A novel COVID-19 vaccine (BriLife®) has been developed by the Israel Institute for Biological Research (IIBR) to prevent the spread of the SARS-CoV-2 virus throughout the population in Israel. One of the components in the vaccine formulation is tris(hydroxymethyl)aminomethane (tromethamine, TRIS), a buffering agent. TRIS is a commonly used excipient in various approved parenteral medicinal products, including the mRNA COVID-19 vaccines produced by Pfizer/BioNtech and Moderna. TRIS is a hydrophilic basic compound that does not contain any chromophores/fluorophores and hence cannot be retained and detected by reverse-phase liquid chromatography (RPLC)-ultraviolet (UV)/fluorescence methods. Among the few extant methods for TRIS determination, all exhibit a lack of selectivity and/or sensitivity and require laborious sample treatment. In this study, LC–mass spectrometry (MS) with its inherent selectivity and sensitivity in the multiple reaction monitoring (MRM) mode was utilized, for the first time, as an alternative method for TRIS quantitation. Extensive validation of the developed method demonstrated suitable specificity, linearity, precision, accuracy and robustness over the investigated concentration range (1.2–4.8 mg/mL). Specifically, the R2 of the standard curve was >0.999, the recovery was >92%, and the coefficient of variance (%CV) was <12% and <6% for repeatability and intermediate precision, respectively. Moreover, the method was validated in accordance with strict Good Manufacturing Practice (GMP) guidelines. The developed method provides valuable tools that pharmaceutical companies can use for TRIS quantitation in vaccines and other pharmaceutical products.
The chemical derivatization to enhance the signal intensity and signal-to-noise (S/N) of several organophosphorus (OP) acids in liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) is illustrated. The OP class of compounds represents the environmental degradants of OP nerve agents and pesticides. N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) was utilized to derivatize a panel of eight acids consisting of five alkyl methylphosphonic acids (ethyl-, isopropyl-, isobutyl-, cyclohexyl-, and pinacolyl-methylphosphonic acid) along with three dialkylphosphate analogs (diethyl-, dibutyl-, and diethyl thio-phosphate). The derivatization reaction with CAX-B was conducted in acetonitrile in the presence of potassium carbonate at 70 °C for 1 h. The resulting acid derivatives were analyzed with an LC-Orbitrap-ESI-MS/MS, and their dissociation processes were investigated. It was found that the derivatization procedure increased the limits of identification (LOIs) by one to over two orders of magnitude from the range of 1 to 10 ng/mL for the intact OP-acids to the range of 0.02–0.2 ng/mL for the derivatized acids utilizing an LC-MS(QqQ) in MRM mode, regardless of the sample matrix (hair, concrete, or plant extracts). The interpretation of the corresponding ESI-MS/MS spectra for each type of derivatized sub-OP family revealed the formation of characteristic neutral losses and a characteristic ion for the organophosphorus core. This derivatization is beneficial and useful for screening and identifying target and “unknown” OP acids.
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