Improvised method for urinary p-cresol detection and measurement using high performance liquid chromatography/mass spectrometry

Gut microbiota has been implicated in many disorders including Autism Spectrum Disorder (ASD). ASD is a neurodevelopmental brain disorder affecting individuals leading to restricted and repetitive pattern of behaviour and disruption of communication and social interactions. Altered microbiome and the presence or absence of key species capable of affecting specific responses in levels of their fermentation products are reflected in the urinary metabolite profile of patients. The aim of our study is to develop an improvised method for the detection and quantification of urinary p-cresol levels which could serve as an indicator for GI microbial dysbiosis. The p-cresol analysis was achieved using HPLC by a reverse phase C18 column with mobile phase composition of Acetonitrile/water/formic acid (10:90:0.05, v/v/v) in an isocratic mode of elution with a flow rate of 1.0 mL/min. The mass analysis of p-cresol was performed using LC-MS [Triple Quadrupole Liquid Chromatography Mass Spectrometer] in negative ESI mode with electron multiplier detector. p-cresol was eluted at a retention time of approximately 3.4 min. The standard calibration curves had a superior regression coefficient of greater than 0.99 (R(2) > 0.99) and were linear over a range from 0.0005 mg/mL to 0.015 mg/mL. The method was validated by analysis of six replicates with 0.08% relative standard deviation and method detection and quantification limits were 20 ng/mL and 50 ng/mL respectively. Further validation of method on real urine samples from two groups of children (Control population:< 10 years of age; 5M: 3F and ASD individuals: <10 years of age; All males) showed that detection was effective over a wide range of metabolite at levels as high as 149.73 μg/mL to as low as 0.897 μg/mL. This study reports a rapid, validated and sensitive method for the detection of p-cresol in urine samples.