Development of the LC-MS/MS method for determining the p-cresol level in plasma
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.
Section snippets
Chemicals
Reference standard p-cresol was purchased from Sigma-Aldrich (Darmstadt, Germany). p-Cresol sulfate and p-cresol-d7 (pC-D7) were supplied by Toronto Research Chemicals (Toronto, Canada). Dansyl chloride (5-dimethylamino-1-naphthalenesulfonyl chloride, Dns-Cl) was purchased from Life Technologies (Eugene, OR, US) and sodium carbonate was purchased from Chempur (Piekary Śląskie, Poland). Ammonium acetate ULC-MS optigrade was purchased from LGC Standards (Wesel, Germany). Solvents, HPLC gradient
Direct vs. indirect detection of p-cresol
Direct detection of pC (1 μg mL−1) resulted in high value of LOD (S/N = 29.4) (Fig. A.1.). The IDL was calculated as 510 pg.
In case of dansyl chloride-based derivatization of pC (10 ng mL−1) (S/N = 316.8) we obtained IDL as 0.9 pg (Fig. A.2). Therefore, further experiments were performed using indirect detection of p-cresol.
Method optimization
During method optimization, various conditions of dansyl chloride-based derivatization process were tested (see Supplementary data – Section A.2.5). Dansyl chloride is a
Conclusion
The novel, accurate LC–MS/MS method of p-cresol determination in plasma was developed and validated. The IDL (0.9 pg) was determined on the lowest level compared with previously described methods (more than 50 times). The selected LLOQ was 0.85 ng mL−1 for plasma. The method was applied for the analysis of this compound in samples of people with MODS and undergoing dialysis. The results have shown that concentration of pC in human plasma samples is on detectable level and that pC is being
Acknowledgments
The authors are grateful to Ryszard Marszałek for the technical assistance during LC–MS/MS analysis.
Conflicts of interest
The authors declare no conflict of interest.
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