Development, Validation, and Comparison of Two Mass Spectrometry Methods (LC-MS/HRMS and LC-MS/MS) for the Quantification of Rituximab in Human Plasma

Rituximab is a chimeric immunoglobulin G1-kappa (IgG1κ) antibody targeting the CD20 antigen on B-lymphocytes. Its applications are various, such as for the treatment of chronic lymphoid leukemia or non-Hodgkin’s lymphoma in oncology, and it can also be used in the treatment of certain autoimmune diseases. Several studies support the interest in therapeutic drug monitoring to optimize dosing regimens of rituximab. Thus, two different laboratories have developed accurate and reproductive methods to quantify rituximab in human plasma: one using liquid chromatography quadripolar tandem mass spectrometer (LC-MS/MS) and the other, liquid chromatography orbitrap tandem mass spectrometer (LC-MS/HRMS). For both assays, quantification was based on albumin depletion or IgG-immunocapture, surrogate peptide analysis, and full-length stable isotope-labeled rituximab. With LC-MS/MS, the concentration range was from 5 to 500 µg/mL, the within- and between-run precisions were <8.5%, and the limit of quantitation was 5 µg/mL. With LC-MS/HRMS, the concentration range was from 10 to 200 µg/mL, the within- and between-run accuracy were <11.5%, and the limit of quantitation was 2 µg/mL. Rituximab plasma concentrations from 63 patients treated for vasculitis were compared. Bland–Altman analysis and Passing–Bablok regression showed the interchangeability between these two methods. Overall, these methods were robust and reliable and could be applied to routine clinical samples.

IN VITRO ANALYTICAL EVALUATION OF NITROSAMINE – A CARCINOGENIC IMPURITIES IN OLMESARTAN MEDOXIMIL BY GC MS/MS METHOD

Objective: A simple and sensitive method development and validation for the simultaneous determination of the N-nitrosamine dimethylamine (NDMA) and N Nitrosamine diethylamine (NDEA) in Olmesartan medoxomil (OLM) API and formulations by a tandem mass spectrometer (GC-MS/MS). Methods: Gas chromatography with a programmed oven temperature controller, Elite Wax (30 m × 0.25 mm × 0.5 μm) column, Helium as carrier gas and hyphenated to the tandem mass spectrometer powered with triple quadrupole mass analyzer, and photomultiplier tube detector. The method was validated as per the United States Food and Drug Administration (USFDA) guidelines. Results: With the selected GC-MS/MS conditions, the NDMA and NDEA 0.08 μg/ml (80 ng/ml) and 0.16 μg/ml (160 ng/ml) injected and Rt. for NDMA 5.634 and NDEA 6.516 min, respectively. A linear/range lies in between 0.024 and 0.120 μg/ml and 0.048 and 0.240 μg/ml for NDMA and NDEA with r2 >0.99. The precision, accuracy, and system suitability are established as per USFDA and ICH guidelines, the sensitivity of NDMA limit of detection and limit of quantification 0.08, 0.024 and NDEA 0.16, 0.048. Conclusion: Other nitrosamine impurities are not involved in the determination of NDMA and NDEA in the OLM using GC-MS/MS and the method is simple, sensitive, rapid, accurate, and precise.

Center-of-Mass iso-Energetic Collision-Induced Decomposition in Tandem Triple Quadrupole Mass Spectrometry

Two scan modes of the triple quadrupole tandem mass spectrometer, namely Collision Induced Dissociation Precursor Ion scan and Neutral Loss scan, allow selectively pinpointing, in a complex mixture, compounds that feature specific chemical groups, which yield characteristic fragment ions or are lost as distinctive neutral fragments. This feature of the triple quadrupole tandem mass spectrometer allows the non-target screening of mixtures for classes of components. The effective (center-of-mass) energy to achieve specific fragmentation depends on the inter-quadrupole voltage (laboratory-frame collision energy) and on the masses of the precursor molecular ion and of the collision gas, through a non-linear relationship. Thus, in a class of homologous compounds, precursor ions activated at the same laboratory-frame collision energy face different center-of-mass collision energy, and therefore the same fragmentation channel operates with different degrees of efficiency. This article reports a linear equation to calculate the laboratory-frame collision energy necessary to operate Collision-Induced Dissociation at the same center-of-mass energy on closely related compounds with different molecular mass. A routine triple quadrupole tandem mass spectrometer can operate this novel feature (iso-energetic collision-induced dissociation scan; i-CID) to analyze mixtures of endogenous metabolites by Precursor Ion and Neutral Loss scans. The latter experiment also entails the hitherto unprecedented synchronized scanning of all three quadrupoles of the triple quadrupole tandem mass spectrometer. To exemplify the application of this technique, this article shows two proof-of-principle approaches to the determination of biological mixtures, one by Precursor Ion analysis on alpha amino acid derivatized with a popular chromophore, and the other on modified nucleosides with a Neutral Fragment Loss scan.