Search Results (32)

Early pregnancy diagnosis is a key component of reproductive management in swine production systems. Accurate identification of pregnant and non-pregnant sows within the first 30 days after insemination allows timely reproductive decisions and reduces non-productive days. The present systematic review evaluates the diagnostic efficiency of ultrasonographic and progesterone-based methods used for early detection of pregnancy in sows. A structured literature search was conducted in accordance with the PRISMA Statement guidelines, using major scientific databases. Studies evaluating pregnancy diagnosis in sows within the first 30 days after insemination were included. Diagnostic approaches were analyzed with respect to methodological design, timing of examination, biological sample matrix, and reported indicators of diagnostic accuracy. Ultrasonographic techniques have evolved from early acoustic detection in A-mode to real-time imaging in B-mode and more recently algorithm-assisted interpretation of ultrasound images. Real-time ultrasonography allows direct visualization of gestational structures; in one study, diagnostic accuracy above 95% was reported after approximately 23–24 days of pregnancy under optimal examination conditions. Progesterone-based analyses evaluate luteal endocrine activity and are particularly useful for early identification of non-pregnant animals after luteolysis. The diagnostic efficiency of hormonal assays depends strongly on the timing of sampling and the biological matrix used for analysis, including plasma, serum, dried blood spots, saliva, or feces. The comparative analysis shows that ultrasonography provides morphological confirmation of pregnancy, whereas progesterone analyses serve mainly as functional indicators of luteal activity. These methods play complementary roles in reproductive management. Ultrasonography remains the most reliable method for confirming pregnancy, while progesterone-based analyses are valuable tools for early reproductive screening and identification of non-pregnant sows. Full article

Methotrexate (MTX) remains the first-choice treatment for rheumatoid arthritis (RA), but individual variability in response and adherence underscores the need for reliable biomarkers of long-term drug exposure. Intracellular methotrexate polyglutamates (MTXPGs), typically measured in red blood cells (RBCs), fulfill this role but require invasive venous sampling. This study aimed to develop and validate a multi-matrix LC–MS/MS method for measuring MTXPGs in capillary blood samples obtained via volumetric absorptive microsampling (VAMS) and dried blood spots (DBS), and to compare these methods with traditional matrices. The method was validated in accordance with ICH M10 guidelines across RBC, whole blood (WB), VAMS, and DBS samples. MTX and MTXPG_2–5 and total MTXPG were measured in 40 matched clinical samples. MTXPG_6–7 were not detected across the tested clinical samples. Validation using Passing–Bablok regression, Bland–Altman analysis, and Spearman correlation showed strong agreement between VAMS and DBS (slopes 0.95–1.07; bias −4.21% to 0.36%; SRCC ≥ 0.969), with up to 100% of samples within ±20% of the agreement limits for total MTXPG. Significant differences were observed between capillary matrices and RBCs, with higher MTXPG levels in erythrocytes (bias up to −28%). Whole blood showed closer agreement with microsampling methods. ISR pass rates ranged from 84% to 95%, and stability tests indicated matrix- and chain length-dependent degradation, particularly for long-chain MTXPGs. These findings show that VAMS and DBS yield comparable results and can be considered interchangeable within a capillary-sampling framework. However, interpretation must account for matrix-specific differences when relating measurements to RBC-based reference values. This validated method could support the analytical feasibility of decentralized MTXPG monitoring in RA. However, prospective studies linking matrix-specific thresholds with disease activity, adherence, and toxicity are required before implementation for therapeutic decision-making. Full article

The use of dried matrix spots (DMSs) has recently re-emerged as a useful sample storage technique and analytical platform along with the increased adoption of and general preference for ambient ionization mass-spectrometric methods. However, challenges associated with precise liquid confinement and sample targeting persist. In this paper, we present a laser micromachining-based approach to prepare DMSs on hydrophobic paper substrates that include visual recognition elements, or reticles, around surface energy traps (SETs). This targeted DMS substrate is combined with direct mass spectrometric analyses, namely liquid microjunction–surface sampling probe–mass spectrometry (LMJ-SSP-MS) and flowing atmospheric-pressure afterglow–mass spectrometry (FAPA-MS). With the laser-based micromachining approach, DMSs flanked by crosshairs for enhanced visualization are prepared on SETs as small as 0.55 mm in diameter, which offers an approximately 12-fold reduction in size compared to traditional DMS preparations. The DMSs prepared on these targeting SETs are demonstrated with the detection of caffeine in model aqueous and artificial urine solutions using LMJ-SSP-MS and FAPA-MS, respectively. With further refinement, this approach could be automated using computer vision and robotics to broaden the scope of DMSs and improve the analytical workflow. Full article

Background: β-thalassemia is a rare genetic disorder affecting 1–5% of the global population and poses a health burden due to migration of individuals from endemic regions. Identifying asymptomatic β-thalassemia carriers is essential to prevent the birth of thalassemic babies. A simple, sensitive method compatible with self-sampling could enhance the detection of β-thalassemia in the population. Methods: Capillary blood was collected via dried blood spot (DBS) and dried blood matrix (DBM) from 18 members (52.9%, 18/34) of a three-generation family. Hemoglobin was extracted, and globin chains were analyzed on a triple quadrupole mass spectrometer (TQMS). δ/β (%) was utilized as a biomarker to identify β-thalassemia. Venous blood collected from positive and negative individuals (n = 11) was further tested to confirm the findings and validated with complete blood count (CBC) and Capillary Electrophoresis (CE). Results: β-thalassemia was detected in seven individuals: three from generation I, three from generation II, and one from generation III. CBC showed thalassemia indices, while CE demonstrated elevated HbA2 consistent with β-thalassemia. Molecular sequencing of two samples confirmed the heterozygous c.92 + 5 G > C mutation in the β-globin gene. The overall prevalence of β-thalassemia in the family was 20.6% (7/34). High clinical performance was achieved across sample types, with 100% sensitivity for DBS, 100% specificity for DBM, and an overall accuracy of 91% when compared with CE. Conclusions: TQMS in combination with CBC parameters successfully identified asymptomatic heterozygous β-thalassemia carriers using self-sampling techniques. Cascade screening within affected families emerges as a possible strategy for early detection of β-thalassemia pending comprehensive validation. Full article

Background/Objectives: MDMA and MDA are among the stimulant drugs most frequently encountered in forensic casework, and oral fluid represents a practical biological matrix for their detection. However, liquid oral fluid requires refrigeration, is susceptible to degradation, and can be logistically demanding for routine laboratories. Dried Oral Fluid Spots (DOFS) offer a low-cost and stable alternative. This study aimed to develop and validate a DOFS-based analytical workflow for quantifying MDMA and MDA using liquid chromatography and a diode-array detector. Methods: Watercolor paper was selected as the substrate and pretreated with diluted nitric acid to improve analyte desorption. DOFS were prepared using 150 µL of pooled oral fluid, dried for 4 h, and extracted with methanol. Chromatographic separation was performed on a phenyl column using aqueous TFA and acetonitrile mobile phase. Method validation followed the ICH M10 criteria. Results: The method showed linear responses between 12.5 and 5000 ng mL⁻¹, with LOD and LLOQ of 6 and 12 ng mL⁻¹ for both analytes, respectively. Precision and accuracy met acceptance criteria across all QC levels. Recoveries ranged from 84% to98%. DOFS samples demonstrated adequate stability under multiple storage and handling conditions. Conclusions: The optimized DOFS–LC–DAD workflow offers a robust, low-cost, and flexible approach for the analysis of MDMA and MDA in oral fluid for laboratory-based or semi-controlled collection environments. Its compatibility with both LC- and GC-based detectors enhances applicability in diverse forensic laboratory settings. Full article

Background/Objectives: Therapeutic drug monitoring (TDM) of antipsychotic medications plays an important role in optimizing treatment efficacy, reducing adverse effects, and supporting adherence. While Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS) has long been the gold standard for antipsychotic quantification, recent advances in automated platforms and microsampling raise questions about its current clinical practicality. This systematic review evaluated the clinical applicability and analytical performance of UHPLC-based methods for monitoring antipsychotic drugs, focusing on precision, recovery, matrix effects, and suitability across various biological matrices. Methods: A systematic search of PubMed, Scopus, and Web of Science was conducted for studies published between 2013 and 2024 involving UHPLC-based quantification of antipsychotics in clinical samples from adult patients. Data on analytical parameters, sample matrices, and study characteristics were extracted. A custom quality checklist was used to assess methodological rigor. In addition to qualitative synthesis, non-traditional quantitative approaches were applied, including descriptive aggregation of recovery, matrix effects, and precision across studies, as well as correlation analyses to explore relationships among performance parameters. Results: Twelve studies were included, spanning a range of typical and atypical antipsychotics and metabolites. Plasma and serum demonstrated the highest analytical reliability (recovery >90%, minimal matrix effects), while dried blood spots (DBSs), whole blood, and oral fluid showed greater variability. Clinically, UHPLC–MS/MS enabled more accurate dose adjustments and identification of non-adherence, outperforming immunoassays in sensitivity, specificity, and metabolite detection. Microsampling methods showed promise for outpatient and decentralized care but require further clinical validation. Conclusions: UHPLC–MS/MS remains the most robust and reliable method for TDM of antipsychotics, especially when quantification of active metabolites is required. While logistical barriers remain, technological advances may enhance feasibility and support broader integration into routine psychiatric care. Full article

Dried matrix spot (DMS) techniques have gained increasing attention in bioanalytical and forensic toxicology for the detection of opiates and opioids, offering minimally invasive sampling, enhanced sample stability, and simplified storage and transport. This review provides a critical overview of recent methodological advances and applications of DMS across multiple biological matrices, including blood, plasma, urine, and oral fluid. Particular focus is given to sample preparation protocols, extraction strategies, analytical instrumentation, and method performance. Dried blood spots (DBS) remain the most established format; however, alternative matrices such as dried plasma, urine, and saliva spots (DPS, DUS, DSS) are expanding the scope of DMS, particularly in decentralised and point-of-care contexts. Despite clear advantages, such as reduced biohazard risk and compatibility with high-throughput workflows, several limitations persist, including low sample volumes, matrix-specific recovery issues, and lack of standardised procedures. Future efforts should aim to optimise paper substrates, improve solvent–matrix compatibility, and integrate DMS workflows with automated or miniaturised mass spectrometry platforms. Overall, DMS techniques represent a versatile and evolving analytical platform with strong potential for reliable opioid monitoring in both clinical and forensic settings. Full article

Deoxynivalenol (DON) and zearalenone (ZEN) are common mycotoxins in animal feeds, and their metabolites can be detected in exposed animals. Traditional methods focus on mycotoxin detection in feed, whereas biomarker-based approaches are used for evaluating individual exposure. This study aimed to develop and validate a multi-analyte method for the detection of biomarkers of ZEN, DON, and their metabolites α-zearalanol (α-ZAL), zearalanone (ZAN), deepoxy-DON (DOM-1), and 3-acetyl-DON (3-ADON) in swine using dried blood spots (DBSs) on qualitative filter paper. Analysis was performed using high-performance liquid chromatography–tandem mass spectrometry. Blank blood samples from three male pigs were fortified with 20, 40, and 60 μg/L of each analyte. Aliquots of 40 μL were spotted onto filter paper and then extracted and analyzed. Method validation included evaluating limits of detection and quantification, linearity, matrix effects, recovery, repeatability, intermediate precision, and selectivity. All analytes were detectable in DBS. Also, ZEN, ZAN, DON, and DOM-1 met all validation criteria, with recovery values of 89.10%, 79.79%, 101.50%, and 79.50%, respectively. Both α-ZAL and 3-ADON showed lower recoveries (74.66% and 58.66%). The method was successfully validated for simultaneous analysis of ZEN, ZAN, DON, and DOM-1 in swine DBS, offering a practical and minimally invasive tool for biomonitoring mycotoxin exposure. Full article

The sphingolipidoses Fabry disease, Gaucher disease and Acid sphingomyelinase deficiency (ASMD) are the three most common lysosomal storage diseases for which treatment is currently available. Timely diagnosis with estimation of the disease severity and possibilities of follow-up of patients, whether or not under therapy, is crucial for providing good care and for the prevention of possible lethal complications. With this research we provide an efficient and sensitive detection method; its implementation in clinical practice could optimize the diagnosis and follow-up of patients with Gaucher, Fabry and ASMD. This detection method on dried blood spots (DBS) was validated according to the international Clinical and Laboratory Standards Institute (CLSI) guidelines, looking at reproducibility, linearity, carry-over and lower limit of quantification. Analogously, validation and subsequent comparison of the method validation results using another matrix, the Capitainer blood sampling cards (Capitainers), was fulfilled. The results showed that this detection method is fully applicable clinically when using DBS as well as Capitainers. In addition, even additional improvements of some validation parameters were found when using the Capitainers. Twenty-six patient samples and fifteen healthy samples were analyzed for case finding control. All patient cases were detected without ambiguity. We present a high-resolution mass spectrometry method that provides an accurate analysis for targeted screening, aiming for improved/accelerated diagnosis when added in the diagnostic pathway and monitoring of Fabry, Gaucher and ASMD in DBS as well as in Capitainers, with the main advantages of a small volume of blood samples, guaranteeing stability and easy transportation from the collection site to the laboratory. Full article

Everolimus (EVE), an mTOR inhibitor, is widely used in solid organ transplantation (SOT) because of its immunosuppressive properties. Due to its narrow therapeutic window and significant pharmacokinetic variability, therapeutic drug monitoring (TDM) is essential for achieving optimal outcomes. We developed and thoroughly validated a robust LC-MS/MS method to measure EVE levels in venous whole blood (WB) and capillary blood collected using two microsampling devices: Mitra™ (volumetric absorptive microsampling, VAMS) and Capitainer^® (quantitative dried blood spot, qDBS). The validation followed EMA and IATDMCT guidelines, assessing linearity (1.27–64.80 ng/mL for WB and 0.50–60 ng/mL for VAMS/qDBS), as well as selectivity, accuracy, precision, matrix effects, recovery, stability, and incurred sample reanalysis. Clinical validation involved 66 matched samples from 33 adult SOT recipients. The method demonstrated high accuracy and precision across all matrices, with no significant carryover or matrix interference. Statistical analysis using Passing–Bablok regression and Bland–Altman plots showed excellent agreement between the microsampling methods and the venous reference. Hematocrit effects were tested both in laboratory conditions and on clinical samples and were found to be negligible. This study provides the first comprehensive analytical and clinical validation of the Mitra and Capitainer devices for EVE monitoring. The validated LC-MS/MS microsampling method supports decentralized, patient-centred TDM, offering a reliable alternative to conventional blood sampling in transplant care. Full article

Background: The combination of ivacaftor, tezacaftor and elexacaftor (ETI) is approved for patients with cystic fibrosis (CF) aged two years and older and at least one F508del mutation in the CFTR gene. Variability in ETI treatment response has been repeatedly reported, and its reasons are unclear and understudied. Objectives: We present a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid and simultaneous quantification of ETI in plasma, dried plasma spots (DPS), and whole blood volumetric absorptive microsampling (VAMS). Methods: The method utilizes a rapid extraction protocol with 200 μL methanol after the addition of deuterated internal standards. Chromatographic separation was achieved using a reversed-phase Hypersil Gold aQ column (Thermo Fisher Scientific). The method was validated according to ICH (International Council on Harmonisation) guidelines M10 for bioanalytical method validation, demonstrating linearity in the concentration range 0.020–12.000 µg/mL. It was also proved accurate and reproducible with no matrix effect. This method was applied to anonymized samples from patients undergoing ETI treatment: eight plasma and DPS and five VAMS samples were analyzed. Results: ETI concentrations measured in plasma and DPS were interchangeable, whereas ETI concentrations in VAMS were lower than in plasma, as expected for molecules with high plasma protein binding (99%). A correction factor based on the hematocrit value was used to calculate the equivalent plasma concentration from VAMS concentrations. Conclusions: This method is suitable for pharmacokinetic (PK) studies and could facilitate the centralization of samples to specialized laboratories, supporting multicenter studies. Full article

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure–toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95–106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22–55%),with high recovery rates (81–93%) and minimal matrix effects (ME 0.9–1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented. Full article

The spotted lanternfly (SLF) is an invasive species native to China. It was first discovered in the United States in Pennsylvania in 2014. It is known to cause great economic damage by destroying various crops, specifically grape vines, and therefore, several efforts have been made to control and mitigate its spread from the Northeast. Canine detection is a useful detection tool; however, it is crucial to understand the volatile organic compounds emitting by this pest to better direct canine training paradigms to prevent false alerts and to understand potential volatile markers of importance indicative of this species. The purpose of this study is to address the gap in research regarding the volatile organic compound (VOC) profile of SLF to better inform pest control mitigation strategies. Instrumental analysis was performed utilizing SPME-GC/MS on cold-killed SLF eggs, dried crickets, and tree bark. Differences in detected VOCs within each sample set depicted distinctive odor profiles for each matrix tested. Storage of these samples also depicted VOC accumulation variation as a function of time, thereby providing implications for long-term storage and sample handling for these types of training aids in canine applications. Full article

Neglected tropical diseases (NTDs) are a group of illnesses which usually present with a chronic clinical picture. NTDs can lead to permanent disability and are often associated with social stigma. In many developing countries where NTDs are endemic, there are no diagnostic tools for the safe storage and transport of biological samples, and there are no specialist diagnostic centers where the samples could be processed. The transport of biological samples (blood, urine) collected in field conditions and brought to laboratories located in developed countries requires the maintenance of the cold chain during transportation. Ensuring temperature control during transport could be problematic or even impossible to achieve; it is also expensive. A helpful solution to this problem is to use the dried matrix spot (DMS) technique, which seems to be a reliable method for collecting biological samples to be used for screening purposes and conducting epidemiological surveillance of NTDs in developing countries. This article is an overview of how DMSs can be used in the diagnosis of most neglected tropical diseases. Full article

Background: The antiepileptic drug lamotrigine (LTG) shows high pharmacokinetic variability due to genotype influence and concomitant use of glucuronidation inducers and inhibitors, both of which may be frequently taken by elderly patients. Our goal was to develop a reliable quantification method for lamotrigine and its main glucuronide metabolite lamotrigine-N2-glucuronide (LTG-N2-GLU) in dried blood spots (DBS) to enable routine therapeutic drug monitoring and to identify altered metabolic activity for early detection of drug interactions possibly leading to suboptimal drug response. Results: The analytical method was validated in terms of selectivity, accuracy, precision, matrix effects, haematocrit, blood spot volume influence, and stability. It was applied to a clinical study, and the DBS results were compared to the concentrations determined in plasma samples. A good correlation was established for both analytes in DBS and plasma samples, taking into account the haematocrit and blood cell-to-plasma partition coefficients. It was demonstrated that the method is suitable for the determination of the metabolite-to-parent ratio to reveal the metabolic status of individual patients. Conclusions: The clinical validation performed confirmed that the DBS technique is a reliable alternative for plasma lamotrigine and its glucuronide determination. Full article