Development of the LC-MS/MS method for determining the p-cresol level in plasma

Highlights

• First LC–MS/MS method of p-cresol determination in plasma.

• The IDL is 50 times lower compared with existing methods.

• The analyzed validation requirements were fulfilled.

• The results have shown that pC is being removed from blood by dialysis.

Abstract

p-Cresol is a protein-bound uremic retention solute that originates in the intestine through bacterial metabolism and accumulates throughout the body in case of kidney failure. To date, there has been no method to analyze unconjugated p-cresol concentration in the blood with a limit of detection lower than 75 pg. Thus, the aim of this study was to develop and validate a novel liquid chromatography-tandem mass spectrometry method for the determination of unconjugated p-cresol in plasma with a lower detection limit than what has been determined using previously described methods. Sample preparation included derivatization of p-cresol with dansyl chloride (derivatization reagent) showed to be a better approach to analyze the compound. The method optimization involved various pH, time of the reaction, and concentration of derivatization reagent. The validation process was performed according to the procedures prescribed by the European Medicines Agency. All analyzed validation criteria were fulfilled. The novel validated method was applied to compare the level of p-cresol in patients with chronic renal failure before and after dialysis (n = 24). Additionally, the concentration of p-cresol was determined in patients with multiple organ dysfunction syndrome (n = 23). The established method can be used for determination of p-cresol in the plasma in further clinical research.

Introduction

p-Cresol (4-methylphenol, 108.1 g mol⁻¹) is a protein-bound uremic retention solute produced from tyrosine, phenylalanine, and phenol in the intestine via bacterial metabolism, for example, Clostridium difficile [[1], [2], [3]]. Approximately 95% of p-cresol (pC) is converted to p-cresol sulfate (pCS) through sulfonation in the colon and liver. Approximately 3–4% of pC is metabolized in the liver to p-cresol glucuronide (pCG) through glucuronidation. Only 0.5–1% of pC is present in free form in the bloodstream [1]. Other possible metabolites of pC have been identified in vitro as 4-hydroxybenzaldehyde, 4-methyl-ortho-hydroquinone and their corresponding GSH adducts [4].

p-Cresol and its metabolites, pCS and pCG, are efficiently removed from a body by the healthy kidney [1,5]. In case of kidney failure, uremic toxins accumulate in the blood [5,6]. Many in vitro experiments have shown that unconjugated pC causes various toxic effects, for example, induces disruption in the gap junctions of cardiomyocytes [7] and promotes autophagy in renal proximal tubular cells [8]. In vivo studies have associated pC and its metabolites with renal failure [6], as well as with cardiovascular disease in patients treated with hemodialysis [9] and autism spectrum disorder in children [1].

Clearance of total pC (conjugated and unconjugated) via hemodialysis is limited because of its binding to protein [2]. Therefore, several therapeutic strategies have been tested to decrease its concentration, for example, supplementation of sevelamer hydrochloride (a synthetic phosphate binder) to reduce pCS uptake in the intestine [5] or synbiotic treatment to normalize bowel habits [10].

There are numerous methods to determine pCS and pCG in different matrices. Sulfate conjugate of pC is determined in plasma [2], saliva [11], and urine [12]. The concentration of glucuronide conjugate of pC is measured in blood [3] and urine [12]. The majority of methods for pC determination in biological matrices use a sample preparation with a heat/acid precipitation, which causes deconjugation of pCS and pCG (Table A.1). Finally, measured concentration refers not only to pC but also to its metabolites [2,3,13]. To date, only two methods avoid hydrolysis of pCS and pCG during sample preparation: high-performance liquid chromatography with fluorescence detection (HPLC-FL) described by Martinez et al. [2] and gas chromatography-mass spectrometry method (GC–MS) described by de Loor et al. [3]. Both methods used organic solvents (acetone, methanol), which do not cause deconjugation of pC metabolites. However, neither of these methods described the validation results, thus their analytical performance is unknown. Due to utilized devices, a high level of limit of detection (LOD) of both methods was determined, for GC–MS: LOD = 75 pg and for HPLC-FL: LOD around 2000 pg. As a result, because of the low concentration of unconjugated pC in blood, the compound was not detected in most of the samples [2,3]. Therefore, the clearance of unconjugated pC through dialysis and its significance in various clinical states is still unclear.

The objective of this study was to develop and validate a high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method, with the lowest LOD among the previously described methods, for the analysis of p-cresol in plasma. The method was applied to analyze plasma samples of patients with multiple organ dysfunction syndrome (MODS) and dialyzed patients (before and after dialysis) with chronic kidney disease. To the best of our knowledge, this is the first paper describing a reproducible LC–MS/MS method to determine unconjugated pC concentration in human plasma.