Ocular pharmacokinetics and toxicity of nanoparticular acetazolamide: In vivo distribution and safety of PHBV-ACZ nanoparticle.

Diabetic macular edema (DME) is defined as fluid accumulation in the macular region, between the retinal layers, due to many diseases, especially diabetes. DME is one of the major complications of diabetic retinopathy (DRP). Carbonic anhydrase inhibitors (CAI) are a pharmaceutical agent used in different fields, especially glaucoma treatment. Acetazolamide (ACZ), which is a CAI, is an active substance that has been used off-label for many years in the treatment of macular edema due to diabetes and many other diseases. The low solubility and bioavailability of ACZ limit its use in the treatment of DME. In this study, a nanoparticulate formulation was developed that would increase the solubility and bioavailability of ACZ and allow it to be administered intravitreally. ACZ was loaded on poly(3-hydroxybutyrate-co-3-Hydroxyvalerate) (PHBV) nanoparticles and the loading efficiency was 71.58 ± 1.22%. Toxicity of nanoparticles after intravitreal application was evaluated with anterior segment and posterior segment examination findings, intraocular pressure (IOP) measurements and electrophysiological tests. At the end of the 3-month follow-up, electroretinography (ERG) measurements demonstrated that ACZ loaded PHBV (PHBV-ACZ) nanoparticles did not cause loss of function in retinal cells. On histological examination, rare degenerative changes were observed in several cell groups. In addition, pharmacokinetic studies were performed to determine the tissue distribution of ACZ at various periods. ACZ was identified in vitreous humor and retina at the highest concentration. Based on our results, the prepared nanoparticle formulation can release long-term CAI for DRP therapy and accordingly can reduce the need for monthly intravitreal injections.

Utilizing linkage-specific ethyl-esterification approach to perform in-depth analysis of sialylated N-glycans present on milk whey glycoproteins.

Glycosylation of milk whey proteins, specifically the presence of sialic acid-containing glycan residues, causes functional changes in these proteins. This study aimed to analyze the N-glycome of milk whey glycoproteins from various milk sources using a linkage-specific ethyl esterification approach with MALDI-MS (matrix-assisted laser desorption/ionization-mass spectrometry). The results showed that the N-glycan profiles of bovine and buffalo whey mostly overlapped. Acetylated N-glycans were only detected in donkey milk whey at a rate of 16.06%. a2,6-linked N-Acetylneuraminic acid (a2,6-linked NeuAc, E) was found to be the predominant sialylation type in human milk whey (65.16%). The amount of a2,6-linked NeuAc in bovine, buffalo, goat, and donkey whey glycoproteomes was 42.33%, 44.16%, 39.00%, and 34.86%, respectively. The relative abundances of a2,6-linked N-Glycolylneuraminic acid (a2,6-linked NeuGc, Ge) in bovine, buffalo, goat, and donkey whey were 7.52%, 5.41%, 28.24%, and 17.31%, respectively. Goat whey exhibited the highest amount of a2,3-linked N-Glycolylneuraminic acid (a2,3-linked NeuGc, Gl, 8.62%), while bovine and donkey whey contained only 2.14% and 1.11%, respectively.

Chemometrics assisted untargeted metabolomic analysis to explore metabolic alterations in chronic urticaria via LC/Q-TOF/MS/MS.

Chronic urticaria (CU) is a common disease characterized by the development of recurrent itchy blisters and/or angioedema lasting longer than 6 weeks. The evidence-based diagnosis of CU is described in the most recent urticaria guideline. Metabolomics has the potential to offer diagnostic biomarkers for the detection and prognosis of diseases and predict the efficacy and safety of pharmaceutical interventions. Determining the variation in metabolites found in the plasma of CU patients (n = 20) and 20 controls has therefore been the goal of this investigation. Samples were analyzed using liquid chromatography quadrupole time-of-flight mass spectrometry after applying acetonitrile precipitation. For the purpose of identifying and characterizing metabolites, the METLIN database was utilized. According to results, 21 metabolites were found to be significantly (VIP score > 0.7, p < .05 and fold analysis >1.5) altered. Differentiations between each group were successful via both OPLS-DA and ROC analysis. While plasma allantoate, homogentisate, indole acetate, proline, phenylalanine levels decreased in CU patients compared to healthy subjects, tryptophan, spermidine, phenyl pyruvate, oleic acid, lysine, valine, ornithine, histidine, glutamate, leucine, kynurenine, hypoxanthine, tyrosine, glucose, creatine and cortisol levels were significantly increased. Diagnosis of CU could be achieved by evaluating the metabolic profile of patients.

Determination and pharmacokinetic study of riociguat by UPLC-MS/MS in human plasma.

Pulmonary hypertension (PH) is frequent in the general population and is linked to an increased risk of death. Riociguat is a kind of endothelin receptor antagonist that is often used to treat PH. For pharmacokinetic studies and the determination of riociguat in PH patients, a new, quick, easy, and sensitive UPLC-MS/MS approach was designed and validated. Riociguat and irbesartan (IS) were detected using ESI in positive ion and multiple reaction monitoring mode, respectively, by monitoring the mass transitions m/z 423.0 â†’ 391.0 and 429.1 â†’ 206.9. A reverse-phase C18 column (5 µm, 4.6 × 150 mm) was used with an isocratic mobile phase of water containing 0.1 % formic acid-acetonitrile (25:75, v/v) at a flow rate of 1 ml/min for chromatographic separation. In the range of 5-400 ng/ml, the calibration curve was linear and had a good correlation coefficient (0.9972). This is the first UPLC-MS/MS technique that has been developed and validated for determining riociguat from human plasma. The developed analytical method was extensively validated for linearity, selectivity, specificity, accuracy, precision, sensitivity, stability, matrix effect and recovery, according to FDA criteria. The devised approach was successfully used for a pharmacokinetic research and riociguat determination in PH patients.

Evaluation of Serum Trace Element Levels and Biochemical Parameters of COVID-19 Patients According to Disease Severity.

While the COVID-19 disease progresses mildly or asymptomatically in some people, its progression is severe and symptomatic in others, and it is an issue that requires a scientific response regarding the disease. The present study includes 60 people infected with COVID-19, and the cases were divided into the following groups: asymptomatic, mild, moderate, and severe. Serum Zn, Se, and Cu levels of these groups were analyzed by ICP-MS. All measurements in the patients were compared with those of 32 healthy individuals. When the patient group is compared with the control group, the serum Zn and Se concentrations were statistically low (p < 0.001) in the patient group. Serum Zn level decreased significantly in 4 different patient groups compared to the control group. Although the serum Se level decreased in all four patient groups compared to the control group, the change in Se level was statistically significant only in the severe and mild patient groups. This study examined serum Zn, Se concentrations, and biochemical parameters in patients with different severity of COVID-19, compared them with healthy individuals, and revealed new targets for diagnosis and treatment by revealing those data that may be important.

The serum amino acid profile in COVID-19.

The pandemic of the coronavirus disease (COVID-19) caused by SARS-CoV-2 affects millions of people worldwide. There are still many unknown aspects to this infection which affects the whole world. In addition, the potential impacts caused by this infection are still unclear. Amino acid metabolism, in particular, contains significant clues in terms of the development and prevention of many diseases. Therefore, this study aimed to compare amino acid profile of COVID-19 and healthy subject. In this study, the amino acid profiles of patients with asymptomatic, mild, moderate, and severe/critical SARS-CoV-2 infection were scanned with LC-MS/MS. The amino acid profile encompassing 30 amino acids in 142 people including 30 control and 112 COVID-19 patients was examined. 20 amino acids showed significant differences when compared to the control group in COVID-19 patient groups with different levels of severity in the statistical analyses conducted. It was detected that the branched-chain amino acids (BCAAs) changed in correlation with one another, and L-2-aminobutyric acid and L-phenylalanine had biomarker potential for COVID-19. Moreover, it was concluded that L-2-aminobutyric acid could provide prognostic information about the course of the disease. We believe that a new viewpoint will develop regarding the diagnosis, treatment, and prognosis as a result of the evaluation of the serum amino acid profiles of COVID-19 patients. Determining L-phenylalanine and L-2-aminobutyric levels can be used in laboratories as a COVID-19-biomarker. Also, supplementing COVID patients with taurine and BCAAs can be beneficial for treatment protocols.

A Sensitive UPLC-MS/MS Method for the Determination of Flurbiprofen in Rat Plasma: Application to Real Sample.

For the quantification of flurbiprofen in rat plasma, a simple UPLC-MS/MS method with high sensitivity and short retention time for flurbiprofen was developed and validated using specific parameters. Etodolac was used as internal standard. The transitions (precursor to the product) of flurbiprofen and internal standard were obtained using the electrospray ionization in the negative ion multiple reaction monitoring mode, 243.2 â†’ 199.2, 286.2 â†’ 212.1, respectively. For chromatographic separation, C18 column was used for the stationary phase and gradient elution was used for the mobile phase. This mobile phase consisted of a methanol (A) and a 5 mM ammonium formate solution (B), which varied at a flow rate of 0.4 mL/min. For flurbiprofen, LLOQ was determined as 5 ng/mL. Quantification of flurbiprofen in the rat plasma with a linear calibration curve of 5-5000 ng/mL (r > 0.9991 for plasma) is possible with a retention time of 1.89 min. The total analysis time of the method was 3 min. The proposed method was validated. The intraday and inter-day precision (RSD%) and accuracy (RE%) were within 10% in all cases for flurbiprofen. The stability of flurbiprofen was evaluated under conditions such as short-term, long-term, autosampler and freeze/thaw. After method validation, flurbiprofen was succesfully quantified in real rat plasma samples.

Differential N-glycosylation profiling of formalin-fixed paraffin-embedded (FFPE) invasive ductal carcinoma tissues using MALDI-TOF-MS.

Invasive ductal carcinoma (IDC) is the most common type of breast cancer. As dynamic changes of the glycome are closely associated with complex diseases, they have become a focal point of cancer research involving predictive and prognostic markers. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are representative of the tumor environment and are thus utilized in studies on cancer related research and biomarker discovery. Further studies on differential N-glycosylation profiling of IDC cancer tissues are necessary in order to understand the biological role of glycans in cancer and to evaluate their predictive ability. In this study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted for determining differential N-glycosylation patterns of IDC. Two different derivatization methods, namely, 2-aminobenzoic acid (2-AA) labeling and linkage-specific sialic acid esterification, were used for the analysis of N-glycans. Forty-seven 2-AA labeled and fifty ethyl esterified N-glycans were identified by MALDI-MS. In statistical analyses conducted for 2-AA-labeled N-glycans, the relative amounts of 32 N-glycans and prevalence of 15 N-glycan traits showed significant (p < 0.05) differences between cancer and normal tissues; and in such analyses for the ethyl-esterified N-glycans, the relative amounts of 27 N-glycans and prevalence of 17 N-glycan traits showed significant (p < 0.05) differences between them. It was found that mainly high mannose N-glycans, including H5N2, H6N2, and H7N2, and two fucosylated compositions (H3N3F1 and H5N5F1) showed strong discrimination between IDC and controls. In addition, compared with the controls, high mannose N-glycans were observed to be up-regulated in IDC whereas bisecting N-glycans were down-regulated.

Metabolomic profiling of liver tissues after acute administration of vardenafil in rats.

Erectile dysfunction (ED) diseases have almost affected 100 million men all over the world. Orally administered phosphodiesterase 5 (PDE 5) inhibitors are the most used pharmaceutical formulations for the treatment of ED. In this study, it is aimed to investigate the metabolomics feature of orally administered vardenafil in rats. To carry out the experimental procedure eight male Wistar albino rats were used. Their livers were gently removed and metabolomics profiles of each sample were determined by UPLC Q-TOF MS. Identification of metabolites was achieved by the METLIN database. Cluster analysis was also performed via Principle Component Analysis. Several metabolites were identified and results were evaluated by XCMS software. UPLC Q-TOF MS could be successfully applied to profile biomarkers and help us understand the molecular mechanisms of vardenafil usage. It was concluded that the level of some metabolites, responsible for the collagen synthesis and Kreb's cycle, has been statistically significant after the vardenafil administration.

N-glycan profiling of papillary thyroid carcinoma tissues by MALDI-TOF-MS.

Papillary thyroid carcinoma (PTC) is a type of thyroid cancer whose incidence rate has increased recently all over the world. Glycosylation is a crucial post-translational modification (PTM) for the regulation of thyroid hormone synthesis in thyroid glands. However, our knowledge regarding the N-glycosylation change in PTC is limited. To the best of our knowledge, this is the first study to profile glycans in PTC tissues by mass spectrometry. Herein, we have analyzed the N-glycans of formalin-fixed paraffin-embedded (FFPE) tissues of patients diagnosed with PTC in a matched case-control study. Using MALDI-TOF(/TOF)-MS, 35 enzymatically released N-glycan compositions were characterized. The statistical analyses showed significant differences including six N-glycan compositions (p < 0.001) between patients and controls. It was determined that four of them (H5N4E1, H5N4F1E1, H5N4F1L1E1 and H5N4F1E2, E: α2,6-linked sialic acid; L: α2,3-linked sialic acid) were up-regulated in PTC tissues, whereas two N-glycans (H8N2 and H9N2) found to be down-regulated. Besides, a significant difference was found in six different N-glycan traits. Variants of PTC (follicular, classical, hurtle cell) were also studied to define specific N-glycan change for each variant.

Determination of olanzapine for therapeutic drug monitoring in schizophrenia patients by LC/MS method.

Olanzapine is an atypical antipsychotic drug from the thienobenzodiazepine family which displays efficacy in patients with schizophrenia and related psychoses. A novel LC/MS method was developed and validated for determination of olanzapine in schizophrenia patients' plasma. A liquid-liquid extraction procedure was carried out using 5 mL diethyl ether-diisopropyl ether mixture (1:1, v/v). Average recovery of the extraction procedure was 94.8%. Chromatographic separation was performed on reversed-phase C18 column (250 × 2.0 mm, 5 µm) using mixture of deionized water (trifluoro acetic acid 0.1%)-acetonitrile (20:80, v/v) as mobile phase at a flow rate of 1 mL/min. Irbesartan was used as internal standart and total run time was 2.5 min. Mass spectrometric analysis were carried out in selective-ion montoring mode, and detected olanzapine at m/z 313.1 and IS at m/z 429.4 in all forms of the ions. The calibration curve of olanzapine was linear in the range 2-300 ng/mL (r2 > 0.9993). The interday and intraday precisions (RSD) were <7.55%, and accuracy was >7.59% (n = 6). The proposed study was successfully validated with respect to the US Food and Drug Administration guidelines.

A new spectrofluorimetric method for determination of losartan potassium in rabbit plasma and its application to pharmacokinetic study.

A new spectrofluorimetric method to determine losartan potassium (LP) in rabbit plasma is described. The method was based on measuring the native fluorescence of LP in acidic medium. Optimum excitation and emission wavelengths were found to be 248 nm and 410 nm, respectively, in methanol that was diluted with a sulfurous acid solution LP was extracted from rabbit plasma by methyl-tertiary-butyl-ether in acidic media and then back extracted with NaOH. The calibration curves were linear between 0.025 and 0.5 µg/mL with a lower limit of detection 0.004 µg/mL. Precision and accuracy values of the method were calculated as lower than 4.97% and ± 5.68, respectively and the recovery of LP from rabbit plasma was higher than 91.1%. In addition, stability studies of LP in rabbit plasma were carried out and demonstrated its good stability at - 20 °C and at room temperature. The developed and validated method was successfully applied for estimating the pharmacokinetic parameters of LP following oral administrations of a single 10 mg LP/kg to rabbits and it could be concluded that the method can be applied to clinical trials.

Optimization of a spectrofluorimetric method based on a central composite design for the determination of potassium losartan in pharmaceutical products

Here, a spectrofluorimetric method for the determination of potassium losartan (PL) in pharmaceutical products is described. The effects of critical parameters, pH, acid molarity, and temperature, on the fluorescence intensity of PL were analyzed, and these parameters were optimized using a central composite design (CCD). The highest fluorescent intensity at excitation (λex) and emission (λem) wavelengths of 248 nm and 410 nm, respectively, was achieved using 0.01 M sulfurous acid (pH 2) at 21.6 °C. Under optimum conditions, the method was linear from 0.025-0.5 µg/mL, with a reasonably high correlation coefficient (0.9993). Furthermore, the method was very sensitive (LOQ, 0.006), accurate (RE, ≤7.06), and precise (%RSD, ≤6.51). After development and validation of the method, samples containing PL were analyzed with this method, and the obtained data were statistically compared with those obtained with a previously published reference method using a two one-sided equivalence test (TOST). According to the data, the results from the proposed and reference assays were equivalent.

Descreve-se método espectrofluorométrico para a determinação de losartana potássica (PL) em produtos farmacêuticos. Os efeitos de parâmetros críticos (pH, molaridade ácida e temperatura) na intensidade da fluorecência foram otimizados usando o planejamento de componente central (DCC). A mais alta intensidade fluorescente com λex=248 nm e λem= 410 nm foi obtida usando ácido sulfúrico 0.01 M (pH 2) e 21.6 ºC. Nas condições ideais, a linearidade do método foi estabelecida na faixa de concentração de 0.025-0.5 µg/mL com coeficiente de correlação bastante elevado (0.9993). Além disso, o método foi muito sensível com valor de LOQ 0.006, exato (RE≤7.06) e preciso (RSD%≤6.51). Depois do desenvolvimento e validação do método, amostras de medicamentos contendo PL foram analisadas com este método e os resultados obtidos foram comparados estatisticamente com método de referência, publicado anteriormente, usando o Teste de equivalência TOST (Teste de Equivalência Unilateral). De acordo com os dados estatísticos, os resultados do ensaio de referência e do método proposto foram equivalentes.