A UPLC-MS/MS method for simultaneous determination of eight special-grade antimicrobials in human plasma and application in TDM.

Linezolid, vancomycin, teicoplanin, tigecycline, imipenem, meropenem, voriconazole, and micafungin are eight special-grade antimicrobials commonly used for patients with severe infections. Changes in the pharmacodynamics and pharmacokinetics of critically ill patients severely affect the efficacy of antimicrobial drugs. Therefore, conventional or standard dosing regimens do not achieve satisfactory anti-infective effects. In the current study a simple and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneously determining the concentrations of the above-mentioned eight antimicrobials in human plasma only 3 min after one-step magnetic solid phase extraction pre-treatment. Multiple-reaction monitoring and positive ion modes were used for detection. The calibration curves were established over a concentration range of 0.1-25.0 µg/mL for teicoplanin, linezolid, micafungin, voriconazole, imipenem, igecyclin, and meropenem, and 0.2-50.0 µg/mL for vancomycin; the coefficient of correlation was > 0.9971 for all the compounds. The inter- and intra-day coefficients of variation were < 6.88% at the lower limit of quantification and quality control (QC) levels (low concentration-QC, medium concentration-QC, and high-concentration QC). The UPLC-MS/MS method was successfully used for clinical therapeutic drug monitoring of linezolid, vancomycin, teicoplanin, tigecycline, imipenem, meropenem, voriconazole, and micafungin for critically ill patients.

Recent Advances in the Discovery and Function of Antimicrobial Molecules in Platelets.

The conventional function described for platelets is maintaining vascular integrity. Nevertheless, increasing evidence reveals that platelets can additionally play a crucial role in responding against microorganisms. Activated platelets release molecules with antimicrobial activity. This ability was first demonstrated in rabbit serum after coagulation and later in rabbit platelets stimulated with thrombin. Currently, multiple discoveries have allowed the identification and characterization of PMPs (platelet microbicidal proteins) and opened the way to identify kinocidins and CHDPs (cationic host defense peptides) in human platelets. These molecules are endowed with microbicidal activity through different mechanisms that broaden the platelet participation in normal and pathologic conditions. Therefore, this review aims to integrate the currently described platelet molecules with antimicrobial properties by summarizing the pathways towards their identification, characterization, and functional evaluation that have promoted new avenues for studying platelets based on kinocidins and CHDPs secretion.

Platelet activation and antimicrobial activity of L-PRF: a preliminary study.

Leukocyte and platelet rich fibrin (L-PRF) is one of the platelet concentrates used to support regeneration and healing process. Many studies showed possible immunological and antibacterial properties of L-PRF. We perform an in vitro study to analyze the effect of L-PRF on platelet activation, platelet-leukocytes interactions and antimicrobial activity, important components in the healing process. Molecular biomarkers related with platelet activation and platelet-leukocyte interactions were analyzed by means of flow cytometry when L-PRF exudate was added to whole blood platelets. L-PRF membrane was used to evaluate antimicrobial activity using Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC 90028). Our experimental design allows to evaluate platelet activation and analyze molecular biomarkers of other immune cells and platelet-leukocyte interactions. From the results obtained we can conclude that L-PRF can be a valuable tool in healing process, efficient in activating platelets of whole blood and inhibiting microbial growth. In our opinion, the use of L-PRF exudate, in addition to L-PRF membrane, presents some advantages that have to be considered in clinical trials. Additional research on the characterization and quantification of cells and its products present in the L-PRF exudate, as well as on the temporal factor released. Also, further studies using strains isolated from clinical cases are needed.

Serum vitamin D, calcium, and zinc levels in patients with COVID-19.

BACKGROUND AND AIM: COVID-19 is a global public health concern. As no standard treatment has been found for it yet, several minerals and vitamins with antioxidants, immunomodulators, and antimicrobials roles can be sufficient for the immune response against the disease. The present study evaluates the serum vitamin D, calcium, and Zinc levels in patients with COVID-19. MATERIALS & METHODS: This research is a case-control study performed in May 2020 on 93 patients with COVID-19 hospitalized in a Shoushtar city hospital and on 186 healthy subjects with no symptoms of COVID-19. The serum vitamin D, calcium, and zinc levels were collected and analyzed using correlation coefficient and independent t-test via SPSS 18. RESULTS: Vitamin D levels had a significant difference between the case and control groups (p = 0.008). Serum calcium and serum zinc levels also had statistically significant differences between the two groups (p < 0.001). CONCLUSION: The research results showed that serum zinc, calcium, and vitamin D levels in COVID-19 patients are lower than in the control group. The supplementation with such nutrients is a safe and low-cost measure that can help cope with the increased demand for these nutrients in risk of acquiring the COVID-19 virus.

Pharmacokinetics of anti-infective agents during CytoSorb hemoadsorption.

Cytokine hemoadsorption might be beneficial in patients with sepsis. However, its effect on anti-infective agents' disposition remains largely unknown. We sought to determine the influence of hemoadsorption on the pharmacokinetics of common anti-infective agents. This is an interventional experimental study, conducted in 24 healthy pigs. Animals were randomly allocated to either hemoadsorption (cases) or sham extracorporeal circuit (controls) and to drug combinations (3 cases and 3 controls for each combination). Hemoadsorption was performed with CytoSorb (CytoSorbents Corporation, USA). We evaluated 17 drugs (clindamycin, fluconazole, linezolid, meropenem, piperacillin, anidulafungin, ganciclovir, clarithromycin, posaconazole, teicoplanin, tobramycin, ceftriaxone, ciprofloxacin, metronidazole, liposomal amphotericin B, flucloxacillin and cefepime). Repeated blood sampling from the extracorporeal circulation (adsorber inlet/outlet, sham circuit) was performed over six hours following administration. Total clearance and adsorber-specific clearance were computed. Hemoadsorption was associated with increased clearance of all study drugs, except ganciclovir. Its impact on total body clearance was considered as moderate for fluconazole (282%) and linezolid (115%), mild for liposomal amphotericin B (75%), posaconazole (32%) and teicoplanine (31%) and negligible for all other drugs. Hemoadsorber clearance declined over time, with even delayed desorption for beta-lactams. It was moderately correlated with drug's lipophilicity (p = 0.01; r2 = 0.43). Hemoadsorption with CytoSorb appears to increase to a clinically significant extent the clearance of five among 17 tested anti-infectives. Studies in human patients are required to confirm the need for dosage adjustment of these agents.

Simultaneous quantification of plasma levels of 12 antimicrobial agents including carbapenem, anti-methicillin-resistant Staphylococcus aureus agent, quinolone and azole used in intensive care unit using UHPLC-MS/MS method.

OBJECTIVES: Critically ill patients in intensive care unit (ICU) are susceptible to infectious diseases, thus empirical therapy is recommended. However, the therapeutic effect in ICU patients is difficult to predict due to fluctuation in pharmacokinetics because of various factors. This problem can be solved by developing personalized medicine through therapeutic drug monitoring. However, when different measurement systems are used for various drugs, measurements are complicated and time consuming in clinical practice. In this study, we aimed to develop an assay using ultra-high performance liquid chromatography coupled with tandem mass spectrometry for simultaneous quantification of 12 antimicrobial agents commonly used in ICU: doripenem, meropenem, linezolid, tedizolid, daptomycin, ciprofloxacin, levofloxacin, pazufloxacin, fluconazole, voriconazole, voriconazole N-oxide which is a major metabolite of voriconazole, and posaconazole. DESIGN & METHODS: Plasma protein was precipitated by adding acetonitrile and 50% MeOH containing standard and labeled IS. The analytes were separated with an ACQUITY UHPLC CSH C18 column, under a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid and 2 mM ammonium formate. RESULTS: The method fulfilled the criteria of US Food and Drug Administration for assay validation. The recovery rate was more than 84.8%. Matrix effect ranged from 79.1% to 119.3%. All the calibration curves showed good linearity (back calculation of calibrators: relative error ≤ 15%) over wide concentration ranges, which allowed determination of Cmax and Ctrough. Clinical applicability of the novel method was confirmed. CONCLUSIONS: We have developed an assay for simultaneous quantification of 12 antimicrobial agents using a small sample volume of 50 µL with a short assay time of 7 min. Our novel method may contribute to simultaneous calculation of pharmacokinetic and pharmacodynamic parameters.

Development and validation of an LC-MS/MS method for quantifying nine antimicrobials in human serum and its application to study the exposure of Chinese pregnant women to antimicrobials.

BACKGROUND: To study the prevalence of the exposure of pregnant women to antimicrobials, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine nine antimicrobials, namely sulfadimidine, sulfapyridine, sulfadiazine, sulfathiazole, ofloxacin, ciprofloxacin, norfloxacin, tetracycline, and lincomycin, in human serum. METHODS: The sample preparation procedure included protein precipitation followed by a cleanup step with solid phase extraction (SPE). Separation was carried out using a CORTECS T3 column (100 × 2.1 mm, 2.7 µm) by gradient elution with a runtime of 8.0 min. Detection was performed on a triple quadruple tandem mass spectrometer with scheduled multiple reaction monitoring (sMRM) in positive ion scan mode. RESULTS: The calibration curves were linear over the concentration range of 0.5-50 ng/ml, and the limit of quantitation was between 0.01 and 0.2 ng/ml. For each level of quality control samples, the inter- and intra-assay precision values were less than 12.0%, and the accuracy ranged from 86.1% to 109.0%. No significant matrix effect or carryover was observed. The antimicrobials of interest were stable under all investigated conditions. The validated method was applied to analyze clinical samples from pregnant women in China, and 10 out of 500 samples showed the presence of antimicrobial residues. Moreover, compared with the time-resolved fluoro-immunoassay (TRFIA) method, the developed method showed greater sensitivity and specificity. CONCLUSION: This study provides a simple and rapid LC-MS/MS method for the simultaneous measurement of nine antimicrobials in serum samples, which could be a useful tool in clinical utilization.

Serum hepcidin concentrations in relation to iron status in children with type 1 diabetes.

Chronic low-grade inflammation in type 1 diabetes (T1D) might increase hepcidin synthesis, possibly resulting in functional iron deficiency (FID). We hypothesized that in T1D children with FID, hepcidin concentrations are increased compared to those with normal iron status and those with absolute iron deficiency (AID). We evaluated hepcidin concentrations in T1D children in relation to iron status, and investigated whether hepcidin is useful in assessing FID. A cross-sectional study was conducted. FID was defined as elevated zinc protoporphyrin/heme ratio and/or red blood cell distribution width, and AID as low serum ferritin concentration. Post-hoc analyses with different definitions of FID were performed, using transferrin saturation and reticulocyte hemoglobin content. Serum hepcidin concentrations were measured using mass-spectrometry. The IRODIAB-study is registered at www.trialregister.nl (NTR4642). This study included 215 T1D children with a median age of 13.7 years (Q1-Q3: 10.1-16.3). The median (Q1-Q3) hepcidin concentration in patients with normal iron status was 1.8 nmol/l (0.9-3.3), in AID-patients, 0.4 nmol/l (0.4-0.4) and in FID-patients, 1.6 nmol/l (0.7-3.5). Hepcidin concentrations in FID-patients were significantly higher than in AID-patients (p < 0.001). Irrespective of FID-definition used, hepcidin concentrations did not differ between FID-patients and patients with normal iron status. This might be explained by the influence of various factors on hepcidin concentrations, and/or by differences in response of iron parameters over time. Single hepcidin measurements do not seem useful in assessing FID in T1D children. Multiple hepcidin measurements over time in future studies, however, might prove to be more useful in assessing FID in children with T1D.

High-Density Lipoproteins as Homeostatic Nanoparticles of Blood Plasma.

It is well known that blood lipoproteins (LPs) are multimolecular complexes of lipids and proteins that play a crucial role in lipid transport. High-density lipoproteins (HDL) are a class of blood plasma LPs that mediate reverse cholesterol transport (RCT)-cholesterol transport from the peripheral tissues to the liver. Due to this ability to promote cholesterol uptake from cell membranes, HDL possess antiatherogenic properties. This function was first observed at the end of the 1970s to the beginning of the 1980s, resulting in high interest in this class of LPs. It was shown that HDL are the prevalent class of LPs in several types of living organisms (from fishes to monkeys) with high resistance to atherosclerosis and cardiovascular disorders. Lately, understanding of the mechanisms of the antiatherogenic properties of HDL has significantly expanded. Besides the contribution to RCT, HDL have been shown to modulate inflammatory processes, blood clotting, and vasomotor responses. These particles also possess antioxidant properties and contribute to immune reactions and intercellular signaling. Herein, we review data on the structure and mechanisms of the pleiotropic biological functions of HDL from the point of view of their evolutionary role and complex dynamic nature.

Improving sensitivity of antimicrobial drug nitrofurazone detection in food and biological samples based on nanostructured anatase-titania sheathed reduced graphene oxide.

In this study, we have prepared anatase titanium (IV) oxide warped reduced graphene oxide nanocomposites (TiO2-rGO NC) using ultrasonic methodology. The morphology of the TiO2-rGO NC was studied using FESEM and TEM. In addition, XRD, Raman, thermogravimetric analysis (TGA) and XPS are used to analyze the crystallinity and chemical composition of the TiO2-rGO NC. We have also investigated the electrochemical behavior of the as-prepared NCs with electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and different pulse voltammetry techniques (DPV). The TiO2-rGO NC modified electrode shows the lower charge transfer resistance (R ct ) of 62.87 Ω. Next, the glassy carbon electrode (GCE) was modified with sonochemically prepared TiO2-rGO NC and used to determine the electrocatalytic reduction of nitrofurazone (NTF). Thus, the proposed sensor established the wider covering range (WCR) of 0.01 to 380 µM and an excellent detection limit of 2.28 nM. Finally, the TiO2-rGO NC/GCE was applied to determine the NTF in real samples, including crayfish and human blood serum samples, which acquired good found and recovery values.

Quantification of metronidazole in human bile fluid and plasma by liquid chromatography-tandem mass spectrometry.

This study was conducted to develop a highly selective, sensitive, and validated method for quantifying metronidazole in human plasma and bile fluid. Metronidazole and metronidazole-d4 (internal standard) were extracted from 100 µL of plasma and bile fluid by liquid-liquid extraction. Liquid chromatography with a Hydrosphere C18 column (50 × 2.0 mm) was performed using 10 mM ammonium formate (pH 4.0) and acetonitrile (20:80, v/v) as the mobile phase. Triple quadrupole mass spectrometry was operated with an electrospray ionization interface in multiple reaction monitoring and positive ion modes. The calibration curves were linear for bile and plasma samples over the range of 50-20,000 ng/mL (r2 > 0.999). The intra- and inter-day coefficients of variation (CVs) for plasma ranged from 2.50% to 7.85% and 3.11% to 16.9%, respectively; for bile, the intra-and inter-run precision (CVs) ranged from 2.76% to 13.2% and 3.16% to 11.5%, respectively. The mean extraction recovery for metronidazole ranged from 76.5% to 82.1% in plasma and from 78.8% to 87.8% in bile, respectively. Our proposed analytical method was successfully applied to determine metronidazole concentrations in bile as well as in plasma at multiple time points in a patient with acute cholangitis.

Increased Arbekacin Clearance in Patients With Febrile Neutropenia.

BACKGROUND: Arbekacin (ABK) is used to treat infections caused by methicillin-resistant Staphylococcus aureus and is used widely for the treatment of febrile neutropenia (FN). As ABK has a narrow therapeutic concentration window, the dosage must be adjusted via therapeutic drug monitoring. However, the influence of the physiology of patients with FN on the pharmacokinetic (PK) parameters of ABK remains unclear. Therefore, we examined this influence on ABK PK parameters. METHOD: We performed a retrospective cohort study using data from patients with a hematologic malignancy who were ≥18 years and had been administered ABK. We excluded patients who did not receive therapeutic drug monitoring and had an estimated glomerular filtration rate (eGFR) of <30 mL/min, because clinically sufficient data would not be available. RESULT: Of the 99 enrolled patients, 25 did not have FN and 74 had FN. Arbekacin clearance (CLabk) was shown to correlate with eGFR in patients with FN (r = 0.32, P = 0.0062) and without FN (r = 0.50, P = 0.01). CLabk was higher in patients with FN than in those without FN. In addition, in the eGFR of <100 mL/min group (normal renal function), CLabk and CLabk/eGFR were also higher in patients with FN than in those without FN. CONCLUSIONS: CLabk was increased in patients with FN and normal renal function; therefore, we propose an increased ABK dose for patients with FN and normal renal function.

Role of Oxidative Stress in Hypersensitivity Reactions to Sulfonamides.

Antimicrobial sulfonamides are important medications. However, their use is associated with major immune-mediated drug hypersensitivity reactions with a rate that ranges from 3% to 4% in the general population. The pathophysiology of sulfa-induced drug hypersensitivity reactions is not well understood, but accumulation of reactive metabolites (sulfamethoxazole [SMX] hydroxylamine [SMX-HA] and SMX N-nitrosamine [SMX-NO]) is thought to be a major factor. These reactive metabolites contribute to the formation of reactive oxygen species (ROS) known to cause cellular damage and induce cell death through apoptosis and necroptosis. ROS can also serve as "danger signals," priming immune cells to mount an immunological reaction. We recruited 26 sulfa-hypersensitive (HS) patients, 19 healthy control subjects, and 6 sulfa-tolerant patients to this study. Peripheral blood monocytes and platelets were isolated from blood samples and analyzed for in vitro cytotoxicity, ROS and carbonyl protein formation, lipid peroxidation, and GSH (glutathione) content after challenge with SMX-HA. When challenged with SMX-HA, cells isolated from sulfa-HS patients exhibited significantly (P ≤ .05) higher cell death, ROS and carbonyl protein formation, and lipid peroxidation. In addition, there was a high correlation between cell death in PBMCs and ROS levels. There was also depletion of GSH and lower GSH/GSSG ratios in peripheral blood mononuclear cells from sulfa-HS patients. The amount of ROS formed was negatively correlated with intracellular GSH content. The data demonstrate a major role for oxidative stress in in vitro cytotoxicity of SMX reactive metabolites and indicate increased vulnerability of cells from sulfa-HS patients to the in vitro challenge.

Population Pharmacokinetics of Danofloxacin After Single Intramuscular Dose Administration in California Brown Pelicans (Pelecanus occidentalis californicus).

The pharmacokinetics of danofloxacin was investigated in rehabilitated California brown pelicans (Pelecanus occidentalis californicus) after a single intramuscular injection at a dose of 15 mg/kg body weight. The concentration of the drug in plasma was assayed by high-pressure liquid chromatography. A sparse-sampling design was used to reduce the number of samples (1-4 venipunctures) obtained from 24 brown pelicans. A population pharmacokinetic analysis with nonlinear mixed-effects modeling was used to accommodate the sparse-sampling strategy. The nonlinear mixed-effects modeling approach measured both fixed effects (typical values for the population) and random effects (between-subject variability) for this population. A 1-compartment model best represented the concentration-versus-time data after injection. After injection, the elimination half-life, peak concentration, area under the curve, and volume of distribution were 2.76 hours, 2.5 µg/mL, 13.75 µg/h/mL, and 4.35 L/kg, respectively. Rate of absorption was highly variable among the birds. The intramuscular injection of danofloxacin in pelicans at this dose produced plasma concentrations that meet therapeutic targets for bacteria with a minimum inhibitory concentration of ≤0.25 µg/mL. This dose can be used for future studies to evaluate the efficacy of danofloxacin for treating susceptible bacteria.

Pharmacokinetics of sodium baicalin following intravenous and intramuscular administration to piglets.

The purpose of this study was to determine the pharmacokinetics of baicalin after intravenous and intramuscular administration of sodium baicalin at 50 mg/kg to piglets. Plasma baicalin levels were determined by high-performance liquid chromatography. The plasma concentration-time data of baicalin for both administration routes were best described by two-compartmental open model. The area under the plasma concentration-time curve and the elimination half-lives were 77.47 ± 6.14 µg/ml × h and 1.73 ± 0.16 hr for intravenous and 64.85 ± 5.67 µg/ml × h and 2.42 ± 0.15 hr for intramuscular administration, respectively. The apparent volume of distribution and body clearance were 1.63 ± 0.23 L/kg and 2.74 ± 0.30 L h-1  kg-1 for intravenous and 0.51 ± 0.10 L/kg and 0.78 ± 0.08 L h-1  kg-1 for intramuscular routes, respectively. An intramuscular injection of sodium baicalin in piglets resulted in rapid and complete absorption, with a mean maximal plasma concentration of 77.28 ± 7.40 µg/ml at 0.17 hr and a high absolute bioavailability of 83.73 ± 5.53%.

CuCo2O4/N-Doped CNTs loaded with molecularly imprinted polymer for electrochemical sensor: Preparation, characterization and detection of metronidazole.

CuCo2O4 nanoparticles modified with nitrogen doped carbon nanotubes (CuCo2O4/N-CNTs) have high specific surface area and good electrical conductivity. Herein, a novel electrochemical sensor based on CuCo2O4/N-CNTs loaded molecularly imprinted polymer (MIP) modified glassy carbon electrode (GCE) is proposed for rapid and ultrasensitive detection of metronidazole (MNZ). The composite of CuCo2O4/N-CNTs with MIP significantly enhances the electrical signal. The electrochemical polymerization was performed with MNZ as template and aniline as functional monomer by cyclic voltammetry (CV), and differential pulse voltammetry (DPV) was used to detect MNZ. Factors that affect sensor response were optimized. Under the optimal experimental conditions, the DPV current response shows two linearity ranges for MNZ in the range of 0.005-0.1 µM and 0.1-100 µM with very low limit of detection (LOD) of 0.48 nM (S/N = 3). This electrochemical sensing system has high sensitivity, selectivity, excellent reproducibility, repeatability and stability. The recovery (95.9%-100.9%) and reasonable relative standard deviation (RSD) (3.2%-4.8%) for determination of real samples indicate the practicality of the sensing system. This sensing system has high potential for rapid determination of MNZ in samples such as metronidazole tablets, human serum and urine.

Pharmacokinetics of intravenous and intramuscular danofloxacin in red-eared slider turtles (Trachemys scripta elegans).

This study aimed to investigate the pharmacokinetics of danofloxacin in red-eared slider turtle (Trachemys scripta elegans) following a single intravenous (IV) and intramuscular (IM) administrations of 6 mg/kg, using a two-way crossover study with 30-day washout period. Eight clinically healthy red-eared slider turtle weighing 410-600 g (mean 490 g) were used for the study. Danofloxacin concentrations were measured using the reversed-phase high-performance liquid chromatography. The plasma concentration-time data were evaluated by a non-compartmental method. After IV administration, the elimination half-life (t1/2ʎz), mean residence time (MRT0-∞), area under the concentration-time curve (AUC0-∞), volume of distribution at steady state and total body clearance in plasma were 24.17 hr, 30.64 hr, 143.31 hr·µg/ml, 1.29 l/kg and 0.04 l/hr/kg, respectively. Following IM administration, t1/2ʎz, MRT0-∞, AUC0-∞, peak concentration (Cmax), time to reach Cmax, and bioavailability in plasma were 32.00 hr, 41.15 hr, 198.23 hr·µg/ml, 8.75 µg/ml, 1.5 hr and 139.89%, respectively. Danofloxacin has clinically superior pharmacokinetic properties, including the complete IM absorption, slow elimination and wide volume of distribution in red-eared slider turtles. However, further pharmacokinetics/pharmacodynamics studies are necessary for the treatment of diseases caused by susceptible bacteria with known minimum inhibitory concentration values in red-eared slider turtles.