Standard stock solution (100 mg/L) was prepared

Standard stock solution (100 mg/L) was prepared sellckchem by dissolving an appropriate amount of 1-hydroxypyrene in a few drops of methanol before making it up to the 1 L mark. Working solutions were prepared by an appropriate dilution of the stock solutions with pure methanol. All solutions were stored in the refrigerator (4 ��C) to avoid degradation. Mode of exposure to generator fumes The test animals were weighed and divided into four groups. Group A, B and C had nine animals each while group D had ten. The animals in group A were placed one meter away from the base of the generator exhaust. The animals in group B were placed two meters away from the generator exhaust. The animals in group C were placed three meters away from the exhaust of the generator.

The ten rats in group D were used as controls hence they were not exposed to the generator fume. This represents the usual distance between the generator and most people that make use of it for their work place. The generator was left on for 8 hours daily for a period of 42 days. Most workers that generate their own power are exposed for 8 hours daily. Mortality rate The mortality number was checked daily and percentage casualties were recorded after exposing the animals for a period 42 days (8 hr/day) to generator fumes. Extraction of 1-hydroxypyrene After 42 days of exposure of the rats to 8 hours of generator fumes daily, the rats underwent cervical dislocation and their blood samples were collected by ocular puncture via heparinized capillary tubes into well-labeled heparinized bottles.

The whole blood was centrifuged (4000 ��g, 10 min) in a Cencom bench centrifuge and then 2 mL of clear plasma supernatant collected. 1 mL of 2000 units of the beta glucuronidase was added to the decanted supernatant and incubated for 14 hr at 37 ��C. This step was to enable the complete hydrolysis of the conjugated 1-hydroxypyrene glucuronide to yield the analyte of interest: 1-hydroxypyrene. Thereafter, 2.5 mL of acetonitrile (ACN) was added to the incubated sample, shaken for 2 min, allowed to settle for 10 min, centrifuged again, and decanted into a clean measuring cylinder. This step was repeated twice. The combined clear supernatants obtained were made up to the 7.5 mL with acetonitrile. 1-hydroxypyrene determination By HPLC An Agilent 1100 series HPLC machine with a UV detector was used for the determination of 1-hydroxypyrene in the blood samples of the exposed rats.

AV-951 An X-bridge C-18 column (150 �� 4.6 mm) 5 ��m was used as the stationary phase. The mobile phase composition was Methanol:Water (88:12) at a flow rate of 1.2 mL/min in an isocratic run with a 3 min run time. The separation was carried out at an injection volume of 5 ��L, wavelength 250 nm and temperature at 30 ��C. This method was used for both the standards and the samples.

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