Mixed-mode Stationary Phases Without Aromatic Rings:Preparation,Evaluation And Applications In Pharmaceutical Analysis

Mixed-mode chromatography based on reversed-phase/strongly cationic exchanger（RP/SCX）is widely used for separation of basic compounds due to its superior retentivity and selectivity.However,the synthesis of the stationary phases suffers from several problems such as excessive consumption of organic solvents harmful to the environment,poor reproducibility due to reactions difficult to control,and broadening peaks observed with the stationary phases that contain aromatic rings.On the other hand,fixed-dose combination drugs（FDCs）have been increasingly prescribed as a remedy to various illness and diseases.Because of the vastly different properties of the active ingredients,their analysis often requires separate methods for different components,contributing to the cost of their quality control.To address these issues,this study was aimed to develop an environment-friendly procedure for preparation of a RP/SCX stationary phase without aromatic rings and to apply it to the separation of fixed-dose combination drugs which otherwise require multi-cycles of analysis for different components.To this end,a comprehensive study was conducted,involving preparation,evaluation and applications of the new stationary phases.Principal results obtained are summarized as follows.Firstly,an epoxy bonded silica prepared by vapor deposition method was used as an intermediate phase and the sulfonic acid and octadecyl groups were chemically bonded onto the epoxy groups to obtain the mixed-mode stationary phase(C₁₈/SO₃Na).Four stationary phases with diol modified silica（Diol）,octadecyl ester modified silica(C₁₈),sulfonic acid modified silica（SO₃Na）and mixed-bed(C₁₈-SO₃Na)were prepared for comparative.The resultant phases were characterized by Fourier transform infrared spectroscopy（FTIR）,（13）^C solid-state nuclear magnetic resonance（（13）^C NMR）spectroscopy and elemental analysis.Secondly,the influence factors of mixed-mode retention were investigated.Alkylaniline homologues which have both ionic group and hydrophobic group were separated on the five different stationary phases with varying of salt and organic solvent concentrations in the mobile phase.The effects of the mobile phase composition including salt concentration,organic phase ratio and both of two factors on the retention behavior of alkylaniline homologues on SO₃Na stationary phase were investigated.The retention behaviors of alkylaniline homologues and phenylamine homologues which have different structures and same ionic groups and hydrophobic groups were compared to reveal the effects of solute structure on retention.The results show that among the phases studied,the C₁₈/SO₃ Na stationary phase exhibits strongest retention for alkylaniline homologues under comparable elution conditions.The SO₃Na stationary phase can display mixed-mode behavior as well,depending upon the mobile phase composition:reversed-phase/ion exchange mixed-mode（RP/IEX）in low organic phase and hydrophilic/ion exchange mixed-mode（HILIC/IEX）in high organic phase.Ion exchange retention mechanism exists in different organic phase conditions,while hydrophobic retention is affected by organic phase ratio.Therefore,the retention mode can be switched by adjusting the organic phase ratio.Thirdly,if the sample is capable of simultaneous interaction with the hydrophobic group and ion exchange group of the stationary phase such as C₁₈/SO₃Na phase,an enhancement in retention is observed.And the different ion exchange group and different hydrophobic group also have effect on the mixed-mode retention,which the smaller gap between ion exchange retention and reversed-phase retention will make mixed-mode interaction between the sample and stationary phase larger.Finally,C₁₈/SO₃Na stationary phase was applied to separate compound methoxynamine capsules,compound dihydralazine sulfate tablets,compound antihypertensive tablets and compound reserpine tablets.Besides,compound reserpine tablets were separated and detected by liquid chromatography-mass spectrometry（LC/MS）.The results show that C₁₈/SO₃Na stationary phase can be successfully applied to the separation of FDCs.Compared to traditional C₁₈ stationary phase,the C₁₈/SO₃Na exhibits higher selectivity and better peak shape for the separation of compound methoxyphenamine capsules.In the separation of compound dihydrazine sulfate tablets,three components can be separated simultaneously on C₁₈/SO₃Na stationary phase,which reduces the number of times of the analysis comparing with traditional methods.Four components of compound antihypertensive tablets can be separated on the mixed-mode stationary phase simultaneously.Six components of compound reserpine tablets can be separated simultaneously on C₁₈/SO₃Na stationary phase by optimizing separation conditions.Because the buffer salts used in the mixed-mode chromatography can be replaced with volatile salts,so it is compatible with mass spectrometry.Seven components of compound reserpine tablets can be separated and detected simultaneously by LC/MS.It can be concluded that the mixed-mode stationary phase without aromatic rings can separate multi components of FDCs simultaneously.For more complex separation,optimization can be achieved by adjusting the ratio of functional groups and the type of functional groups to regulate the mixed-mode effect between the stationary phase and specific samples,which will be an alternative chromatography type for separation of complex samples.