Pre‐column Derivatization and Ultra‐High‐Performance Liquid Chromatography‐Tandem Mass Spectrometry‐based Method Development of Gemcitabine in Reversed‐Phase Column for Pharmaceutical Applications

Abstract Gemcitabine (Gem) is a Food and Drug Administration‐approved chemotherapeutic agent used for the treatment of a wide variety of cancers. Yet, its clinical application is constrained by poor pharmacokinetics and early degradation in biological environments, thus creating the need for developing a sensitive, specific, and reliable analytical method to quantify it. The present study illustrates a validated ultra‐high‐performance liquid chromatography‐tandem mass spectrometry (UHPLC–MS/MS) method duly incorporating a new pre‐column derivatization step for enhanced detection and retention. The derivatization was carried out using 9‐fluorenylmethyloxycarbonyl chloride, which selectively reacts with the primary amine group of Gem and forms a stable, non‐toxic, monosubstituted derivative. This method provides higher specificity and stability compared to the earlier reported methods. Indomethacin has been utilized as an internal standard to ensure maintaining analytical continuity for accurate quantitation of Gem. Chromatographic separation was done on an Acquity UPLC BEH C18 reversed‐phase column (2.1 × 100 mm, 1.7 µm particle size) with the help of a linear gradient elution program. The Waters Acquity UPLC‐MS/MS system (XEVO‐TQD, Milford, MA, USA), having a triple quadrupole mass analyzer and operating in multiple reaction monitoring mode with positive electrospray ionization, was utilized for detection purposes with a total run time of 8 min. This method illustrated excellent linearity within the concentration limit of 3.9–2000 ng/mL and correlation coefficient (R 2 ) of 0.9997, which conforms to the ICH Q2(R2) guidelines. It demonstrated high precision and had relative standard deviation values under 2%. The limit of detection and limit of quantification were found to be at 3.9 and 13 ng/mL, respectively. As the present method was found to be simple, fast, and precise with high levels of sensitivity, it proves to be a well‐suited approach for the quantitative analysis of Gem in a variety of pharmaceutical and clinical research applications.