Comparison of Various Chromatographic Systems for Analysis of Cytisine in Human Serum, Saliva and Pharmaceutical Formulation by HPLC with Diode Array, Fluorescence or Mass Spectrometry Detection.

Background: Identification and quantitative determination of cytisine, especially in biological samples and pharmaceutical formulations, is still a difficult analytical task. Cytisine is an alkaloid with a small and very polar molecule. For this reason, it is very weakly retained on reversed phase (RP) stationary phases, such as commonly used alkyl-bonded phases. The very weak retention of cytisine causes it to be eluted together with the components of biological matrices. Objective: Comparison and evaluation of various chromatographic systems for analysis of cytisine in different matrices-serum, saliva and pharmaceutical formulation-by high performance liquid chromatography (HPLC) with diode array (DAD), fluorescence (FLD) and mass spectrometry (MS) detection. Methods: The analyses were performed using HPLC in reversed phase (RP), hydrophilic interaction liquid chromatography (HILIC) and ion exchange chromatography (IEC) modes. Different sample pre-treatment methods were tested: Protein precipitation (with acetone, methanol (MeOH) or acetonitrile (ACN), and solid phase extraction (SPE) using cartridges with octadecyl (C18), hydrophilic-lipophilic balanced copolymer (HLB) or strong cation exchange sorbents (Strata X-C). Conclusion: Significant differences were observed in retention parameters with a change of the used chromatographic system. The various properties of stationary phases resulted in differences in analyte retention, peaks' shape and systems' efficiency. The weakest retention was observed using RP systems; however, the use of the Polar RP phase can be an alternative for application in green chromatography. In the strongest retention was observed using a strong cation exchange (SCX) phase. The most optimal systems were chosen for the analysis of cytisine in the pharmaceutical preparation, serum and saliva after sample pre-treatment with the new SPE procedure. Due to the sensitivity, the use of HPLC-DAD or HPLC-FLD is the most optimal for drug analysis in pharmaceutical preparations, whereas HPLC-MS is suitable for analysis of cytisine in biological samples.