A validated surrogate analyte LC-MS/MS assay for quantification of endogenous cortisol in human whole blood.

Cortisol is a steroid hormone that is frequently measured as a marker of stress, inflammation, and immune function. While commonly analyzed in saliva, hair, blood plasma and urine, a recent trend towards whole blood-based at-home collection devices has emerged, which necessitates development of more sensitive assays for cortisol in whole blood. To support the implementation of a patient-centric sampling approach in a drug development program, a fit-for-purpose surrogate analyte-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for cortisol in whole blood was developed using 13C3-cortisol as a surrogate analyte and cortisol-d6 as the internal standard. The surrogate analyte approach was chosen due to a lack of available cortisol-free whole blood and the absence of appropriately representative surrogate matrices. Samples were prepared using supported liquid extraction, and the LC-MS/MS analysis consisted of a 4.00 min analytical run. The method demonstrated linearity between 0.500 and 500 ng/mL of 13C3-cortisol, and accuracy, precision and robustness were all acceptable per current regulatory guidance for bioanalytical method validation of chromatographic assays for cortisol- and 13C3-cortisol-based quality control (QC) samples when quantified against a 13C3-cortisol calibration curve. The acceptable robustness of cortisol-based QCs when quantified against a 13C3-cortisol-based calibration curve also suggests parallelism between the analytes. These results indicate a viable surrogate analyte method, that is fit-for-purpose to analyze whole blood cortisol levels using a surrogate analyte LC-MS/MS approach. Evaluation of patient samples showed very promising comparability between whole blood and plasma cortisol concentrations, suggesting that whole blood could be used in place of or in addition to a plasma-based sampling protocol in clinical trials analyzing cortisol. Overall, this method presents a novel tool that is a first step in supporting the trend towards sample miniaturization and at-home sample collection, and may be readily used in clinical and diagnostic settings.

Assignment of Core versus Antenna Fucosylation Types in Protein N-Glycosylation via Procainamide Labeling and Tandem Mass Spectrometry.

Fucosylation is an important feature of protein N-glycosylation as it has been reported to influence the efficacy of therapeutic proteins and as a potential disease biomarker. A common approach for characterizing protein N-glycans is to analyze the native glycans via tandem mass spectrometry (MS). However, tandem MS analysis of native N-glycans typically results in proton migration, which in turn leads to fucose residue migration from the glycan core to the antenna and vice versa. This phenomenon ultimately leads to ambiguous assignment of N-glycan fucosylation. Although the use of specific fucosidases has been successfully employed for assigning fucosylation, such strategies are often too cumbersome, expensive, and time-consuming for routine N-glycan analysis. As an alternative, we explore the influence of labeling N-glycans with procainamide hydrochloride to inhibit fucose migration during tandem MS analysis. The labeled N-glycan pool was separated and analyzed using ultraperformance liquid chromatography and a hydrophobic interaction liquid chromatography column coupled to a quadrupole time-of-flight mass spectrometer (UPLC-HILIC-QTOF-MS). The observation of the m/z 587.3 core fucose diagnostic peak corresponding to [GlcNAc + Fucose + Procainamide + H](+) in the tandem MS data of fucosylated N-glycans rapidly verifies core fucosylation while its absence signifies antennae fucosylation. This unique approach is here validated with human IgG (for core fucosylation) and human alpha-1-acid-glycoprotein (for antenna fucosylation). We further present a useful application toward the rapid verification of fucosylation types in a therapeutic protein (Rituximab).

Glycosylation characterization of Human IgA1 with differential deglycosylation by UPLC-ESI TOF MS.

Differential deglycosylation was introduced as an effective technique to characterize glycosylation in glycoprotein containing both N-linked and O-linked glycans at both protein and peptide levels. Human IgA1 was used as a model glycoprotein to demonstrate this technique. The glycans attached to Human IgA1 were removed from their attachment sites by an array of enzymes. After reduction by DTT, the resulting deglycoproteins were analyzed by UPLC-ESI TOF MS to estimate the numbers of N-glycan and O-glycan sites through differential masses. The deglycoproteins and unmodified glycoprotein were further digested to deglycopeptide through trypsin digestion. The glycopeptides and deglycopeptides were identified by UPLC-ESI TOF MS. Two N-glycan and four O-glycan sites were identified and confirmed at peptide levels. These results matched those from deglycoproteins. The N-glycosylation site and N-glycan sequence confirmation were also demonstrated in this study.

The evaluation of a novel approach for the profiling and identification of N-linked glycan with a procainamide tag by HPLC with fluorescent and mass spectrometric detection.

Procainamide was investigated as a multifunctional oligosaccharide label for glycan profiling and identification in a HPLC-FL/ESI-QTOF system. Addition of this aromatic amine to glycans through reductive amination improves fluorescence detection and ESI ionization efficiency. Both procainamide and 2-AB derivatives of N-linked glycans released from three glycoproteins (Human IgG, Mouse IgG, and RNase B) were quantitatively profiled with HPLC-FL and identified with ESI-QTOF. The procainamide derivatives produced FL glycan profiles comparable to the 2-AB derivatives, but with a few extra minor peaks, which suggests better labeling efficiency for procainamide derivatives for minor peaks. The procainamide derivatives also improve ESI ionization efficiency by 10-50 times over the respective 2-AB derivatives and the ESI-QTOF method sensitivity is at the low picomole to high femtomole level. Using the procainamide tag, all N-linked glycans released from three tested glycoproteins can be quantitatively detected with HPLC-FL and identified with ESI-QTOF at the same time. Monosaccharide sequence confirmation was also demonstrated in this study.