interest in OS and the associated parameters to measure the degree of OS in biofluids. An increasing number of reports using LC-MS/MS methods for the analysis of OS biomarkers can be found. Since bioanalysis is usually complicated by matrix effects, various types of cleanup procedures are used to effectively separate the biomarkers from the matrix. This is an essential part of the analysis to prepare a reproducible and homogenous solution suitable for injection onto the column. The present review gives a summary of the chromatographic methods used for the determination of OS biomarkers in both urine and plasma, serum, and whole blood samples. The first part mainly describes the biological background of the different OS biomarkers, while the second part reports examples of chromatographic methods for the analysis of different metabolites connected with OS in biofluids, covering a period from 2015 till early 2020. The selected examples mainly include LC-MS/MS methods for isoprostanes, oxidized proteins, oxidized lipoproteins, and DNA/RNA biomarkers. The last part explains the clinical relevance of this review.

Oxidative stress in autosomal dominant polycystic kidney disease: player and/or early predictor for disease progression?

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in PKD1 or PKD2 genes, is the most common hereditary renal disease. Renal manifestations of ADPKD are gradual cyst development and kidney enlargement ultimately leading to end-stage renal disease. ADPKD also causes extrarenal manifestations, including endothelial dysfunction and hypertension. Both of these complications are linked with reduced nitric oxide levels related to excessive oxidative stress (OS). OS, defined as disturbances in the prooxidant/antioxidant balance, is harmful to cells due to the excessive generation of highly reactive oxygen and nitrogen free radicals. Next to endothelial dysfunction and hypertension, there is cumulative evidence that OS occurs in the early stages of ADPKD. In the current review, we aim to summarize the cardiovascular complications and the relevance of OS in ADPKD and, more specifically, in the early stages of the disease. First, we will briefly introduce the link between ADPKD and the early cardiovascular complications including hypertension. Secondly, we will describe the potential role of OS in the early stages of ADPKD and its possible importance beyond the chronic kidney disease (CKD) effect. Finally, we will discuss some pharmacological agents capable of reducing reactive oxygen species and OS, which might represent potential treatment targets for ADPKD.

Oxidative stress in chronic kidney disease.

Oxidative stress (OS), defined as disturbances in the pro-/antioxidant balance, is harmful to cells due to the excessive generation of highly reactive oxygen (ROS) and nitrogen (RNS) species. When the balance is not disturbed, OS has a role in physiological adaptations and signal transduction. However, an excessive amount of ROS and RNS results in the oxidation of biological molecules such as lipids, proteins, and DNA. Oxidative stress has been reported in kidney disease, due to both antioxidant depletions as well as increased ROS production. The kidney is a highly metabolic organ, rich in oxidation reactions in mitochondria, which makes it vulnerable to damage caused by OS, and several studies have shown that OS can accelerate kidney disease progression. Also, in patients at advanced stages of chronic kidney disease (CKD), increased OS is associated with complications such as hypertension, atherosclerosis, inflammation, and anemia. In this review, we aim to describe OS and its influence on CKD progression and its complications. We also discuss the potential role of various antioxidants and pharmacological agents, which may represent potential therapeutic targets to reduce OS in both pediatric and adult CKD patients.

Simultaneous determination of allantoin and adenosine in human urine using liquid chromatography - UV detection.

We report a HPLC-UV method for the quantitative determination of allantoin and adenosine in human urine, validated according to the acceptance criteria of both the USA Food and Drug Administration (FDA) guideline for bioanalytical method validation and the European Medicines Agency (EMA) validation guidelines. Both allantoins and adenosine are compounds of the purine catabolic pathway. Adenosine is situated at the top as a uric acid (UA) precursor, while allantoin is the best-known degradation product of UA. These two compounds are endogenously present in human urine. Chromatographic separation was achieved with a gradient elution at 0.6 mL/min using a Zorbax SB-Aq column coupled to a Zorbax SB-Aq guard column. Three different mobile phases were used: mobile phase A consisted of 10 mM KH2PO4 (pH 4.7) in milli-Q water, mobile phase B was 12.5 mM KH2PO4 (pH 4.7) - ACN (80:20) and mobile phase C consisted of ACN - H2O (50:50). The linear response range in human urine was 14-800 µM for allantoin and 1.25-50 µM for adenosine. The recoveries of allantoin, adenosine and the internal standard were greater than 93.8%. The intra-day accuracy ranged between 99.5 and 104.9% for allantoin and between 96.6 and 107.3% for adenosine, while the inter-day accuracy ranged respectively from 91.2 to 103.0% and from 94.5 to 107.8%. The intra-day precision range was from 0.8 to 6.2% RSD for allantoin and from 0.6 to 15.0% for adenosine. The inter-day precision ranged from 2.1-17.5% for allantoin and from 2.9-17.9% for adenosine. This method was successfully applied to analyze both compounds in urine samples of healthy volunteers. In conclusion, an accurate, precise and stable HPLC-UV method was developed and validated to quantify the endogenously present compounds allantoin and adenosine in human urine samples.