Search Results (72)

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

Background: Ustekinumab (UST) is a monoclonal antibody (mAb) used in the treatment of inflammatory bowel disease (IBD). Elevated serum concentrations are typically associated with improved therapeutic outcomes, and therapeutic drug monitoring (TDM) is a useful tool for guiding mAbs treatment. This study aimed to develop a dried blood spot (DBS) method for TDM of UST in patients with IBD. Methods: The commercial enzyme-linked immunosorbent assay for plasma samples was optimized for DBS samples and subsequently validated according to international guidelines for classical and DBS-specific validation parameters. It was then applied to analyze serum and DBS samples obtained from venous and capillary blood of IBD patients undergoing UST therapy. Results: The method was linear (3–12 mg/L) with acceptable inter-day accuracy (90.1–106%) and precision (<12%). We confirmed that there was no hematocrit effect and that DBS samples were stable for one month under room conditions. A linear model was developed between venous DBS and serum UST concentrations, which showed no systemic bias, and 71% of the samples were within ±20% of the mean. In addition, a linear correlation between venous DBS and capillary DBS samples was established, showing no significant bias, with 84% of samples within ±20% of the mean. Finally, a novel strategy was developed to overcome the limitations of poor-quality samples (irregular shapes) based on area image analysis. Conclusions: The newly developed DBS method is the first to enable reliable measurement of UST in capillary blood, appropriate clinical interpretation of the measured concentrations, and remote monitoring of patients in the early phase of therapy. Full article

Dried blood spot (DBS) sampling has emerged as a promising microsampling technique in biomedical and clinical research, offering advantages such as reduced invasiveness, minimal blood volume requirements, and enhanced analyte stability. Although well established in human medicine for neonatal screening and diagnostic applications, its potential in veterinary pharmacology remains underexplored. This study investigated the feasibility of using DBS samples to quantify anesthetic agents—ketamine and medetomidine in cats and lidocaine in horses—during routine surgical procedures at a veterinary teaching hospital. A standardized DBS collection protocol was developed, and LC-MS/MS methods were validated for the quantification of target analytes in both DBS and plasma samples. These methods were subsequently applied to real samples collected during anesthesia to conduct pharmacokinetic analyses. Comparative evaluations, including Bland–Altman analysis, assessed the suitability of DBS samples for pharmacokinetic studies in veterinary medicine. Preliminary results indicated satisfactory agreement for medetomidine, meeting EMA guidelines, with 75.6% of mean values falling within ±20% of paired measurements. Results for ketamine (46.9%) were promising but require further optimization, while those for lidocaine (21.4%) highlighted the need for additional investigation. These findings underscore the potential of DBS sampling as a minimally invasive alternative for pharmacokinetic studies in veterinary medicine, particularly for medetomidine, while identifying areas for further methodological refinement. Future research should optimize DBS techniques and expand their application to other drugs and species, broadening their impact on veterinary pharmacology. Full article

The analysis of biological fluids plays a crucial role in biomarker discovery, disease diagnostics, and precision medicine. Dried sample carriers—such as dried blood spots, dried plasma, serum, saliva, tears, and urine—have emerged as powerful tools, offering advantages in sample collection, storage, and transport, particularly in remote and resource-limited settings. Recent advances in proteomic methodologies have expanded the potential of these dried matrices, yet challenges related to protein stability, sensitivity, and standardization persist. This review critically examines the current state of proteomic investigations using dried biological fluids. Furthermore, we compare proteomics’ progress in this field with other omics approaches, such as metabolomics, to contextualize its development and integration potential. While dried fluid proteomics is promising for non-invasive diagnostics and large-scale epidemiological studies, addressing technical limitations will be essential for its broader adoption in clinical and translational research. 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

The GeneXpert HBV Viral Load test is a simplified tool to scale up screening and HBV monitoring in resource-limited settings, where HBV is endemic and where molecular techniques to quantify HBV DNA are expensive and scarce. However, the accuracy of field diagnostics compared to gold standard assays in HBV-endemic African countries has not been well understood. We aim to validate the diagnostic performance of the GeneXpert HBV Viral Load test in freshly collected and stored plasma and dried blood spot (DBS) samples to assess turn-around-time (TAT) for sample processing and treatment initiation, to map GeneXpert machines and to determine limitations to its use in The Gambia. Freshly collected paired plasma and DBS samples (n = 56) were analyzed by the GeneXpert test. Similarly, stored plasma and DBS samples (n = 306, n = 91) were analyzed using the GeneXpert HBV test, in-house qPCR and COBAS TaqMan Roche. The correlation between freshly collected plasma and DBS is r = 0.88 with a mean bias of −1.4. The GeneXpert HBV test had the highest quantifiable HBV DNA viremia of 81.4% (n = 249/306), and the lowest was detected by in-house qPCR at 37.9% (n = 116/306) for stored plasma samples. Bland–Altman plots show strong correlation between GeneXpert and COBAS TaqMan and between GeneXpert and in-house qPCR with a mean bias of +0.316 and −1.173 log₁₀ IU/mL, respectively. However, paired stored plasma and DBS samples had a lower mean bias of 1.831 log₁₀ IU/mL, which is almost significant (95% limits of agreement: 0.66–3.001). Patients (n = 3) were enrolled in the study within a TAT of 6 days. The GeneXpert HBV test displayed excellent diagnostic accuracy by detecting HBV viremia in less than 10 IU/mL. Full article

Background: Dried blood spots (DBSs) enable the study of serological markers of various pathogens without the need to obtain serum/plasma through venipuncture. Methods: Sixty-four blood samples were prepared on Whatman™ 903 cards using specimens obtained by venipuncture to study the detection of serological markers of diphtheria, tetanus, and pertussis in DBSs, and their stability 4 months post-collection. An automated chemiluminescent immunoassay was used to investigate diphtheria, tetanus, and pertussis IgG levels from both DBSs and plasma samples. Results: An optimal cut-off value for DBSs was calculated to improve the performance of diphtheria and tetanus serological markers in DBSs, achieving high sensitivity (95% and 98%, respectively) and specificity (91.7% and 92.3%, respectively). No protection against pertussis was found in the population studied. The correlation observed between the plasma and the DBSs processed after sample collection was high (0.967–0.970) for all antibodies studied except pertussis (0.753), both considering hematocrit before sample elution or not. The correlation between DBSs and plasma for diphtheria and tetanus remained strong following a 4-month delay in DBS processing at 4 °C (0.925–0.964) and −20 °C (0.924–0.966), with only a minor decrease observed for diphtheria at room temperature (0.889), while maintaining a strong correlation for tetanus (0.960). For pertussis, the correlation between DBSs and plasma was drastically reduced after delaying its processing for 4 months at any temperature. Conclusions: To summarize, hematocrit levels within the normal range do not affect the processing of DBSs in the study of serological markers of diphtheria, tetanus, and pertussis. The DBS stability for serological diagnosis of diphtheria and tetanus is adequate when samples are stored at −20 °C for a period of 4 months. The pertussis serological marker does not appear to remain stable after 4 months, even when the DBS is stored frozen at −20 °C. 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 frequency of virus-associated cancers is growing worldwide, especially in resource-limited settings. One of the biggest challenges in cancer research among people living with HIV (PLWH) has been understanding how infection with both HIV and Kaposi sarcoma-associated herpesvirus (KSHV) promotes the pathogenesis of Kaposi sarcoma (KS), the most common cancer among PLWH worldwide and a significant public health problem in regions with high prevalence of HIV such as Sub-Saharan Africa (SSA). The AIDS and Cancer Specimen Resource (ACSR) provides samples for research, including dried blood spots (DBS) that were collected from large clinical epidemiology studies of KSHV and KS in PLWH conducted more than a decade ago in SSA. Here, we validated the quality of DNA derived from DBS samples from SSA studies and provided evidence of quantitative recovery of inflammatory cytokines using these DBS samples through comparison with paired frozen plasma. Significant differences in DNA, protein yields, and inflammatory biomarker levels were also observed between PLWH with/without KS. Establishing the fitness of DBS samples for studies of KS pathogenesis extends the number of projects that can be supported by these ACSR special collections and provides evidence that DBS collection for future KS research is a practical option in resource-limited settings. Full article

Hereditary tyrosinemia type I (HT1), or hepatorenal tyrosinemia, is an amino acid disorder which may cause hepatic failure as well as renal and neurologic comorbidities. Early detection of this disorder is possible with newborn screening (NBS). The objective of this study is to describe the clinical, biochemical, and molecular characteristics of Filipino patients diagnosed with HT1 through the expansion of the Philippine NBS program in 2014. There were a total of 16 patients with confirmed HT1 from then until September 2022. Clinical and biochemical data during confirmation and initial evaluation, as well as molecular data, were obtained from the patients’ medical records. The cohort included children between the ages of 18 and 54 months at the time of data collection. The mean age at treatment initiation was 26.8 days. The mean succinylacetone level from dried blood spot sampling using tandem mass spectrometry (MS) was 11.1 µmol/L. Biochemical confirmatory tests via plasma amino acid analysis showed mean levels of tyrosine, phenylalanine, and methionine of 506.1 µmol/L, 111.5 µmol/L, and 125.4 µmol/L, respectively. Upon urine organic acid (UOA) analysis, succinylacetone was detected in all except for one patient, who was managed prior to UOA analysis. The most common clinical characteristics were abnormal clotting times (62.5%), elevated alpha fetoprotein (37.5%), anemia (31.3%), and metabolic acidosis (31.3%). The most common genotype was homozygous c.122T>C p.Leu41Pro in 64.3% of patients. The allelic frequency of this pathogenic variant is 71.4%. The inclusion of HT1 in the Philippine NBS program allowed early diagnosis and management of HT1 patients. Full article

Despite a higher safety profile compared to vitamin K antagonists, rivaroxaban therapy is still connected with multiple adverse effects, such as a high risk of bleeding. Thus, therapeutic drug monitoring (TDM) of rivaroxaban concentrations is suggested. An alternative to plasma samples can be dried blood spots (DBS), which minimize the cost of sample storage and transport. In this study, we developed a UPLC-MS/MS method for the analysis of rivaroxaban in DBS and plasma samples. Chromatographic separation was achieved on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm; 3.5 µm, Agilent Technologies Inc., Santa Clara, CA, USA) with a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. The analytes were detected using a positive ionization mode by multiple reaction monitoring. We validated the method according to ICH guidelines. The precision and accuracy were satisfactory. Extraction recovery was approximately 57% and 66% for DBS and plasma samples, respectively. A high correlation between rivaroxaban concentrations in plasma and DBS samples collected from patients was confirmed with Deming regression. The suitability of both sampling techniques for the rivaroxaban TDM was also verified by Bland–Altman plots based on DBS-predicted and observed plasma concentrations. In addition, we found a significant relationship between rivaroxaban concentrations and coagulation parameters, including prothrombin time (PT) and international normalized ratio (INR). Full article

The stability of antibiotic preanalytical samples is a critical factor in therapeutic drug monitoring (TDM), a practice of undoubted importance for the proper therapeutic use of antibiotics, especially in complex management patients, such as pediatrics. This review aims to analyze the data in the literature regarding the preanalytical stability of some of the antibiotics for which TDM is most frequently requested. The literature regarding the preanalytical stability of amikacin, ampicillin, cefepime, ceftazidime, ciprofloxacin, daptomycin, gentamicin, levofloxacin, linezolid, meropenem, piperacillin, teicoplanin, and vancomycin in plasma, serum, whole blood, and dried blood/plasma spot samples was analyzed. Various storage temperatures (room temperature, 4 °C, −20 °C, and −80 °C) and various storage times (from 1 h up to 12 months) as well as subjecting to multiple freeze–thaw cycles were considered. The collected data showed that the non-beta-lactam antibiotics analyzed were generally stable under the normal storage conditions used in analytical laboratories. Beta-lactam antibiotics have more pronounced instability, particularly meropenem, piperacillin, cefepime, and ceftazidime. For this class of antibiotics, we suggest that storage at room temperature should be limited to a maximum of 4 h, storage at 2–8 °C should be limited to a maximum of 24 h, and storage at −20 °C should be limited to a maximum of 7 days; while, for longer storage, freezing at −80 °C is suggested. Full article

Background: Fabry disease (FD) is a rare X-linked lysosomal storage disorder that commonly manifests cardiovascular complications. We aimed to assess the prevalence of FD in a Chinese population with left ventricular hypertrophy (LVH) whilst implementing a gender-specific screening approach. Methods: Patients with LVH, defined as a maximum thickness of the left ventricular septal/posterior wall ≥ 13 mm, were considered eligible. All patients with hypertrophic cardiomyopathy (HCM) were excluded. Plasma α-galactosidase (α-GLA) enzyme activity was assessed using a dried blood spot test. Males with low enzyme activity underwent genetic testing to confirm a diagnosis of FD whereas females were screened for both α-GLA and globotriaosylsphingosine concentration and underwent genetic analysis of the GLA gene only if testing positive for ≥1 parameter. Results: 426 unrelated patients (age = 64.6 ± 13.0 years; female: male = 113:313) were evaluated. FD was diagnosed in 3 unrelated patients (age = 69.0 ± 3.5 years, female: male = 1:2) and 1 related female subject (age = 43 years). Genetic analyses confirmed the late-onset cardiac variant GLA c.640-801G>A (n = 3) and the missense variant c.869T>C associated with classic FD (n = 1). Cardiac complications were the only significant findings associated with the late-onset c.640-801G>A mutation, manifesting as mild or severe concentric LVH. In contrast, the classic c.869T>C mutation FD exhibited multisystemic manifestations in addition to severe concentric LVH. Conclusions: The prevalence of FD is lower in Chinese patients with LVH when HCM is excluded. The pathological variant c.640-801G>A remains the most common cause of late-onset FD, while the detection of FD in females can be improved by utilizing a gender-specific screening method. Full article

Millions of individuals throughout the world suffer from lymphatic filariasis (LF), which is a morbid disease caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori. These infections belong to tissue-invading nematodes and are one of the major neglected tropical diseases that often result in permanent and enduring disability among individuals in endemic regions. Due to combination therapy, LF eradication has drastically decreased infections globally. The development of blood micro-sampling techniques allowing precise quantitation of drugs in blood would facilitate pharmacokinetic (PK) studies in remote populations. Therefore, an LC-MS/MS bioanalytical method was utilized to analyze albendazole (ABZ), albendazole sulfone (ABZ-ON), albendazole sulfoxide (ABZ-OX), and probenecid (PR) in plasma and dried blood spots. Solid-phase extraction was utilized to extract the analyte from both plasma and blood-spiked DBS. Analytes of interest were eluted with a gradient mobile system using 0.05% formic acid in water (A) and 0.05% formic acid in methanol (B) and separated using a reversed-phase Acquity ^®BEH C18 UPLC column (100 × 2.1 mm, 1.7 µm). Precision and accuracy at each QC level were within the acceptable limit, i.e., ±15% for all analytes in both the matrices. Tests for stability under laboratory and storage conditions indicated that no notable changes were observed for plasma and DBS. The LC-MS/MS method demonstrated its capability to consistently identify all target analytes (ABZ, ABZ-ON, ABZ-OX, and PR) at low concentrations, even at the small specimen volumes obtained from DBS cards. This confirms the efficacy and durability of DBS cards as a micro-sampling technique. Moreover, it enhances collection efforts for therapeutic drug monitoring in remote locations for patients infected with lymphatic filariasis. 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