| Objective:To screen the Q-markers in traditional Chinese medicines（TCMs）from the perspective of effectiveness,we propose a Phosphatidylcholine specific phospholipase C（PC-PLC）-based hollow fiber target fishing,which combined with high performance liquid chromatography-ultraviolet detector（HPLC-UVD）to realize the preliminary screening and identification of Q-marker in Curcumae Longae Rhizoma.To improve the conventional dispersive liquid-phase microextraction mode,new extractant can be applied to establish more convenient,efficient and environmental-friendly methods for the concentration and determination of the Q-markers in TCMs with high determination sensitivity and purification.Methods:The hollow fiber was used as a carrier with heptanol in the wall pores and PC-PLC solution in lumen was placed in the sample phase solution,which was stirred at a constant temperature for screening of Q-markers.The factors affecting screening efficiency were optimized and methodology evaluation was conducted.With the optimized screening conditions,the Q-marker screening in Curcumae Longae Rhizoma was conducted.Additionally,the inhibition of PC-PLC activity by Q-marker was exlored.A deep eutectic solvent（DES） synthesized from tetrabutylammonium chloride and decanoic acid as an extractant was added to the sample solution.After finishing the extraction at the water bath,the extractant was collected by freezing for further analysis.Parameters influcing the extraction were optimized and methodology was investigated under the optimal conditions.The developed method was applied to analyze quantitatively the Q-marker in Curcumae Longae Rhizoma and active compounds in turmeric tea combined with HPLC-UVD.To confirm the suitability of this method for the extraction of target compounds in complex samples,the results of the proposed method were compared with those of the method in Chinese pharmacopoeia 2015 edition（Ch.P.2015）.The hydrophobic natural deep eutectic solvent（L-menthol-lactic acid）was added to the sample phase solution containing the analytes as the extractant instead of the traditional low-density organic solvent.The dispersion was promoted by manually shaking,and the two phases were separated by adding demulsifier for further analysis.Traditional one-factor-at-a-time method combined with response surface methodology was utilized to optimize parameters,and the interaction between variables was elucidated.In addition,the methodological evaluation was conducted under the optimal conditions.The novel method was applied for the determination of the Q-marker in Curcumae Longae Rhizoma and active compounds in turmeric tea coupled with HPLC.Results:The optimal extraction conditions of hollow fiber target fishing method were as follows:the solvent in wall pores was heptanol;the sample phase was 6 mL（pH 6.8）;the target concentration was 1 mg/ml（pH 6.0）;the screening was conducted on45℃ for 90 min;the optimized concentration of bisdemethoxycurcumin,demethoxycurcumin and curcumin were 6mg/mL,4mg/mL,and 5.3mg/mL respectively.The chromatographic peak areas of the active compounds of hollow fiber with PC-PLC solution were significantly greater than those of the hollow fiber with phosphate buffer and blank control.In this screening and measurement conditions（420 nm）,the captured active compound was bisdemethoxycurcumin（tR14.164）,demethoxycurcumin（tR16.242）,curcumin（tR18.652）.The binding capacity of certain component in different origin was similar.Curcumin could be used as a inhibitor of PC-PLC.The optimal extraction conditions for liquid-phase microextraction based on solidification of floating deep eutectic solvent drop were as follows:the extractant was 70μL of tetrabutylammonium chloride-decanoic acid（1:1,n:n）;the sample phase pH was 3.0;the extraction was conducted on 40℃ for 10 min at 1100 rpm.Under the optimum conditions,the enrichment factors were 608,621 and 848 for bisdemethoxycurcumin,demethoxycurcumin and curcumin.The linear ranges were 0.001-0.7mg/m L,0.0015-0.75mg/mL,0.002-0.8mg/mL respectively with correlation coefficients higher than 0.9900.Limits of detection and limits of quantitation were respectively 7.0×10-5-9.0×10-5mg/mL and 2.0×10-4-3.0×10-4mg/mL.The relative standard deviations for intra-day precision and inter-day precision respectively were 2.4%-4.2%and 1.4%-3.7%.The average recovery was in the range of 84.6%-116.8%.The average contents of three curcuminoids were 2.95,2.42 and 5.12 mg/g in Curcumae Longae Rhizoma and 1.40,1.54 and 4.15 mg/g in turmeric tea.The optimal extraction conditions for natural deep eutectic solvent based solvent terminated liquid-liquid microextraction:the extractant was 160μL of L-menthol-lactic acid（1:2,n:n）;the sample phase pH was 7.0 with 18%of NaCl;the manual shaking time was 30s;the demulsifier was 800mL of methanol;the demulsification time was 230 s;and the sample phase was 8.0 mL.Under the optimum conditions,the enrichment factors were 279,300 and 350 for bisdemethoxycurcumin,demethoxycurcumin and curcumin.The linear ranges were0.002-0.8mg/mL,0.001-0.7mg/mL,0.0015-0.75mg/mL respectively with correlation coefficients higher than 0.9900;limits of detection and limits of quantitation were respectively 1.0×10-4-4.0×10-4mg/mL and 4.0×10-4-1.2×10-3mg/mL.The relative standard deviations for intra-day precision and inter-day precision respectively were3.0%-9.2% and 2.2%-6.2%.The average recovery was in the range of 85.3%-108.9%.The average contents of three curcuminoids were 5.18,2.4 and 6.19 mg/g in Curcumae Longae Rhizoma and 2.24,1.43 and 4.7 mg/g in turmeric tea,respectively.Conclusions:In the research,a PC-PLC-based hollow fiber target fishing method was established,which provided a convenient method for the identification of Q-marker in TCMs.Meanwhile,DES has been successfully applied to different modes of dispersive liquid-phase microextraction,which expanded the application of DES in liquid-phase microextraction,and provided simpler,greener and more efficient pretreatment methods for the enrichment and determination of Q-marker in TCMs. |
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