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).