| Objective:Through research on the general character, identification, impurities check and assay of KM, to establish a series of methods for controlling the quality of it and provide experimental basis and research conditions for stability test of KM, thus an accurate and reliable quality standards for KM was developed.Methods:According to the legend and appendix of Ch.P 2010 version, the characters (containing appearance, smell, the solubility, melting point, specific optical rotation), identification (including IR and HPLC retention time identification), impurities inspection (consisting of the moisture, chloride, sulfate salt, acidity, residue on ignition, heavy metal, arsenic salt, related substances and residual solvents), and the content about the drug was studied based on HPLC, GC and other methods. Then we also investigated changes of every index in stability test (containing factors test, long-term test, accelerated test), such as characters, acidity, residue on ignition, enantiomers, related substances, content and so on.The HPLC chromatographic system I was established to detect content in APIs. The separation was achieved on a Symmetrix ODS-R C18 (4.6x250mm,5μm) column by a mobile phase of acetonitrile-water (6:94). The detection wavelength was 254 nm. The detection column temperature was 35℃ and the flow rate was 1.0 mL·min-1. The injection volume was 10μL.The HPLC chromatographic system II was used to detect related substances in APIs. All separations were achieved on a Symmetrix ODS-AQ C18(4.6x250mm,5μm) column by 10μL injection volume where solvent A= acetonitrile/water (3:97) and solvent B= acetonitrile (gradient elution). The detection wavelength was 254 nm. The detection column temperature was 35℃ and the flow rate was 1.0 mL·min-1.The chromatographic system III was established to detect enantiomer in APIs and it was taken as a special impurity. The separation was achieved on a Daicel CHIRALPAK AD (4.6x250mm, 10μm) column through a mobile phase of hexane-ethanol (53:47). An isocratic elution at a flow rate of 1.3 mL·min-1 was employed, UV chromatograms were processed at 254 nm (for KM Enantiomer). The column temperature was 40℃ at the volume of 10.0 μLThe GC chromatographic system I was used to detect benzyl alcohol and benzyl chloride in APIs. All separations were achieved on a HP-5 (30m×0.32mm×0.25μm) column. The carrier gas flow rate (nitrogen) was 1.0 mL·min-1 while split ratio=3:1. The column temperature was 115 ℃ and constant in 6 mins, the detector temperature was 300℃ and the inlet temperature was 260℃ at the same time. The injection modes were manual injection.The four residual solvent (methanol, tetrahydrofuran, ethyl acetate and dichloromethane) in its APIs were detected by GC chromatographic system Ⅱ.The separation was achieved on a DB-624 (30m×0.32mm×0.18μm) column. The carrier gas flow rate (nitrogen) was 1.0 mL·min-1 while split ratio=5:1. The column temperature was 60℃ and constant in 10 mins, then the temperature was raised to 220℃ at a rate of 30 ℃·min-1, and constant in 5 mins. While the detector temperature was 250℃ and the inlet temperature was 200℃. The injection modes were automatic headspace. The conditions of headspace injection was established. The equilibrium temperature was 80℃ and the equilibrium time was 30 mins, while transfer tube temperature was 130℃, the pressing time was 6s with the 1.0 mL injection volume.Results:1. KM appears white crystalline powder and odorless, soluble in dimerthylformamide (DMF), hydrochloric acid (0.1mol/L) and sodium hydroxide (0.1mol/L), slightly soluble in ethanol and methanol, very slightly soluble in water, insoluble in acetonitrile. KM started to be melt at about 248 ℃, but its melting point is more higher and longer, yet it has not been completely melted at 270 ℃, and this is same to the results of thermal analysis, its specific optical rotation is+25° to+29° at 25℃ .2. The retention time of HPLC and IR absorption Spectrum showed that the sample of KM was in accordance with that of the reference.3. The result was compared for the two methods of HPLC and potentiometric titration, we finally choose HPLC method to determine KM in APIs considering with the cost, feasibility and operability. The HPLC chromatographic system I was achieved on a Symmetrix ODS-R C18 (4.6×250mm,5μm) column. The mobile phase was acetonitrile-water (6:94, v/v) with a flow rate of 1.0 mL·min-1. The detection wavelength was 254 nm. The column temperature was 35℃ and the injection volume was 10μL.4. The water content in KM was 5.9% to 6.5%, chloride content was less than 0.02%, sulfates content was less than 0.04%, pH was 6.2 to 6.3, the residue content on ignition were not more than 0.1%, the heavy metals content was less than 20 ppm, arsenic salt was not checked out and Arsenic spot was obviously showed with lug arsenic. In addition, we used four chromatographic systems to inspect the related substances, while optical isomer and residual solvents and the methods were validated. The results of related substances test were as follows:known impurities (non-enantiomer) were not detected, the single largest impurity was not more than 0.1%, total impurity were not more than 0.3%. The residual solvent check were as follow:benzyl chloride, ethyl acetate, methylene chloride, tetrahydrofuran were not detected, while methanol were detected in three batches, and its residues were less than 0.002%,which was in accordance with the regulations of ICH and Ch.P 2010.5. The indicators of character, acidity, water, enantiomer, related substances and assay were mainly investigated in influential factor test, accelerate test and long-term test. Those changes were compared at 0 days, 5 days and 10 days in Influential factor test, at 0,1,2,3 and 6 months in accelerate test, at 0,3 and 6 months in long-term test, the changes were not significant in all indicators, thus the quality of KM was basically stability.Conclusion:In our studies, the methods which matched the regulations of ICH and Ch.P 2010 were established. The methods and data we obtained could provide technical and data support for quality standard and promote the development of quality control and enrich the mode of thinking for KM and similar drugs. |
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