Bioanalysis of Stress Biomarkers through Sensitive HILIC-MS/MS Method: A Stride toward Accurate Quantification of MDA, ACR, and CTA.

Quantifying reactive aldehyde biomarkers, such as malondialdehyde, acrolein, and crotonaldehyde, is the most preferred approach to determine oxidative stress. However, reported analytical methods lack specificity for accurately quantifying these aldehydes as certain methodologies may produce false positive results due to harsh experimental conditions. Thus, in this research work, a novel HILIC-MS/MS method with endogenous histidine derivatization is developed, which proves to have higher specificity and reproducibility in quantifying these aldehydes from the biological matrix. To overcome the reactivity of aldehyde, endogenous histidine is used for its derivatization. The generated adduct is orthogonally characterized by NMR and LC-HRMS. The method employed a hydrophilic HILIC column and multiple reaction monitoring (MRM) to accurately quantify these reactive aldehydes. The developed method is an unequivocal solution for quantifying stress in in vivo and in vitro studies.

Hyphenated liquid chromatography - diode array detection - charged aerosol detection - high resolution - multistage mass spectrometry with online hydrogen/deuterium exchange: One stop solution for pharmaceutical impurity profiling.

Hyphenation of different analytical techniques has always been advantageous in structural characterization as it saves time, money and resources. In the pharmaceutical sector, chromatography-based impurity profiling, including identification, characterization, and quantification in drug substances or finished products, is of utmost importance to comply with quality, patient safety and regulatory requirements. These impurities are monitored using LC-UV/DAD and identified and/or characterized using HRMS and MS/MS. LC analysis usually yields the area percent purity of the targeted peak, however, this is not sufficient for pharmaceutical purposes; where the regulatory requirement is to report impurities in percent weight by weight. Unfortunately, the non-availability of impurity standards and relative response factors at an early stage of drug development, risks the product quality due to the inability of the method to differentiate percent purity, and percent weight by weight. Hence, there is a need for a distinctive way of determining the relative response factor. In the current study, a unique hyphenation has been employed by integrating LC with DAD, CAD, and HRMSn with hydrogen-deuterium exchange. The LC flow, post-DAD detection has been diverted to CAD with an inverse gradient for relative response factor determination and MS Orbitrap for exact mass, and MSn fragmentation. A separate infusion pump has been incorporated to infuse D2O on a need basis, which can perform partial hydrogen deuterium exchange for determining the number of labile hydrogens in the impurity structure. This hyphenation has been validated with four model compounds and a total of nineteen chromatographic peaks. The technique provides ample information for their qualitative analysis along with percent weight-by-weight values, which fulfils the regulatory requirements and can be used as one-stop solution for impurity profiling.