Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine.

Tuberculosis (TB) remains one of the leading global causes of mortality. Several methods have been established to detect anti-TB agents in human plasma and serum. However, there is a notable absence of studies analyzing TB drugs in urine. Thus, our objective was to validate a method for quantifying first-line anti-TB agents: isoniazid (INH), pyrazinamide (PZA), ethambutol (ETH), and rifampicin (RIF), along with its metabolite 25-desacetylrifampicin, and degradation products: rifampicin quinone and 3-formyl-rifampicin in 10 µL of urine. Chromatographic separation was achieved using a Kinetex Polar C18 analytical column with gradient elution (5 mM ammonium acetate and acetonitrile with 0.1% formic acid). Mass spectrometry detection was carried out using a triple-quadrupole tandem mass spectrometer operating in positive ion mode. The lower limit of quantification (LLOQ) was 0.5 µg/mL for INH, PZA, ETH, and RIF, and 0.1 µg/mL for RIF's metabolites and degradation products. The method was validated following FDA guidance criteria and successfully applied to the analysis of the studied compounds in urine of TB patients. Additionally, we conducted a stability study of the anti-TB agents under various pH and temperature conditions to mimic the urine collection process in different settings (peripheral clinics or central laboratories).

Simplified urine-based method to detect rifampin underexposure in adults with tuberculosis: a prospective diagnostic accuracy study.

Variable pharmacokinetics of rifampin in tuberculosis (TB) treatment can lead to poor outcomes. Urine spectrophotometry is simpler and more accessible than recommended serum-based drug monitoring, but its optimal efficacy in predicting serum rifampin underexposure in adults with TB remains uncertain. Adult TB patients in New Jersey and Virginia receiving rifampin-containing regimens were enrolled. Serum and urine samples were collected over 24 h. Rifampin serum concentrations were measured using validated liquid chromatography-tandem mass spectrometry, and total exposure (area under the concentration-time curve) over 24 h (AUC0-24) was determined through noncompartmental analysis. The Sunahara method was used to extract total rifamycins, and rifampin urine excretion was measured by spectrophotometry. An analysis of 58 eligible participants, including 15 (26%) with type 2 diabetes mellitus, demonstrated that urine spectrophotometry accurately identified subtarget rifampin AUC0-24 at 0-4, 0-8, and 0-24 h. The area under the receiver operator characteristic curve (AUC ROC) values were 0.80 (95% CI 0.67-0.90), 0.84 (95% CI 0.72-0.94), and 0.83 (95% CI 0.72-0.93), respectively. These values were comparable to the AUC ROC of 2 h serum concentrations commonly used for therapeutic monitoring (0.82 [95% CI 0.71-0.92], P = 0.6). Diabetes status did not significantly affect the AUC ROCs for urine in predicting subtarget rifampin serum exposure (P = 0.67-0.92). Spectrophotometric measurement of urine rifampin excretion within the first 4 or 8 h after dosing is a simple and cost-effective test that accurately predicts rifampin underexposure. This test provides critical information for optimizing tuberculosis treatment outcomes by facilitating appropriate dose adjustments.

New approach to rifampicin stability and first-line anti-tubercular drug pharmacokinetics by UPLC-MS/MS.

Successful tuberculosis (TB) therapy requires achieving sufficient exposure to multiple drugs. Limited stability of several first-line anti-TB drugs might compromise reliable therapeutic drug monitoring (TDM). We developed and validated a sensitive and selective UPLC-MS/MS method for simultaneous quantification of isoniazid (INH), pyrazinamide (PZA), rifampicin (RIF), its metabolite 25-desacetylrifampicin and degradation products: rifampicin quinone and 3-formyl-rifampicin. Analysis was completed from a very small plasma volume (20 µL) using only protein precipitation with methanol. Chromatographic separation was achieved on a Kinetex Polar C18 column (2.6 µm; 150 × 3 mm) with a mobile phase consisting of 5 mM ammonium acetate and acetonitrile, both containing 0.1 % formic acid, in gradient elution. The analytes were detected using a positive ionization mode by multiple reaction monitoring. The LLOQ for RIF and its degradation products was 0.1 µg/mL, 0.05 µg/mL for INH, and 0.2 µg/mL for PZA. The method was validated based on the FDA guidance. The application of the method was confirmed in the analysis of RIF, INH, and PZA, as well as RIF metabolism/degradation products in plasma samples of patients with TB. Based on the detailed stability study of the analyzed compounds at various storage conditions, we proposed recommendations for handling the plasma and serum samples in TDM and other pharmacokinetic studies.

Designing negative feedback loops in enzymatic coacervate droplets.

Membraneless organelles within the living cell use phase separation of biomolecules coupled with enzymatic reactions to regulate cellular processes. The diverse functions of these biomolecular condensates motivate the pursuit of simpler in vitro models that exhibit primitive forms of self-regulation based on internal feedback mechanisms. Here, we investigate one such model based on complex coacervation of the enzyme catalase with an oppositely charge polyelectrolyte DEAE-dextran to form pH-responsive catalytic droplets. Upon addition of hydrogen peroxide "fuel", enzyme activity localized within the droplets causes a rapid increase in the pH. Under appropriate conditions, this reaction-induced pH change triggers coacervate dissolution owing to its pH-responsive phase behavior. Notably, this destabilizing effect of the enzymatic reaction on phase separation depends on droplet size owing to the diffusive delivery and removal of reaction components. Reaction-diffusion models informed by the experimental data show that larger drops support larger changes in the local pH thereby enhancing their dissolution relative to smaller droplets. Together, these results provide a basis for achieving droplet size control based on negative feedback between pH-dependent phase separation and pH-changing enzymatic reactions.

Alternative Methods for Therapeutic Drug Monitoring and Dose Adjustment of Tuberculosis Treatment in Clinical Settings: A Systematic Review.

BACKGROUND AND OBJECTIVE: Quantifying exposure to drugs for personalized dose adjustment is of critical importance in patients with tuberculosis who may be at risk of treatment failure or toxicity due to individual variability in pharmacokinetics. Traditionally, serum or plasma samples have been used for drug monitoring, which only poses collection and logistical challenges in high-tuberculosis burden/low-resourced areas. Less invasive and lower cost tests using alternative biomatrices other than serum or plasma may improve the feasibility of therapeutic drug monitoring. METHODS: A systematic review was conducted to include studies reporting anti-tuberculosis drug concentration measurements in dried blood spots, urine, saliva, and hair. Reports were screened to include study design, population, analytical methods, relevant pharmacokinetic parameters, and risk of bias. RESULTS: A total of 75 reports encompassing all four biomatrices were included. Dried blood spots reduced the sample volume requirement and cut shipping costs whereas simpler laboratory methods to test the presence of drug in urine can allow point-of-care testing in high-burden settings. Minimal pre-processing requirements with saliva samples may further increase acceptability for laboratory staff. Multi-analyte panels have been tested in hair with the capacity to test a wide range of drugs and some of their metabolites. CONCLUSIONS: Reported data were mostly from small-scale studies and alternative biomatrices need to be qualified in large and diverse populations for the demonstration of feasibility in operational settings. High-quality interventional studies will improve the uptake of alternative biomatrices in guidelines and accelerate implementation in programmatic tuberculosis treatment.

An unusual case of paediatric plasmablastic lymphoma presenting with malignant effusion and the challenges in its diagnosis.

Plasmablastic lymphoma (PBL) is an aggressive non-Hodgkin lymphoma occurring commonly in the oral mucosa and jaw of human immunodeficiency virus (HIV) positive adult males. PBL is not a common occurrence in children and a presentation with malignant effusion is rarely reported. Herein, we share our experience in the challenges confronted in the diagnosis of PBL in a 6-year-old, HIV positive boy presenting with malignant pleural and peritoneal effusions along with gum hypertrophy, lymphadenopathy and paranasal sinus mass. Amenability of pleural effusion to exfoliative cytology led to an initial cytological examination demonstrating large atypical lymphoid cells with plasmacytoid morphology and a plasmablastic variant of Burkitt lymphoma was initially considered. However immunophenotyping by flowcytometry (FCM) and a cell block immunohistochemical evaluation of the serous effusion suggested a plasma cell immunophenotype and a diagnosis of PBL was favored. A subsequent biopsy from the paranasal sinus mass confirmed the diagnosis of PBL but showed tumour cell angiocentricity on morphology and CD45 expression on immunohistochemistry (IHC), both unusual features in PBL. A CD20 negative/MUM-1 positive immunoprofile and presence of a solid tumour mass in a typical location in addition to malignant effusion substantiated the diagnosis of PBL. The patient was offered HAART (highly active antiretroviral therapy) and chemotherapy and is on follow-up. Paediatric PBL with malignant effusion is rarely reported and this case stresses the importance of use of a multimodality diagnostic approach for an accurate diagnosis.