Marwa Fathy Bakr Ali

A novel lophine-based fluorescence probe and its binding to human serum albumin

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510nm. The binding constant and binding number determined by Scatchard plot was 3.65×10(6)M(-1) and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.

Ultrasound assisted dispersive liquid-liquid microextraction coupled with high performance liquid chromatography designated for bioavailability studies of felodipine combinations in rat plasma

Felodipine (FLD), a calcium channel antagonist, is commonly prescribed for the treatment of hypertension either with Metoprolol (MET) or Ramipril (RAM) in two different drug combinations. FLD has high plasma protein binding ability affecting its extraction recoveries from plasma samples. Hence, a specific ultrasound assisted dispersive liquid-liquid microextraction (UA-DLLME) method coupled with HPLC using photodiode array detector was developed and validated for the simultaneous determination of FLD, MET and RAM in rat plasma after oral administration of these combinations. The factors affecting UA-DLLME were carefully optimized. In this study, UA-DLLME method could provide simple and efficient plasma extraction procedures with superior recovery results. Under optimum condition, all target drugs were separated within 13min. The validation procedures was carried out in agreement with US-FDA guidelines and shown to be suitable for anticipated purposes. Linear calibration ranges were obtained in the range 0.05-2.0μgmL-1 for FLD and MET and 0.1-2.0μgmL-1 for RAM with detection limits of 0.013-0.031μgmL-1 for all the studied drug combinations. The%RSD for inter-day and intra-day precisions was in range of 0.63-3.85% and the accuracy results were in the range of 92.13-100.5%. The validated UA-DLLME-HPLC method was successfully applied for the bioavailability studies of FLD, MET and RAM. The pharmacokinetic parameters were calculated for all the investigated drugs in rats after single-dose administrations of two different drug combinations. Although FLD was bioequivalent in the two formulations, a small increase in plasma levels of MET and RAM was found in the presence of FLD.

Determination of 4-hydroxy-2-nonenal in serum by high-performance liquid chromatography with fluorescence detection after pre-column derivatization using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole.

4-Hydroxy-2-nonenal (4HNE) is a major aldehyde generated during lipid peroxidation. The clinical monitoring of 4HNE in biological fluids should be useful for the early diagnosis of several diseases involving lipid peroxidation, such as rheumatoid arthritis, Parkinsons disease and cancer. In this study, an HPLC with fluorescence detection method was developed for the determination of 4HNE in human serum. The proposed method involves the extraction of 4HNE from human serum by sub-zero temperature extraction and fluorescent labeling of 4HNE with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2, 1,3-benzoxadiazole. The lower detection limit (signal-to-noise ratio = 3) of the method was 0.06 μm in serum. The proposed method was successfully applied to the measurement of 4HNE in sera obtained from patients with rheumatoid arthritis.

Micelle and inclusion complex enhanced spectrofluorimetric methods for determination of Retigabine: Application in pharmaceutical and biological analysis

Two new, simple, selective, and highly sensitive spectrofluorimetric methods were developed and validated for the determination of the antiepileptic drug; retigabine (RTG). The first method (Method-I) depends on enhancement of the weak native fluorescence of RTG via the use of an organized medium; sodium dodecyl sulphate (SDS) in acetate buffer (pH 3.74). The second method (Method-II) depends on the enhancement of RTG weak native fluorescence through complexation with a macromolecule; beta cyclodextrin (β-CD) in phosphate buffer (pH 3.20). A full study of different experimental parameters influencing the fluorescence intensity was carried out. In addition, a thorough investigation of the fluorescence quantum yield, fluorophore brightness and mechanism of fluorescence enhancement was performed. A seven-fold improvement in the fluorescence intensity was brought by the first method, whereas a six and half-fold enhancement of the fluorescence intensity was obtained by the second one. Linearity was achieved over wide ranges (0.05-12.5 μg mL-1) and (0.05-15 μg mL-1) with low limits of detection (LOD) of 10.6 and 14.3 ng mL-1, and limits of quantification (LOQ) of 32.0 and 43.2 ng mL-1 for (Method-I) and (Method-II), respectively. The proposed methods were validated according to ICH and US-FDA guidelines. The applicability of the proposed methods was tested for determination of RTG in its pharmaceutical dosage forms, and to study the stability of RTG under different stress conditions according to ICH guidelines including alkaline, acidic, oxidative, thermal, and photolytic stress conditions. Moreover, the high sensitivity achieved by the proposed methods permitted the determination and detection of RTG in both spiked and real rabbit plasma samples utilizing a simple protein precipitation step followed by liquid-liquid extraction method. Percentage recoveries from rabbit plasma samples were within the acceptable limits; (93.47-104.74%) and (91.33-105.70%) for (Method-I) and (Method-II), respectively.

3-Amino-5-pyridin-3-yl-1,2,4-triazole, a novel fluorescence probe for trace analysis of silymarin in bulk material, pharmaceutical dosage forms and human plasma: Further insights on reaction mechanism using computational molecular modeling and NMR spectroscopy

A rapid, highly sensitive and roubst spectrofluorimetric method was developed for trace analysis of silymarin (SLM) in active pharmaceutical ingredient (API), pharmaceutical preparations and human plasma. The proposed method is based on reaction of SLM with a novel reagent; 3-amino-5-pyridin-3-yl-1,2,4-triazole (3-APT); in the presence of 0.04 M sodium hydroxide. The formed fluorescent product was formed within 5 min and was measured at 504 nm after excitation at 390 nm. All reaction parameters were optimized and the proposed method was validated according to ICH guidelines. The developed method was linearly correlated at the concentration range of 0.05-8 μg mL-1 with good correlation coefficient 0.9993, limit of detection 10.79 ng mL-1 and limit of quantitation 32.71 ng mL-1. The relative standard deviations %RSD values were 1.59-2.69% and 1.47-2.62% in case of intra- and inter-day precision, respectively. Computational molecular modeling and NMR spectroscopy were used to identify the reaction mechanism between SLM and 3-APT. The proposed method was employed for determination of SLM in API or bulk material, pharmaceutical capsules and sachets. Further, the method was sensitive enough to be applied for analysis of the free (unconjugated) SLM flavonolignans in human plasma samples.

Ultrasonographic Reference Values of Kidney Dimensions and Clinicopathological Findings Associating the Transcutaneous Ultrasound-Guided Renal Biopsy in Donkeys (Equus asinus)

Abstract This study aimed to establish normal ultrasonographic reference values of kidney dimensions in donkeys (Equus asinus) and to describe and evaluate the clinicopathological variations associated with ultrasound‐guided renal biopsy. The ultrasonographic dimensions of the right and left kidneys were conducted on 16 donkeys, which were then divided into two groups; eight each for biopsy of the right kidney (RK) and left kidney (LK). Three ultrasonographic cineloops were obtained at 17th intercostal space daily for 3 consecutive days. Renal length, width, and dimensions of the cortex, medulla, and pelvis for both the kidneys in each donkey were recorded. Maximal dimensions were obtained for the RK (length 10 ± 8 cm, width 4.9 ± 1 cm, thickness 4.2 ± 0.4 cm) and LK (length 8.9 ± 0.9 cm, width 4.7 ± 0.8 cm, thickness 3.5 ± 0.7 cm) with good‐to‐excellent repeatability for all measurements. Follow‐up ultrasonography revealed development of postbiopsy subcapsular hematomas, which were confirmed postmortem, of mild (volume < 20 mL), moderate (volume from 20 to 40 mL), and severe degrees (volume > 40 mL). Gross hematuria had been observed till 24 hours after biopsy, and then microscopic hematuria was noticed thereafter. Variable clinicopathological changes were noticed in blood and urine. All the biopsy specimens were adequate for histopathological assessment. Postmortem histopathological examination revealed various kidney changes. In conclusion, kidney dimensions can be used by veterinarians for accurate diagnosis and management of renal diseases. Ultrasound‐guided renal biopsy is a relatively safe procedure; however, some complications may develop. Renal biopsy is commonly associated with clinicopathological variations; thus, caution should be taken during interpretation of these variables. HighlightsEstablishment of normal reference ranges of kidneys may be helpful in the diagnosis and prognosis of renal diseases.Ultrasound‐guided renal biopsy is a relatively safe procedure; however, some complications may develop.Histopathological information obtained from renal biopsy specimens has a clinical relevance in confirmation of some obscure renal problems.Gross or microscopic hematuria and subcapsular renal hematoma may occur after kidney biopsy, but they are generally self‐limiting.Renal biopsy is commonly associated with clinicopathological variations; thus, caution should be taken during interpretation of these variables.

Development and Complications of Blind and Ultrasound-Guided Percutaneous Liver Biopsy Techniques in Donkeys (Equus asinus)

Abstract The present study was designed to develop blind (BL) and ultrasound‐guided (UG) liver biopsy (LB) techniques in donkeys (Equus asinus) and assess the complication rates of both techniques. Forty donkeys were included in this study. Based on the LB technique and biopsy site, the animals were allocated into four groups as BL biopsy at the 13th intercostal space (ICS) (BL13, n = 10), BL14 (n = 10), UG13 (n = 10), and UG14 (n = 10). All animals underwent liver ultrasonography prior to the biopsy procedure and then kept for 2‐day observation period postbiopsy. The ultrasonographic thickness of the liver was higher at the 13th and 14th ICSs than the 12th and 15th ICSs (P < .05). The liver was partially covered by the lung at the most upper part of the 13th ICS. No mortality or major complications were observed. Follow‐up ultrasonography revealed no serious complications, except a case in BL13 group exhibited a subcutaneous hematoma. The mean values of erythrocytic count and packed cell volume were significantly lowered in the BL13 group in comparison with their prebiopsy values (P < .05). All the biopsy specimens (40/40) were adequate for histopathological assessment. Mean specimen length and tissue fragmentation were significantly higher in the BL groups than UG ones (P < .001). In conclusion, percutaneous UG LB is a safe and accurate procedure and can be performed easily in donkeys. Blind LB at the 13th ICS is not recommended; however, it can be done safely at the 14th ICS. HighlightsLiver biopsy (LB) techniques in donkeys can be used for practical and research purposes.Ultrasound‐guided LB is a safe and accurate procedure and can be performed easily in donkeys.Some complications may develop as a result of blind LB.Blind LB at 13th intercostal space may be unsuitable in donkeys.

Sensitive spectrofluorimetric methods for determination of sitagliptin phosphate, dipeptidyl peptidase-4 inhibitor, in pharmaceutical tablets and spiked human urine

Background: Sitagliptin (SITA), is an oral anti-diabetic drug of dipeptidyl peptidase-4 (DPP-4) inhibitor class used as a monotherapy or in association with metformin to treat type II Diabetes Mellitus. Methods: Two new, sensitive and precise spectrofluorimetric methods have been developed for the determination of SITA in tablets and spiked human urine samples. SITA has a native fluorescence that was enhanced by 2 folds after addition of β-cyclodextrin (method A). The fluoregenic product was measured at 296 nm after excitation at 267 nm. In addition, SITA was derivatized by 7-chloro-4nitrobenzofurazon (NBD-Cl) to a highly sensitive fluoregenic product was measured at 532 nm after excitation at 466 nm (method B). Results: The fluorescence intensities were directly proportional to the concentration over the range 0.35-10 μg mL-1 and 0.1-3 μg mL-1 for method A and B, respectively. The %RSD for inter-day and intra-day precisions was in a range of 0.160-0.915% and the accuracy results were in the range of 96.28% -100.03%. Conclusion: Successful applications of the developed methods, for drug determination in pharmaceutical tablet and spiked human urine samples, were performed with high accuracy and precision.

Characterization and Comparison of Methacrylic Acid with 2-Acrylamido-2-methyl-1-propanesulfonic Acid in the Preparation of Monolithic Column for Capillary Electrochromatography

Butyl methacrylate (BMA)-ethylene dimethacrylate (EDMA)-methacrylic acid (MAA) and BMA-EDMA-2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monolithic columns were prepared by varying the percentage of ionic monomers for capillary electrochromatography. Monolithic columns with a higher content of ionic monomers provided better column efficiency, and the performance of BMA-EDMA-MAA monoliths was better than BMA-EDMA-AMPS. To characterize and optimize BMA-EDMA-MAA monoliths, the effects of the content of cross-linker and the total monomer in the polymerization mixture on column performance were also studied. Plate heights of 8.2 µm for the unretained solute (thiourea) and 12.6 µm for the retained solute (naphthalene) were achieved with a monolithic column using 2.5% MAA (Column I).