Simultaneous HPLC-APCI-MS/MS Quantification Of Endocannabinoids And Glucocorticoids In Hair

The endocannabinoid and glucocorticoid systems are very essential and well organized central regulatory structures that affect a wide range of biological processes such as antinociception, hypothermia, analgesia, energy and appetite regulation as well as glucose and lipid metabolism. The glucocorticoid system together with the endogenous endocannabinoids (a group of endogenous lipid ligands of the ECS) plays a pivotal role in the assessment of a myriad of aberrant human physiological conditions such as obesity. Of the many types of endocannabinoids discovered far and the most investigated are N-arachidonoyl-ethanolamine (AEA) and 2-arachydonyl-gylcerol (2-AG) whilst cortisol and cortisone are some of the major types of glucocorticoids known. Hair matrix could retrospectively record the association of endocannabinoids and glucocorticoids in a diverse of physiological functions. However, spontaneous isomerization of 2-AG to 1-arachidonoylglycerol (1-AG) and depending on the extraction conditions, the possible rearrangement of O-arachidonoyl ethanolamine (OAEA) to AEA in various sample matrices could be major obstacles encountered in the detection of both 2-AG and AEA.This study aimed to develop a novel method for simultaneous quantification of 2-AG, AEA, cortisol and cortisone in hair. Analysis was carried out on a fairly huge sample size of 473 multi-gender research participants which aided in carrying out correlation studies with regards to the concentration of hair AEA and 2-AG with Body Mass Index (BMI), glucocorticoids as well as gender.Methanol was used as the incubation solution and an acidic mixture of methanol and de-ionized water utilized as mobile phase in order to avert possible rearrangements of both OAEA and 2-AG. The analyses were performed using a high-performance liquid chromatography tandem mass spectrometer with atmosphere pressure chemical ionization in positive mode. Agilent 1200 HPLC system fixed with a 5 μm,150 mm X 4.6 mm plastil ODS C18 column was utilized for chromatographic separation of compounds. Mobile phase was a mixture of methanol and de-ionized water ratio 90:10 (v:v) containing 2mmole of ammonium acetate and flow rate of 500μL/min. For the mass spectrometric analysis, a Q trap 3200 mass spectrometer equipped with APCI interface in positive mode was employed. MRM mode was used in the detection of ions.The method showed good linearity in the range of 2.5-250 pg/mg for AEA,15.0-1250 pg/mg for 2-AG and 1-250 pg/mg for cortisol and cortisone. Limits of detection were 1.5 pg/mg for AEA,6 pg/mg for 2-AG and 0.5 pg/mg for cortisol and cortisone. For all four analytes, intra and inter-day coefficients of variation were less than 20% and recovery above 90%. Population analyses in 473 hair samples established that 2-AG was significantly correlated with AEA.2-AG was significantly and positively correlated with cortisol and cortisone. There was a significant positive correlation of AEA with cortisol, but not with cortisone. Obese participants showed a significantly higher concentration of cortisone and 2-AG. Males showed significantly higher 2-AG and cortisone levels but significantly lower AEA levels than females.A new method was developed for application in the detection of endocannabinoids and glucocorticoids in hair. The newly developed assay is discernibly fast, simple and quite sensitive. The assay showed good performances and was precise for the quantification of 2-AG, AEA, cortisol and cortisone in human hair. Despite having limited it to the quantification of only four compounds, it would be recommended that the developed method be validated and utilized to analyze many other analytes in hair.