Establishment of a High-Resolution Liquid Chromatography-Mass Spectrometry Spectral Library for Screening Toxic Natural Products.

Many natural products have biological effects on humans and animals. Poisoning caused by natural products is common in clinical toxicology cases. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) has recently emerged as a powerful analytical tool for large-scale target screening, and the application of LC-HRMS can be expanded to evaluate potential natural product poisoning in clinical cases. We report the construction of an LC-HRMS spectral library of 95 natural products commonly implicated in poisoning, and an LC-HRMS assay was validated for definitive detection of natural products in urine and serum samples. For each compound, the limit of detection was determined in the analytical range of 1.0-1,000 ng/mL for urine samples and 0.50-500 ng/mL for serum samples. The mean (SD) values of matrix effects for urine samples and that for serum samples were both -21% (22%), and the mean (SD) value of recovery for serum samples was 89% (26%). The LC-HRMS assay was successfully applied to identify natural products in clinical cases. The spectral library parameters of each compound are provided in the supplementary material to aid other laboratories in identification of unknown natural toxins and development of similar methods on different mass spectrometry platforms.

A SARS-CoV-2 Label-Free Surrogate Virus Neutralization Test and a Longitudinal Study of Antibody Characteristics in COVID-19 Patients.

Methods designed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humoral response include virus neutralization tests to determine antibody neutralization activity. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockage of molecular interactions have been proposed. A surrogate virus neutralization test was established on a label-free immunoassay platform (LF-sVNT). The LF-sVNT analyzes the binding ability of SARS-CoV-2 spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) after neutralizing RBD with antibodies in serum. The LF-sVNT neutralizing antibody titers (50% inhibitory concentration [IC50]) were determined from serum samples (n = 246) from coronavirus disease 2019 (COVID-19) patients (n = 113), as well as the IgG concentrations and the IgG avidity indices. Although there was variability in the kinetics of the IgG concentrations and neutralizing antibody titers between individuals, there was an initial rise, plateau, and then in some cases a gradual decline at later time points after 40 days after symptom onset. The IgG avidity indices, in the same cases, plateaued after an initial rise and did not show a decline. The LF-sVNT can be a valuable tool in research and clinical laboratories for the assessment of the presence of neutralizing antibodies to COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days post-symptom onset. Despite the decline of IgG concentration and neutralizing antibody titer, IgG avidity index increases, reaches a plateau, and then remains constant up to 8 months postinfection. The decline of antibody neutralization activity can be attributed to the reduction in antibody quantity rather than the deterioration of antibody quality, as measured by antibody avidity.

Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Avidity Maturation and Association with Disease Severity.

The kinetics of IgG avidity maturation during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was studied. The IgG avidity assay, using a novel label-free immunoassay technology, revealed a strong correlation between IgG avidity and days since symptom onset. Peak readings were significantly higher in severe than mild disease cases.

Development of Label-Free Immunoassays as Novel Solutions for the Measurement of Monoclonal Antibody Drugs and Antidrug Antibodies.

BACKGROUND: Immunoassays based on label-free technologies (label-free immunoassay [LFIA]) offer an innovative approach to clinical diagnostics and demonstrate great promise for therapeutic drug monitoring (TDM) of monoclonal antibody (mAb) drugs. An LFIA measures immunocomplex formation in real time and allows for quantification on initial binding rate, which facilitates fast measurement within a few minutes. METHODS: Based on thin-film interferometry (TFI) technology, open-access LFIAs were developed for the quantification of the mAb drugs adalimumab (ADL) and infliximab (IFX) and for the detection of the antidrug antibodies (ADAs) to the mAb drugs (ADL-ADAs and IFX-ADAs). RESULTS: The LFIAs for active mAb drugs (ADL and IFX) and for ADAs (ADL-ADAs and IFX-ADAs) were validated. The analytical measurement range (AMR) for both ADL and IFX was from 2 to 100 µg/mL. The AMR for ADL-ADAs was from 5 to 100 µg/mL and for IFX-ADAs was 10 to 100 µg/mL. In the comparison of LFIAs and reporter gene assays, the correlation coefficient was 0.972 for the quantification of ADL and 0.940 for the quantification of IFX. The concordance rate was 90% for the detection of ADL-ADAs and 76% for the detection of IFX-ADAs. CONCLUSIONS: The LFIAs for active mAb drugs and ADAs were appropriate for the TDM of ADL and IFX. The TFI technology has unique advantages compared with other technologies used for the measurement of mAb drugs. Label-free technologies, especially those allowing for open-access LFIAs, have great potential for clinical diagnostics.

A case of unexplained duodenal ulcer and massive gastrointestinal bleed.

A 73-year-old man was displaying symptoms of massive gastrointestinal (GI) bleed. Surgical actions were performed to control the bleed caused by an erosive duodenal ulcer with duodenal perforation. When investigating the culprit of this case, the pain medications prescribed two weeks prior by a traditional Chinese medicine doctor raised attention. The patient's admission serum sample and the pain medications from unknown sources were analyzed using a clinically validated liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method. The NSAIDs diclofenac, piroxicam, and indomethacin were identified, as well as some other synthetic drugs and natural products. The patient's concurrent exposure to multiple NSAIDs significantly increased the risk of upper GI complications. It is reasonable to argue that the high-dose use of the NSAIDs was a major cause of the duodenal ulcer and GI bleed. In addition, the identified natural products such as atropine and ephedrine have well-documented toxicities. It is important to increase the visibility of unregulated medications, and the capability to perform untargeted mass spectrometry analysis provides a unique diagnostic advantage in cases where exposure to toxic substances is possible.

A thin-film interferometry-based label-free immunoassay for the detection of daratumumab interference in serum protein electrophoresis.

BACKGROUND: Daratumumab (DARA) is a fully human anti-CD38 IgG1-κ monoclonal antibody drug used in the treatment of multiple myeloma (MM). While serum protein electrophoresis (SPEP) is an important assay for diagnosis and monitoring of patients with MM, DARA can appear in the γ-region as a single band and interfere with the interpretation of SPEP results. An approach to detect the interference is measuring the quantity of DARA in serum samples and assessing its impact on SPEP results. Immunoassays based on label-free technologies, i.e. label-free immunoassays (LFIA's), can achieve real-time immunometric measurement without attaching a reporter molecule (enzyme, fluorophore, etc.) to the immunocomplex. The recorded time course of the immunocomplex formation allows for quantitation on initial binding rate, which facilitates rapid measurement within a few minutes. Based on the thin-film interferometry (TFI) technology, a rapid LFIA was established for the quantitation of DARA in serum samples. METHODS: The TFI-based LFIA for DARA was validated for imprecision (CV), accuracy, limit of quantitation (LOQ), and analytical measurement range (AMR). Interference to the LFIA was evaluated using a group of protein samples, as well as hemolytic, lipemic, and icteric clinical samples. RESULTS: The precision of the TFI-based LFIA's for DARA ranged from 6.5% to 10.7% (within-run CV), and 7.4% to 11.6% (between-run CV), with a bias of -2.1% to 10.1%. The LOQ was 10 µg/ml (n = 4, CV 9.8%), with an AMR ranging from the LOQ to 1000 µg/ml. The LFIA was used to measure 37 patient samples submitted for SPEP testing. The LFIA results were 100% consistent with the history of DARA use as documented in the medical record. CONCLUSIONS: The TFI-based LFIA was successful at accurately identifying DARA in serum samples and can be used to identify DARA interference in SPEP testing. This work demonstrates the applicability of label-free technologies, particularly the TFI technology, to clinical diagnostic needs. Given the simplicity and the speed of the testing process, the TFI technology provides a unique testing approach for the measurement of proteins in clinical samples.

Quantitation of Cannabinoids in Breath Samples Using a Novel Derivatization LC-MS/MS Assay with Ultra-High Sensitivity.

As the legalization of medical and recreational marijuana use expands, measurement of tetrahydrocannabinol (THC) in human breath has become an area of interest. The presence and concentration of cannabinoids in breath have been shown to correlate with recent marijuana use and may be correlated with impairment. Given the low concentration of THC in human breath, sensitive analytical methods are required to further evaluate its utility and window of detection. This paper describes a novel derivatization method based on an azo coupling reaction that significantly increases the ionization efficiency of cannabinoids for LC-MS/MS analysis. This derivatization reaction allows for a direct derivatization reaction with neat samples and does not require further sample clean-up after derivatization, thus facilitating an easy and rapid "derivatize & shoot" sample preparation. The derivatization assay allowed for limits of quantitation (LOQ's) in the sub-pg/mL to pg/mL range for the five cannabinoids in breath samples, i.e., only 5~50 femtograms of an analyte was required for quantitation in a single analysis. This ultrahigh sensitivity allowed for the quantitation of cannabinoids in all breath samples collected within 3 hours of smoking cannabis (n = 180). A linear correlation between THC and cannabinol (CBN) in human breath was observed, supporting the hypothesis that CBN is converted from THC during the combustion of cannabis. The derivatization method was also applied to the analysis of cannabinoids in whole blood samples, achieving LOQ's at ten-pg/mL to sub-ng/mL level. This azo coupling-based derivatization approach provided the needed analytical sensitivity for the analysis of THC in human breath samples using LC-MS/MS and could be a valuable tool for the analysis of other aromatic compounds in the future.

Azo coupling-based derivatization method for high-sensitivity liquid chromatography-tandem mass spectrometry analysis of tetrahydrocannabinol and other aromatic compounds.

An azo coupling-based derivatization method is reported for high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitation of tetrahydrocannabinol (THC) and other aromatic compounds, i.e. phenols and amines. Through the azo coupling of a diazonium to an analyte, it produces a derivatized analyte which has enhanced ionization efficiency and results in high-response fragments in tandem mass spectrometry. The derivatization method was applied to six typical aromatic compounds using three different diazonium salts as derivatization reagents, demonstrating its applicability to a variety of analytes and reagents. The derivatization reaction can be directly carried out in neat samples, and after derivatization the samples can be immediately sent to the LC-MS/MS instrument for analysis. These advantages facilitate a one-step sample preparation procedure that can be completed in less than one hour, allowing for a "derivatize & shoot" lab workflow. The derivatization method was applied to establish an LC-MS/MS assay for the quantitation of THC in human breath samples. The derivatization conditions were studied in this application, including the effects of acidity, organic solvent, and diazonium concentration in the reaction. The THC derivatization assay was validated and achieved a limit of quantitation (LOQ) of 0.50 pg/ml using either of the two regio-isomers of the azo-derivative of THC (THC-DRV). To prove that the derivatization method has compatibility with complex-matrix samples, a THC derivatization assay for serum samples was established, in which the azo coupling reaction was directly carried out in crude protein-precipitated supernatants. An LOQ of 5.0 pg/ml was achieved. In addition, excellent correlation between THC derivatization and non-derivatization assays was found in the analysis of whole blood samples.