A Simple, Specific, Mass Compatible and Validated Gas Chromatographic Method for the Estimation of Piperidine-3-amine Content in Linagliptin Finished and Stability Samples without Derivatization

A new simple, selective, highly sensitive, specific (stability indicating), robust, rugged and masscompatible gas chromatographic method and sample with direct injection-mode was developed forthe quantitative determination of amino-3-piperidine (3-aminopiperidine, 3-AMP) in linagliptin. Asthe CAD-LC method did not proved for specificity and pre-derivitizations are challenging task for qualitycontrol (QC), a simple GC method has been developed. Compared to LC-CAD method, which is notproved for specificity and pre-column derivitization methods having the limitations to analyze thereaction monitoring in-process samples and degradation samples, the present method is selective,simple, cost effective, QC friendly, widely available GC technique with direct injection and high insensitivity. Also this method is mass compatible, specificity proved by forced degradation, methodwas validated as per ICH guidelines. Mass balance was proved by analyzing the stressed samples fornet degradation by HPLC and assay by HPLC methods. This GC method also provides an advantageto analyze the in-process samples to monitor the progress of the synthetic process, where the reactionmonitoring samples are unpurified or non-isolated samples and contains many process related impurities(reference) and solvents, which will have interference with 3-AMP in LC-CAD and pre-columnderivitization methods. This method involves simple sample preparation process and direct injectionwith GC-FID technique. Hence, this method can be used to analyze the finished product samples,degradation samples, stability samples and reaction monitoring samples. The method was developedwith widely available GC column (diphenyl dimethyl polysiloxane as stationary phase, 30 m length,0.53 mm internal diameter & 5.0 μm thickness), helium as carries gas, FID detector set at 240 ºC,column oven starts at 200 ºC and starts increases after 2 min with 20 ºC/min and holds up to 11 min,which will ensure the column bake. The solvents used for the process were well separated from 3-AMPpeak. Mass balance was reported > 99%. The limit of quantification and limit of detection values for3-AMP were 0.002% (0.4 μg/mL) and 0.007% (1.4 μg/mL) of targeted concentration (20 mg/mL),respectively. The method was precise at LOQ and at specification level with %RSD values 2.8 and4.7, respectively. Linearity was established in the range of 0.0014 mg/mL (LOQ) to 0.045 mg/mL for3-AMP with correlation coefficient (r2 > 0.9995). The percentage recoveries were obtained between99.9% and 104.4%. The range of the method was established from LOQ (0.0014 mg/mL) to 150%(0.045 mg/mL) of the specification targeted limit of 0.15% (0.03 mg/mL). The standard and spikedsample solutions were studied up to 2 days and are stable at room temperature. The forced degradationstudies were performed by using HCl, NaOH, H2O2 thermal, UV radiation and water. A mild degradationbout 0.25% was observed in base degradation condition, which was confirmed with mass number byGC-MS analysis. Validation parameters were evaluated according to the International Conference onHarmonization (ICH) Q2 guidelines.