Stability In Whole Blood Research Articles

Is a “toxic” death possible with gliclazide, an oral hypoglycemic drug, found at therapeutic concentration?

Summary An unexpected death of a 46-year-old woman presenting a type II diabetes was observed and the Prosecutor requested an autopsy followed by toxicological investigations. Autopsy findings (labial ecchymosis, multiviscera congestion, asphyxia syndrome, moderate cerebral edema) were in accordance with a possible hypoglycemia death. Toxicological analyses revealed the presence of gliclazide in femoral blood at 2.2 mg/L, which is in the range of published therapeutic concentrations. During a meeting with the pathologist, 3 possible explanations were discussed: 1. unknown cause of death (other than a toxic death), 2. death due to gliclazide which could have been degraded due to chemical instability during the 6-weeks interval between death and analysis (at the time of death the blood concentration could have been much higher), and 3. blood concentration was enough to produce fatal hypoglycemia in a non-compliant patient. According to the pathologist, no traumatic cause of death could be established. Testing for gliclazide stability in whole blood over a period that matches the delay of the toxicological analyses demonstrated instability (loss of about 65% after 6 weeks storage at +4 °C), but this was found irrelevant for a massive overdose. Hair tested positive for gliclazide in the 3 × 2 cm segments, at 7, 8 and 3 pg/mg, and were highly indicative of non-compliant use of the medicine. It was therefore concluded by the pathologist that the cause of death of the subject was more likely than not an inappropriate use of gliclazide, which can be responsible of a fatal glycemic disorder.

Performance of 6 routine coagulation assays on the new Roche Cobas t711 analyzer

ObjectivesFor new analyzers or tests, analytical evaluation is required before implementation in the clinical laboratory. We evaluated the novel Roche Cobas t711 analyzer with six newly developed coagulation assays: the activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), fibrinogen, d-dimer and anti-Xa. The evaluation included imprecision experiments, method comparison with the currently used Stago STA-R Evolution, monitoring of unfractionated heparin (UFH) with aPTT, a fast centrifugation protocol to improve turn-around time, and determination of sample stability in whole blood and plasma. Design and methodsImprecision and method comparison were assessed using commercial quality control samples and patient samples, respectively. For dose monitoring of UFH with the aPTT, samples from patients treated with UFH were used. Samples from healthy volunteers were collected for evaluation of the fast centrifugation protocol (5’ 2750×g) and for investigating sample stability over 6–8 h. ResultsResults for between-run precision were within the desirable specification. Method comparison showed an excellent agreement for fibrinogen, d-dimer and anti-Xa. For aPTT, PT and INR, a good correlation was found, but results were significantly lower on the t711 compared to the STA-R Evolution, which is caused by different coagulation activators. Results from the fast centrifugation protocol differed not significantly from the standard protocol (15’ 2500×g). Blood and plasma samples were stable at room temperature up to 6 and 8 h, respectively. ConclusionsThe t711 coagulation analyzer with 6 novel tests is suitable for routine use in clinical laboratories.

Evaluation of Diabetes Care parameters in capillary blood collected with a novel sampling device

ObjectivesThe aim of this study was to determine whether the Hem-Col method of obtaining and storing blood is an acceptable alternative to venepuncture for measuring Diabetes Care parameters. Design and methods: Hem-Col is a novel blood collection device that is designed to collect capillary blood drawn with a finger prick. Hem-Col is a microtube containing an anticoagulant and a preservation buffer to enhance analyte stability in whole blood. The Diabetes Care parameters cholesterol, creatinine, HbA1c, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, and triglycerides were measured both in blood/plasma collected via Hem-Col and blood/plasma collected with venepuncture. The results were compared to assess the agreement between the two methods. Results: HbA1c shows agreement after storage for up to 120 hours at temperatures ranging from 4 to 37 °C. Cholesterol, HDL cholesterol, LDL cholesterol, triglycerides and creatinine can be measured after 120 hours of storage in Hem-Col buffer, if high temperatures are avoided, and with the use of correction factors or adaptations to reported reference intervals. Conclusion: Hem-Col is suitable for the measurement of HbA1c after storage for up to 120 hours at temperatures ranging from 4 to 37 °C. Cholesterol, creatinine, HDL cholesterol, LDL cholesterol and triglycerides can be measured after 120 hours of storage in Hem-Col buffer, if high temperatures are avoided. Further studies are required to determine whether Hem-Col can replace the venepuncture for the Diabetes Care parameters.

Preanalytical Stability of Piperacillin, Tazobactam, Meropenem, and Ceftazidime in Plasma and Whole Blood Using Liquid Chromatography-Tandem Mass Spectrometry.

Therapeutic drug monitoring (TDM) is increasingly used to optimize the dosing of beta-lactam antibiotics in critically ill patients. However, beta-lactams are inherently unstable and degrade over time. Hence, patient samples need to be appropriately handled and stored before analysis to generate valid results for TDM. The appropriate handling and storage conditions are not established, with few and conflicting studies on the stability of beta-lactam antibiotics in clinical samples. The aim of this study was to assess the preanalytical stability of piperacillin, tazobactam, meropenem, and ceftazidime in human plasma and whole blood using a liquid chromatography-tandem mass spectrometry method for simultaneous quantification. A reverse phase liquid chromatography-tandem mass spectrometry method for the quantification of piperacillin, tazobactam, meropenem, and ceftazidime in plasma after protein precipitation was developed and validated. The preanalytical stability of these beta-lactams was assessed in EDTA- and citrate-anticoagulated plasma at 24, 4, and -20°C. The whole blood stability of the analytes in EDTA-anticoagulated tubes was assessed at 24°C. Stability was determined by nonlinear regression analysis defined by the lower limit of the 95th confidence interval of the time to 15% of degradation. Based on the lower limit of the 95th confidence interval of the time to 15% of degradation, piperacillin, tazobactam, meropenem, and ceftazidime were stable in EDTA-anticoagulated plasma for at least 6 hours at 24°C, 3 days at 4°C, and 4 days at -20°C. Stability in EDTA- and citrate-anticoagulated plasma was similar. Stability in whole blood was similar to plasma at 24°C. Plasma samples for the TDM of piperacillin, tazobactam, meropenem, and ceftazidime should be processed within 6 hours if kept at room temperature and within 3 days if kept at 4°C. All long-term storage of samples should be at -80°C.

Evolution and Antibacterial Evaluation of 8-Hydroxy-cycloberberine Derivatives as a Novel Family of Antibacterial Agents Against MRSA.

Twenty-five new derivatives of 8-hydroxycycloberberine (1) were synthesized and evaluated for their activities against Gram-positive bacteria, taking 1 as the lead. Part of them displayed satisfactory antibacterial activities against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-intermediate Staphylococcus aureus (VISA). Especially, compound 15a displayed an excellent anti-MRSA activity with MICs (minimum inhibitory concentrations) of 0.25–0.5 μg/mL, better than that of 1. It also displayed high stability in liver microsomes and whole blood, and the LD50 value of over 65.6 mg·kg−1 in mice via intravenous route, suggesting a good druglike feature. The mode of action showed that 15a could effectively suppress topo IV-mediated decatenation activity at the concentration of 7.5 μg/mL, through binding a different active pocket of bacterial topo IV from quinolones. Taken together, the derivatives of 1 constituted a promising kind of anti-MRSA agents with a unique chemical scaffold and a specific biological mechanism, and compound 15a has been chosen for the next investigation.

Stability of hematological parameters of canine blood samples stored with citrate phosphate dextrose adenine-1 anticoagulated plastic vacutainers.

Aim:The effect of citrate phosphate dextrose adenine-1 on the hematological parameters of stored Nigerian indigenous dog’s blood with plastic vacutainer was investigated. This was done in view of determining the viability and stability of the studied parameters for blood banking purpose. Till date, there is no literature on the stability of whole blood of Nigerian indigenous dogs for blood banking purposes.Materials and Methods:A total of six apparently adult healthy dogs were sampled, and their blood was stored at 4°C and analyzed for their packed cell volume (PCV), hemoglobin (Hgb) concentration, red blood cell (RBC) count, total and differential white blood cell (WBC) count, platelet count (PC), mean corpuscular values (mean corpuscular Hgb [MCH], MCH concentration, and mean corpuscular volume [MCV]), blood potency of hydrogen (pH), and erythrocyte sedimentation rate (ESR) over a period of 14 days.Results:Significant changes were observed in some of the studied parameters. Of the 14 days’ study period, PCV, Hgb concentration, total WBC count, PC, and neutrophil count showed no significant (p≥0.05) difference until day 2 post-storage (PS), while RBC count, ESR, MCV, and lymphocyte count showed no significant (p≥0.05) variation up until day 3 PS. Blood pH showed no significant (p≥0.05) variation within 24-h PS but was significantly (p≤0.05) higher than obtained values on days 1, 3, 7, 10, and 14 PS.Conclusion:Based on our finding, we could suggest that hematological laboratories and hemotherapists could use canine blood stored in a plastic vacutainer for 2-3 days.

Impact of clinical sample handling and processing on ultra-low level measurements of plasma cytokines

ObjectivesIn this study, we evaluated the impact of clinical sample handling and processing on IL-6, IL-10, IFNγ, and IL-2 measurements in plasma. Design and methodsWe collected whole blood samples and analyzed various pre-analytical parameters. We assessed the following: 1) cytokine stability in whole blood that was stored over a ten-hour period at room temperature and 4 °C; 2) cytokine stability in plasma over 6 h; 3) vigorous sample handling including repeated dropping and transport through a pneumatic transport system; and 4) freeze-thaw stability of cytokines in plasma. To ensure ability to measure IL-6, IL-10, IFNγ, and IL-2 levels in plasma, we used Simoa, an ultra-sensitive immunoassay platform. ResultsWe show that whole blood storage at room temperature results in decreased cytokine levels and that whole blood storage at 4 °C results in greater cytokine stability. We also show that cytokines are stable when whole blood samples are subjected to vigorous sample handling. Lastly, we show that cytokines are stable in plasma over three freeze-thaw cycles. ConclusionsClinical sample handling and processing can affect measurements of IL-6, IL-10, IFNγ, and IL-2 in plasma. We believe this study will be a useful reference for future studies in which these cytokines are used as potential biomarkers.

Stability of mephedrone and five of its phase I metabolites in human whole blood.

Mephedrone is a new psychoactive substance known to be unstable in biological matrices stored at room temperature or refrigerated. While the instability of mephedrone has been investigated before, there is currently no data regarding the stability of mephedrone metabolites. In this study, a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of mephedrone and five of its phase I metabolites (dihydro-mephedrone, nor-mephedrone, hydroxytolyl-mephedrone, 4-carboxy-mephedrone and dihydro-nor-mephedrone) in human whole blood has been developed and validated. Samples were extracted by a mixed mode solid-phase extraction and analyzed on a pentafluorophenylpropyl column. The method was successfully validated for selectivity, linearity (0.2-2 to 10-100ng/mL), limits of detection (50-500pg/mL) and quantification (200-2000pg/mL), precision (0.924-8.27%), accuracy (86.6-115%), carryover, recovery (32.5-88.3%), and matrix effects (71.0-108%). Analyte stability in human whole blood preserved with sodium fluoride/potassium oxalate was assessed at +4°C and-20°C after 24 hours, 48hours, 4 days, and 10days of storage. Instability was observed in samples stored at +4°C: nor-mephedrone and 4-carboxy-mephedrone lost 40.2±6.7% and 48.1±4.8%, respectively, of their initial concentration at low concentration level and 33.8±4.2% and 44.6±6.5%, respectively, at high concentration level after 10days. All analytes were more stable at -20°C where the highest loss of 22.6±6.9% was observed for 4-carboxy-mephedrone after 10days. This is the first time stability of mephedrone metabolites in human whole blood has been assessed, indicating -20°C to be the recommended storage condition for all analytes in clinical settings.

Abstract 5554: Preclinical characterization of GSK532, a novel STING agonist with potent anti-tumor activity

Abstract The cGAS-STING pathway is the major cytosolic DNA sensing pathway to induce downstream innate immune responses against viral infection as well as tumorigenesis. Recently it has been demonstrated that cGAS-STING pathway takes central stage in the immune sensing of tumor. STING activation induces the production of type I interferons (IFNα and β) and pro-inflammatory cytokines that collectively stimulate tumor antigen cross presentation to CD8 T cells and primes the adaptive immune response against tumor. In order to evaluate the potential of STING agonism to activate the anti-tumor immune response, we developed GSK532, a novel cyclic dinucleotide (CDN). Unlike cGAMP, the endogenous CDN ligand for STING, GSK532 potently activates STING both in vitro and in vivo. In vitro functional assays demonstrated the ability of GSK532 to induce cytokine responses in a panel of human PBMC samples with various STING haplotypes. Furthermore, GSK532 was shown to activate STING orthologs from preclinical species including cyno, minipig, dog, rat and mice. GSK532 demonstrated high stability in human whole blood with minimal degradation observed over 52 hrs. When dosed intratumorally, GSK532 induced a strong anti-tumor effect in the CT26 murine syngeneic model in both the injected (primary) and uninjected (distal) tumors. The cured mice were resistant to rechallenge with same tumor cell line indicating the involvement of an adaptive immune response. Further evidence of the development of anti-tumor adaptive immune response comes from the observation that the efficacy of GSK532 was significantly attenuated when CD8+ T cells were depleted in the murine model. Consistent with the anti-tumor efficacy, pharmacokinetic analysis demonstrated high exposure and long retention of GSK532 in both injected and distal tumors and pharmacodynamic studies showed induction of IFNβ and several inflammatory cytokines (TNFα, IL-6, etc) in both tumors. Currently, GSK532 is being progressed towards clinical investigation. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Jingsong Yang, Michael Adam, Jim Clemens, Katrina Creech, Jess Schneck, Kishore Pasikanti, Jean-Luc Tran, Devika Joglekar, Chris Hopson, Scott Pesiridis, Joshi Ramanjulu, Yiqian Lian, Jerry L. Adams, James Smothers, Axel Hoos. Preclinical characterization of GSK532, a novel STING agonist with potent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5554.

Free oxysterols and bile acids including conjugates - Simultaneous quantification in human plasma and cerebrospinal fluid by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K3-stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort.A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 μL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8–250 ng mL−1 for free hydroxycholesterols, 0.2–10 ng mL−1 for dihydroxycholesterols, 0.2–500 ng mL−1 for bile acids and 16–2000 μg mL−1 for cholesterol with acceptable accuracy and precision.In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained.In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified.

Ketamine and norketamine stability in whole blood at ambient and 4°C conditions.

A study was implemented to describe the pharmacokinetics (PK) of ketamine (K) and its metabolite norketamine (NK) in critically ill adults. Conducting studies in these subjects is hindered by the immediate need to process and freeze samples obtained in a busy intensive care setting. The ability to store unprocessed samples at room temperature for an extended time period would overcome this barrier. Stability and blood to plasma partitioning of K and NK were investigated in whole blood for up to 120 h at room temperature and 4°C. Whole blood was spiked with K and NK (1000 ng/mL each). Blood samples were aliquoted at different time points (0-120 h), extracted and analyzed using a validated high-performance liquid chromatography tandem mass spectrometry assay. The study demonstrated the stability of both K and NK in whole blood up to 120 h. These in vitro studies suggest that the concentrations of K and NK measured in the PK samples are reliable. The established stability results were successfully employed to investigate K and NK pharmacology studies in critically ill adults.

Rapid Homogeneous Immunoassay to Quantify Gemcitabine in Plasma for Therapeutic Drug Monitoring

Gemcitabine (2',2'-difluoro-2'-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors. Several studies have shown a relationship between gemcitabine peak plasma concentration (Cmax) and hematological toxicity. An immunoassay for gemcitabine in plasma was developed and validated to facilitate therapeutic drug monitoring (TDM) by providing an economical, robust method for automated chemistry analyzers. A monoclonal antibody was coated on nanoparticles to develop a homogenous agglutination inhibition assay. To prevent ex vivo degradation of gemcitabine in blood, tetrahydrouridine was used as a sample stabilizer. Validation was conducted for precision, recovery, cross-reactivity, and linearity on a Beckman Coulter AU480. Verification was performed on an AU5800 in a hospital laboratory. A method comparison was performed with (LC-MS/MS) liquid chromatography tandem mass spectrometry using clinical samples. Selectivity was demonstrated by testing cross-reactivity of the major metabolite, 2',2'-difluorodeoxyuridine. Coefficients of variation for repeatability and within-laboratory precision were <8%. The deviation between measured and assigned values was <3%. Linear range was from 0.40 to 33.02 μ/mL (1.5-125.5 μM). Correlation with validated LC-MS/MS methods was R = 0.977. The assay was specific for gemcitabine: there was no cross-reactivity to 2',2'-difluorodeoxyuridine, chemotherapeutics, concomitant, or common medications tested. Tetrahydrouridine was packaged in single-use syringes. Gemcitabine stability in whole blood was extended to 8 hours (at room temperature) and in plasma to 8 days (2-8°C). The assay demonstrated the selectivity, test range, precision, and linearity to perform reliable measurements of gemcitabine in plasma. The addition of stabilizer improved the sample handling. Using general clinical chemistry analyzers, gemcitabine could be measured for TDM.

Preparation and preliminary characterization of recombinant neurolysin for in vivo studies

The goal of this study was to produce milligram quantities of pure, catalytically active, endotoxin-free recombinant neurolysin (rNln) in standard laboratory conditions for use as a research tool. To this end, we transformed E. coli cells with a plasmid construct for polyhistidine-tagged rNln, selected a high-expressing clone and determined the optimal time-point for translation of rNln. rNln was purified to homogeneity from the soluble pool of the cell lysate using Ni-NTA affinity and size-exclusion chromatography, followed by removal of endotoxins. Using this protocol ∼3mg pure, catalytically active and nearly endotoxin-free (≈0.003EU/μg protein) rNln was reproducibly obtained from 1l of culture. Lack of cytotoxicity of rNln preparation was documented in cultured mouse cells, whereas stability in whole mouse blood. Intraperitonealy administered rNln in mice reached the systemic circulation in intact and enzymatically active form with Tmax of 1h and T1/2 of ∼30min. Administration of rNln (2 and 10mg/kg) did not alter arterial blood pressure, heart rate, body temperature and blood glucose levels in mice. These studies demonstrate that the rNln preparation is suitable for cell culture and in vivo studies and can serve as a research tool to investigate the (patho)physiological function of this peptidase.

Development and validation of a UHPLC-UV method for the determination of a prostate secretory protein 94-derived synthetic peptide (PCK3145) in human plasma and assessment of its stability in human plasma.

PCK3145 is a synthetic peptide, derived from the Prostate Secreted Protein 94 (PSP94), with promising in vitro and animal in vivo results in prostate cancer. The aim of the present study was to develop and validate a fast and robust ultra-high-performance liquid chromatography with ultraviolet detection for the determination of PCK3145 in human plasma which would be suitable for the assessment of PCK3145 stability to proteolytic degradation. Following protein precipitation, chromatographic separation was carried out on an Aeris Peptide C18 column with mobile phase consisting of acetonitrile-water at a flow-rate of 0.50 mL/min. The calibration curve was linear over the range 0.50-20.00 μg/mL. Intra- and inter-day percentage relative standard deviation and relative error were ≤10%. The limit of detection and the lower limit of quantification were 0.15 and 0.50 μg/mL, respectively. Recovery of PCK3145 from human plasma was ≥96%. The peptide presented high stability in whole blood and in human plasma (>98% intact peptide after 24 h incubation at 37°C in human plasma), which represents a distinctive advantage in the therapeutic use of the compound. This is the first validated UHPLC method for the determination of PCK3145 reported, and it was successfully applied in the study of the proteolytic stability of PCK3145 in human plasma ex vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Stability of Routine Biochemical Analytes in Whole Blood and Plasma From Lithium Heparin Gel Tubes During 6-hr Storage.

The stability of biochemical analytes has already been investigated, but results strongly differ depending on parameters, methodologies, and sample storage times. We investigated the stability for many biochemical parameters after different storage times of both whole blood and plasma, in order to define acceptable pre- and postcentrifugation delays in hospital laboratories. Twenty-four analytes were measured (Modular® Roche analyzer) in plasma obtained from blood collected into lithium heparin gel tubes, after 2-6 hr of storage at room temperature either before (n = 28: stability in whole blood) or after (n = 21: stability in plasma) centrifugation. Variations in concentrations were expressed as mean bias from baseline, using the analytical change limit (ACL%) or the reference change value (RCV%) as acceptance limit. In tubes stored before centrifugation, mean plasma concentrations significantly decreased after 3 hr for phosphorus (-6.1% [95% CI: -7.4 to -4.7%]; ACL 4.62%) and lactate dehydrogenase (LDH; -5.7% [95% CI: -7.4 to -4.1%]; ACL 5.17%), and slightly decreased after 6 hr for potassium (-2.9% [95% CI: -5.3 to -0.5%]; ACL 4.13%). In plasma stored after centrifugation, mean concentrations decreased after 6 hr for bicarbonates (-19.7% [95% CI: -22.9 to -16.5%]; ACL 15.4%), and moderately increased after 4 hr for LDH (+6.0% [95% CI: +4.3 to +7.6%]; ACL 5.17%). Based on RCV, all the analytes can be considered stable up to 6 hr, whether before or after centrifugation. This study proposes acceptable delays for most biochemical tests on lithium heparin gel tubes arriving at the laboratory or needing to be reanalyzed.

Analytical challenges in quantitative analysis (LC/MS/MS) of abiraterone: A validated assay to determine abiraterone in human plasma.

329 Background: Abiraterone acetate is rapidly hydrolyzed to the active abiraterone in vivo. In order to study the relation between drug exposure and therapeutic effect an assay to determine plasma concentrations of abiraterone is required. A quantitative bioanalytical liquid chromatography tandem mass spectrometric (LC-MS/MS) assay with a BEH C18(2.1 × 50 mm, 1.7 µm) column was developed. Several analytical challenges were encountered. Methods: Protein precipitation with acetonitrile was used to pretreat the EDTA plasma samples. A stable isotope (D4-abiraterone) was used as the internal standard. During validation five quality controls were used (LLOQ, LQ, MQ, HQ, ULOQ). The method was validated according to EMA guidelines. Results: The LC-MS/MS method was validated over a concentration range from 1-500 ng/mL. Within – and between day precisions and accuracies were &lt; 5%. The LLOQ was 1 ng/ml and the ULOQ was 499 ng/mL. Abiraterone is stable in plasma for at least 7 days at ambient temperature and at 2-8°C. Stability at -40°C was demonstrated for at least 6 months. Abiraterone is only stable for 3 hours in whole blood at ambient temperature. Stock solution stability was demonstrated for at least 1 year. The concentration levels of the LQ, MQ and HQ were 5, 300 and 400 ng/mL, respectively. During method development several analytical challenges were encountered and solved: abiraterone adsorption on glass containers, limited stability in citrate plasma and whole blood, carry-over issues, very limited stability of abiraterone acetate in whole blood was demonstrated, curiously with proven stability in plasma. Throughout the method polypropylene vials and utilities were used. EDTA plasma was used for development and validation. The needle wash was adjusted and the runtime was prolonged with a longer time at 100% acetonitrile to address the carry over issues. Whole blood samples were treated on ice. The quantification of abiraterone acetate was abandoned. Conclusions: After different hurdles the method is successfully validated and applied to determine plasma concentrations of abiraterone in clinical studies and in regular patient care in patients with prostate cancer.

Delivery of tissue plasminogen activator and streptokinase magnetic nanoparticles to target vascular diseases

Thrombolytic therapy for acute myocardial infarction standardly makes use of the medications streptokinase (SK) and tissue plasminogen activator (tPA). In this study, the potential of silica-coated magnetic nanoparticles (SiO2-MNPs) as nanocarriers clinical thrombolytic therapy was investigated. SiO2-MNPs for use in targeted therapeutic delivery of tPA and SK were prepared using a combined technique incorporating controlled precipitation and hydrothermal methods. Response surface methodology (RSM) was employed to evaluate the efficiency of the SiO2-MNPs. The production of SK secreted from Streptococcus equi was enhanced using random mutagenesis. The tPA and SK A were encapsulated by means of a silanizing agent with a surface rich in 3-aminopropyltrimethoxysilane layered around the SiO2-MNPs. Blood clot lysis assays and fibrin-containing agarose plates were used to carry out in vitro thrombolysis testing. The optimum conditions for producing MNPs were found to be at pH=13 and at a temperature of 75°C for 45min. Culture conditions of 2.75% NaCl concentration at initial pH=7.5 for 90s under UV resulted in maximum SK activity. The tPA/SK-conjugated SiO2-MNPs (SiO2-MNP-tPA-SK) increased operating stability in whole blood and storage stability in a buffer by 92%. More effective thrombolysis using magnetic targeting was indicated by a 38% reduction in blood clot lysis time achieved with SiO2-MNP-tPA-SK compared to administering the SiO2-MNPs without guidance. The silica-coated magnetic nanocarriers developed in this study show potential for improved clinical thrombolytic therapy.

Pre-analytical stability of adrenocorticotrophic hormone from healthy horses in whole blood, plasma and frozen plasma samples

The stability of equine adrenocorticotrophic hormone (ACTH) in blood samples is not fully known. The study objectives were to determine ACTH stability (1) in whole blood and plasma over 72 h at either 4 or 21 °C, and (2) in plasma frozen at either −20 or −80 °C over 30 days. Nine horses were sampled and ACTH concentration were measured after storage as whole blood or plasma, at 4, 21, −20 and −80 °C for up to 30 days. The ACTH concentration was significantly reduced at 24 h but remained stable when plasma was frozen at −20 and −80 °C for 30 days. Beyond 24 h, samples stored at 21 °C showed a greater reduction in ACTH concentrations than those stored at 4 °C. Therefore, samples can be stored for 8 h without centrifugation, or frozen for 30 days without appreciable reductions in ACTH concentrations.

Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds.

BackgroundHIV viral load (VL) testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures.Methods and FindingsUsing a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence) in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence), with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (<3,000 copies/mL) environments.ConclusionsWhole blood and plasma samples in EDTA may remain stable under conditions exceeding current manufacturer recommendations for HIV VL testing. However, given the limited number of studies addressing this question, especially at low levels of viremia, additional evaluations on HIV RNA stability in EDTA tubes and PPT in field conditions are needed.

Pt(IV) prodrugs designed to bind non-covalently to human serum albumin for drug delivery.

Albumin is the most abundant protein in human serum and drugs that are administered intravenously inevitably interact with it. We present here a series of platinum(IV) prodrugs designed specifically to enhance interaction with human serum albumin (HSA) for drug delivery. This goal is achieved by asymmetrically functionalizing the axial ligands of the prodrug so as to mimic the overall features of a fatty acid. Systematic variation of the length of the aliphatic tail tunes the cellular uptake and, consequently, the cytotoxicity of cis,cis,trans-[Pt(NH3)2Cl2(O2CCH2CH2COOH)(OCONHR)], 4, where R is a linear alkyl group. Investigation of an analogue bearing a fluorophore conjugated to the succinate ligand confirmed that these compounds are reduced by biological reductants with loss of the axial ligands. Intracellular release of cisplatin from 4 was further confirmed by observing the characteristic effects of cisplatin on the cell cycle and morphology following treatment with the prodrug. The most potent member of series 4, for which R is a hexadecyl chain, interacts with HSA in a 1:1 stoichiometry to form the platinum-protein complex 7. The interaction is non-covalent and extraction with octanol completely removes the prodrug from an aqueous solution of HSA. Construct 7 is robust and can be isolated following fast protein liquid chromatography. The nature of the tight interaction was investigated computationally, and these studies suggest that the prodrug is buried below the surface of the protein. Consequently, complexation to HSA is able to reduce the rate of reduction of the prodrug by ascorbate. The lead compound from series 4 also exhibited significant stability in whole human blood, attributed to its interaction with HSA. This favorable redox profile, in conjunction with the established nonimmunogenicity, biocompatibility, and enhanced tumor accumulation of HSA, produces a system that holds significant therapeutic potential.