Determination of daptomycin in human plasma and breast milk by UPLC/MS-MS

During the lactation, the choice of a proper antibiotic is crucial since the drug can cross into breast milk causing toxicity to the infant. Therefore, an extraction protocol and LC/MS-MS method for the determination of daptomycin in human milk and plasma were developed, validated and applied to a case of a breastfeeding mother affected by a purulent acute soft skin infection treated with daptomycin. Because of daptomycin high protein binding and its high molecular weight, the optimisation of the extraction protocol and analytical conditions were deeply investigated, and several parameters were taken into account: in particular the type of extraction, internal standard, the type of organic modifier, pH of the aqueous solution, and gradient. The use of a protein precipitation protocol coupled to a C8-reverse phase LC-MS/MS allows for a reliable quantification of daptomycin in both plasma (in the range of 19\u2013199 \u3bcg/mL) and breast milk (in the range of 0.12\u20130.32 \u3bcg/mL). The determination of milk/plasma (M/P) ratio, which ranged from 0.002 to 0.006, allowed to assess that daptomycin, effective for the mother, was contemporarily safe for the breastfed newborn

A Novel Approach by SPME-GC/MS for the Determination of gammahydroxybutyric acid (GHB) in Urine Samples after Conversion into gamma-butyrolactone (GBL)

The quantitative determination of gamma-hydroxybutyric acid (GHB) in urine samples is very important to assess illicit intake or administration. To this end we evaluated several analytical methods: headspace gas-chromatography coupled to flame ionization detection (HS-GC/FID), headspace gas-chromatography coupled to mass spectrometry (HS-GC/MS), headspace gas-chromatography coupled to solid phase microextraction and mass spectrometry (HS-SPME-GC/MS). All these methods were endowed with a not sufficient sensitivity, and then we moved to solid phase microextraction coupled to gas-chromatography with mass spectrometry detection (SPME-GC/MS). At first, GHB was extracted from urine with an organic solvent and analyzed after derivatization. Under these conditions, however, there was a partial overlapping between the chromatographic peak of GHB and that of urea, also extracted by the organic solvent. Then we decided to change analytical approach and to convert GHB to gamma-butyrolactone (GBL), which is not an endogenous compound. A SPME method was optimized and validated for the determination of GBL. The limit of detection (LOD) of the method resulted to be 0.25 \u3bcg/mL for GBL, corresponding to 0.5 \u3bcg/mL for GHB. The lower limit of quantification (LLOQ) was 0.4 \u3bcg/mL for GBL and 0.8 \u3bcg/mL for GHB. The LLOQ of the method resulted 10 times lower than the endogenous level, thus allowing to distinguish between physiological conditions and exogenous assumption

Raised homocystein plasma concentration in patients with Heart Failure: clinical significance

Elevated plasma levels of homocysteine is associated with increased risk of thrombotic and atherosclerotic vascular disease. Several studies have demonstrated that hyperhomocysteinemia is an indipendent risk factor for vascular disease and is associated to heart failure. However there are no data regarding the association between homocysteine and various objective as well as subjective measures of heart failure. We hypothesized that plasma homocysteine is associated with clinical and echocardiographic signs of heart failure. On this ground we have analysed levels of homocysteine in patients with heart failure and possible correlation between these levels and clinical-functional pattern (NYHA class and ejection fraction). Methods: Plasma homocysteine levels were determined in 123 patients with dilated cardiomyopathy (59 males, 64 females, mean age 67±10 years, mean EF 31±11% and mean NYHA 2.4±0.9, 47 idiopatic and 76 postischemic cardiomyopathy) and 85 healthy control subjects (homogeneus group for sex and age). Patients with chronic renal failure, vitamin B12 and folate deficiency or factors affecting homocysteine plasma levels were escluded from this study. Homocysteine levels were determined in coded plasma samples by immunoenzimatic methods. Results: Patients with heart failure had a higher homocysteine level (mcg/L) than control subjects (21.72±10.28 vs 12.9±6.86, p<0,001) both postischemic (20.89±9.6 vs 12.9±6.86, p<0,001) and idiopatic cardiomiopathy (23.0±11.2 vs 12.9±6.86, p<0,001). A significant correlation was observed between homocysteine and NYHA functional class (p<0,001), age (p<0,001), creatinine (p<0,001), colesterol (p<0,05) while no correlations were observed with hemodynamic (HR, BP), functional (ejection fraction) and other metabolic parameters (triglycerides). Serum homocysteine was lowest in control and increased with increasing NYHA class. In idiopatic cardiomiopathy the correlation between homocysteine and NYHA functional class, creatinine (p<0,001), fibrinogen (p<0,05) was confirmed; in postischemic cardiomiopathy a significant correlation with creatinine and NYHA class (p<0,001) and with triglycerides (p<0,05) was also found. Conclusion: Plasma homocysteine was directly related to NYHA class. This observation may underline the strong relations of plasma homocysteine to congestive heart failure. Further research is indicated to evaluate a causal or noncausal mechanism for this association

Myriocin Effect on Tvrm4 Retina, an Autosomal Dominant Pattern of Retinitis Pigmentosa

Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary pre-requisites for translation into human therapy to slow down RP

Screening of new psychoactive substances (NPS)by gas-chromatography/time of flight mass spectrometry (GC/MS-TOF)and application to 63 cases of judicial seizure

A screening method for the separation and identification of more than fifty NPS is proposed. The method is based on fast gas-chromatography/time of flight mass spectrometry (FAST-GC/MS-TOF). Thanks to the shorter and narrower capillary column and to the rapid acquisition of the TOF detector a huge number of compounds are separated in a very short time of analysis (10 min). Only a few peaks were overlapped. The possibility to apply deconvolution by the software of the GC/MS-TOF instrument allowed the unequivocal identification also for the superimposed peaks. Linearity and LOD was studied and the method was applied to 63 cases of powders seized by the judicial authority at the airport of Milano Malpensa in Northern Italy in the period 2014\u20132017

Determination of polycyclic aromatic hydrocarbons in lipstick by gas-chromatography coupled to mass spectrometry: A case history

A suitable extraction protocol based on an liquid-liquid extraction with hexane/dimethyl sulfoxide and a GC/MS method were developed and validated to determine the concentration of six prohibited Polycyclic Aromatic Hydrocarbons (PAHs; benzo[a]pyrene; dibenz[a,h]anthracene; benz[a]anthracene; benzo[j]fluoranthene; benzo[k]fluoranthene; chrysene) in lipsticks commissioned by a cosmetic company to a manufacturer. The lipsticks were produced in four different colors. Analyses confirmed the presence of benz[a]anthracene and chrysene only in two colors in a concentration of 9.3\u20139.4 ng/g. The concentration of PAHs was 250 times lower than what is considered a toxic level on the basis of what reported in the litaraure and guidances for cosmetic ingredients; therefore we could assume that the risk for consumer health was negligeble

Sodium-glucose cotransporter 2 inhibitors antagonize lipotoxicity in human myeloid angiogenic cells and ADP-dependent activation in human platelets: Potential relevance to prevention of cardiovascular events

Background: The clear evidence of cardiovascular benefits in cardiovascular outcome trials of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in type 2 diabetes might suggest an effect on atherosclerotic plaque vulnerability and/or thrombosis, in which myeloid angiogenic cells (MAC) and platelets (PLT) are implicated. We tested the effects of SGLT2i on inflammation and oxidant stress in a model of stearic acid (SA)-induced lipotoxicity in MAC and on PLT activation. The possible involvement of the Na+/H+ exchanger (NHE) was also explored. Method: MAC and PLT were isolated from peripheral blood of healthy subjects and incubated with/without SGLT2i [empagliflozin (EMPA) and dapagliflozin (DAPA) 1-100 μM] to assess their effects on SA (100 μM)-induced readouts of inflammation, oxidant stress and apoptosis in MAC and on expression of PLT activation markers by flow-cytometry after ADP-stimulation. Potential NHE involvement was tested with amiloride (aspecific NHE inhibitor) or cariporide (NHE1 inhibitor). Differences among culture conditions were identified using one-way ANOVA or Friedman test. Results: NHE isoforms (1,5-9), but not SGLT2 expression, were expressed in MAC and PLT. EMPA and DAPA (100 μM) significantly reduced SA-induced inflammation (IL1β, TNFα, MCP1), oxidant stress (SOD2, TXN, HO1), but not apoptosis in MAC. EMPA and DAPA (both 1 μM) reduced PLT activation (CD62p and PAC1 expression). SGLT2i effects were mimicked by amiloride, and only partially by cariporide, in MAC, and by both inhibitors in PLT. Conclusions: EMPA and DAPA ameliorated lipotoxic damage in stearate-treated MAC, and reduced ADP-stimulated PLT activation, potentially via NHE-inhibition, thereby pointing to plaque stabilization and/or thrombosis inhibition as potential mechanism(s) involved in SGLT2i-mediated cardiovascular protection