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
Authors: Vuyyuru Nagi Reddy , Reddy A. Malleswara , Ramadevi B. , Kumar Y. Ravindra , Durga Prasad B. Janaki 
Published: September 2020

A new simple, selective, highly sensitive, specific (stability indicating), robust, rugged and mass
compatible gas chromatographic method and sample with direct injection-mode was developed for
the quantitative determination of amino-3-piperidine (3-aminopiperidine, 3-AMP) in linagliptin. As
the CAD-LC method did not proved for specificity and pre-derivitizations are challenging task for quality
control (QC), a simple GC method has been developed. Compared to LC-CAD method, which is not
proved for specificity and pre-column derivitization methods having the limitations to analyze the
reaction 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 in
sensitivity. Also this method is mass compatible, specificity proved by forced degradation, method
was validated as per ICH guidelines. Mass balance was proved by analyzing the stressed samples for
net degradation by HPLC and assay by HPLC methods. This GC method also provides an advantage
to analyze the in-process samples to monitor the progress of the synthetic process, where the reaction
monitoring 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-column
derivitization methods. This method involves simple sample preparation process and direct injection
with 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 developed
with 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-AMP
peak. Mass balance was reported > 99%. The limit of quantification and limit of detection values for
3-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 and
4.7, respectively. Linearity was established in the range of 0.0014 mg/mL (LOQ) to 0.045 mg/mL for
3-AMP with correlation coefficient (r2 > 0.9995). The percentage recoveries were obtained between
99.9% 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 spiked
sample solutions were studied up to 2 days and are stable at room temperature. The forced degradation
studies were performed by using HCl, NaOH, H2O2 thermal, UV radiation and water. A mild degradation
bout 0.25% was observed in base degradation condition, which was confirmed with mass number by
GC-MS analysis. Validation parameters were evaluated according to the International Conference on
Harmonization (ICH) Q2 guidelines.