Spectrophotometric Determination of Polyvinyl Pyrrolidone in Pure and Pharmaceutical Dosage Form.

BACKGROUND: Recently, functional polymers have attracted significant attention in the areas of pharmaceuticals and biomedical applications, so it is important to develop simple techniques to analyze functional polymers in their pharmaceutical dosage forms. OBJECTIVE: Three simple, accurate, and sensitive UV spectrophotometric methods have been developed and validated for determination of polyvinyl pyrrolidone (PVP) in the presence of benzalkonium chloride (BZ) and sodium lactate in ternary mixtures. METHOD: Method A is a derivative ratio spectra zero-crossing (DRSZ) method which measures PVP peak amplitude at 303.1 nm. Method B is a double divisor ratio derivative (DDRD), used for determination of both PVP and BZ in the presence of sodium lactate at 272.6 and 271.5 nm, respectively. Method C is a double divisor ratio derivative-ratio difference spectrophotometric method (DDRD-RDSM), a new and hybrid method of double divisor and ratio difference that hasn't been applied before. It measures peak amplitude difference of the ratio spectra at ΔP278 - 252.4 and ΔP260.9 - 213 for PVP and BZ, respectively. RESULTS: Linear ranges for PVP (5.00-35.00, 10.00-40.00, and 10.00-40.00 µg/mL) was obtained by using DRSZ, DDRD, and DDRD-RDSM, respectively. While the linear range for BZ (5.00-60 µg/mL) was obtained by using both DDRD and DDRD-RDSM. CONCLUSIONS: All results were statistically compared with reported methods. No significant differences were observed. The developed methods were applied to the analysis of the investigated drugs in pure and pharmaceutical dosage forms. HIGHLIGHTS: The proposed methods are of great value, improving the efficiency of routine analysis of PVP and BZ in their pharmaceutical dosage forms.

Validated RP-HPLC Method for Simultaneous Determination of Ribavirin, Sofosbuvir and Daclatasvir in Human Plasma: A Treatment Protocol Administered to HCV Patients in Egypt.

Egypt has the highest prevalence of hepatitis C virus (HCV) in the world thus it launched a national program for eliminating HCV aiming to treat 300,000 HCV patients per year. Three anti-HCV co-administered drugs; ribavirin (RBV), sofosbuvir (SF) daclatasvir (DAC) were simultaneously determined in human plasma by a validated, simple and sensitive RP-HPLC method using propyl paraben as an internal standard. Liquid-liquid extraction using ethyl acetate was used for samples extraction. Chromatographic separation was achieved on Scharlau® C18 column (250 × 4.6 mm2, 5 µm). Gradient elution was employed with a mobile phase mixture of water and acetonitrile at a flow rate 1 mL/min. UV detection using photodiode array detector was carried out at 207, 260 and 312 nm for RBV, SF and DAC, respectively. Method validation was performed according to the FDA guidelines for bioanalytical method validation. The calibration curves were linear over the ranges (0.5-80, 0.1-40 and 0.5-80 µg/mL) with average recoveries (100.64-108.28%, 98.48-105.91% and 97.68-101.38%) for RBV, SF and DAC, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Stability assays revealed that the three studied analytes were stable during sample storage, preparation and injection. The method can be successfully applied in routine analysis of plasma of HCV patients treated with this combination therapy which aids in therapeutic drug monitoring and patients' follow-up especially in Egypt and other developing countries fighting HCV.

Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry.

Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3'- and 5'-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences. Graphical abstract Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS.

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities.

Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, owing to their complex structure and multistep synthesis and purification processes, generation of low-level impurities and degradation products are common. Therefore, they require significant investment in quality control and impurity identification. This requires the development of advanced methods for analysis, characterization and quantitation. In addition, the presence of the PS linkage leads to the formation of chiral centers which can affect their biological properties and therapeutic efficiency. In this review, the different types of oligonucleotide impurities and degradation products, with an emphasis on their origin, mechanism of formation and methods to reduce, prevent or even eliminate their production, will be extensively discussed. This review will focus mainly on the application of chromatographic techniques to determine these impurities but will also discuss other approaches such as mass spectrometry, capillary electrophoresis and nuclear magnetic resonance spectroscopy. Finally, the chirality and formation of diastereomer mixtures of PS oligonucleotides will be covered as well as approaches used for their characterization and the application for the development of stereochemically-controlled PS oligonucleotides.

Precise simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples following their successive i.p. administration.

A sensitive high-performance liquid chromatography (HPLC) assay with dual UV detection has been developed and validated for the simultaneous quantification of methadone and cocaine in rat serum and brain tissue samples. Liquid-liquid extraction using hexanes was applied for samples extraction with Levo-Tetrahydropalmatine (L-THP) as the internal standard. Chromatographic separation of the analytes was achieved on a reversed-phase Waters Symmetry® C18 column (150mm×4.6mm, 5µm). A gradient elution was employed with a mobile phase consisting of 5mM potassium phosphate containing 0.1% triethylamine (pH=6.5) (A) and acetonitrile (B) with a flow rate of 1mL/min. UV detection was employed at 215nm and 235nm for the determination of methadone and cocaine, respectively. The calibration curves were linear over the range of 0.05-10µg/mL for both methadone and cocaine. The assay was validated according to FDA guidelines for bioanalytical method validation and results were satisfactory and met FDA criteria. Inter-day accuracy values of serum and brain samples ranged from 96.97 to 105.59% while intra-day accuracy values ranged from 91.49 to 111.92%. Stability assays showed that both methadone and cocaine were stable during sample storage, preparation, and analytical procedures. The method was successfully used to analyze biological samples obtained from a drug- drug interaction pharmacokinetics (PK) study conducted in rats to investigate the effect of methadone on cocaine PK. Our method not only can be used for bioanalysis of samples obtained from rats but also can potentially be applied to human biological serum samples to monitor compliance to methadone maintenance therapy (MMT) and to detect possible cocaine-methadone co-abuse.

Ovarian leiomyoma with myxoid stroma.

Ovarian smooth muscle tumours are rare. Notable myxoid change in smooth muscle tumours is uncommon, and raises diagnostic issues that need to be considered on evaluating a spindle cell lesion with notable myxoid change. There is only one case of myxoid leiomyoma of the ovary previously reported. We here report a case of ovarian leiomyoma with areas of myxoid stroma and discuss the relevant differential diagnosis and histological features to be assessed in such a lesion.

Spectrophotometric and chemometric methods for determination of imipenem, ciprofloxacin hydrochloride, dexamethasone sodium phosphate, paracetamol and cilastatin sodium in human urine.

New accurate, sensitive and selective spectrophotometric and chemometric methods were developed and subsequently validated for determination of Imipenem (IMP), ciprofloxacin hydrochloride (CIPRO), dexamethasone sodium phosphate (DEX), paracetamol (PAR) and cilastatin sodium (CIL) in human urine. These methods include a new derivative ratio method, namely extended derivative ratio (EDR), principal component regression (PCR) and partial least-squares (PLS) methods. A novel EDR method was developed for the determination of these drugs, where each component in the mixture was determined by using a mixture of the other four components as divisor. Peak amplitudes were recorded at 293.0 nm, 284.0 nm, 276.0 nm, 257.0 nm and 221.0 nm within linear concentration ranges 3.00-45.00, 1.00-15.00, 4.00-40.00, 1.50-25.00 and 4.00-50.00 µg mL(-1) for IMP, CIPRO, DEX, PAR and CIL, respectively. PCR and PLS-2 models were established for simultaneous determination of the studied drugs in the range of 3.00-15.00, 1.00-13.00, 4.00-12.00, 1.50-9.50, and 4.00-12.00 µg mL(-1) for IMP, CIPRO, DEX, PAR and CIL, respectively, by using eighteen mixtures as calibration set and seven mixtures as validation set. The suggested methods were validated according to the International Conference of Harmonization (ICH) guidelines and the results revealed that they were accurate, precise and reproducible. The obtained results were statistically compared with those of the published methods and there was no significant difference.

Sensitive spectrofluorimetric methods for determination of ethopabate and amprolium hydrochloride in chicken plasma and their residues in food samples.

Two sensitive and selective spectrofluorimetric methods are proposed to determine ethopabate (ETH) and amprolium hydrochloride (AMP). First derivative synchronous spectrofluorimetry determines the natively fluorescent ethopabate at 288 nm in presence of amprolium hydrochloride which is a non fluorescent quaternary compound with average recovery 100.54±0.721 over a concentration range of 0.01-0.8 µg/mL. Limits of detection (LOD) and quantification (LOQ) are 0.002 and 0.007 µg/mL, respectively. The second method is direct synchronous spectrofluorimetry for determining amprolium hydrochloride at 362 nm after a reaction with 5% NaOH and 0.08% potassium ferricyanide that is optimized by a two-level factorial design. This method is linear over a concentration range of 0.01-0.65 µg/mL with average recovery 99.4±1.28. Limits of detection (LOD) and quantification (LOQ) are 0.002 and 0.006 µg/mL, respectively. The proposed methods are found to be valid and applicable for the analysis of ETH and AMP in their veterinary formulation. They are successfully applied to determine the studied drugs in chicken plasma and their residues in chicken muscle, liver, egg and chicken-based baby food product with recoveries in the ranges of 95.71-108.73% and 97.36-111.89% and for ETH and AMP, respectively.

Application of new spectrofluorometric techniques for determination of atorvastatin and ezetimibe in combined tablet dosage form.

Two accurate, reliable, and highly sensitive spectrofluorometric methods were developed for simultaneous determination of the binary mixture of Atorvastatin and Ezetimibe without prior separation steps. The first method is based on double scan synchronous fluorescence spectrometry. Each of Atorvastatin and Ezetimibe can be determined independent of the other when scanned at Δλ=100 nm and 40 nm, respectively. The relative fluorescence intensity-concentration plots at two wavelengths, 272 (Δλ=100 nm) and 266 nm (Δλ=40 nm) were rectilinear over the range of 0.4-8 µg/mL (for Atorvastatin) and 0.6-8 µg/mL (for Ezetimibe), respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which two equations are solved simultaneously after using a single excitation wavelength of 273 nm and λEm1=380 nm of Atorvastatin and λEm2=301 nm of Ezetimibe. Under the optimum conditions, linear relationships were found between the relative fluorescence intensity and the concentrations of the investigated drugs in the range of 0.4-8 µg/mL (for Atorvastatin) 0.6-8 µg/mL (for Ezetimibe). The different experimental parameters affecting the fluorescence intensities of the two drugs were carefully studied and optimized. The proposed methods were successfully applied for the determination of the investigated drugs in pure form, dosage form and in synthetic mixtures with good recovery and the results obtained were favorably compared to those obtained with a reference method.

Screening and Optimization of the Reaction of Polymyxin B Sulphate with NBD-Cl for the Synchronous Spectrofluorimetric Determination of Polymyxin B Sulphate in Human Plasma.

An accurate and sensitive synchronous spectrofluorimetric method has been developed for the determination of Polymyxin B sulphate (Poly B) in human plasma. The method is based on the reaction of non-fluorescent Poly B with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 7 producing a yellow color with maximum relative fluorescence at 440 nm using a constant wavelength difference Δλ = 80 nm. Reaction conditions and other analytical parameters were studied and optimized using factorial design. Three level factorial designs have been employed for the screening, optimization of all experimental variables and determination of their interactions on the final product formation. The variables under investigation were: pH of borate buffer, volume of buffer, volume of NBD-Cl, temperature, time of heating and volume of sulfuric acid. A linear plot between relative fluorescence and concentration was obtained over the concentration range 100.00-1200.00 ng mL(-1). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 10.31 and 31.24 ng mL(-1), respectively. The proposed method was validated according to ICH guidelines and successfully applied for the determination of Poly B in human plasma, where satisfactory results were obtained. The results obtained were statistically compared with those of a published method, where no significant difference was observed.

Five different spectrophotometric methods for determination of Amprolium hydrochloride and Ethopabate binary mixture.

Five simple, specific, accurate and precise UV-spectrophotometric methods are adopted for the simultaneous determination of Amprolium hydrochloride (AMP) and Ethopabate (ETH), a binary mixture with overlapping spectra, without preliminary separation. The first method is first derivative of the ratio spectra ((1)DD) for determination of AMP and ETH at 234.7nm and 306.8nm respectively with mean percentage recoveries 99.76±0.907 and 100.29±0.842 respectively. The second method is the mean centering of the ratio spectra for determination of AMP and ETH at 238.8nm and 313nm respectively with mean percentage recoveries 100.26±1.018 and 99.94±1.286 respectively. The third method is based on dual wavelength selection for determination of AMP and ETH at 235.3nm & 308nm and 244nm & 268.4nm respectively with mean percentage recoveries 99.30±1.097 and 100.03±1.065 respectively. The fourth method is ratio difference method for determination of AMP and ETH at 239nm & 310nm and 239nm & 313nm respectively with mean percentage recoveries 99.27±0.892 and 100.40±1.814 respectively. The fifth one is area under the curve (AUC) method where the areas between 235.6-243nm and 268.3-275nm are selected for determination of AMP and ETH with mean percentage recoveries 100.35±1.031 and 100.39±0.956 respectively. These methods are tested by analyzing synthetic mixtures of the two drugs and they are applied to their pharmaceutical veterinary preparation. Methods are validated according to the ICH guidelines and accuracy, precision and repeatability are found to be within the acceptable limit.

Novel spectroscopic methods for determination of Cromolyn sodium and Oxymetazoline hydrochloride in binary mixture.

New accurate, sensitive and selective spectrophotometric and spectrofluorimetric methods were developed and subsequently validated for determination of Cromolyn sodium (CS) and Oxymetazoline HCl (OXY) in binary mixture. These methods include 'H-point standard addition method (HPSAM) and area under the curve (AUC)' spectrophotometric method and first derivative synchronous fluorescence spectroscopic (FDSFS) method. For spectrophotometric methods, absorbances were recorded at 241.5nm and 274.9nm for HPSAM and the wavelength was selected in ranges 232.0-254.0nm and 216.0-229.0nm for AUC method, where the concentration was obtained by applying Cramer's rule. For FDSFS method, the first-derivative synchronous fluorescence signal was measured at 290.0nm, using Δλ=145.0nm. The suggested methods were validated according to International Conference of Harmonization (ICH) guidelines and the results revealed that they were precise and reproducible. All the obtained results were statistically compared with those of the reported method and there was no significant difference.

Primary myxoid chondrosarcoma of the breast.

Primary breast chondrosarcoma has been rarely reported in the literature. Conservative breast surgery has never been part of the management of previously reported cases. Surgery remains the mainstay management of such a disease as it is resistant to chemotherapy and radiotherapy. In this report, we present a case of rare primary myxoid chondrosarcoma of the breast that was managed successfully with a conservative approach.

Assessment of the pharmacological properties of 5-methoxyindole derivatives at 5-HT4 receptors.

OBJECTIVES: The aim was to examine the biological activity of 5-methoxytryptamine derivatives at the 5-hydroxytryptamine (5-HT)(4) receptor to explore the effect of substitution on the aliphatic amine of the 5-methoxyamine scaffold. METHODS: Three compounds were tested for affinity at the 5-HT(4) receptor by radioligand binding and functional activity using guinea-pig ileum and human colon circular muscle preparations and also in the mouse whole gut transit test. KEY FINDINGS: The three compounds all had agonist properties at the 5-HT(4) receptor but their efficacy differed in the different functional tests. Compound 3 had the highest affinity for the 5-HT(4) receptor and was a full agonist at relaxing human colon circular muscle with efficacy closest to 5-HT. Compounds 1 and 2 were partial agonists in this assay with lower efficacies; compound 2 was a full agonist in the guinea-pig ileum assay whereas compound 3 was a partial agonist. Compounds 1 and 2 also showed activity in the mouse gut transit assay while compound 3 had no activity. CONCLUSIONS: Of the compounds tested, compound 3 was the most promising 5-HT(4) receptor agonist and the results highlight the value of using human tissue in functional tests when assessing compounds for potential activity.

Synthesis, testing and structure-activity studies on a library of 5-HT4 ligands.

Several indole derivatives and analogues comprising a range of related structural classes were designed, synthesized and tested as ligands for the 5-HT4 receptor. Within each series, binding experiments showed compounds with good affinity demonstrating high percentage displacement values at 1 µM. The most potent of these (20) had a pKi of 8.54 demonstrating very good affinity. These indole analogues were combined with 55 ligands that were previously produced in our laboratory to explore the structure-activity relationships of these 5-HT4 ligands. A CoMFA (Comparative Molecular Field Analysis) analysis was used to extend an earlier simple pharmacophore to suggest two new molecular features beyond the primary amino binding site. The pharmacophore confirmed that a newly described tetrahydroquinoline analogue was able to match the basic requirements of the model and the pharmacology of this molecule is provided in more detail.

Differential entrainment distinguishes atrioventricular nodal reentry tachycardia from atrioventricular reentrant tachycardia.

BACKGROUND: Entrainment from the right ventricular (RV) apex and the base has been used to distinguish atrioventricular reentrant tachycardia (AVRT) from atrioventricular nodal reentry tachycardia (AVNRT). The difference in the entrainment response from the RV apex in comparison with the RV base has not been tested. METHODS: Fifty-nine consecutive patients referred for ablation of supraventricular tachycardia (SVT) were included. Entrainment of SVT was performed from the RV apex and base, pacing at 10-40-ms faster than the tachycardia cycle length. SA interval was calculated from stimulus to earliest atrial electrogram. Ventricle to atrium (VA) interval was measured from the RV electrogram (apex and base) to the earliest atrial electrogram during tachycardia. The SA-VA interval from apex and base was measured and the difference between them was calculated. RESULTS: Thirty-six AVNRT and 23 AVRT patients were enrolled. Mean age was 44 ± 12 years; 52% were male. The [SA-VA]apex-[SA-VA]base was demonstrable in 84.7% of patients and measured -9.4 ± 6.6 in AVNRT and 10 ± 11.3 in AVRT, P < 0.001. The difference was negative for all AVNRT cases and positive for all septal accessory pathways (APs). CONCLUSION: The difference between entrainment from the apex and base is readily performed and is diagnostic for all AVNRTs and septal APs.

Stability studies of oxazolidine-based compounds using 1H NMR spectroscopy.

A series of oxazolidine-based compounds with a variety of substituents in positions 2 and 3 was synthesized and their stability studied. Ring opened intermediates formed on addition of limiting amounts of D(2)O to oxazolidine solutions, as observed by NMR. As the hydrolysis reactions proceeded, a series of novel dimeric beta-amino alcohol compounds formed via an internal reaction between ephedrine and the ring opened intermediates. 2-Phenyl substituted oxazolidine compounds containing electron withdrawing nitro substituents were more rapidly hydrolyzed than the unsubstituted derivative and methoxy substituted compounds, with the nitro substituents appearing to stabilize the ring opened intermediates. Two oxazolidine derivatives, with a methyl and proton at position 2, were found to be more stable to oxazolidine hydrolysis than the 2-phenyl substituted compounds. Oxazolidines incorporating phenyl substituents at position 3 were synthesized and found to be less stable than those incorporating a methyl substituent at position 3. These fundamental structure-activity relationships may be useful when choosing oxazolidine derivatives as synthetic intermediates and as prodrugs for the delivery of compounds containing either beta-amino alcohol or aldehyde components.

A technique for the rapid diagnosis of wide complex tachycardia with 1:1 AV relationship in the electrophysiology laboratory.

BACKGROUND: The differential diagnosis of wide complex tachycardia (WCT) with 1:1 atrioventricular (AV) relationship is broad. Accurate identification of the tachycardia mechanism is essential for successful ablation. We suggest a simple pacing maneuver that can immediately clarify the tachycardia mechanism in the electrophysiology laboratory. METHODS: Eight consecutive patients (four males, 32 +/- 14 years) demonstrating stable sustained WCT with persistent 1:1 AV relationship during electrophysiologic testing were included in this study. During the tachycardia, atrial overdrive pacing was performed. The following responses were observed: (1) a change of the QRS morphology during atrial pacing and (2) the first return electrogram of the tachycardia, whether occurring in the atrium (AVA response) or in the ventricle (AVVA response). RESULTS: Atrial overdrive pacing was successfully performed in all patients. It was associated with either a change or narrowing of the QRS in all ventricular tachycardia (VT) patients but not in supraventricular tachycardia (SVT) patients. All VT patients had an AVVA response upon cessation of atrial overdrive pacing as opposed to AVA response in SVT patients, P = 0.029. CONCLUSION: The response to atrial overdrive pacing during WCT with 1:1 AV relationship can rapidly diagnose or rule out VT as a mechanism of tachycardia.

Synthesis and preliminary screening of novel indole-3-methanamines as 5-HT4 receptor ligands.

Twenty-three indole-3-methanamines were designed, synthesized and evaluated as ligands for the 5-HT(4) receptor. Compounds I-d, I-j, I-o, I-q and I-u showed good affinity at 100 microM and I-o was found to be only 5-fold less potent than the agonists serotonin (1) and 5-methoxytryptamine (2). Substitution on the 3-methanamine nitrogen clearly influenced activity with docking experiments into a homology model of the 5-HT(4) receptor showing a range of interactions with these side chain substituents. This modelling work together with the SAR determined in this study has provided promising ideas for future synthetic work.