Synthesis Of Acridone Sulfonyl Chloride Derivative Reagent And Its Uses In HPLC Analysis

Background:Aminoglycoside antibiotics and aliphatic amines molecules lack any chromophores capable of giving a general and reliable signal in the UV region, and almost no UV absorption and fluorescence. Liquid chromatography in conjunction with pre-and post-column derivatization is perhaps the most used technique for aminoglycoside antibiotics determination, and then detected by UV or Fluorescence detector. To our knowledge, derivatization agents which have been reported are not perfect, and especially,detection sensitivity are low. It does not apply to the trace determination of amino drugs. Therefore, it is significent to find new derivative agents and HPLC trace determinations on amino drugsObjective:To Synthesize N-methyl/ethyl-acridone-3-chloride (MASC, EASC), and the derivate production identifications were performed by RP-HPLC system for content determination of amikacin, its impurities A(K29), kanamycin A, its impurities kanamycin B and aliphatic amines.Method:1. To synthesize EASC and MASC by the reaction of alkylation on N position and chlorosulfonation taking acridone as raw materials with chlorosulfonic acid. The structures of compouds were characterized by UV-Vis, NMR and MS techniques.2. Methods were performed by RP-HPLC system for analysis of amikacin and its impurities A(K29) with MASC and EASC as precolumn derivatization reagents respectively. Chemical derivatizations were performed in buffer solution KH2PO4-NaOH (pH10.0) at60℃in waterbath. Chromatographic conditions:At LiCHROM C18column (200mm×4.6mm,5μm). Mobile phase A:0.3%triethylamine(TAE)-acetic acid(HAc)-water(pH3.20), mobile phase B:CAN, the gradient protocol was applied, detection wavelength:λMASC=257nm,λEASC=257nm. The flow rate was1.0mL·min-1; the column temperature was30℃3. Methods were performed by RP-HPLC system for analysis of kanamycin, kanamycin B with MASC, EASC and danayl chloride (DNS-Cl) as precolumn derivatization reagents respectively in KH2PO4-NaOH buffer solution with waterbath (MASC:pH9.0,55℃; EASC:pH9.5,60℃;DNS-Cl:pH9.0,50℃). Chromatographic conditions:At LiCHROM Ci8column (200mm×4.6mm,5μm). Mobile phase A:0.3%TAE-HAc-water(pH3.20), mobile phase B:CAN, the gradient protocol was applied, detection wavelength: λMASC=257nm, λEASC=257nm,λDNS-Cl=323nm. The flow rate was1.OmL·min-1; the column temperature was30℃.4. Eleven aliphatic amines were analysized by RP-HPLC with MASC as a pre-column derivatization reagent in buffer solution KH2PO4-NaOH (pH10.0) at45℃in waterbath. Chromatographic conditions:At LiCHROM C18column (200mm×4.6mm,5μm). Mobile phase A:water, mobile phase B:CAN, the gradient protocol was applied (detection at257nm). The flow rate was1.OmL·min-1; the column temperature was30℃.Result:1. The products of synthesis were target compouds, MASC and EASC, by UV-Vis, NMR and MS techniques2. The linears range for the quantitation of amikacin were over1.56～50.0μg·mL-1, rMASC=0.9997, rEAsc=0.9998, n=6. The detection limit (signal to noise ratio=3) were3.08ng·L-1(MASC) and5.0ng·L-1(EASC), average recoveries were101.6%(MASC) and101.7%(EASC), the RSD of precisions were lower than1.5%.3. The linears range for the quantitation of kanamycin A were3.12～100μg·mL-1with MASC and EASC as pre-column derivatization reagents, rMASC=0.9999, rEASC=0.9999, n=6; the linears range was15.62～500μg·mL-1with DNS-Cl, rDNS-Cl=0.9999, n=6. The detection limit (signal to noise ratio=3) were3.10ng· L-1MASC),3.13ng·L-1EASC) and14.29ng·L-1DNS-Cl), average recoveries were from100.0%to100.7%, the RSD of precisions were lower than1.5%.4. The method was fast, accurate and good reproducibility with MASC as a as pre-column derivatization reagent to determinaten of aliphatic amines, the linears range were1.0×10-4～6.5×10-8mol·L/-1, r=0.9994. The detection limit (signal to noise ratio=3) were over1.02×10-10～6.10×10-9mol·L-1.Conclusion:The methods are high sensitivity, reproducibility and low detection limit for the quality control of aliphatic amines and aminoglycoside antibiotics and its related impurities, and can be used to actual samples. These provide some new approachs for the determination of these compounds and provides a reference for similar compounds, and can be used widly.