Current Issue : April-June Volume : 2025 Issue Number : 2 Articles : 5 Articles
Tryptophan (TRP) is an essential amino acid crucial for the production of many bioactive compounds. Disturbances in TRP metabolism have been revealed in various diseases, many of which are closely related to the immune system. In recent years, we have focused on finding blood-based biomarkers of successful immunotherapy in cancer. Thus, we aimed to develop a robust liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for TRP and its metabolites that could be used in biomarker studies. Although analyzing TRP derivatives in biological matrices is not a new topic, we encountered multiple challenges during method development. One of them was the phenomenon of cross-interferences between the analyzed molecules, which has not been explored in most published papers. We noticed that injecting a pure single-compound solution often generated a signal in the other compounds’ MS/MS channels. Specifically, TRP generated unexpected peaks in the channel for kynurenine, kynurenic acid, and xanthurenic acid, while kynurenine generated peaks in the channel for kynurenic acid. We also recorded a mutual cross-talk between kynurenine and isotope-labeled TRP. Different origins of the observed cross-signal contribution were proposed. This paper draws attention to investigating cross-interferences in LC-MS/MS, especially when structurally related compounds will be analyzed. Despite all the challenges, the method was successfully validated according to international guidelines (EMA/ICH), and its applicability was confirmed in a pilot study including 20 patients with lung cancer undergoing chemoimmunotherapy....
Several studies have highlighted the essential role of taurine in maintaining the health of small animals, particularly dogs. Taurine deficiency has been linked to various health issues, especially in certain dog breeds. Therefore, accurately assessing taurine levels in canine blood is crucial for diagnosing and monitoring these conditions. In this study, we present the development of a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for rapidly quantifying taurine concentrations in dog whole blood and plasma. The method was validated according to current guidelines, showing excellent accuracy, precision, and sensitivity across a wide concentration range. Specifically, the limit of quantification was set at 80 nmol/mL for whole blood and 8 nmol/mL for plasma, ensuring the method’s reliability for both matrices. The application of this validated technique to blood samples of healthy dogs allowed for the establishment of reference intervals for taurine concentrations (148 to 495 nmol/mL for whole blood; 42 to 183 nmol/mL for plasma). Due to its robustness and simplicity, this method represents a valuable tool, supporting its routine use in health assessments and enabling more effective monitoring of taurine status in dogs....
Screening for inborn metabolic disorders (IMDs) in newborns is an important way to prevent serious metabolic and developmental difficulties that can result in lasting disabilities or even death. Electrospray ionization tandem mass spectrometry (MS/MS) provides an efficacious newborn blood spot screening (NBS) mechanism for analyzing dried blood spot specimens (DBSs) for biochemical markers for these conditions. Where possible, the elimination of derivatization in specimen preparation can simplify and streamline analysis. The Paya Hamsan Technologies Underivatized Newborn Screening Assay (PHUNSA) is an underivatized MS/MS test kit for IMD NBS. Validation of the accuracy, precision, linearity, and stability was based on the ISO 15189 standard and the CLSI NBS04 guideline. The PHUNSA kit demonstrated suitable performance along with acceptable recovery rates and negligible bias for many IMD analytes. Assay sensitivity was demonstrated through acceptable limits of detection (LOD) and lower limits of quantification (LLOQ). Specimen preparation times were decreased, the coefficients of variation were consistently below 10%, and accuracy and stability were demonstrated under various testing conditions, including prolonged storage and transportation. The PHUNSA kit provides a simplified, efficient, and reliable approach to IMD NBS with the potential to enhance NBS in Iran and other locations by providing a scalable, cost-effective, and streamlined option for early IMD detection and management....
Background: Quantifying urinary catecholamines and metanephrines is essential for the clinical screening and diagnosis of neuroendocrine tumours. HPLC with electrochemical detection (HPLC-ECD) is commonly used for this type of analysis but requires extensive sample cleanup. Simple and rapid dilute-and-shoot LC–multiple-reaction monitoring (MRM)-MS assays have been developed for quantitating these analytes in urine but have not yet been validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Methods: A simple dilute-and-shoot sample preparation without derivatization was used. C18 RP-UPLC-MRM-MS and positive-ion ESI were used, usually with two transitions per analyte being monitored. Certified deuterated internal standards were used for each analyte. Results: This assay was validated according to the CLSI C62-A guidelines, including accuracy/trueness, imprecision, sensitivity, specificity, carryover, stability, and linearity. The final MRM-MS method was compared to the established HPLCECD clinical chemistry reference method. The run time was reduced from 25 min to 5 min. Conclusions: A simple, robust, rapid, and cost-effective LC-MRM-MS assay for measuring urinary catecholamines and metanephrines was developed and validated according to the CLSI guidelines. This validated method requires minimal sample manipulation before analysis and provides sensitivity, specificity, and improved precision. The implementation of this assay in clinical laboratories will facilitate early and accurate diagnosis....
Background/Objectives: This study evaluates the applicability of a comprehensive twodimensional gas chromatography−flame ionisation detection (GC×GC−FID) approach for the simultaneous determination of 12 underivatised psychoactive drugs, including new psychoactive substances, that comprised of amphetamine, methamphetamine, mephedrone, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, α-pyrrolidinovalerophenone, n-ethylpentylone (ephylone), norketamine, ketamine, 3,4-methylenedioxypyrovalerone, methadone, and cocaine. Methods: Separation was effected using a non-polar first dimension (1D) and a polar second dimension (2D) column, demonstrating an improved separation of drug compounds compared to a polar/non-polar column configuration. Interference-free baseline separation of all psychoactive compounds in a urine matrix was achieved within 8 min. The GC×GC−FID method was validated according to the guidelines defined by Standard Practices for Method Validation in Forensic Toxicology. Results: The calibration curves for the 12 psychoactive drugs were well correlated (r2 > 0.99) within the concentration ranges of 50–1500 ng mL−1. Detection limits of 10–20 ng mL−1 were obtained, and good repeatability and reproducibility (CV < 11.4%) were attained for retention times and peak areas. Method recoveries for the small-scale solvent extraction procedure ranged from 96.9 to 114.5%, and bias was between −3.1% and 14.5%. Conclusions: The validated approach was successfully applied for the determination of these illicit compounds in spiked urine samples of different concentrations, highlighting its potential for rapid forensic drug screening....
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