Acetaminophen (paracetamol, N-(4-hydroxyphenyl) acetamide) is one of the most prescribed drugs for the pain management, minor aches, and treatment of fever. Quantification of acetaminophen in pre-term and term neonates and small children requires the availability of highly sensitive assays in small volume blood samples. We developed and validated an LC-MS/MS assay for the quantification of acetaminophen in human whole blood. The drug molecule was extracted from whole blood samples followed by protein precipitation extraction technique followed by LC-MS/MS analysis. An excellent analyte separation was achieved by using a phenomenex Gemini® C18 column (50× 3.0 mm, 3 μm) using mobile phase A-Water:Formic Acid (100:0.1) and Acetonitrile:Formic acid (100:0.1) as mobile phase B. With 3.40 minutes as analytical run time, this analytical method demonstrated linearity between 50.0 to 50,000 ng/mL and the achieved correlation coefficient (r2) was greater than 0.9996. Accuracy, precision, matrix effect and robustness were all acceptable according to FDA guidelines. In addition, acetaminophen was stable up to 179 days at both -20°C and -70°C temperatures. Hence, the acquired results proved that the LC-MS/MS was precise and accurate for quantification of acetaminophen in whole blood, and eventually this method can be applied to micro-sampling technique which is the recent trend towards sample miniaturization, collection of the sample from home and sending the devices for laboratory testing.

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