Identification of Degradation Products and a Stability-Indicating RP-HPLC Method for the Determination of Flupirtine Maleate in Pharmaceutical Dosage Forms

In this stability-indicating, reversed-phase high-performance liquid chromato-graphic method for flupiritine maleate, forced degradation has been employed and the formed degradants were separated on a C18 column with a 80:20% v/v mixture of methanol-water containing 0.2% (v/v) triethylamine; the pH was adjusted to 3.1. The flow rate was 1 mLmin−1 and the photodiode array detection wavelength was 254 nm. Forced degradation of the drug was carried out under acidic, basic, thermal, photolytic, peroxide, and neutral conditions. Chromatographic peak purity data indicated no co-eluting peaks with the main peaks. This method resulted in the detection of seven degradation products (D1–D7). Among these, three major degradation products from acidic and basic hydrolysis were identified and characterized by 1H-NMR, 13C-NMR, and mass spectral data. The method was validated as per International Conference on Harmonization guidelines (Q2). The linearity of the method was in the concentration range of 20–120 μgmL−1. The relative standard deviations for intra- and interday precision were below 1.5%. The specificity of the method is suitable for the stability-indicating assay

Validation of liquid biopsy: plasma cell-free DNA testing in clinical management of advanced non-small cell lung cancer

Vidya H Veldore,1,* Anuradha Choughule,2,* Tejaswi Routhu,1 Nitin Mandloi,1 Vanita Noronha,2 Amit Joshi,2 Amit Dutt,3 Ravi Gupta,1 Ramprasad Vedam,1 Kumar Prabhash2 1MedGenome Labs Private Ltd,, Bangalore, India; 2Tata Memorial Centre, Parel, Mumbai, India; 3The Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi Mumbai, Maharashtra, India *These authors contributed equally to this work Abstract: Plasma cell-free tumor DNA, or circulating tumor DNA (ctDNA), from liquid biopsy is a potential source of tumor genetic material, in the absence of tissue biopsy, for EGFR testing. Our validation study reiterates the clinical utility of ctDNA next generation sequencing (NGS) for EGFR mutation testing in non-small cell lung cancer (NSCLC). A total of 163 NSCLC cases were included in the validation, of which 132 patients had paired tissue biopsy and ctDNA. We chose to validate ctDNA using deep sequencing with custom designed bioinformatics methods that could detect somatic mutations at allele frequencies as low as 0.01%. Benchmarking allele specific real time PCR as one of the standard methods for tissue-based EGFR mutation testing, the ctDNA NGS test was validated on all the plasma derived cell-free DNA samples. We observed a high concordance (96.96%) between tissue biopsy and ctDNA for oncogenic driver mutations in Exon 19 and Exon 21 of the EGFR gene. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the assay were 91.1%, 100% 100%, 95.6%, and 97%, respectively. A false negative rate of 3% was observed. A subset of mutations was also verified on droplet digital PCR. Sixteen percent EGFR mutation positivity was observed in patients where only liquid biopsy was available, thus creating options for targeted therapy. This is the first and largest study from India, demonstrating successful validation of circulating cell-free DNA as a clinically useful material for molecular testing in NSCLC. Keywords: liquid biopsy, NSCLC, EGFR, ctDNA, NG