Meropenem assay:
The samples were analyzed at the clinical pharmacy laboratory of SBMU.
The plasma concentration of meropenem was determined by a validated
High-Performance Liquid Chromatography (HPLC) according to a previously
reported procedure with some minor modifications [20]. In brief,
sample preparation involves two-step plasma protein precipitation with
acetonitrile and dichloromethane. Initially, 950 µl of plasma was added
to 50 µl of acetaminophen (40 µg/mL) following the addition of 1000 µl
of acetonitrile. After shaking for 10 min by Vortex Mixer and 10 min
centrifugation at 1000 g respectively, a 1000 µl of supernatant was
added to 1000 µl methylene chloride. Finally, a 20 µl of the aliquot of
the upper aqueous layer was injected into the C18 analytical column
(250×4.6 mm with 3.5 µm spherical particles) after 10 min shaking by
Vortex Mixer and 10 min centrifugation at 1000 g in turn. The mobile
phase consisted of 10.53 mmol/L ammonium acetate: acetonitrile (91:9,
v/v) (pH=4) pumped at 1ml/min. The UV detector was adjusted at 298 nm.
The meropenem calibration curve was linear over the concentration range
0.25-20 mg/L with the correlation coefficient (r2)
=0.999. Intra-assay accuracy ranged from +1.38% to +8.50 % and
precision was less than .3.06%. Inter-assay accuracy ranged from
-1.28% to +2.17% and precision was less than .5.42%. The lower limit
of quantification was 0.125 mg/L.