A simple and cost-effective method was developed to determine fluorouracil (5-FU) and phenobarbital (PHB) in human body fluids. A coating based on a zeolitic imidazole framework-67 (ZIF-67) template cobalt nanoporous carbon (Co-NPC) was synthesized by one-step carbonization of ZIF-67 without using any other carbon precursors in the present study. The prepared Co-NPC was then fabricated on the surface of a prepared stir bar to be used as stir bar sorptive extraction (SBSE) method. The extracted analytes were then analyzed via high performance liquid chromatography-ultraviolet detection (HPLC-UV). Plackett–Burman design was employed for screening the experimental factors. The effective factors were then optimized using Central Composite Design (CCD). Under the optimum conditions, limits of detections (LODs) of 5-FU were 0.36, 0.41 and 1.2 μg L−1 in water, urine and plasma samples, respectively. Limits of quantifications (LOQs) were also 1.2, 1.3 and 4.5 μg L−1 in water, urine and plasma samples, respectively. Relative standard deviations (RSDs) at a spiked concentration of 10 μg L−1 were 8.1, 7.9 and 8.6% (as intra-day RSD) in water, urine and plasma samples, respectively. The linear dynamic ranges (LDRs) were in the range of 1–200 μg L−1. Moreover, LODs of PHB were 0.21, 0.32 and 1.4 μg L−1 in water, urine and plasma samples, respectively. LOQs were also 0.9, 1.1 and 4.6 μg L−1 in water, urine and plasma samples, respectively. Relative standard deviations (RSDs) at a spiked concentration of 10 μg L−1 were 6.3, 6.5 and 6.9% (as intra-day RSD) in water, urine and plasma samples, respectively. The linear dynamic ranges (LDRs) were in the range of 1–500 μg L−1. The applicability of the method was investigated by the extraction and determination of the target analytes in different biological fluids including urine and plasma samples.
