Validated, Stability-Indicating LC Method for Analysis of Pipenzolate Bromide and Its Hydrolysis Products

A liquid chromatographic method has been developed and validated for quantitative analysis of pipenzolate bromide (PP), its hydrolysis products, and phenobarbitone, sodium benzoate, and sodium saccharine. A 5-μm particle ODS column was used with acetonitrile–KH₂PO₄ (10 mm, pH 3.5) 40:60 (v/v), containing 5 mm heptanesulfonic acid sodium salt, as mobile phase. Quantitation was achieved by UV detection at 210 nm, on the basis of peak area. Forced degradation studies were performed on a bulk sample of PP using 0.1 M hydrochloric acid, 0.01 M sodium hydroxide, 0.33% hydrogen peroxide, heat (70 °C), and photolytic degradation. The proposed LC method was used to study the kinetics of acidic hydrolysis and pH-rate profiles of hydrolysis of PP in Britton–Robinson buffer solutions.     

Boronate-Containing Copolymer Grafted on Eupergit C as Matrix for Affinity Chromatography: Isotherms and Kinetics Study

A stability-indicating HPLC method has been developed and subsequently validated for the simultaneous determination of domperidone and pantoprazole in commercial tablets. The proposed HPLC method utilizes Phenomenex^® Gemini C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) and mobile phase consisting of methanol-acetonitrile-20 mM dipotassium hydrogen phosphate and phosphoric acid buffer pH 7.0 (20:33:47, v/v/v) at a flow rate of 1.19 mL min^?1. Quantitation was achieved with UV detection at 285 nm based on peak area with linear calibration curves at concentration ranges 0.5–5.0 μg mL^?1 for domperidone and 1.0–10 μg mL^?1 for pantoprazole (R ² > 0.999 for both drugs). The method was validated in terms of accuracy, precision, linearity, limits of detection, limits of quantitation and robustness. This method has been successively applied to pharmaceutical formulation and no interference from the tablet excipients was found. Domperidone, pantoprazole and their combination drug product were exposed to acid, base and neutral hydrolysis, oxidation, dry heat and photolytic stress conditions and the stressed samples were analyzed by the proposed method. As the proposed method could effectively separate the drug from its degradation products, it can be employed as stability-indicating method for the determination of instability of these drugs in bulk and commercial products.     

Quantification of an intact monoclonal antibody, rituximab, by (RP)HPLC/DAD in compliance with ICH guidelines

We studied the quantification of an intact therapeutic monoclonal antibody (mAb), rituximab (RTX), using (reverse-phase) high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD). To this end, we developed a chromatographic method and validated it as stability-indicating in accordance with the International Conference on Harmonization guidelines (ICH). A 300-Å C8 column (250 mm?×?4.6 mm, 5 μm) was used to perform the analysis, and the temperature was maintained at 70 °C. Although only one mAb was analyzed, it was necessary to apply a gradient to elute it with a complex organic mixture. Chromatograms were registered at several wavelengths, with λ?=?214 nm employed for quantification purposes. The method was developed to quantify marketed RTX under typical hospital administration conditions. Further dilution was avoided in order to prevent additional mAb modification, and in this way the method was shown to be linear from 60 to 5000 mg/L. The precision of the method (repeatability and intermediate precision, estimated as the relative standard deviation, RSD %), was less than 1.0 %. Accuracy, specificity, robustness, and system suitability were also evaluated as specified in the ICH guidelines. We conducted a comprehensive chromatographic analysis by submitting RTX to several informative stress conditions. These forced degradation studies were conducted for two reasons: to estimate the specificity of the method, and to evaluate the robustness of the mAb formulation against external stress factors when handling it in preparation for administration. Thus, we investigated the effects of acid, base, oxidation, ionic strength, temperature, and UV light. Although a slight modification to the intact mAb could not be distinguished chromatographically in the stress studies we conducted, the procedure proposed here to evaluate peak purity enabled us to detect it with a satisfactory level of confidence. The proposed method could therefore be considered stability-indicating for quantyfying the intact mAb since it is qualified to detect its degradation/modification. Finally, the method was used to evaluate RTX in a long-term stability study performed under hospital conditions of use.     

Validated Optimized Method for Simultaneous Analysis of Active Silymarin Components and Dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene Dioxybiphenyl-2,2′-dicarboxylate in a Pharmaceutical Preparation by Use of a Monolithic Silica C18 Column

A simple, rapid, and sensitive isocratic reversed-phase LC method using a monolithic column has been developed and validated for simultaneous analysis of the active components of silymarin [taxifolin, silydianin, silychristin, diastereomers of silybin (silybin A and B), and diastereomers of isosilybin (isosilybin A and B)] and dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxybiphenyl-2,2′-dicarboxylate in a commercial formulation. The mobile phase was a 45:55 (v/v) mixture of methanol and 5 mM NaH₂PO₄ (adjusted to pH 2.75 with phosphoric acid) at a flow rate of 1.5 mL min^?1. UV detection was performed at 288 nm and quantification was based on peak area. The method was validated for linearity, accuracy, precision, selectivity, and robustness.     

Validated Stability-Indicating HPLC–DAD Determination of the Antihypertensive Binary Mixture of Carvedilol and Hydrochlorothiazide in Tablet Dosage Forms

A simple and selective HPLC with diode array detection stability-indicating method was developed for the simultaneous determination of the antihypertensive drugs carvedilol (CRV) and hydrochlorothiazide (HCT) in their combined formulations. Effective chromatographic separation was achieved using Zorbax SB-C8 column (4.6 × 250 mm, 5 μm) with gradient elution of the mobile phase composed of 0.025 M phosphoric acid and acetonitrile at a flow rate of 1 mL min^?1. The multiple wavelength detector was set at 242 nm for measurement of CRV and 271 nm for HCT. Quantification was based on measuring the peak areas. The cited drugs were resolved with retention times 4.9 and 6.7 min for HCT and CRV, respectively. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 5–300 and 5–200 μg mL^?1 for CRV and HCT, respectively, with correlation coefficients >0.9996. Both drugs were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the drugs from their forced degradation products. Moreover, specificity of the method was verified by resolution of both drugs from more than 20 pharmaceutical compounds of various medicinal categories. The validated HPLC method was applied to the analysis of the cited antihypertensive drugs in their combined tablet dosage forms. The proposed method made use of DAD as a tool for peak identity and purity confirmation.     

Analysis of Temocillin and Impurities by Reversed Phase Liquid Chromatography: Development and Validation of the Method

A robust, specific, precise and sensitive high-performance liquid chromatographic method has been described for purity control of temocillin. Chromatographic separation was achieved using a Symmetry C₁₈ (150 × 4.6 mm, 5 µm) column kept at 30 °C. The mobile phase consisted of a gradient mixture of mobile phases A (5 g/L solution of Na₂HPO₄·2H₂O, pH 7) and B (ACN-MeOH-H₂O, 50:10:40 v/v/v) pumped at a flow rate of 1.0 mL/min. UV detection was performed at 235 nm. The developed method was validated according to the ICH guidelines for its robustness, selectivity, sensitivity, precision and linearity. An experimental design was applied for the robustness study. Linearity was assessed both at impurity level in the range from LOQ to 10 % and assay level from 25 % to 150 % (0.6 mg/mL = 100 %). It is the first liquid chromatographic method described for the separation of temocillin and its potential impurities. It was possible to identify four degradation products from the forced degradation studies. The degradants do not interfere with the main peak and other known impurities showing that the method is specific and stability-indicating.     

Development and Validation of a Reversed-Phase HPLC Method for the Determination of Deflazacort in Pharmaceutical Dosage Forms

A reversed-phase high-performance liquid chromatographic method has been developed and validated for quantitative determination of deflazacort in tablets and in compounded capsules. Isocratic chromatography was performed on a C₁₈ column with acetonitrile–water 80:20 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. UV detection was at 240 nm. The linearity of the method was good (r > 0.999), as also were intra-day and inter-day precision (RSD <2%) and accuracy (recovery >98%). The method was also validated for specificity and robustness. The results showed the proposed method is suitable for its intended use.     

Validated Stability-Indicating HPLC–DAD Determination of the Antihypertensive Binary Mixture of Carvedilol and Hydrochlorothiazide in Tablet Dosage Forms

A simple and selective HPLC with diode array detection stability-indicating method was developed for the simultaneous determination of the antihypertensive drugs carvedilol (CRV) and hydrochlorothiazide (HCT) in their combined formulations. Effective chromatographic separation was achieved using Zorbax SB-C8 column (4.6 × 250 mm, 5 μm) with gradient elution of the mobile phase composed of 0.025 M phosphoric acid and acetonitrile at a flow rate of 1 mL min⁻¹. The multiple wavelength detector was set at 242 nm for measurement of CRV and 271 nm for HCT. Quantification was based on measuring the peak areas. The cited drugs were resolved with retention times 4.9 and 6.7 min for HCT and CRV, respectively. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 5–300 and 5–200 μg mL⁻¹ for CRV and HCT, respectively, with correlation coefficients >0.9996. Both drugs were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the drugs from their forced degradation products. Moreover, specificity of the method was verified by resolution of both drugs from more than 20 pharmaceutical compounds of various medicinal categories. The validated HPLC method was applied to the analysis of the cited antihypertensive drugs in their combined tablet dosage forms. The proposed method made use of DAD as a tool for peak identity and purity confirmation.

     

LC–DAD–MS Method for Analysis of Pomiferin and Osajin, Major Isoflavones in Maclura pomifera (Rafin.) Schneider

Pomiferin and osajin are major bioactive isoflavones found in Maclura pomifera (Rafin.) Schneider (Moraceae). A simple, reproducible, and sensitive method for analysis of pomiferin and osajin in different parts of M. pomifera has been developed and validated. The compounds were separated on a C₈ column by gradient elution at a flow rate at 1.0 mL min^?1. Column eluent was monitored at 280 nm by use of a diode-array detector. Linear range, peak purity, selectivity, system performance, precision, accuracy, and robustness were shown to be acceptable. LC–MS with atmospheric pressure electrospray ionization was used for identification of the compounds; use of positive-ion mode resulted in protonated pseudomolecular ions of the compounds.     

Validation of an HPLC Method for the Determination of Valdecoxib and its Degradation Product: a Mixture of α- and β-n-Lactosyl Sulfonamide Anomers

An HPLC method has been developed for the separation of valdecoxib and a degradation product consisting of α and β-N-lactosyl sulfonamide, i.e. α and β anomers (SC-77852). Best results were achieved with a Chromolith Performance RP-18e column (100 mm × 4.6 mm), macropore size 2 μm, mesopore size 13 nm, with an eluent of methanol:water containing a 1% solution of TEA (36:64 v/v), pH 7.4 (adjusted with 85% orthophosphoric acid), at 22 °C. Detection was at 220 nm. The method was validated for its selectivity, linearity, precision (repeatability) and robustness. Quantitation and detection limits were determined for both valdecoxib and SC-77852. Method robustness was further evaluated by performing 2³ full factorial design experiments. The final step, optimisation of the variables, was performed using response surface design. The validated method was used for assay of valdecoxib and SC-77852 in Bextra^® film-coated tablets.     

Enantiomeric Separation of Moxifloxacin and Its (R,R)-Isomer by Ligand-Exchange Chiral Chromatography

A new stereospecific LC method for the separation and quantification of moxifloxacin and its (R,R)-enantiomer in bulk drug was developed and validated by ligand-exchange liquid chromatography on a reversed phase column using aqueous mobile phase containing the chiral reagent l-isoleucine-Cu(II). The UV detector was operated at 293 nm. The flow rate of mobile phase was set at 0.9 mL min^?1. The achiral ODS column offers good separation of the two enantiomers in less than 20 min. The test concentration was 1,000 μg mL^?1 in the mobile phase. This method was capable of detecting the (R,R)-enantiomer of moxifloxacin up to 0.1 μg mL^?1 for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with the (R,R)-enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the (R,R)-enantiomer in bulk drug samples ranged from 98.1 to 104.4%. The test solution was found to be stable in the mobile phase for 48 h after preparation.     

A Rapid and Sensitive LC Method for Determination of Diastereomeric Purity of Tenofovir Alafenamide

A rapid and sensitive LC method was developed and validated for the determination of diastereomeric purity of tenofovir alafenamide (GS-7340). Baseline separation with resolution >2.8 was achieved within 17 min on a CHIRALPAK AD-3 (250 × 4.6 mm; particle size 3 μm) column using n-hexane:2-propanol (60:40 v/v) as the mobile phase at a flow rate of 1 mL min^?1. The analytes were detected by UV absorbance at 260 nm. The effects of ethanol, 2-propanol, and temperature on diastereomeric selectivity and resolution of diastereomerism were evaluated. The method was extensively validated and proved to be robust. The recoveries were between 98.17 and 102.84 % with <1.93 % relative standard deviation. The limit of detection and limit of quantitation for GS-7339 were 0.77 and 2.56 μg mL^?1 and for GS-7340 were 0.61 and 2.04 μg mL^?1, respectively. This method was extensively proved to be accurate, stable, rapid, and sensitive for the determination of diastereomeric purity of tenofovir alafenamide (GS-7340) in bulk samples.     

A validated chiral LC method for the enantiomeric separation of AT13148 on polysaccharide-based chiral stationary phase

A sensitive and accurate LC method for the determination of AT13148 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.8 was accomplished within 15 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane: 2-propanol: diethylamine (85:15:0.1, v/v) as mobile phase at a flow rate of 1 mL min^?1. Eluted analytes were monitored by UV absorption at 254 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 7 and 500 μg mL^?1 (n = 11), and the recoveries between 98.24 and 100.99% were obtained, with relative standard deviation lower than 1.32%. LOD and LOQ for AT13148 were 2.46 and 7.38 μg mL^?1 and for its enantiomer were 2.54 and 7.49 μg mL^?1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AT13148, especially for the analysis of bulk samples.     

Qualitative and Quantitative Studies on Impurities in Schizonepetin, a Novel Antiviral Agent, Using HPLC, NMR and MS

Schizonepetin is a new naturally occurring monoterpene with antiviral activity. Three unknown related impurities were observed in analysis of schizonepetin bulk drug. A semi-preparative liquid chromatography was used for isolation of the three impurities. Three impurities were characterized as (?)-mintlactone (Impurity I), (R)-(+)-pulegone (Impurity II) and (+)-menthofuran (Impurity III) by a variety of spectral data (IR, UV, MS, ¹H-NMR, ¹³C-NMR, DEPT and 2D-NMR). The simultaneous quantitative determination of schizonepetin and its impurities (Imp-I, II and III) was performed by reversed-phase HPLC method with UV detection at 220 nm. The method shows good precision, sensitivity, linearity (between 0.025 and 1.0 mg mL^?1) and robustness.     

Hyphenated HSCCC–DPPH· for Rapid Preparative Isolation and Screening of Antioxidants from Selaginella moellendorffii

A method for rapid preparative isolation and screening of antioxidants has been developed by combining preparative HSCCC with on-line radical scavenging detection by use of DPPH· (1,1-diphenyl-2-picrylhydrazyl) as model radical. The HSCCC effluent was split into two parts. The major part was collected and the rest was introduced, through a switch valve, directly into a reaction coil for on-line evaluation of antioxidant activity. Each of the antioxidants separated by HSCCC reacted postcolumn with the DPPH· solution in methanol and was observed as a negative peak at 517 nm of size corresponding to its antioxidant activity. The on-line method was successfully used for preparative isolation and screening of antioxidant components from an ethyl acetate extract of the traditional Chinese medicine Selaginella moellendorffii. A total of 114.7 mg amentoflavone, 12.4 mg hinokiflavone, 7.2 mg podocarpusflavone A, and 1.1 mg ginkgetin, identified as the main radical scavengers, were obtained with purity of 99.4, 98.6, 98.9, and 99.2%, respectively, as determined by LC, from 1.0 g ethyl acetate extract. Their chemical structures were identified by MS and by UV and NMR spectroscopy.     

Determination of AR-42 enantiomeric purity by HPLC on chiral stationary phase

A sensitive and accurate liquid chromatographic method for the determination of AR-42 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.9 was accomplished within 10 min using a CHIRALPAK AD column (250 mm × 4.6 mm; particle size 5 μm) and n-hexane/2-propanol/diethylamine (75:25:0.1 v/v/v) as mobile phase at a flow rate of 1 mL min^?1. Eluted analytes were monitored by UV absorption at 260 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.001 and 0.5 mg mL^?1 (n = 10), and the recoveries between 98.23 and 101.87% were obtained, with relative standard deviation lower than 1.31%. Limit of detection and limit of quantitation for AR-42 were 0.39 and 1.28 μg mL^?1 and for its enantiomer were 0.36 and 1.19 μg mL^?1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AR-42, especially for the analysis of bulk samples.     

Effect of Laser Pulse Energy on the Characteristics of Cu Nanoparticles Produced by Laser Ablation Method in Acetone

The effect of laser fluence on the characteristics of Cu nanoparticles, prepared by laser ablation method, is investigated experimentally. 1–6 nm Cu nanoparticles were synthesized by the pulsed laser ablation of a high purity copper bulk in acetone. Effect of laser fluence on the size, morphology and structure of produced nanoparticles has been studied. Pulses of a Q-switched Nd:YAG laser of 1,064 nm wavelengths at 7 ns pulse width at different fluences was employed to irradiate the Cu target in acetone. The UV–Vis–NIR absorption spectra of nanoparticles exhibit surface plasmon resonance absorption peak in the visible region. TEM and SEM micrographs indicate that with increasing the laser fluence the average size of spherical Cu nanoparticles is decreased and only the sample which is produced with the highest fluence shows exceptional behavior. It is found that Cu nanoparticles exhibit photoluminescence emission with single peak due to its interband transition.     

Development and Validation of an RP–LC–UV Method for the Determination of Ondansetron: Application to Pharmaceutical Dosage Forms

A new, simple, rapid, sensitive and specific isocratic RP–LC–UV method was developed and validated for the determination of ondansetron in pharmaceutical dosage forms of orally disintegrating tablets, oral solution and injection. The LC separation was achieved on a Hypersil C4 column (250 × 4.6 mm, 5 μm) using a mobile phase of 50 mM potassium dihydrogen phosphate anhydrous adjusted to pH 3.5 with orthophosphoric acid and acetonitrile (30:70, v/v) at a flow rate of 1.0 mL min^?1 and UV detection at 310 nm. The method was validated for specificity, linearity, precision, accuracy, limit of quantification, limit of detection, robustness and solution stability. The calibration curve was linear over a concentration range of 100–1,000 ng mL^?1 (r ²  = 0.9996) with limit of detection and limit of quantification 50 and 100 ng mL^?1, respectively. The intra-day and inter-day precision and accuracy were between 0.79 and 2.37% and ?0.64 and 1.65%, respectively. The method was successfully applied for analysis of ondansetron in the presence of excipients in commercially available pharmaceutical dosage forms.     

A Stability Indicating LC Method for Deferasirox in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of deferasirox, its related impurities in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at 245 nm. The mobile phase consisted of buffer, acetonitrile and methanol (50:45:5, v/v) delivered at a flow rate of 1.0 mL min^?1. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 3.0 by using orthophosphoric acid. In the developed LC method the resolution (R _s ) between deferasirox and its four potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.999 for deferasirox and its four impurities. This method was capable to detect all four impurities of deferasirox at a level of 0.002% with respect to test concentration of 0.5 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for all four impurities and for deferasirox was found to be within 2.0% RSD. The method showed good and consistent recoveries for deferasirox in bulk drugs (98.3–101.1%), pharmaceutical dosage forms (100.2–103.1%) and for its all the four impurities (99.7–102.1%). The test solution was found to be stable in methanol for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.95%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog

Nitrosylcobalamin (NO-Cbl), a novel vitamin B₁₂ analog and anti-tumor agent, functions as a biologic ‘Trojan horse’, utilizing the vitamin B₁₂ transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. A stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an Ascentis^® RP-Amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min^?1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. Additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B₁₂ therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.