Objective  To establish a method for the simultaneous determination of the contents of cotinine and 3-hydroxycotinine in serum using automatic liquid-liquid extraction-high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in a solid medium.

Methods  The sample was pretreated with liquid-liquid extraction by adding 50 μL of potassium hydroxide solution and 50 μL of isotope internal standard. The solution was mixed well and added into a 96-well plate used for liquid-liquid extraction in a solid medium. After 750 μL of extraction solvent (5% isopropanol dichloromethane mixture) was added twice at an interval of time, the extraction solution was collected and blow-dried with nitrogen gas. The sample was reconstituted with 100 μL of pure water and then loaded onto the machine for determination. The compounds of interest were separated on an HSS T3 liquid-phase column (2.1 mm×100 mm, 1.8 μm), with 0.05% ammonia solution and acetonitrile used as a mobile phase for gradient elution separation. The sample underwent positive electrospray ionization and multiple reaction monitoring. The retention time and ion pairs were measured using qualitative method, and quantitative measurements were performed using the internal standard method.

Results  The two compounds of interest had a good linear relationship within the ranges of 0.1-50 and 5-300 μg/L, with a correlation coefficient (r) of greater than 0.999 0. The limits of detection of cotinine and 3-hydroxycotinine were 0.016 and 0.023 μg/L, respectively. The average recoveries of the two compounds were between 86.4% and 117.2%, with relative standard deviations falling between 2.03% and 8.03%, when the spiking levels were 2.0, 15.0, and 40.0 μg/L.

Conclusion  The pretreatment of samples using this method consumes less organic reagents, most steps can be automated, and it is accurate, precise, and sensitive, making it suitable for the determination of the content of cotinine and 3-hydroxycotinine in the serum of smokers and non-smokers.