Development and validation of a method to quantify acetaminophen in plasma from small rodents for the study of gastric emptying alterations

Factors that affect drugs plasma levels, and consequently their kinetics, such as diet, diseases and pregnancy, can influence the safety and efficacy of treatments. Depending on whether gastric emptying is physiologic, accelerated or retarded and on the place where drug absorption takes place (stomach or intestines) drug absorption can be modified. Acetaminophen, an analgesic and antipyretic drug, is absorbed almost exclusively in the intestine and the rate of its absorption depends on the time of gastric emptying. Therefore, investigations on the gastric emptying rate can be done indirectly by measuring acetaminophen blood plasma levels. The aim of this work was to develop and validate a methodology for the analysis of acetaminophen on plasma sample from small rodents (mice and rats) to infer about gastric emptying of testing drugs/ preparations such as new synthetic compounds or phytomedicines. The methodology used was High Performance Liquid Chromatography coupled to Diode-Array Detector and visualization on Ultraviolet range (HPLCDAD-UV), using a Shimadzu Nexera equipment. In this study, DBN2 mice, Swiss mice and Wistar rats were oral treated with 50 mg.kg-1 of saline acetaminophen solution alone or in combination with metoclopramide or morphine sulfate (10 mg.kg-1) , previously administered by sub-cutaneous route. Acetaminophen analysis method development and validation in blood plasma were in accordance with recommendation of the Brazilian National Health Surveillance Agency (Resolution 899/93). The extraction of acetaminophen from blood plasm (only 50 !lL) was procedure with protein precipitation, subsequent supernatant recovery and directly analysis by HPLC-DAD-UV. Acetaminophen recovery rates with this methodology were above 90%. Best analysis parameters to analyze acetaminophen were achieved with silica-modified ACE® 5 C18 column (250 mm x 4.6 mm id, 5 !liD particle size); mobile phase composed of acidified ultrapure water (pH 3.0) and HPLC grade acetonitrile in the ratio of 80:20; flow rate at 1.0 mL.min-1; oven temperature of 50° C. The developed and validated method showed to be selective for analysis of acetaminophen in plasma is short time (6 minutes, including differentiating it from morphine and metoclopramide retention time), as well as being linear, accurate, precise and robustness. The detection and quantification limits were 30 ng.mL-1 and 45 ng.mL-1, respectively. The test chosen for the study of gastric emptying was adequate to detect both acceleration ( metoclopramide) and retard (morphine) of drug absorption using acetaminophen as marker. This developed test is great for pre-clinical studies of synthetic or natural compounds/ extracts since it is fast, employs low amount of plasma blood sample and spends low amounts of acetonitrile in the mobile phase.