2.6. Biochemical Analysis
The animals’ blood samples were centrifuged for 10 minutes at 2000 rpm. The serum samples were placed in tubes and stored in the freezer (at a temperature of -80 °C). Frozen materials were transported to a +4°C unit one day before biochemical analysis to dissolve. Then, troponin-I, myoglobin, pro-brain natriuretic peptide (pro-BNP), low density lipoprotein (LDL), high density lipoprotein (HDL), triglyceride, total cholesterol parameters were analysed.
2.7. Quantification of JWH-018 and its metabolites byLiquid Chromatography Tandem Mass Spectrometry in the heart tissue
Liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of  JWH-018 and JWH-018’s metabolites in rat heart tissue was developed. The method was validated in rat heart homogenates and was significantly sensitive to quantitate the concentrations of JWH-018 and JWH-018’s metabolites. The method determined by Poklis et al. was used in the preparation of heart tissue samples (28,29).
2.7.1 Heart Tissue Calibration Method
Low quantity limit 10ng/g, medium quantity limit of 80 ng/g and high quantity limit of 100 ng/g to JWH-018 metabolites in cardiac tissue and calibration samples of JWH-018-free (1:4 tissue:water) was prepared using rat heart tissue homogenates. Tissues taken from rats were homogenized with a glass homogenizer and diluted ¼ percent with pure water. The samples were kept overnight after being vortexed and added while being vortexed drop by drop with 2 ml of cold acetonitrile on the following day. Then, the samples were centrifuged at 3500 rpm for 10 minutes and kept at -40oC for at least 2 hours. The upper part containing acetonitrile was taken with a disposable pipette and transferred to a clean tube. The extracts were then dried using Teknosem TAB-40-WEL Evaporator. The residue was dissolved in 100µL mobile phase. The samples were taken into insert vials and made ready for analysis. A 7-8-fold difference between the peak areas of JWH-018 and its metabolites, and the metabolites that are difficult to separate from each other, were properly separated by solid phase extraction method.
2.7.2. Solid Phase Extraction
The heart tissue samples were prepared using OASIS HLB cartridges. The cartridges were conditioned with ethyl acetate, methanol and water,respectively . The tissue sample was weighed and diluted with water in a ratio of 1: 4, and then homogenized for 1 min at 4400 rpm. On the homogenate, 5 ml of water was added and after centrifugation at 4400 rpm for 10 min, the upper clear part was placed in the conditioned cartridge. It was washed with 5% methanol solution by volume. It was dried under high vacuum for at least 30 min. The elution step was completed twice with 0.5 ml methanol and 0.5 ml ethyl acetate. It was centrifuged at 14000 rpm for 10 min and placed in 0.2ml LC-MS/MS inserts. JWH-018 and its N-(2-hydroxypentyl), N-(3-hydroxypentyl), N-(4-hydroxypentyl), N-(5-hydroxypentyl) and N-pentanoic acid metabolites were quantificated in the heart tissue by LCMS-8040 (Shimadzu Scientific Instruments, Columbia, MD).