| RP-HPLC is one of the most important method in the control of drug related substance. The separation ability of chromatographic system should be verified in establishing the RP-HPLC method. The effectiveness of the HPLC method can be evaluated by the correlation with other analytical methods of different principles, such as NP-HPLC,HPTLC,HPCE.This paper is to study the validation method for the separation ability of RP-HPLC system with HPTLC as a complementary method,by means of the correlation research between HPTLC and RP-HPLC in analysis of quinolone antibiotics related substance.The content is sumarized as follows:1,Establishment of the correlation research method between HPLC and HPTLC with gatifloxacin and its related substance as examples(1)The establishment of HPTLC separation method for gatifloxacin and its 9 related substance.Based on the experimental design, the HPTLC method was established and optimized.The preliminary screening developing system was carried out by a homogeneous design from the 22 kinds of common TLC solvents.The percentage of the developing system was optimized by a central composite design. The optimalizing developing solvent was methanol:1,2-dichloroethane:ammonia water(15%):acetonitrile (V/V/V/V)=2.8:7.2:0.5:0.5.The HPTLC method was as follows: the solid phase is the MERK HPTLC plate(10×10cm)prewashed by methanol and activatied at 110。C for 1h. The spot was 3mm with 10μL sample. It was developed at room temperature.The wavelength was 292nm in the mode of UV detection.(2)HPTLC-MS method for the analysis of gatifloxacin related substance.HPTLC-MS method was established by the TLC-MS interface technology,collecting the Q1 MS data. The quasi-molecular ion was determined and the mean peaks were corresponded.HPTLC-MS method was carried out by ESI in positive ion mode with the heating temperature at 420℃.The spraying voltage was 4.5kV and the cone voltage was 120V. The scanning range of m/z is 100～500.The mobile phase: was water:methanol(V/V)=50:50 with the flow rate at 0.3ml/min.90s were sufficient for MS analysis of each substance.(3)HPLC-MS method for the analysis of gatifloxacin related substance.HPLC-MS method was established ,collecting the Q1 MS data. The quasi-molecular ion was determined and the mean peaks were corresponded.The chromatographic condition:The column was Phenomenex C18 (150×4.6 mm, 5μm) and the temperature was 30℃;The mobile phase was 10 mmol/L ammonium acetate solution(adjust PH=4.3 with acetic acid):methanil(V/V)=78∶22,with the flow rate at 0.4mL/min;The detection wavelength was 292 nm;The injection size was 10μL.The MS condition was ESI in positive ion mode with the heating temperature at 420℃.The spraying voltage was 4.5kV and the cone voltage was 120V. The scanning range of m/z is 100～500.(4) Establishment of correlation between HPTLC-MS and HPLC-MS data by MassWorks softwareHPTLC-MS and HPLC-MS quasi-molecular ion data was calculated and corrected by MassWorks software to get the lowest semblance value of isotopic peak of the same substance as the threshold of the correlation between HPTLC and HPLC(97%),which means the correlation of the substance with the same elements in the HPLC and HPTLC.2,Further validation of the correlation research method between HPLC and HPTLC by the analysis of lomefloxacin and ciprofloxacin degradation products.The HPTLC-MS and HPLC-MS methods were establiashed for the degradation solutions of lomefloxacin and ciprofloxacin respectively. The quasi-molecular ion data of each impurity was calculated and corrected by MassWorks to get the semblance value of isotopic peak. Lomefloxacin and 9 degradation products could be corresponded in the HPTLC and HPLC chromatogram. 3 related substances of ciprofloxacin detected by HPTLC-MS and HPLC-MS could be corresponed too.This paper illustrated that by means of HPTLC-MS and HPLC-MS ,peaks in HPTLC and HPLC could be corresponded withMassWorks software,which provide the key to verify the separation ability of RP-HPLC with HPTLC.
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