The identification of tuberculosis biomarkers in human urine samples.

We aimed to determine whether shotgun proteomic approaches could be used to identify tuberculosis (TB)-specific biomarkers in the urine of well-characterised patients with active TB versus no TB. Patients with suspected TB (n=63) were classified as: definite TB (Mycobacterium tuberculosis positive culture, n=21); presumed latent-TB infection (LTBI) (M. tuberculosis negative culture, no radiological features of active TB, a positive QuantiFERON-TB Gold In-Tube (QFT-IT) test and a positive T-SPOT.TB test, n=24); and presumed non-TB/non-LTBI (M. tuberculosis negative culture, no radiological features of active TB, a negative QFT-IT test and a negative T-SPOT.TB test, n=18). Urine proteins, in the range of 3-50 kDa, were collected, separated by a one-dimensional SDS-PAGE gel and digested using trypsin, after which high-performance liquid chromatography-tandem mass spectrometry was used to identify the urinary proteome. 10 mycobacterial proteins were observed exclusively in the urine of definite TB patients, while six mycobacterial proteins were found exclusively in the urine of presumed LTBI patients. In addition, a gene ontology enrichment analysis identified a panel of 20 human proteins that were significant discriminators (p<0.05) for TB disease compared to no TB disease. Furthermore, seven common human proteins were differentially over- or under-expressed in the TB versus the non-TB group. These biomarkers hold promise for the development of new point-of-care diagnostics for TB.

A rapid Dilute-and-Shoot LC-MS/MS method for quantifying THC-COOH and THC-COO(Gluc) in urine.

Cannabis remains one of the most commonly used psychotropes. Cannabis use is frequently evaluated via the testing of suspected patient samples. Thus, there is a high demand for simple, accurate and fast assays to support the increasing needs for testing. This report highlights a reliable, simple and fast liquid chromatography - tandem mass spectrometry assay that quantifies the cannabis metabolites THC-COOH and THC-COO(Gluc) in human urine. The assay employs a direct dilute-and-shoot approach, whereby urine samples are diluted 10X before being directly injected on the liquid chromatography and mass spectrometer. The assay quantification is based on an internal calibration approach that used deuterated analogues for THC-COOH and THC-COO(Gluc) as internal standards. The assay's analysis time was 5 min. The quantification was valid over a wide linear range (25 - 8,000 ng/mL) for both analytes and was free of matrix interferences. The within-day and between-day precision was determined to be ≤ 15 % CV for both analytes. The assay was validated based on the College of American Pathologists (CAP) and Clinical Laboratory Standards Institute (CLSI) guidelines.

Chiral purity assay for Flindokalner using tandem mass spectrometry: method development, validation, and benchmarking.

The present work demonstrates the application and validation of a mass spectrometry method for quantitative chiral purity determination. The particular compound analyzed is Flindokalner, a Bristol-Myers Squibb drug candidate for post-stroke neuroprotection. Chiral quantification of Flindokalner was achieved using tandem mass spectrometry (MS/MS) and the kinetic method, a gas phase method used for thermochemical and chiral determinations. The MS/MS method was validated and benchmarked against two separate chromatographic techniques, chiral high performance liquid chromatography with ultra-violet detection (LC/UV) and achiral high performance liquid chromatography with circular dichroism detection (LC/CD). The chiral purity determination of Flindokalner using MS/MS proved to be rapid (3 min run time for each sample) and to have accuracy and precision comparable to the chiral LC/UV and achiral LC/CD methods. This method represents an alternative to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful in rapid screening experiments.