Quality assurance of add-on testing in plasma samples: stability limit for 29 biochemical analytes.

Introduction: Clinical laboratories should guarantee sample stability in specific storage conditions for further analysis. The aim of this study is to evaluate the stability of plasma samples under refrigeration for 29 common biochemical analytes usually ordered within an emergency context, in order to determine the maximum allowable period for conducting add-on testing. Materials and methods: A total of 20 patient samples were collected in lithium heparin tubes without gel separator. All analyses were performed using Alinity systems (Abbott Laboratories, Abbott Park, USA) and samples were stored at 2-8 °C. Measurements were conducted in primary plasma tubes at specific time points up to 48 hours, with an additional stability study in plasma aliquots extending the time storage up to 96 hours. The stability limit was estimated considering the total limit of change criteria. Results: Of the 29 studied parameters, 24 demonstrated stabilities within a 48-hour storage period in primary plasma tubes. However, five analytes: aspartate aminotransferase, glucose, lactate dehydrogenase, inorganic phosphate and potassium evidenced instability at different time points (7.9 hours, 2.7 hours, 2.9 hours, 6.2 hours and 4.7 hours, respectively). The stability study in plasma aliquots showed that all parameters remained stable for 96 hours, except lactate dehydrogenase, with a stability limit of 63 hours. Conclusions: A reduced stability of primary plasma samples was observed for five common biochemical analytes ordered in an emergency context. To ensure the quality of add-on testing for these samples, plasma aliquots provide stability for a longer period.

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma.

INTRODUCTION: Untargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the study purpose. OBJECTIVES: The current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices. METHODS: Separation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C18, and Gold C18), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others. RESULTS: All three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column. CONCLUSION: The results underline that the outcome of this untargeted toxicometabolomic study LC-HRMS metabolomic study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix.

Plasma and serum metabolic analysis of healthy adults shows characteristic profiles by subjects' sex and age.

INTRODUCTION: Pre-analytical factors like sex, age, and blood processing methods introduce variability and bias, compromising data integrity, and thus deserve close attention. OBJECTIVES: This study aimed to explore the influence of participant characteristics (age and sex) and blood processing methods on the metabolic profile. METHOD: A Thermo UPLC-TSQ-Quantiva-QQQ Mass Spectrometer was used to analyze 175 metabolites across 9 classes in 208 paired serum and lithium heparin plasma samples from 51 females and 53 males. RESULTS: Comparing paired serum and plasma samples from the same cohort, out of the 13 metabolites that showed significant changes, 4 compounds related to amino acids and derivatives had lower levels in plasma, and 5 other compounds had higher levels in plasma. Sex-based analysis revealed 12 significantly different metabolites, among which most amino acids and derivatives and nitrogen-containing compounds were higher in males, and other compounds were elevated in females. Interestingly, the volcano plot also confirms the similar patterns of amino acids and derivatives higher in males. The age-based analysis suggested that metabolites may undergo substantial alterations during the 25-35-year age range, indicating a potential metabolic turning point associated with the age group. Moreover, a more distinct difference between the 25-35 and above 35 age groups compared to the below 25 and 25-35 age groups was observed, with the most significant compound decreased in the above 35 age groups. CONCLUSION: These findings may contribute to the development of comprehensive metabolomics analyses with confounding factor-based adjustment and enhance the reliability and interpretability of future large-scale investigations.

Integrating a Multi-label Deep Learning Approach with Protein Information to Compare Bioactive Peptides in Brain and Plasma.

Peptide therapeutics is gaining momentum. Advances in the field of peptidomics have enabled researchers to harvest vital information from various organisms and tissue types concerning peptide existence, expression and function. The development of mass spectrometry techniques for high-throughput peptide quantitation has paved the way for the identification and discovery of numerous known and novel peptides. Though much has been achieved, scientists are still facing difficulties when it comes to reducing the search space of the large mass spectrometry-generated peptidomics datasets and focusing on the subset of functionally relevant peptides. Moreover, there is currently no straightforward way to analytically compare the distributions of bioactive peptides in distinct biological samples, which may reveal much useful information when seeking to characterize tissue- or fluid-specific peptidomes. In this chapter, we demonstrate how to identify, rank, and compare predicted bioactive peptides and bioactivity distributions from extensive peptidomics datasets. To aid this task, we utilize MultiPep, a multi-label deep learning approach designed for classifying peptide bioactivities, to identify bioactive peptides. The predicted bioactivities are synergistically combined with protein information from the UniProt database, which assist in navigating through the jungle of putative therapeutic peptides and relevant peptide leads.

Multiomics plasma effects of switching from triple antiretroviral regimens to dolutegravir plus lamivudine.

INTRODUCTION: The DOLAM trial revealed that switching from triple antiretroviral therapy (three-drug regimen; 3DR) to dolutegravir plus lamivudine (two-drug regimen; 2DR) was virologically non-inferior to continuing 3DR after 48 weeks of follow-up. Weight increased with 2DR relative to 3DR but it did not impact on metabolic parameters. METHODS: Multiomics plasma profile was performed to gain further insight into whether this therapy switch might affect specific biological pathways. DOLAM (EudraCT 201500027435) is a Phase 4, randomized, open-label, non-inferiority trial in which virologically suppressed persons with HIV treated with 3DR were assigned (1:1) to switch to 2DR or to continue 3DR for 48 weeks. Untargeted proteomics, metabolomics and lipidomics analyses were performed at baseline and at 48 weeks. Univariate and multivariate analyses were performed to identify changes in key molecules between both therapy arms. RESULTS: Switching from 3DR to 2DR showed a multiomic impact on circulating plasma concentration of N-acetylmuramoyl-L-alanine amidase (Q96PD5), insulin-like growth factor-binding protein 3 (A6XND0), alanine and triglyceride (TG) (48:0). Correlation analyses identified an association among the up-regulation of these four molecules in persons treated with 2DR. CONCLUSIONS: Untargeted multiomics profiling studies identified molecular changes potentially associated with inflammation immune pathways, and with lipid and glucose metabolism. Although these changes could be associated with potential metabolic or cardiovascular consequences, their clinical significance remains uncertain. Further work is needed to confirm these findings and to assess their long-term clinical consequences.

Human biomonitoring of dioxins, furans, and non-ortho dioxin-like polychlorinated biphenyls (PCBs) in blood plasma from Old Crow, Yukon, Canada (2019).

Dioxins, furans, and dioxin-like polychlorinated biphenyls (PCBs) are a group of persistent and toxic chemicals that are known to have human health effects at low levels. These chemicals have been produced for commercial use (PCBs) or unintentionally as by-products of industry or natural processes (PCBs, dioxins, and furans). Additionally, dioxin-like PCBs were formerly used in electrical applications before being banned internationally (2004). These chemicals are widely dispersed in the environment as they can contaminate air and travel hundreds to thousands of kilometers before depositing on land or water, thereafter, potentially entering food chains. Community concerns surrounding the safety of traditional foods prompted a human biomonitoring project in Old Crow, Yukon Territory (YT), Canada (2019). Through collaborative community engagement, dioxins and like compounds were identified as a priority for exposure assessment from biobanked samples. In 2022, biobanked plasma samples (n = 54) collected in Old Crow were used to measure exposures to seven dioxins, ten furans, and four dioxin-like PCBs. 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,4,7,8-PeCDF, 1,2,3,6,7,8-HxCDF, PCB 126, and PCB 169 were detected in at least 50 % of samples. Among these analytes, the only congener at elevated levels was PCB 169, which was approximately ∼2-fold higher than the general population of Canada. No significant sex-based or body mass index (BMI) differences in biomarker concentrations were observed. Generally, the concentrations of the detected congeners increased with age, except for 1,2,3,4,6,7,8-HpCDD. For the first time, this research measures dioxin and like-compound exposures in Old Crow, advancing the information available on chemical exposures in the Arctic. Further research could be directed towards the investigation of PCB 169 exposure sources and temporal monitoring of exposures and determinants.

Analysis of free cisplatin in microdialysates and plasma ultrafiltrate by liquid chromatography-tandem mass spectrometry.

Cisplatin is a potent cytotoxic agent used in the treatment of various malignancies and exerts its antitumor effect through malignant cell DNA damage and apoptosis induction. Evaluation of systemic delivery of cisplatin is important in optimization of cisplatin treatment. However, accurate quantification of systemic cisplatin is challenging due to its various forms in circulation. This study aimed to develop a sensitive (LOQ < 0.1 µg/mL) and precise Ultra Performance Liquid Chromatography (UPLC) - Tandem Mass Spectrometry (MS/MS) method for quantifying free cisplatin in microdialysates and plasma. Furthermore the aim was to compare free cisplatin concentrations measured in standard plasma samples with those obtained from intravenous microdialysis catheters in a porcine model. The method developed utilizes dichloro(ethylenediamine)platinum(II) as an internal standard that co-elutes with cisplatin, ensuring precise correction for ion suppression/enhancement effects. The method was validated, demonstrating linearity up to 100 µg/mL and good intermediate precision (CV% < 6 %) in the range of 1.0-100 µg/mL, with an LOQ of 0.03 µg/mL. The pharmacokinetic parameters (AUC0-last, Cmax, T1/2, and Tmax) showed no significant differences between the two sampling methods. This validated LC-MS/MS method provides a reliable tool for quantifying systemic free cisplatin concentrations, facilitating future systemic and local pharmacokinetic evaluations for optimization of cisplatin-based cancer treatments.

HILIC/MS quantitation of low-abundant phospholipids and sphingolipids in human plasma and serum: Dysregulation in pancreatic cancer.

A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC.

Label-free screening of common urinary system tumors from blood plasma based on surface-enhanced Raman spectroscopy.

BACKGROUND: The incidence of common urinary system tumors has been rising rapidly in recent years, and most urinary system-derived tumors lack specific biomarkers. OBJECTIVES: To explore the efficacy of surface-enhanced Raman spectroscopy (SERS) of blood plasma in screening three common urinary system tumors, including bladder cancer (BC), prostate cancer (PCa), and renal cell carcinoma (RCC). METHODS: SERS plasma spectra from 125 plasma samples, including 25 PCa, 38 RCC, 24 BC patients, and 38 normal volunteers, were collected. All candidates had no other comorbidities. The Diagnosis was based on the combination of Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA), and the effectiveness of the diagnostic algorithms was verified using the Receiver Operating Characteristic Curve (ROC). RESULTS: There are significant differences in SERS signals between PCa, BC, RCC, and normal plasma, especially at 639, 889, 1010, 1136, and 1205 cm-1. The PCA-LDA results show that high sensitivity (100 %), specificity (100 %), and accuracy (100 %) could be achieved for screening the PCa, RCC, BC group vs. the normal group, the PCa group vs. the BC and RCC group, respectively. The diagnostic sensitivity, specificity, and accuracy for the BC group vs. the RCC group are 79.2 %, 71.1 %, and 75.15 %, respectively. The integrated area under the ROC curve (AUC) is 1.0, 1.0, and 1.0 for the PCa, RCC, and BC group vs. the normal group, respectively. The AUC of the PCa group vs. the BC group and RCC group and the BC group vs. the RCC group are 1.0, 1.0, and 0.842, respectively. CONCLUSIONS: Label-free plasma-SERS technology with PCA-LDA analysis could be a useful screening method for detecting urinary system tumors (PCa, RCC, and BC) in this exploratory study.

New plasma protein C and protein S concentrate: A synergy for therapeutic purposes.

BACKGROUND AND OBJECTIVES: Deficiencies of protein C (PC) or protein S (PS) are rare diseases, characterized by mutations in the PC or PS genes, which encode plasma serine proteases with anti-coagulant activity. Severe PC or PS deficiencies manifest in early life as neonatal purpura fulminans, a life-threatening heamorrhagic condition requiring immediate treatment. First-line treatment involves replacement therapy, followed by maintenance with anti-coagulants. Replacement therapy with specific protein concentrates is currently only limited to PC, and therefore, a PC + PS concentrate represents a useful addition to therapeutic options, particularly for severe PS deficiency. Further, the production of a PC + PS concentrate from unused plasma fractionation intermediates would impact favourably on manufacturing costs, and consequently therapy prices for patients and health systems. MATERIALS AND METHODS: Several chromatographic runs were performed on the same unused plasma fractionation intermediates using different supports to obtain a PC/PS concentrate. The best chromatographic mediums were chosen, in terms of specific activity and recovery. A full process of purification including virus inactivation/removal and lyophilization steps was set up. RESULTS: The final freeze-dried product had a mean PC concentration of 47.75 IU/mL with 11% of PS, and a mean specific activity of 202.5 IU/mg protein, corresponding to over 12,000-fold purification from plasma. CONCLUSION: The development of a novel concentrated PC/PS mixture obtained from a waste fraction of other commercial products could be used for its potential therapeutic role in the management of neonatal purpura fulminans pathology.

Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens.

The foundation for integrating mass spectrometry (MS)-based proteomics into systems medicine is the development of standardized start-to-finish and fit-for-purpose workflows for clinical specimens. An essential step in this pursuit is to highlight the common ground in a diverse landscape of different sample preparation techniques and liquid chromatography-mass spectrometry (LC-MS) setups. With the aim to benchmark and improve the current best practices among the proteomics MS laboratories of the CLINSPECT-M consortium, we performed two consecutive round-robin studies with full freedom to operate in terms of sample preparation and MS measurements. The six study partners were provided with two clinically relevant sample matrices: plasma and cerebrospinal fluid (CSF). In the first round, each laboratory applied their current best practice protocol for the respective matrix. Based on the achieved results and following a transparent exchange of all lab-specific protocols within the consortium, each laboratory could advance their methods before measuring the same samples in the second acquisition round. Both time points are compared with respect to identifications (IDs), data completeness, and precision, as well as reproducibility. As a result, the individual performances of participating study centers were improved in the second measurement, emphasizing the effect and importance of the expert-driven exchange of best practices for direct practical improvements.

Understanding protein-nanoparticle interactions leading to protein corona formation: In vitro - in vivo correlation study.

Nanoparticles (NPs) in contact with biological fluids form a biomolecular corona through interactions with proteins, lipids, and sugars, acquiring new physicochemical properties. This work explores the interaction between selected proteins (hemoglobin and fetuin-A) that may alter NP circulation time and NPs of different surface charges (neutral, positive, and negative). The interaction with key proteins albumin and transferrin, the two of the most abundant proteins in plasma was also studied. Binding affinity was investigated using quartz crystal microbalance and fluorescence quenching, while circular dichroism assessed potential conformational changes. The data obtained from in vitro experiments were compared to in vivo protein corona data. The results indicate that electrostatic interactions primarily drive protein-NP interactions, and higher binding affinity does not necessarily translate into more significant structural changes. In vitro and single protein-NP studies provide valuable insights that can be correlated with in vivo observations, opening exciting possibilities for future protein corona studies.

Reductions in plasma and urine mercury concentrations following N,N'bis-(2-mercaptoethyl) isophthalamide (NBMI) therapy: a post hoc analysis of data from a randomized human clinical trial.

Environmental mercury exposure possesses a significant risk to many human populations. At present there are no effective treatments for acute mercury toxicity. A new compound, N,N'bis-(2-mercaptoethyl) isophthalamide (NBMI), a lipophilic chelating agent was created to tightly/irreversibly bind mercury. A post hoc dose-dependent analysis of NBMI therapy was undertaken on data from a randomized controlled NBMI human treatment trial on 36 Ecuadorian gold miners with elevated urinary mercury concentrations. Study subjects were randomly assigned to receive 100 milligram (mg) NBMI/day, 300 mg NBMI/day, or placebo for 14 days. For each study subject daily mg NBMI dose/Kilogram (Kg) bodyweight were determined and plasma and urine mercury concentrations (micrograms (µg)/Liter (L)) on study day 1 (pre-NBMI treatment), 15 (after 14 days of NBMI treatment) and 45 (30 days after NBMI treatment) were correlated with NBMI dosing using the linear regression statistic in SAS. Regression revealed significant inverse correlations between increasing per mg NBMI/Kg bodyweight/day and reduced concentrations of urinary and plasma mercury on study day 15 (reduced by in urine = 18-20 µg/L and plasma = 2 µg/L) and study day 30 (reduced by in urine = 15-20 µg/L and plasma = 4 µg/L) and significant correlations between reductions in mercury concentrations in urine and plasma. Significant 30% reductions in urinary mercury concentrations per mg NBMI/Kg bodyweight/day administered for 14 days were observed. This study supports the dose-dependent ability of NBMI therapy to significantly reduce mercury concentrations, particularly in the urine, in an acutely mercury exposed human population. NBMI therapy should be evaluated in other mercury exposed populations.

Comparative Analysis of Spray-Dried Porcine Plasma and Hydrolyzed Porcine Protein as Animal-Blood-Derived Protein Ingredients for Pet Nutrition.

Spray-dried porcine plasma (SDPP) and hydrolyzed porcine protein (HPP) are promising animal protein ingredients sourced from healthy animal blood that are rich in biomolecules, including immunoglobulins, and can be an appropriate and valuable animal protein ingredient to supply the growing need for ingredients that meet the natural needs of carnivorous pets. The aim of this preliminary study was to analyze the chemical composition and mineral profile of a novel HPP compared with results for SDPP. The basic composition analysis followed AOAC guidelines, and the elemental analysis utilized atomic absorption spectrometry. Statistical comparisons employed an independent Student's t-test (p < 0.05). Both SDPP and HPP are low in moisture (<4.3%) and rich in protein, with SDPP significantly exceeding HPP (75.4% vs. 71.4%). They boast mineral richness indicated by crude ash content (12.7% and 12.5%), featuring Na, K, P, and the trace elements Mo, Fe, and Zn. Notably, SDPP contains elevated molybdenum levels (51.39 mg/100 g vs. 10.93 mg/100 g in HPP), an essential element for diverse animal functions. Quantifying these elements in raw materials aids in achieving optimal nutrient levels in the final product. The study underscores SDPP as an excellent protein source, confirming that its nutritional value is similar to or better than other protein components in pet food.

Optimization for the analysis of 42 per- and polyfluorinated substances in human plasma: A high-throughput method for epidemiological studies.

There is an increasing awareness about the presence of per- and polyfluoroalkyl substances (PFAS) in many environmental and biological compartments, including human biofluids and tissues. However, the increase of PFAS replacements, including alternatives with shorter chain or less bioaccumulative potential, has broaden the exposure and the need for wider identification procedures. Moreover, the low volumes available for human blood or plasma, and the high number of samples needed to assess adequately epidemiologic studies, require particularly fast, reproducible and, if possible, miniaturized protocols. Therefore, accurate and robust analytical methods are still needed to quantify the PFAS's burden in humans and to understand potential health risks. In this study, we have developed and validated the analysis of 42 PFAS in human plasma by means of a Captiva 96-well micro extraction plate and a LC-q-Orbitrap. For the optimization of the analytical workflow, three extraction/clean-up methods were tested, and the selected one was validated using spiked artificial and bovine plasma at four concentration levels. The final method showed high absolute recoveries for the 42 PFAS, ranging from 52% to 130%, instrumental detection limits between 0.001-0.6 ng mL-1, overall good precision (CV < 20% for most of the PFAS) and a low uncertainty (< 30% of relative expanded deviation, k = 2). The method was further validated both with the NIST plasma Standard Reference Material 1950, showing that the accuracy of the provided results was between 63%-101%, and by the proficiency test arranged by the Arctic Monitoring Assessment Program (AMAP, 2022) obtaining satisfactory results within 95% confidence interval of the assigned value.

Identification of Sanguinarine Metabolites in Rats Using UPLC-Q-TOF-MS/MS.

Sanguinarine (SAN), as the main active component of a traditional Chinese veterinary medicine, has been widely used in the animal husbandry and breeding industry. However, the metabolites of SA are still uncertain. Therefore, this research aimed to investigate the metabolites of SA based on rats in vivo. The blood, feces, and urine of rats were collected after the oral administration of 40 mg/kg SAN. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was employed to identify the metabolites of SAN. The elemental composition of sanguinarine metabolites was inferred by analyzing their exact molecular weight, and the structures of the metabolites were predicted based on their fragment ions and cleavage pathways. A total of 12 metabolites were identified, including three metabolites in the plasma, four in the urine, and nine in the feces. According to the possible metabolic pathways deduced in this study, SAN was mainly metabolized through reduction, oxidation, demethylation, hydroxylation, and glucuronidation. This present research has summarized the metabolism of SAN in rats, which is helpful for further studying the metabolic mechanism of SAN in vivo and in vitro.

Quantification of Oligonucleotides Using Tandem Mass Spectrometry with Isobaric Internal Standards.

In recent years, oligonucleotides have become more important in research, drug approvals and medical therapies. Due to this growing interest in pharmaceutical applications, it is essential to develop reliable analytical methods for this substance class. In this work, we present a quantification method using liquid chromatography coupled with tandem mass spectrometry by applying an isobaric oligonucleotide standard. In addition to a proof of principle, we perform a method qualification to assess its readiness for validation according to ICH Q2 guidelines. In addition to good linearity, sensitivity, accuracy and recovery, the method showed no significant matrix effects. Furthermore, we demonstrated the application of the method by applying the quantification in a biological matrix, as well as an exemplary degradation of an oligonucleotide in bovine plasma.