Novel dummy molecularly imprinted polymer for simultaneous solid-phase extraction of stanozolol metabolites from urine.

Stanozolol, a synthetic derivative of testosterone, is one of the common doping drugs among athletes and bodybuilders. It is metabolized to a large extent and metabolites are detected in urine for a longer duration than the parent compound. In this study, a novel dummy molecularly imprinted polymer (DMIP) is developed as a sorbent for solid-phase extraction of stanozolol metabolites from spiked human urine samples. The optimized DMIP is composed of stanozolol as the dummy template, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker in a ratio of 1:10:80. The extracted analytes were quantitively determined using a newly developed and validated ultrahigh-performance liquid chromatography tandem mass spectrometry method, where the limits of detection and quantitation were 0.91 and 1.81 ng mL-1, respectively, fulfilling the minimum required performance limit decided on by the World Anti-Doping Agency. The mean percentage extraction recoveries for 3'-hydroxystanozolol, 4ß-hydroxystanozolol, and 16ß-hydroxystanozolol are 97.80% ± 13.80, 83.16% ± 7.50, and 69.98% ± 2.02, respectively. As such, the developed DMISPE can serve as an efficient cost-effective tool for doping and regulatory agencies for simultaneous clean-up of the stanozolol metabolites prior to their quantification.

Quality-by-design approach for development of aqueous headspace microextraction GC-MS method for targeted metabolomics of small aldehydes in plasma of cardiovascular patients.

Lipid peroxidation products, such as short chain aldehydes, are powerful biomarkers of oxidative stress, due to the advantage of long lifetime compared to other metabolites of the lipidome. This work proposes an advanced combined derivatization/solvent-less extraction procedure from plasma followed by rapid Gas Chromatography with Mass Spectrometric detection (GC-MS). A new sample pretreatment protocol is presented which is based on a combination of aldehyde derivatization with methoxyamine under fully aqueous-based conditions of diluted plasma samples followed by headspace solid-phase microextraction (HS-SPME) which is faster compared to methods in the literature serving the same purpose. Being the smallest oximation reagent, methoxyamine derivatization does not require a silylation step of hydroxyl groups as customary and made it possible to have the shortest run times for this series of aldehydes by GC-MS. A Response Surface Methodology (RSM) is employed to optimize the HS-SPME of the aldehyde methoximes to provide insights into the Design Space (DS) of HS-SPME of aldehydes of variable chain lengths and unsaturation. The workflow includes a Quality by Design (QbD) approach for optimization of sample microextraction and derivatization methodology under fully aqueous conditions, in contrast to all reported non-aqueous tedious and long extraction methods in the literature followed by development of a rapid GC-MS assay. The optimal sample preparation obtained from the RSM, and multiple linear regression procedure involved addition of 15 mg methoxyamine (CH3ONH2) and 160 mg Na2SO4 to 0.5 mL plasma diluted to 1 mL with water in an extraction vial followed by HS-SPME using Polydimethylsiloxane/Divinylbenzene fiber at 750 rpm and 77 °C for 15 min. The developed HS-SPME-GC-MS method was validated according to FDA guidelines in SIM mode and applied for targeted determination of lipid peroxidation aldehyde metabolites in plasma of 24 cardiovascular patients vs 20 healthy controls. The run time of the GCMS method was less than 15 min and the LOQ of the 10 targeted aldehydes were 0.5 nM for decanal, 5 nM for hexanal, heptanal, octanal, citronellal and citral, 7 nM for malondialdehyde, 35 nM for 4- hydroxynonenal, 105 nM for 4- hydroxyhexenal and 500 nM for glyoxal. Hexanal, Malondialdehyde and Hydroxynonenal concentrations were significantly higher in patients (p-value<0.05) in the targeted study, while citral was significantly lower as obtained from the untargeted study. Reporting an aldehydic profile signature -whether predictive or diagnostic-for cardiovascular patients would support proper medical intervention at the initiation or progression phases of the disease when expanded on larger number of subjects.

Quality by design approach for enantioseparation of terbutaline and its sulfate conjugate metabolite for bioanalytical application using supercritical fluid chromatography.

Terbutaline is mainly metabolized by sulfoconjugation stereoselectively, favoring its (S)-(+) enantiomer. Reported chiral separations of Terbutaline enantiomers were achieved by various chromatographic methods. However, the simultaneous enantioseparation of Terbutaline and the monosulfate conjugate metabolites was never reported. This study aims at shedding light on the influential factors and interactions leading to successful enantioseparation of Terbutaline and its monosulfate conjugate pairs by Supercritical Fluid Chromatography (SFC) for the first time within a Quality by Design framework using Design of Experiments. The effect of molarity of mobile phase additive, mobile phase flow rate, column temperature and back pressure were evaluated. Compared to previous reports, the response surface interestingly revealed the favorability of high temperature and high flow rate up to 2.25 ml/min for resolution of the two pairs of enantiomers on polysaccharide chiral stationary phase CHIRALPAK IC. In addition, a switch in the elution order of Terbutaline and the sulfate conjugate peak pairs was observed upon elimination of the mobile phase additive where the sulfate conjugate underwent intra-molecular ionic interactions and the change in elution order was only due to TER behavior. The multifactorial interactions would not have been detected with the common one-factor-at-a-time approach during method development, demonstrating the superiority and importance of the Analytical Quality by Design frame in enantioseparation.

Different Serum, Different Protein Corona! The Impact of the Serum Source on Cellular Targeting of Folic Acid-Modified Chitosan-Based Nanoparticles.

The nanoparticle (NP) protein corona represents an interface between biological components and NPs, dictating their cellular interaction and biological fate. To assess the success of cellular targeting, NPs modified with targeting ligands are incubated with target cells in serum-free culture medium or in the presence of fetal bovine serum (FBS). In the former, the role of the corona is overlooked, and in the latter, the effects of a corona that does not represent the one forming in humans nor the respective disease state are considered. Via proteomic analysis, we demonstrate how the difference in the composition of FBS, sera from healthy human volunteers, and breast cancer patients (BrCr Pt) results in the formation of completely different protein coronas around the same NP. Successful in vitro targeting of breast cancer cells was only observed when NPs were incubated with target cells in the presence of BrCr Pt sera only. In such cases, the success of targeting was not attributed to the targeting ligand itself, but to the adsorption of specific serum proteins that facilitate NP uptake by cancer cells in the presence of BrCr Pt sera. This work therefore demonstrates how the serum source affects the reliability of in vitro experiments assessing NP-cell interactions and the consequent success or failure of active targeting and may in fact indicate an additional reason for the limited clinical success of drug targeting by NPs in cancer.

Multivariate approach for optimization of galactomannan extraction from seeds of Egyptian Trigonella foenum-graecum with insights on its pharmacological activities.

Response surface methodology (RSM), based on the central composite design (CCD), was used for the systemic optimization of galactomannan (GAL) extraction from Trigonella foenum-graecum. GAL was reported to possess a variety of pharmacological effects and is commercialized as adjuvant therapy for diabetes, obesity, and hypercholesterolemia. Seven process variables were evaluated (12 experiments in a Plackett-Burman design) to screen the significant factors affecting the extraction yield. The three most significant variables were evaluated in CCD at two levels (twenty experimental designs) to obtain the utmost percentage yield. The yield of GAL extraction was influenced by the volume of the precipitating solvent to the volume of the soaking water and reached a maximum of 10.1% at a ratio of 3.633:1. Exploring the antioxidant, cytotoxic, and anti-microbial activities of GAL revealed cytotoxic activity against LS174-T colorectal cancer cells, weak antioxidant activity, and moderate antimicrobial activity against Candida tropicalis and Micrococcus species.

Niazimicin: A thiocarbamate glycoside from Moringa oleifera Lam. seeds with a novel neuroprotective activity.

Moringa oleifera (MO) known as the miracle tree is a famous nutritional source in many countries. In this study, the neuroprotective activity of MO seeds was investigated. Fractions of the 70% ethanol seed extract of MO were injected at a dose of 250 mg kg-1  day-1 to albino rats for 15 days, after-which induction of dementia was done using 100 mg/kg AlCl3 over 30 days. Results revealed that all fractions ameliorated the effects of AlCl3 where methylene chloride and ethyl acetate fractions, containing the major bioactive compound niazimicin (NZ), showed the best activities. Biological investigations proved NZ to be a highly potent neuroprotective drug lead as a first report, by causing a decrease in the levels of malondialdehyde, cholinesterase, nitric oxide (NO) and amyloid ß by 47%, 34%, 53% and 59%, respectively, and increasing glutathione levels by 54%. Molecular docking studies suggested NZ neuroprotective effects to be mediated by inhibition of caspase-3 and inducible nitric oxide synthase enzymes. PRACTICAL APPLICATIONS: The current findings present the neuroprotective effect of Moringa oleifera seeds consumed as a food supplement and in daily diet. In addition, niazimicin is a promising lead for the development of novel agents against Alzheimer's disease as seen by the reported results.

An intronic DHCR7 genetic polymorphism associates with vitamin D serum level and incidence of acute coronary syndrome.

INTRODUCTION: Vitamin D deficiency has been linked to cardiovascular pathologies including acute coronary syndrome (ACS). Polymorphisms in vitamin D associated genes have been confounding to vitamin D serum levels and pathological predispositions. 7-hydrocholesterol is a common precursor in cholesterol and vitamin D synthesis. DHCR7/NADSYN1 genetic locus expresses 7-hydrocholesterol reductase (DHCR7), an enzyme that recruits 7-hydrocholesterol in cholesterol biosynthesis, and NAD synthetase 1 (NADSYN1), which participates in the hydroxylation of 25 hydroxyvitamin D. AIM: This study aims to correlate two polymorphisms in the DHCR7/NADSYN1 genetic locus with levels of circulatory vitamin D and the presentation of ACS in an Egyptian population. METHODS: In a case control study, 189 ACS patients and 106 healthy control subjects were genotyped for SNPs rs11606033 of the DHCR7 gene and rs2276360 of the NADSYN1 gene using the amplification-refractory mutation system (ARMS). The levels of 25(OH)D2 and 25(OH)D3 were measured using an in-house developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based protocol. RESULTS: ACS patients have significantly lower levels of circulating vitamin D in comparison to healthy controls. Allele A of the DHCR7 polymorphism was found to correlate with serum vitamin D deficiency and incidence of ACS classes: NSTEMI, STEMI and unstable angina, when compared to allele G. On the other hand, the NADSYN1 polymorphism rs2276360 correlated with serum 25(OH)D3 deficiency. Yet, no significant correlation was found with incidences of ACS. CONCLUSION: We conclude that rs11606033, which is an intronic SNP between exon 4 and exon 5 of the DHCR7 gene, influences vitamin D serum abundance and more importantly ACS incidence.

Point-of-care testing and optimization of sample treatment for fluorometric determination of hydrogen sulphide in plasma of cardiovascular patients.

Introduction: Hydrogen sulphide (H2S) is one of the gasotransmitters that was reported to have a cardioprotective effect at its physiological levels in blood. Previous determinations of H2S levels in cardiovascular disease (CVD) patients suffered from diversity of analytical methods, different targeted chemical forms of the gas, and multitude of matrices assessed. Objectives: In this study, a comparative biological sample preparation study is detailed for optimum selective determination of the unionized form of H2S in blood of CVD patients using a new in-house POCT portable spectrofluorometer together with a Reagent-Analyser system. Methods: Dansyl azide was synthesized to react with hydrogen sulfide in biological matrix to produce the fluorescent dansyl amide. Fluorescence was measured at λex 340 nm and λem 517 nm in the new in-house POCT portable spectrofluorometer. The method was validated according to ICH guidelines. Several blood sample treatments and reaction protocols were compared to achieve maximum fluorescence yield. Results: The H2S Analyser was verified in comparison to a benchtop spectrofluorometer where linearity was confirmed in the range of 3-300 µM, LOD being 1 µM, at λex 340 nm and λem 517 nm. Sample treatment involving blood centrifugation followed by addition of reagent on plasma produced maximum fluorescence yield. Analysis of blood samples of myocardial infarction (MI) patients and controls showed elevated levels of H2S in MI patients (28 µM ± 1.111) vs. controls (23 µM ± 1.036) at p = 0.0015. Conclusion: The study is novel in being a POCT approach for selective determination of H2S molecular form in plasma after simple optimized sample treatment. The study confirms that MI is associated with H2S elevated levels up to 10 hours from emergence of symptoms.

Quality-by-Design Is a Tool for Quality Assurance in the Assessment of Enantioseparation of a Model Active Pharmaceutical Ingredient.

The design of experiments (DoE) is one of the quality-by-design tools valued in analytical method development, not only for cost reduction and time effectiveness, but also for enabling analytical method control and understanding via a systematic workflow, leading to analytical methods with built-in quality. This work aimed at using DoE to enhance method understanding for a developed UHPLC enantioseparation of terbutaline (TER), a model chiral drug, and to define quality assurance parameters associated with using chiral mobile phase additives (CMPA). Within a response surface methodology workflow, the effect of different factors on both chiral resolution and retention was screened and optimized using Plackett-Burman and central composite designs, respectively, followed by multivariate mathematical modeling. This study was able to delimit method robustness and elucidate enantiorecognition mechanisms involved in interactions of TER with the chiral modifiers. Among many CMPAs, successful TER enantioresolution was achieved using hydroxypropyl ß-cyclodextrin (HP-ß-CD) added to the mobile phase as 5.4 mM HP-ß-CD in 52.25 mM ammonium acetate. Yet, limited method robustness was observed upon switching between the different tested CMPA, concluding that quality can only be assured with specific minimal pre-run conditioning time with the CMPA, namely 16-column volume (60 min at 0.1 mL/min). For enantiorecognition understanding, computational molecular modeling revealed hydrogen bonding as the main binding interaction, in addition to dipole-dipole inside the CD cavity for the R enantiomer, while the S enantiomer was less interactive.

Resistance of Biomphalaria alexandrina to Schistosoma mansoni and Bulinus truncatus to Schistosoma haematobium Correlates with Unsaturated Fatty Acid Levels in the Snail Soft Tissue.

Only a fraction of the Biomphalaria and Bulinus snail community shows patent infection with schistosomes despite continuous exposure to the parasite, indicating that a substantial proportion of snails may resist infection. Accordingly, exterminating the schistosome intermediate snail hosts in transmission foci in habitats that may extend to kilometres is cost-prohibitive and damaging to the ecological equilibrium and quality of water and may be superfluous. It may be more cost effective with risk less ecological damage to focus on discovering the parameters governing snail susceptibility and resistance to schistosome infection. Therefore, laboratory bred Biomphalaria alexandrina and Bulinus truncatus snails were exposed to miracidia of laboratory-maintained Schistosoma mansoni and S. haematobium, respectively. Snails were examined for presence or lack of infection association with soft tissue and hemolymph content of proteins, cholesterol, and triglycerides, evaluated using standard biochemical techniques and palmitic, oleic, linoleic, and arachidonic acid, assayed by ultraperformance liquid chromatography-tandem mass spectrometry. Successful schistosome infection of B. alexandrina and B. truncatus consistently and reproducibly correlated with snails showing highly significant (up to P < 0.0001) decrease in soft tissue and hemolymph content of the monounsaturated fatty acid, oleic acid, and the polyunsaturated fatty acids, linoleic, and arachidonic acids as compared to naïve snails. Snails that resisted twice infection had soft tissue content of oleic, linoleic, and arachidonic acid similar to naïve counterparts. High levels of soft tissue and hemolymph oleic, linoleic, and arachidonic acid content appear to interfere with schistosome development in snails. Diet manipulation directed to eliciting excessive increase of polyunsaturated fatty acids in snails may protect them from infection and interrupt disease transmission in a simple and effective manner.

The association of megalin and cubilin genetic variants with serum levels of 25-hydroxvitamin D and the incidence of acute coronary syndrome in Egyptians: A case control study.

Megalin and cubilin are two receptors that mediate endocytosis of 25-hydroxyvitamin D (25(OH)D) for its final activation by hydroxylation. The aim of the present study was to evaluate the association of polymorphisms in megalin (rs2075252 and rs4668123) and cubilin (rs1801222 and rs12766939) with the circulating serum levels of 25(OH)D and with the early incidence of acute coronary syndrome (ACS) in Egyptians. The study included 328 subjects; 185 ACS patients aged between 27 and 60 years, and 143 healthy age-matched controls. Genotyping of cubilin rs12766939 Single Nucleotide Polymorphism (SNP) was performed using Real-Time Polymerase Chain Reaction (qPCR) and for megalin rs4668123 and rs2075252 and cubilin rs1801222 by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP). 25(OH)D levels were measured by Ultra Performance Liquid Chromatography- Tandem Mass Spectroscopy (UPLC-MS/MS). Results showed that vitamin D deficiency was highly linked to ACS incidence (P < 0.0001). The megalin rs4668123 CC, cubilin rs1801222 GG and cubilin rs12766939 GG + GA genotypes are associated with a higher ACS incidence and can be considered risk factors, according to Chi-squared test (P = 0.0003, 0.0442, 0.013 respectively). Conversely, the megalin rs2075252 SNP was not associated with increased ACS incidence. However, after performing multiple logistic regression analysis, only the megalin rs4668123 SNP was considered an independent ACS risk factor. Furthermore, the megalin rs4668123 CC genotype was associated with lower 25(OH)D levels (P = 0.0018). In conclusion, megalin rs4668123 (CC) was linked to lower 25(OH)D levels and can be considered an independent risk factor for incidence of ACS.

Screening and optimization of samarium-assisted complexation for the determination of norfloxacin, levofloxacin and lomefloxacin in their corresponding dosage forms employing spectrofluorimetry.

Multivariate strategy was applied for setting a fluorescent technique for the determination of three fluoroquinolones: norfloxacin (NOR), levofloxacin (LEV) and lomefloxacin (LOM) in their pure powder and dosage forms. Based on their known interaction with lanthanides, and augmented fluorescence intensity obtained by antenna effect at λex/λem = 314/553, 312/553 and 310/556 for NOR, LEV and LOM, respectively, the current research was scrutinized. Four continuous factors were selected for study in the screening step by means of Plackett-Burman Design, where temperature factor was excluded for being non-significant and the other factors as volume of metal ion solution, pH and reaction time were evaluated through Central Composite Design. 3-D surfaces demonstrations and 2-D contour plots designated the factors interactions followed by optimization plots, which defined the best blend for factors conjunction. pH factor was the chief motor force affecting the response as the number of coordinated ligands formed depends on the pH, whereas 1:2 complex is the main species at higher pH values followed by the volume of metal ion solution and ended by little effect of the reaction time. Model verification was monitored, which showed the model superiority for the three fluoroquinolones, where all target points tested were in good agreement with the predicted ones. The linear range for the tested drugs were found to be 0.090-1.280 µg/mL for NOR, 0.068-1.448 µg/mL for LEV and 0.077-1.552 µg/mL in case of LOM, thus approving the suitability of this method for Quality Control testing. Furthermore, applying these conditions to test the fluoroquinolones in their pharmaceuticals was done as well as intra and inter-day effects as to confirm the validity of this technique for routine analysis. Recovery % and RSD were found to be 99.958 ±â€¯0.797, 99.887 ±â€¯0.935 and 100.427 ±â€¯0.698 for NOR, LEV and LOM respectively in their pure powder. While it was calculated to be 100.200 ±â€¯0.785, 100.530 ±â€¯0.396 and 100.620 ±â€¯0.896 for NOR, LEV and LOM in their corresponding dosage forms. This excellent precision and accuracy obtained in results impulse it to be one of the most appropriate methods for further analysis.

A chiral enantioseparation generic strategy for anti-Alzheimer and antifungal drugs by short end injection capillary electrophoresis using an experimental design approach.

The present study describes a generic strategy using capillary electrophoretic (CE) method for chiral enantioseparation of anti-Alzheimer drugs, namely, donepezil (DON), rivastigmine (RIV), and antifungal drugs, namely, ketoconazole (KET), Itraconazole (ITR), fluconazole (FLU), and sertaconazole (SRT) in which these drugs have different basic and acidic properties. Several modified cyclodextrins (CDs) were applied for enantioseparation of racemates such as highly sulfated α, γ CDs, hydroxyl propyl-ß-CD, and Sulfobutyl ether-ß-CD. The starting screening conditions consist of 50-mM phosphate-triethanolamine buffer at pH 2.5, an applied voltage of 15 kV, and a temperature of 25°C. The CE strategy implemented in the separation starts by screening prior to the optimization stage in which an experimental design is applied. The design of experiment (DOE) was based on a full factorial design of the crucial two factors (pH and %CD) at three levels, to make a total of nine (32 ) experiments with high, intermediate, and low values for both factors. Evaluation of the proposed strategy pointed out that best resolution was obtained at pH 2.5 for five racemates using low percentages of HS-γ-CD, while SBE-ß-CD was the most successful chiral selector offering acceptable resolution for all the six racemates, with the best separation at low pH values and at higher %CD within 10-min runtime. Regression study showed that the linear model shows a significant lack of fit for all chiral selectors, anticipating that higher orders of the factors are most likely to be present in the equation with possible interactions.

A Comparative Metabolomics Approach Reveals Early Biomarkers for Metabolic Response to Acute Myocardial Infarction.

Discovery of novel biomarkers is critical for early diagnosis of acute coronary syndrome (ACS). Serum metabolite profiling of ST-elevation myocardial infarction (STEMI), unstable angina (UA) and healthy controls was performed using gas chromatography mass spectrometry (GC/MS), solid-phase microextraction coupled to gas chromatography mass spectrometry (SPME-GC/MS) and nuclear magnetic resonance (1H-NMR). Multivariate data analysis revealed a metabolic signature that could robustly discriminate STEMI patients from both healthy controls and UA patients. This panel of biomarkers consisted of 19 metabolites identified in the serum of STEMI patients. One of the most intriguing biomarkers among these metabolites is hydrogen sulfide (H2S), an endogenous gasotransmitter with profound effect on the heart. Serum H2S absolute levels were further investigated using a quantitative double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). This highly sensitive immunoassay confirmed the elevation of serum H2S in STEMI patients. H2S level discriminated between UA and STEMI groups, providing an initial insight into serum-free H2S bioavailability during ACS. In conclusion, the current study provides a detailed map illustrating the most predominant altered metabolic pathways and the biochemical linkages among the biomarker metabolites identified in STEMI patients. Metabolomics analysis may yield novel predictive biomarkers that will potentially allow for an earlier medical intervention.

Enantiomeric separation of underivatized amino acids: predictability of chiral recognition on ristocetin a chiral stationary phase.

The present work aimed to investigate the predictability of the chromatographic behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R, using a DryLab high-performance liquid chromatography (HPLC) method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. After implementing the basic runs, and judging the predictability via the computed resolution map, it can be deduced that the chiral recognition mechanisms tend towards a hydrophilic interaction chromatography rather than the reversed phase mode, which limits the ability of DryLab software to predict separations on Chirobiotic R.

Interplay of vitamin D and nitric oxide in post-menopausal knee osteoarthritis.

AIM: Mounting evidence has presented nitric oxide (NO) and vitamin D (vitD) as having independently complex roles in osteoarthritis (OA). However, a mechanistic or an observational connection between them has never been investigated in the disease. This study investigates the correlation between circulating 25-hydroxyvitamin D [25(OH)D] and total NO as nitrate/nitrite (NO x ) in patients with knee OA. METHODS: The recruited subjects comprised 36 post-menopausal women with knee OA, ages 50-60 years, as well as 10 healthy males, 20-30 years of age. 25(OH)D and NO x levels were determined using high-performance liquid chromatography and spectrophotometrically using Griess reaction, respectively. RESULTS: The mean (SEM) 25(OH)D and NO x concentrations of OA patients were 25.0 (1.6) ng/mL and 32.45 (2.18) µM, respectively, and 35.4 (2.1) ng/mL and 25.49 (2.23) µM, respectively, for controls. Comparison of mean 25(OH)D and NO x concentrations of OA patients and controls yielded significant results (P = 0.001 and 0.034, respectively). NO notably decreased with decreasing 25(OH)D concentration in patients. However, significant results in terms of mean NO x concentration were observed in the comparison of normal and deficient vitD OA groups (P = 0.048). CONCLUSION: Results suggest that vitD increases NO production and inducible NO synthase expression in osteoarthritic chondrocytes possibly leading to a protective effect.

Insights on vitamin D's role in cardiovascular disease: investigating the association of 25-hydroxyvitamin D with the dimethylated arginines.

Accumulating evidence has stipulated a strong correlation between vitamin D (vitD) deficiency and cardiovascular disease (CVD); however, a mechanistic link is missing. This study investigated the association of vitD with endothelial dysfunction parameters. Subjects comprised male patients with verified coronary artery disease (CAD) (n=69) and age- and sex-matched controls (n=20). 25-Hydroxyvitamin D [25(OH)D] was determined using high performance liquid chromatography with ultraviolet detection whereas asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) were determined by liquid chromatography-mass spectrometry. Nitric oxide (NO) was determined spectrophotometrically and high-sensitivity C-reactive protein (hs-CRP) was determined using enzyme-linked immunosorbent assay (ELISA). Comparison of mean 25(OH)D concentrations of patients and controls yielded a significant result (p=0.0002). 25(OH)D2 was dominant in patients whereas 25(OH)D3 was dominant in controls (p=0.003 and 0.001, respectively). Comparison of mean ADMA and SDMA concentrations of patients exhibiting normal and suboptimal vitD yielded insignificant results (p=0.692 and 0.998, respectively). Significant results were obtained from the comparison of mean hs-CRP and NO concentrations of patients exhibiting normal and suboptimal vitD (p=0.035 and 0.031, respectively). Results suggest involvement of vitD with the NO system, however not via modulation of the dimethylated arginines. A potential anti-inflammatory activity for vitD is also raised.

Predictability of enantiomeric chromatographic behavior on various chiral stationary phases using typical reversed phase modeling software.

Pharmaceutical companies worldwide tend to apply chiral chromatographic separation techniques in their mass production strategy rather than asymmetric synthesis. The present work aims to investigate the predictability of chromatographic behavior of enantiomers using DryLab HPLC method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. Three different types of chiral stationary phases were tested for predictability: macrocyclic antibiotics-based columns (Chirobiotic V and T), polysaccharide-based chiral column (Chiralpak AD-RH), and protein-based chiral column (Ultron ES-OVM). Preliminary basic runs were implemented, then exported to DryLab after peak tracking was accomplished. Prediction of the effect of % organic mobile phase on separation was possible for separations on Chirobiotic V for several probes: racemic propranolol with 97.80% accuracy; mixture of racemates of propranolol and terbutaline sulphate, as well as, racemates of propranolol and salbutamol sulphate with average 90.46% accuracy for the effect of percent organic mobile phase and average 98.39% for the effect of pH; and racemic warfarin with 93.45% accuracy for the effect of percent organic mobile phase and average 99.64% for the effect of pH. It can be concluded that Chirobiotic V reversed phase retention mechanism follows the solvophobic theory.

Determination of the design space of the HPLC analysis of water-soluble vitamins.

Analysis of water-soluble vitamins has been tremendously approached through the last decades. A multitude of HPLC methods have been reported with a variety of advantages/shortcomings, yet, the design space of HPLC analysis of these vitamins was not defined in any of these reports. As per the food and drug administration (FDA), implementing the quality by design approach for the analysis of commercially available mixtures is hypothesized to enhance the pharmaceutical industry via facilitating the process of analytical method development and approval. This work illustrates a multifactorial optimization of three measured plus seven calculated influential HPLC parameters on the analysis of a mixture containing seven common water-soluble vitamins (B1, B2, B6, B12, C, PABA, and PP). These three measured parameters are gradient time, temperature, and ternary eluent composition (B1/B2) and the seven calculated parameters are flow rate, column length, column internal diameter, dwell volume, extracolumn volume, %B (start), and %B (end). The design is based on 12 experiments in which, examining of the multifactorial effects of these 3 + 7 parameters on the critical resolution and selectivity, was carried out by systematical variation of all these parameters simultaneously. The 12 basic runs were based on two different gradient time each at two different temperatures, repeated at three different ternary eluent compositions (methanol or acetonitrile or a mixture of both). Multidimensional robust regions of high critical R(s) were defined and graphically verified. The optimum method was selected based on the best resolution separation in the shortest run time for a synthetic mixture, followed by application on two pharmaceutical preparations available in the market. The predicted retention times of all peaks were found to be in good match with the virtual ones. In conclusion, the presented report offers an accurate determination of the design space for critical resolution in the analysis of water-soluble vitamins by HPLC, which would help the regulatory authorities to judge the validity of presented analytical methods for approval.

Association of suboptimal 25-hydroxyvitamin D levels with knee osteoarthritis incidence in post-menopausal Egyptian women.

The main aim of this study was to assess the vitamin D status of newly diagnosed knee osteoarthritis (OA) patients. Thirty-six post-menopausal Egyptian females of mean age 54.7 years with knee OA were recruited alongside ten healthy males of mean age 25.8 years. The body mass index of all knee OA patients was calculated, and full patient history was gathered to screen for vitamin D status altering conditions or medication. Total 25-hydroxyvitamin D [25(OH)D] was assessed using HPLC which permitted an individualized assessment of both forms of the vitamin's metabolite, 25(OH)D2 and 25(OH)D3. Results showed that mean 25(OH)D ± SEM concentrations were 25.0 ± 1.6 ng/mL and 35.4 ± 2.1 ng/mL for female patients and healthy male participants, respectively. Student's t test statistical comparison yielded a significant result (P = 0.001) when comparing healthy and osteoarthritic participants, and insignificant results when comparing patients of different BMI class, and the different forms of the vitamin's metabolite (P = 0.184 and 0.335, respectively). The 95 % confidence interval associated with knee OA incidence is 21.9-28.1 ng/mL, which is in the vitamin D insufficiency zone. In Conclusion, suboptimal 25(OH)D levels are associated with knee OA incidence in post-menopausal Egyptian females which further fortifies accumulating evidence.