Drug deconjugation-assisted peptide mapping by LC-MS/MS to identify conjugation sites and quantify site occupancy for antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a heterogeneous mixture of conjugated species with varied drug loadings. Depending on conjugation sites, linkers and drugs can exhibit different stability as influenced by the solvent-accessibility and local charge, resulting in different ADC efficacy, pharmacokinetics, and toxicity. Conjugation site analysis is critical for ADC structural characterization to assure product quality and consistency. It enables early conjugation studies at site-specific levels, confirms the absence of unexpected products to support conjugation process development, and aids in ensuring lot-to-lot consistency for comparability studies. Peptide mapping using liquid chromatography-tandem mass spectrometry is the industry standard method for analyzing conjugation sites. However, some concerns remain for this approach as the large and hydrophobic drug moieties often result in poor MS/MS fragmentation quality and impede the identification of conjugation sites. Additionally, the ionization discrepancy between conjugated and unconjugated peptides can lead to a relatively large bias for site occupancy calculation. In this work, we present a simple drug deconjugation-assisted peptide mapping method to identify and quantify the drug conjugation for ADCs with protease-cleavable linkers. Papain-based drug deconjugation was used to remove the highly hydrophobic drug moiety, which significantly improved the quantitation accuracy of conjugation level and the fragmentation quality. Sample preparation conditions were optimized to avoid introducing artificial modifications, allowing the tracking of initial sample status and subsequent changes of quality attributes during process development and stability assessment. This method was applied to analyze thermally-stressed ADC samples to monitor changes of site-specific conjugation levels, DAR, succinimide hydrolysis of the linker, and various PTMs. We believe this is an effective and straightforward tool for conjugation site analysis while simultaneously monitoring multiple quality attributes for ADCs with protease-cleavable linkers.

Migration time correction for dual pressure capillary electrophoresis in semi-targeted metabolomics study.

Migration time fluctuation strongly affects peak alignment and identification of unknown compounds, making migration time correction an essential step in capillary electrophoresis (CE)-based metabolomics. To obtain more reliable information, metabolites with different apparent mobilities are analyzed by tandem mass spectrometry. Applying a small pressure is a common practice for reducing the analysis time of anions in a positive mode CE, known as the pressure-assisted CE. However, applying pressure may reduce the separation efficiency and can be undesirable for cation analysis. A simple way to address this issue is to increase the pressure after a certain time, during the separation. We term this practice as dual pressure CE. However, changing the pressure during the CE separation complicates migration time correction. Previous migration time correction methods were established based on a consistent electroosmotic flow and a constant pressure-driven bulk-flow velocity. We proposed a new correction method to support the peak alignment when dual pressure CE is used. A Python-based script was developed to implement dual pressure CE migration time correction for semi-targeted metabolomics study performed by a multiple reaction monitoring-based method. This script can help select suitable endogenous metabolites as correction markers, perform migration time correction, and conduct peak alignment. A case study showed that migration time precision of 156 metabolites in 32 samples can be improved from 4.8 to 11.4%RSD (relative standard deviation) to less than 1.8%RSD.

Capillary electrophoresis-mass spectrometry analysis of bisphenol A and its analogues in bottled tea beverages with dynamic pH focusing.

A simple and sensitive method for the determination of bisphenol A and its analogues at the ng/mL level in bottled tea beverages is presented. This method utilized a dynamic pH junction to focus the analyte into a more concentrated zone, based on the electrophoretic mobility difference of analytes in the sample matrix and background electrolytes in capillary electrophoresis coupled to mass spectrometry (CE-MS). The optimised analyte focusing led to enhanced signal detection with average peak heights for five bisphenols of 53-170 folds higher than conventional injections. Under optimised conditions, the method showed good linearity in the range of 0.1-100 ng/mL, excellent limits of detection (0.03-0.04 ng/mL), good analyte recovery (80.3-118.1%) with acceptable relative standard deviations (<12%). The limits of quantifications were below the maximum permissible content of bisphenol A set by the European Commission for this product. This method was used to quantitatively analyse bisphenols in six different kinds of bottled tea beverages, making it a promising tool for practical applications.

Enantioselective analysis in complex matrices using capillary electrophoresis-mass spectrometry: A case study of the botanical drug Corydalis Rhizoma.

Enantioselective analysis is critically important in the pharmaceutical and agricultural industries. However, most of the methods reported were developed for the analysis of pure racemates acquired from chemical synthesis or purification. Direct analysis of chiral enantiomers in complex matrices has rarely been reported. This work demonstrated capillary electrophoresis-mass spectrometry (CE-MS) for the enantioselective analysis of botanical drugs for the first time, using a widely used botanical drug, Corydalis Rhizoma, as an example. The method was used for the simultaneous enantioselective analysis of dl-tetrahydropalmatine and (RS)-tetrahydroberberine (canadine) in Corydalis Rhizoma extract. Using (2-hydroxypropyl)-ß-cyclodextrin as the chiral selector, a partial filling technique was used to avoid signal suppression and contamination of the MS detector. Post column organic modifier was used to assist with ionization in the flow through microvial CE-MS interface, therefore, organic solvents was not used in the background electrolyte. The completely aqueous background electrolyte contributed to better chiral separations. The CE-MS method established here can directly determine the analytes in their complex matrix without any pre-purification steps, while also offering high sensitivity and low operational costs (including sample, chiral selector and solvent). In the method validation process, good linearity (r > 0.993), sensitivity and accuracy (recoveries within 89.1-110.0%) were demonstrated. The CE-MS technique was shown to be able to provide good selectivity for the simultaneous chiral separation of multiple pairs of enantiomers in complex matrices.

Determination of urinary prostaglandin E2 as a potential biomarker of ureteral stent associated inflammation.

Ureteral stents are the most widely used surgical implant in urology. However, they may cause adverse effects to patients, including pain, discomfort, and inflammation. In this work, the inflammatory effect of stent placement and the associated elevation of cyclooxygenase-2 (COX-2) expression were observed. Furthermore, a capillary electrophoresis mass spectrometry (CE-MS) based approach was subsequently developed to quantify urinary prostaglandin E2 (PGE2), a COX-2 metabolite known to contribute to inflammatory renal diseases, to further interrogate the role of this pathway. Urine samples were cleaned and preconcentrated by solid-phase extraction (SPE), and an on-line sample stacking method was used for the enrichment of analytes. The accuracy, precision, and specificity of this method were validated. Standard addition methods were performed to assess the reliability of using deuterated internal standards (IS) in compensating the remaining matrix effect after SPE as well as the detector fluctuation. Through the analysis of 32 pig urine samples, a statistically significant increase of PGE2 was observed in the stented group compared to the unstented (P = 0.01) and the recovered (P = 0.004) groups. This work determined that stent placement may contribute to COX-2-dependent inflammation and developed a reliable CE-MS based methodology to quantify PGE2 in stented individuals that may further understand the biology of stent-associated inflammation and inform urologic patient management.

Application of multisegment injection on quantification of creatinine and standard addition analysis of urinary 5-hydroxyindoleacetic acid simultaneously with creatinine normalization.

In this paper, the development of a simple dilute-and-shoot method for quantifying urinary creatinine by CE-ESI-MS was described. The creatinine analysis time was about 7 min/sample by conventional single injection (SI) method and can be significantly reduced to less than 2 min/sample with multi-segment injection (MSI). In addition, the standard addition analysis of 5-hydroxyindole-3-acetic acid (5-HIAA) and creatinine normalization was performed within one run by the MSI technique, and the total analysis time was 14-min faster compared to the SI method for analyzing the same set of samples. The uses of isotopic and non-isotopic internal standards (ISs) were compared. Creatinine-(methyl-13 C) and 5-hydroxyindole-4,6,7-D3 -3-acetic-D2 acid (5-HIAA-D5 ) used as isotopic ISs can provide both accurate and precise results. In contrast, 1,5,5-trimethylhydantoin (1,5,5-TH) used as the non-isotopic IS for creatinine may cause a bias of over 13% in SI method and even worse when the MSI technique was used. Another compound, 2-methyl-3-indoleacetic acid (2-MIAA), was determined not suitable for MSI analysis of 5-HIAA due to endogenous interferences despite its acceptable performance in conventional methods of analysis.

Review of recent developments in determining volatile organic compounds in exhaled breath as biomarkers for lung cancer diagnosis.

Lung cancer is the most common cause of cancer deaths, its global incidence is rising, and continuing rises are predicted. The potential to diagnose lung cancers based on the determination of volatile organic compounds (VOCs) in human breath has been attracting increasing attention with the development of new techniques and methodologies. However, despite many reports of VOC profiling in lung cancer patients, little is known about how specific biomarkers relate to the biochemical pathways involved in lung cancer development, and there is still no reliable method for diagnosing lung cancer at the early stages. This review summarizes some of the latest methods used for monitoring biomarkers in lung cancer patients, which could be applicable for clinical diagnosis. Techniques for capturing and pre-concentrating biomarkers, and the technologies used for subsequently determining them, are also discussed.

Synthesis of Cyclic Vinylidene Complexes and Azavinylidene Complexes by Formal [4+2] Cyclization Reactions.

Reactions of the hydrido-butenylcarbyne complex [OsHCl2(≡CC(PPh3)=CHEt)(PPh3)2]BF4 (1) with nitriles RC≡N (R=2-cyclopropyl-2-oxopropyl, 3-amino-2-oxobutyl) lead to six-membered cyclic vinylidene complexes 3 and azavinylidene complexes 4, that is, iso-osmapyridiniums. Treatment of 1 with excess 2-formylbenzonitrile at reflux temperature in CHCl3 in the presence of air produces a fused osmapyridinium 8, which is first oxidized to the tricyclic iso-osmapyridinium derivative 7, then to iso-osmapyridinium 9, which contains a fused naphthalenone fragment. The conversion of iso-osmapyridinium 9 (with a vinylidene segment) to the iso-osmapyridinium compounds 10 and 11 (with azavinylidene segments) was achieved in the presence of a hydrogen halide, such as HCl or HI. The molecular structures of the complexes synthesized were confirmed by X-ray studies. Moreover, the aromatic stabilization energy and nucleus-independent chemical-shift values of the osmapyridiniums and the strain in the iso-osmapyridinium rings were investigated by DFT calculations.

Conversions of osmabenzyne and isoosmabenzene.

We report herein the first example of the conversion of metallabenzyne II and isometallabenzene III. The osmium hydride vinylidene complex 1 reacts with HC≡CCH(OEt)(2) to give osmabenzyne 3 via isoosmabenzene 2. Compound 3 exhibits high thermal stability in air. Nonetheless, nucleophilic attack at 3 provides isoosmabenzenes 4 a and 4 b, or opens the ring to produce 5 a and 5 b. We propose mechanisms to disclose the intrinsic connection between the six-membered metallacycles, and carry out DFT calculations to rationalize the regioselectivity of the nucleophilic addition reactions.