Current Pharmaceutical Analysis - Volume 12, Issue 1, 2016

Malondialdehyde (MDA) is one of the most common biomarkers of oxidative stress and is used for the diagnosis of many diseases. This work aims to review the reaction used for the derivatization of MDA, the parameters affecting adduct formation, and a number of recently published analytical methods for quantification of MDA in biological samples. A brief discussion of the reliability of MDA as an oxidative stress biomarker is also provided.

The safety of pharmaceuticals is dependent not only on the intrinsic toxicological properties of the active ingredient, but also on the impurities and degradation products that it may contain. The presence of impurities in active ingredients can have a significant impact on the quality, safety and efficacy of pharmaceuticals. Efforts should be made to identify and characterize all unknown impurities in the drug, due to the increasing demand of regulatory agencies to manufacture high-purity drugs. The drug impurity profile is one of the most important issues in modern pharmaceutical analysis during the process of technology development for the production of high-purity substances. The degradation products may be provided through forced degradation studies, which enable development of indicators of stability methods with appropriate specificity and selectivity, purity verification chromatographic peak of active pharmaceutical ingredient in the product, provides information about possible degradation routes of a certain product, evaluation of the factors that may interfere in any way in the drug stability and critical analysis of the drug degradation profile.

Background: The color-change mechanism of bromocresol purple (BCP) bound to a human serum albumin (HSA) molecule can be explained via the following proton-exchange reactions: (i) BCP- + HSA-COO- → BCP2- + HSA-COOH (pH < 6.0; i.e., the pKa of BCP-) and (ii) BCP2- + HSANH3+ → BCP- + HSA-NH2 (pH > 6.0). These can be monitored through pH-dependent experiments and are consistent with the absorbance data calculated using the Henderson-Hasselbalch equation. Methods: The absorbance spectra of the BCP-HSA complex were measured over a range of pH values (2.4–11.6) and HSA concentrations (0–25.6 μmol/L). Results: Six peaks were observed in the absorbance spectra. The HSA-concentration-dependent absorbance change at 430 nm (attributed to BCP) decreased with a simultaneous increase in absorbance at 599 nm at a pH lower than 6.0; the absorbance at 584 nm (attributed to BCP2-) decreased with a simultaneous increase in absorbance at 613 nm at a pH higher than 6.0. The peaks at 599 and 613 nm did not correspond to BCP2- and BCP. Conclusion: The structures of the BCP2- and BCP produced are stabilized by resonance-assisted hydrogen bonds, and reverse reactions do not occur in the two separate (BCP- and BCP2-) binding sites.

Background: Pregnant women who are planned to undergo a cesarean section (CS) may suffer high levels of anxiety. Because glutamate is an excitatory neurotransmitter that is associated with stress conditions and anxiety, it is possible that anxious pregnant women may have altered blood glutamate levels which may influence the wellbeing of the mother and fetus. Objectives: This study was undertaken to examine anxiety levels in pregnant women planned for vaginal delivery (VD) as compared to women scheduled for elective CS, and, to determine the association between severity of anxiety and blood glutamate levels in pre-delivery women. Subjects and Methods: Anxiety was assessed in 3rd trimester, pre-delivery pregnant women (≥18 years old) who met the inclusion criteria of the study and signed a written informed consent to participate. The study was approved by the local Ethical Committee. Anxiety was assessed in 100 women who were scheduled for a spontaneous VD and 100 women who were scheduled for an elective CS using the Hamilton Anxiety Rating Scale (HARS). After completing the HARS, a blood sample was collected for determination of glutamate levels. Mann-Whitney’s test was used to evaluate differences in anxiety levels between the groups. The association between glutamate levels and severity of anxiety was examined using Spearman’s correlation test. Results: Women who underwent VD had a significantly higher level of anxiety as compared to women who underwent a CS (HARS score 12.5 ± 0.73 vs. 6.6 ± 0.7, P < 0.001, respectively). Blood glutamate levels did not differ significantly between the groups (71.9 ± 2.8 vs. 77.8 ± 3.1 μM/L, P = 0.15, respectively). Moreover, there was no association between level of anxiety and glutamate levels in both groups (VD group: R = - 0.084, P = 0.42; CS group: R = 0.124, P = 0.24). Conclusion: The mechanism of pre-delivery anxiety seems to be glutamate-independent. The finding that women who underwent a VD had high levels of anxiety underscores the need for better pre-partum educational programs aimed to reduce anxiety in pregnant women scheduled for a VD.

We focused on a rapid method for the assay of protein unbound indoxyl sulfate (IS) in urine samples by square-wave voltammetry. A sensor based on the electroactivity of commercial graphene screen-printed electrode (GR-SPE) for sensitive estimation of uremic toxin indoxyl sulfate was presented. The behaviors of uremic solute indoxyl sulfate on GR-SP electrodes were explored by cyclic and square-wave voltammetry. The outcomes displayed that the GR-SP electrode exposed perfect electroactivity to uremic toxin indoxyl sulfate and the oxidation of uremic solute IS is a multi-step and irreversible process. The process is pH dependent and takes place with the forging of a main oxidation product that is adsorbed on the GR-SP electrode surface. The calibration curve was achieved in the IS concentration range of 0.5–80 μM and the limit of detection was calculated to be 0.064 μM at a signal-to-noise ratio of 3. The use of the commercially disposable GR-SPE in human serum and urine sample assays obtained satisfactory results, which made it a promising alternative in routine sensing applications.

The risk management system, firstly developed in industrial and transport sectors, has been recently applied also in the healthcare to ameliorate patient safety. Among available methods of risk analysis, the Failure Mode and Effects Analysis-FMEA highlights possible errors before adverse events occur and defines the actions to perform to prevent consequences. This qualitative approach can be implemented by the Failure Modes, Effects and Criticality Analysis-FMECA that quantifies errors. Recently, the FMEA/FMECA has been indicated as an option to adopt to analyze potential risks throughout the entire lifecycle of drugs, also including the class of Advanced Therapy Medicinal Products, such as cell-based medicines. Autologous Chondrocyte Implantation is a widely diffused cell-based treatment that can be used to regenerate articular cartilage. Chondrocytes for Autologous Implantation are classified as Advanced Therapy Medicinal Products (Regulation (EC) No 1394/2007), to be manufactured according to Good Manufacturing Practice. Aim of this study is to apply a FMEA/FMECA analysis on the process of ex vivo Good Manufacturing Practice chondrocyte production in a Facility located in a Public Hospital for Autologous Implantation applications. The analysis evidentiated 26 possible criticalities within the whole process (mostly human errors due to manual activity or inadequate personnel training), leading to different consequences for patients, the most dangerous of which were identified as the possibility of contamination by transmissible pathologies etiological agents, non autologous implantation and microbiological contamination. The improvement actions, implemented within 1 year of production and monitoring, allowed to decrease at least the most critical risks. Even if further evaluations and an increased number of patients are needed in order to confirm our results and reduce method’s subjectivity, this approach revealed to be a useful tool to identify and monitor potential issues during chondrocyte manual ex-vivo manufacturing in the Facility.

This study established a highly effective and reliable method for the separation and quantitative determination of five pharmacodynamic active components of I. sonchifolia using microemulsion electrokinetic chromatography (MEEKC). The MEEKC conditions were investigated. The optimal running buffer (pH 9.25) consisted of 80 mmol/L Brij-35, 0.8% (v/v) n-heptane, 8% (v/v) 1- butanol, 25% (v/v) acetonitrile, 10 mmol/L sodium dihydrogen phosphate–10 mmol/L sodium borate at 25 kV voltage. The regression equations showed good linear relationships, with 0.61–7.76 μg/mL limit of detection and 2.04–25.85 μg/mL limit of quantitation. The established method was also accurate, with intra-day and inter-day relative standard deviations less than 4.78% and less than 6.27%, respectively. Recovery values ranged from 98.61% to 104.76%. The proposed method was successfully applied to determine the five compounds in Kudiezi injection samples. A MEEKC method without adding neutral markers was further established for dissociation constant (pKa*) measurement, and reliable pKa* values were obtained.

In this clinical study, a fast chromatographic (HPLC) method has been established for the quantification of β-estradiol. β-estradiol and the IS (verapamil) were separated from plasma using the deproteinisation procedure, followed by injection of the clear solution of the supernatant into the chromatographic system. For the first time, the proposed method employed a monolithic column in the analysis. The analyte was eluted on a monolithic stationary phase column with a carrier composition of phosphate buffer: acetonitrile (pH 3.5) (90: 10, v/v) at a flow rate of 2.0 mL / min. The target drugs were detected at 280 and 310 nm for fluorescence signal. The proposed method showed a calibration graph over 5 - 1000 ng/ mL, with a low concentration of 1.5 ng/mL. The approach was statistically agreed as regards linearity, accuracy, precision, selectivity and stability according to the FDA criteria. The precision of the method as the intraand inter-day study variation did not exceed 3.0 % from the nominal concentration. The accuracy of β-estradiol was observed to be within ±12% of the theoretical value. The method was good applied to mice plasma.

Background: The application of digoxin in treating patients with heart failure (HF) has been lasted for more than 200 years, however, controversies remain on the effectiveness and safety of digoxin in prognosis of HF patients. Thus, we performed this meta-analysis of clinical trials that examined the digoxin use to evaluate effectiveness and safety. Methods and Result: We searched for the cohort studies that investigated the effect of the digoxin on prognosis of the patients with HF in MEDLINE, EMBASE, Chinese National Knowledge Infrastructure (CNKI), and which were published between January 1993 and October 2013. The outcomes of interest comprised all-cause mortality, HF hospitalization and all-cause hospitalization. In addition, pooled hazard risks (HRs) and 95% confidence intervals (CIs) were calculated to assess the effectiveness and safety of digoxin for HF. Nine cohort studies were retrieved from 1431 citation for the analysis, and in total, 84,692 patients were included in the analysis. After synthesizing data, the meta-analysis showed significant increasing of all cause mortality (HR=1.15, 95%CI=1.04-1.27, p<0.001) in the patients with digoxin treatment compared to the controls, whereas no significant difference was found on HF hospitalization (HR=1.06, 95%CI=0.784-1.431, p<0.001) and all cause hospitalization (HR=0.988, 95%CI=0.720-1.354). Moreover, no altered overall results were found after sensitivity analysis. Conclusion: Therefore, according to the results of our meta-analysis, the use of digoxin could significantly increase the risk of all cause mortality. However, no effect on the HF hospitalization and all-cause hospitalization was found.