Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Stability‐indicating HPTLC method for quantitative estimation of silybin in bulk drug and pharmaceutical dosage form

In the present study a novel stability‐indicating high‐performance thin‐layer chromatography (HPTLC) method for quantitative determination of silybin in bulk drug and nanoemulsion formulation has been developed and validated on silica using solvent chloroform–acetone–formic acid (9 : 2 : 1 v/v/v) (R_f of silybin 0.46 ± 0.05) in the absorbance mode at 296 nm. The method showed a good linear relationship (r² ± 0.999) in the concentration range 25–1500 ng per spot. It was found to be linear, accurate, precise, specific, robust and stability‐indicating and can be applied for quality control and standardization of several multi‐component hepatoprotective formulations as well as for stability testing of different dosage forms. The method proposed was also used to investigate the kinetics of acidic and alkaline degradation processes by quantification of drug at different temperature to calculate the activation energy and half‐life for silymarin degradation. Copyright © 2009 John Wiley & Sons, Ltd     

Microwave‐assisted extraction with water for fast extraction and simultaneous RP‐HPLC determination of phenolic acids in Radix Salviae Miltiorrhizae

An optimized microwave‐assisted extraction method using water (MAE‐W) as the extractant and an efficient HPLC analysis method were first developed for the fast extraction and simultaneous determination of D (+)‐(3,4‐dihydroxyphenyl) lactic acid (Dla), salvianolic acid B (SaB), and lithospermic acid (La) in Radix Salviae Miltiorrhizae. The key parameters of MAE‐W were optimized. It was found that the degradation of SaB was inhibited when using the optimized MAE‐W and the stable content of Dla, La, and SaB in danshen was obtained. Furthermore, compared to the conventional extraction methods, the proposed MAE‐W is a more rapid method with higher yield and lower solvent consumption with a reproducibility (RSD <6%). In addition, using water as extractant is safe and helpful for environment protection, which could be referred to as green extraction. The separation and quantitative determination of the three compounds was carried out by a developed reverse‐phase high‐performance liquid chromatographic (RP‐HPLC) method with UV detection. Highly efficient separation was obtained using gradient solvent system. The optimized HPLC analysis method was validated to have specificity, linearity, precision, and accuracy. The results indicated that MAE‐W followed by HPLC–UV determination is an appropriate alternative to previously proposed method for quality control of Radix Salviae Miltiorrhizae.     

Decarbonylative Radical Coupling of α‐Aminoacyl Tellurides: Single‐Step Preparation of γ‐Amino and α,β‐Diamino Acids and Rapid Synthesis of Gabapentin and Manzacidin A

A new radical‐based coupling method has been developed for the single‐step generation of various γ‐amino acids and α,β‐diamino acids from α‐aminoacyl tellurides. Upon activation by Et₃B and O₂ at ambient temperature, α‐aminoacyl tellurides were readily converted into α‐amino carbon radicals through facile decarbonylation, which then reacted intermolecularly with acrylates or glyoxylic oxime ethers. This mild and powerful method was effectively incorporated into expeditious synthetic routes to the pharmaceutical agent gabapentin and the natural product (?)‐manzacidin A.     

Evaluation of the pattern and kinetics of degradation of adalimumab using a stability‐indicating orthogonal testing protocol

Forced degradation studies are crucial for the evaluation of the stability and biosimilarity. Here, adalimumab was subjected to oxidation, pH, temperature, agitation and repeated freeze–thaw in order to generate all possible degradation products. An orthogonal stability‐indicating testing protocol comprising SE‐HPLC, RP‐HPLC, TapeStation gel electrophoresis, dynamic light scattering (DLS), and functional receptor binding assay was developed and validated. The assay protocol was used for the assessment of the pattern and kinetics of aggregation/degradation of adalimumab. SE‐HPLC and DLS were used to show the formation of aggregates/fragments of adalimumab under nondenaturing conditions. TapeStation electrophoresis was performed under denaturing conditions to reveal the nature of aggregates. Results of the receptor binding assay agreed to those of SE‐HPLC and DLS which indicated that it can be used as an activity‐indicating assay for adalimumab. RP‐HPLC demonstrated excellent selectivity for adalimumab in the presence of its oxidized forms. The kinetics of degradation was studied in each case and the results showed that it followed the first‐order reaction kinetics. Correlation between the results supported the quality assessment of the tested product in industrial and clinical settings. This orthogonal protocol is a useful tool in stability assessment of monoclonal antibodies and a key criterion for the biosimilarity assessment.     

Microwave‐assisted extraction followed by RP‐HPLC for the simultaneous extraction and determination of forsythiaside A,rutin, and phillyrin in the fruits of Forsythia suspensa

An optimized microwave‐assisted extraction (MAE) method and RP‐HPLC method were developed for the simultaneous extraction and determination of rutin, forsythiaside A, and phillyrin in the fruits of Forsythia suspensa. The key parameters of the open‐vessel MAE process were optimized. A mixed solvent of methanol and water (70:30, v/v) was most suitable for the simultaneous extraction of the three components. The sample was soaked for 10 min before extraction. The optimized conditions were: microwave power 400 W, temperature 70°C, solvent‐to‐material ratio 30 mL/g, and extraction time 1 min. Compared to conventional extraction methods, the proposed method can simultaneously extract the three components in high yields and was proved to be a more rapid method with a lower solvent consumption. The optimized HPLC–photodiode array detection analysis was validated to have good linearity, precision, accuracy, and sensitivity. The developed MAE followed by RP‐HPLC is a fast and appropriate method for the simultaneous extraction and determination of rutin, forsythiaside A, and phillyrin in the fruits of F. suspensa.     

Synthesis and Characterization of 3,5‐Diamino‐1,2,4‐triazolium Dinitramide

3,5‐Diamino‐1,2,4‐triazole ( 1 , guanozol) was protonated with diluted hydrochloric acid, nitric acid, as well as perchloric acid forming 3,5‐diamino‐1,2,4‐triazolium chloride hemihydrate ( 2 ), 3,5‐diamino‐1,2,4‐triazolium nitrate ( 3 ) and 3,5‐diamino‐1,2,4‐triazolium perchlorate ( 4 ), respectively. In a second step 4 reacted with potassium dinitramide forming 3,5‐diamino‐1,2,4‐triazolium dinitramide ( 5 ) and low soluble potassium perchlorate. Compounds 2 – 5 were characterized by low temperature single X‐ray diffraction, IR and Raman as well as multinuclear NMR spectroscopy, mass spectrometry and differential scanning calorimetry. The heats of formation of 1 – 5 were calculated by the CBS‐4M method to be 81.1 ( 1 ), 124.7 ( 2 ), –76.1 ( 3 ), –25.2 ( 4 ) and 138.7 ( 5 ) kJ·mol^–1. With these values as well as the X‐ray densities several detonation parameters were calculated using both computer codes EXPLO5.03 and EXPLO5.04. In addition, the sensitivities of 1 – 5 were determined by the BAM drophammer and friction tester as well as a small scale electrical discharge device.     

Improved RP‐HPLC method for determination of bovine lactoferrin and its proteolytic degradation in simulated gastrointestinal fluids

The objective of this study was to qualitatively and quantitatively evaluate bovine lactoferrin (bLf) and its stability using a rapid RP‐HPLC method. bLf could be rapidly detected within 20 min and quantitated at levels down to 5 µg/mL, and the equation of linearity was y = 86.10x + 178.31 with the correlation coefficient (r²) 0.9997. Quantitative data obtained in the present study proved the improved RP‐HPLC method to be a sensitive and accurate analytical tool for bLf determination. The proteolytic cleavage of bLf in simulated human gastrointestinal fluids was further analyzed by RP‐HPLC, and found to follow pseudo‐first‐order kinetics. The typical equation obtained by pepsin was log₁₀ [A_t]/[A₀] = ?0.03x (r² = 0.85), and log₁₀ [A_t]/[A₀] = ?0.01x (r² = 0.81) for trypsin and chymotrypsin combination. Pepsinolysis of bLf in simulated gastric fluid was relatively fast with the half‐life t_1/2 23.1 min. The digestion of bLf in simulated intestinal fluid was slower with about a 3‐fold increase in half‐life (69.3 min). After the complete proteolysis of bLf, small cleaved peptide fragments were fully separated and identified by RP‐HPLC. The proteolytic study indicated that this validated RP‐HPLC was able to evaluate bLf stability though monitoring the derivatization products. Copyright © 2012 John Wiley & Sons, Ltd.     

Study of degradation behavior of besifloxacin,characterization of its degradation products by LC–ESI–QTOF–MS and their in silico toxicity prediction

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth‐generation fluoroquinolone antibiotic, was subjected to different forced‐degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions—DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C₁₈ (250 mm × 4.6 mm, 5 μm) column with 0.1% aqueous formic acid–acetonitrile as a mobile phase. The gradient RP‐HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC–ESI–QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.     

Different stability‐indicating chromatographic methods for specific determination of paracetamol,dantrolene sodium,their toxic impurities and degradation products

A well‐known analgesic (paracetamol, PAR) and skeletal muscle relaxant [dantrolene sodium (DNS)] have been analyzed without interference from their toxic impurities and degradation products. The studied PAR impurities are the genotoxic and nephrotoxic p‐amino phenol (PAP) and the hepatotoxic and nephrotoxic chloroacetanilide, while 5‐(4‐nitrophenyl)‐2‐furaldehyde is reported to be a mutagenic and carcinogenic degradation product of DNS. The five studied components were determined and quantified by TLC–densitometric and RP‐HPLC methods. TLC–densitometry (method 1) used TLC silica gel and chloroform–ethyl acetate–acetic acid–triethylamine (7:3:0.5:0.05, by volume) as the mobile phase with UV scanning at 230 nm, while RP‐HPLC (method 2) was based on separation on a C₁₈ column using methanol–water (55:45, v/v pH 3 with aqueous formic acid) as mobile phase at 1 mL/min and detection at 230 nm. The developed methods were used for determination and quantification of the five studied components in different laboratory‐prepared mixtures. The were also applied for analysis of Dantrelax^® compound capsules where no interference among the studied components with each other or from excipients was observed. The methods were validated as per International Conference on Harmonization guidelines, and they compared favorably with the reported ones.     

Development and validation of a RP‐HPLC method for stability‐indicating assay of gemifloxacin mesylate including identification of related substances by LC‐ESI‐MS/MS, 1H and 13C NMR spectroscopy

A validated stability indicating RP‐HPLC assay of gemifloxacin mesylate was developed by separating its related substances on an Inertsil‐ODS3V‐C₁₈ (4.6 × 250 mm; 5 μm) column using 0.1% trifluoroaceticacid (pH 2.5) and methanol as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min at 27°C. The column effluents were monitored by a photodiode array detector set at 287 nm. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. Forced degradation of gemifloxacin (GFX) was carried out under acidic, basic, thermal, photolysis and peroxide conditions and the degradation products were separated and characterized by ESI‐MS/MS, ¹H and ¹³C NMR spectroscopy. The method was successfully applied to the analysis of bulk drugs and the recoveries of gemifloxacin and impurities were in the range of 97.60–102.90 and 96.99–102.10%, respectively. No previous reports were found in the literature on identification of degradation products of gemifloxacin. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a reversed‐phase high‐performance liquid chromatographic method with solid‐phase extraction for the quantification of hydrochlorothiazide in ex vivo permeation studies

Hydrochlorothiazide (HCT) is a diuretic used to treat hypertension. In order to study its intestinal permeation behavior applying an ex vivo methodology, a rapid, sensitive and selective reversed‐phase liquid chromatography (RP‐HPLC) method coupled with UV detection (RP‐HPLC UV) was developed for the analysis of HCT in TC199 culture medium used as mucosal and serosal solutions in the everted rat intestinal sac model. Also, analytical procedures for the quantification of HCT by RP‐HPLC with UV detection required a sample preparation step by solid‐phase extraction. The method was validated in the concentration range of 8.05 × 10⁻⁷ to 3.22 × 10⁻⁵ m for HCT. Chromatographic parameters, namely carry‐over, lower limit of quantification (1.4491 × 10⁻⁷ m ), limit of detection (3.8325 × 10⁻⁸ m ), selectivity, inter‐ and intraday precision and extraction recovery, were determined and found to be adequate for the intended purposes. The validated method was successfully used for permeability assays across rat intestinal epithelium applying the ex vivo everted rat gut sac methodology to study the permeation behavior of HCT.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Forced degradation studies of clobetasol 17‐propionate in methanol,propylene glycol,as bulk drug and cream formulations by RP‐HPLC

A rapid, simple, stability‐indicating forced degradation study of clobetasol 17‐propionate was conducted using RP‐HPLC. The method was used to analyze clobetasol 17‐propionate in methanol, propylene glycol, and a cream formulation. Isocratic elution of clobetasol and its degradation products was achieved using a Nova‐Pak® 4 μm C₁₈ 150 mm × 3.9 mm id cartridge column and a mobile phase of methanol: water (68:32 v/v) at a flow rate of 0.9 mL min^?1. Quantitation was achieved with UV detection at 239 nm. Nondegraded clobetasol was eluted at a retention time of 6.0 min. Clobetasol 17‐propionate was subjected to different stress conditions viz., acidic, basic, heat, oxidation, light, and neutral hydrolysis. The greatest degradation occurred under strong base and oxidative conditions. Strong base‐degraded clobetasol produced additional peaks at retention times of 1.8, 4.0, 5.0, and 8.0 min and clobetasol oxidation degradation peaks eluted at 2.2 and 24 min. Complete validation was performed for linearity, accuracy, and precision over the concentration range 0.15–15 μg mL^?1. All data were analyzed statistically and this RP‐HPLC method proved to be accurate, precise, linear, and stability indicating for the quantitation of clobetasol 17‐propionate in methanol, propylene glycol, and cream formulations.     

High performance liquid chromatography method for determination of methyl-5-benzoyl-2-benzimidazole carbamate (mebendazole) and its main degradation product in pharmaceutical dosage forms

Methyl-5-benzoyl-2-benzimidazole carbamate (mebendazole) is a drug used as an anthelmintic. A high performance liquid chromatography method has been developed in this study to determine mebendazole and its degradation product in the pharmaceutical dosage forms (tablets and suspension). The expected major degradation product of mebendazole in the dosage forms has been prepared, and identified as 2-amino-5-benzoylbenzimidazole. The proposed HPLC assay was found to be selective, accurate (% recoveries were in the range of 99.9-100.9) for both, mebendazole and the degradation product, repeatable and reproducible (replicate measurements for short and long term measurements showed % RSD of 相似文献   

Chiral separation of amides of amino acid on a (S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamide‐bonded silica using nonaqueous capillary electrochromatography

(S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamine‐bonded silica was used as a chiral stationary phase for separation of a set of racemic π‐acidic and π‐basic α‐amino acid amides in electrolyteless ACN‐water eluents by CEC in the RP and polar organic (PO) modes. The effect of the amount of water in the ACN‐water eluent on chiral separation was examined. As water is added to ACN, retention was shortened but resolution and selectivity deteriorated severely. Retention, enantioselectivity, and resolution factors obtained in 100% ACN were compared with those in an n‐hexane‐isopropanol eluent with a small amount of water by normal phase (NP) CEC. Much shorter retention times with comparable enantioselectivities were observed with 100% ACN, demonstrating the advantage of separation on (S)‐N‐(DNB)leucine‐N‐phenyl‐N‐propylamine‐bonded silica in PO‐CEC over NP‐CEC.     

A new single‐urea‐bound 3,5‐dimethylphenylcarbamoylated β‐cyclodextrin chiral stationary phase and its enhanced separation performance in normal‐phase liquid chromatography

A new single‐urea‐bound chiral stationary phase based on 3,5‐dimethylphenylcarbamoylated β‐cyclodextrin was prepared through the Staudinger reaction of mono (6^A‐azido‐6^A‐deoxy)‐per(3,5‐dimethylphenylcarbamoylated) β‐cyclodextrin and 3‐aminopropyl silica gel under CO₂ atmosphere. The new phase exhibited good enantioseparation performance for 33 analytes using normal‐phase HPLC conditions; 19 of them were baseline separated. Effects of structure of analytes, alcoholic modifiers, and acidic/basic additives on separation performances of this new cyclodextrin chiral stationary phase have been studied in detail. The results showed that the retention and resolution of acidic and basic analytes on the CSP were greatly affected by the additives. Peak symmetry for some analytes could be improved by simultaneously adding acidic and basic additives to the mobile phase. This work expands the potential applications of the cyclodextrin‐based chiral stationary phases in the normal‐phase HPLC.     

New liquid‐crystal alignment agents based on fluorinated polyimides with trifluoromethyl‐substituted benzene or diphenylether in the side chain

Fluorinated copolyimides derived from 4,4′‐oxydiphthalic anhydride (ODPA) with 4,4′‐oxydianline (ODA) and trifluoromethyl‐containing aromatic diamines have been synthesized and characterized. The trifluoromethyl‐containing diamines include 2,4‐diamino‐3′‐trifluoromethylazobenzene, 2,4‐diamino‐1‐[(4′‐trifluoromethylphenoxy) phenyl] aniline, 3,5‐diamino‐1‐[(4′‐trifluoromethylphenoxy) phenyl] benzamide, 3,5‐diamino‐1‐[(3′‐trifluoromethyl) phenyl] benzamide, 1,4‐bis(4′‐aminophenoxy)‐2‐(3′‐trifluoromethylphenyl) benzene, 3,5‐diaminobenzenetrifluoride, 4,4′‐diamino‐4″‐(p‐trifluoromethyl phenoxy) triphenylamine, and 4‐[(4′‐trifluoromethylphenoxy) phenyl]‐2,6‐bis(4″‐aminophenyl)pyridine. Strong and flexible copolyimide films, produced by casting the polyamic acid solution followed by thermal imidization, exhibited great thermal stability and high mechanical properties. The polyimides had an ultraviolet–visible absorption cutoff at 330–340 nm and pretilt angles as high as 20° for nematic liquid crystals, making them great potential candidates for advanced liquid‐crystal display applications. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1583–1593, 2002     

Synthesis of a Novel Double Salt: Ammonium 3,4‐Diamino‐1,2,4‐triazolium Styphnate

Nitrogen‐rich double salt ammonium 3,4‐diamino‐1,2,4‐triazoliumstyphnate (NH₄ · DATr · TNR) ( 2 ) with good thermal stability was successfully synthesized by reacting 3,4‐diamino‐1,2,4‐triazolium chloride aqueous solution with styphnic acid methanol solution under the reaction medium of aqueous ammonia. The title double salt was characterized by elemental analysis (EA), Fourier transformation infrared spectrum (FT‐IR), and X‐ray single‐crystal diffraction. It crystallizes in the monoclinic crystal system with space group P2₁/n. Its density is 1.780 g · cm^–3. Compound 2 is thermal stable below 200 °C by the differential scanning calorimetry (DSC) test. The non‐isothermal kinetics parameters were calculated by the Kissinger's method and Ozawa‐Doyle's method, respectively. In addition, compound 2 showed low friction and impact sensitivities.     

Stability‐indicating chromatographic and chemometric methods for environmentally benign determination of canagliflozin and its major degradation product; A comparative study and greenness assessment

Recently, concepts of sustainable developments, like considering the environmental effect of chemicals used and the amount of hazardous wastes produced, has gained much interest. In this work, a recently approved treatment for type II diabetes mellitus, canagliflozin, was quantified along with its degradation product by two eco‐friendly methods. The first was a specific green HPLC method using a C₁₈ column as a stationary phase and a mobile phase consisting of methanol–water (98:2, v/v) pumped at a flow rate of 1 mL/min with UV detection at 225 nm, and using ibuprofen as an internal standard. The second method was a partial least square chemometric method with the wavelength range 220–320 nm and the data was autoscaled as a preprocessing step for determination of canagliflozin and its degradation product. The greenness profile of the developed methods was studied and compared with the reported methods. The proposed methods were suitable alternatives for the environmentally harmful reported methods for quality control analyses of canagliflozin‐containing samples, analysis of pharmaceutical formulations and sensitive tracing of its possible degradation product. The methods were validated as per International Conference on Harmonization guidelines and statistically compared with the reported HPLC method.