Development and validation of a stability‐indicating HPLC method for topiramate using a mixed‐mode column and charged aerosol detector

The analysis of topiramate in the presence of its main degradation products is challenging due to the absence of chromophore moieties and their wide range of polarity. Mixed‐mode chromatography has been used in such cases because it combines two or more modes of separation. Charged aerosol detector is also an alternative since its detection is independent of optical properties and analyte ionization. This study is aimed to develop and validate two new stability‐indicating methods by high‐performance liquid chromatography for the main degradation products of topiramate using mixed‐mode chromatography and a charged aerosol detector. Method 1 employed an Acclaim Trinity P1® column (3.0 mm × 150 mm, 2.7 μm) with a mobile phase comprising of 80% ammonium acetate buffer (20 mM, pH 4.0) and 20% methanol at a flow rate of 0.5 mL/min at 35°C. Method 2 utilized a C18 Acclaim 120® column (4.6 mm × 250 mm; 5 μm) with ACN/water (50:50) at a flow rate of 0.6 mL/min at 50°C. Validation of the two methods demonstrated excellent performance with respect to linearity, precision, accuracy, and selectivity. The limits of detection for topiramate, fructose, sulfate, sulfamate, and compound A were 2.97, 12.08, 4.02, 13.91, and 3.94 μg/mL, respectively.     

Development and Validation of an HPLC Method for Simultaneous Determination of Ibuprofen and 17 Related Compounds

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for determination of ibuprofen and 17 related compounds (chemical process impurities and degradation products) simultaneously. This method may be used for quality control of ibuprofen-containing substances. A number of chromatographic parameters (column, flow rate, temperature, wavelength, gradient elution, buffer solution, and pH) were evaluated. An Agilent ZORBAX Eclipse Plus C18 (250 × 4.6 mm, 5 μm particle size) column at 40 °C was selected on the basis of its separation efficiency and robustness. The column was eluted at 1.0 mL min^?1 with a gradient using 10 mM sodium phosphate buffer at pH 6.9 as mobile phase A and acetonitrile as mobile phase B. The ultraviolet detector was set at 214 nm. This method was validated to confirm its system suitability, specificity, linearity, precision, accuracy, sensitivity, robustness, and sample stability according to international conference on harmonization (ICH) guidelines. This method was applied to analyze seven batches of ibuprofen drug products from different manufacturers.     

Ultra HPLC Method for Fixed Dose Combination of Azilsartan Medoxomil and Chlorthalidone: Identification and <Emphasis Type="Italic">in silico</Emphasis> Toxicity Prediction of Degradation Products

The fixed dose combination of azilsartan medoxomil potassium and chlorthalidone has been introduced for the effective treatment of hypertension. In the present work a rapid, simple and accurate stability indicating ultra HPLC assay method has been developed. The separation of azilsartan medoxomil, chlorthalidone and their degradation products were accomplished on an Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column using mobile phase combination of 0.02% trifluoroacetic acid in water and acetonitrile in gradient mode. The forced degradation products were identified using liquid chromatography?electrospray ionisation-quadrupole time of flight-tandem mass spectrometry (LC?ESIQTOF–MS/MS) and accurate mass experiments. The in silico toxicities of the degradation products for both the drugs were evaluated. The proposed method was validated as per the ICH Q2 (R1) guideline for selectivity, linearity, precision, accuracy and robustness.     

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

A rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.

Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.     

Development,stability-indicating assessment,and evaluation of influential method conditions using a full factorial design for the determination of Nintedanib esylate-related impurities

The design of an appropriate analytical method for assessing the quality of pharmaceuticals requires a deep understanding of science, and risk evaluation approaches are appreciated. The current study discusses how a related substance method was developed for Nintedanib esylate. The best possible separation between the critical peak pairs was achieved using an X-Select charged surface hybrid Phenyl Hexyl (150 × 4.6) mm, 3.5 μm column. A mixture of water, acetonitrile, and methanol in mobile phase-A (70:20:10) and mobile phase-B (20:70:10), with 0.1% trifluoroacetic acid and 0.05% formic acid in both eluents. The set flow rate, wavelength, and injection volumes were 1.0 ml/min, 285 nm, and 5 μl, respectively, with gradient elution. The method conditions were validated as per regulatory requirements and United States Pharmacopeia general chapter < 1225 >. The correlation coefficient for all impurities from the linearity experiment was found to be > 0.999. The % relative standard deviation from the precision experiments ranged from 0.4 to 3.6. The mean %recovery from the accuracy study ranged from 92.5 to 106.5. Demonstrated the power of the stability-indicating method through degradation studies; the active drug component is more vulnerable to oxidation than other conditions. Final method conditions were further evaluated using a full-factorial design. The robust method conditions were identified using the graphical optimization from the design space.     

Chromatographic analysis of ledipasvir and sofosbuvir: New treatment for chronic hepatitis C infection with application to human plasma

Sofosbuvir (SOF) and ledipasvir (LED) are recently approved and coformulated as directly acting antiviral agents used for treatment of hepatitis C virus (HCV). A reversed phase high performance liquid chromatography - diode array detector (RP-HPLC/DAD) method was developed and validated for the first time for the analysis of newly formulated anti-HCV combination, in pure form, pharmaceutical formulation and in human plasma. In the developed method, separation was performed on Zorbax® Eclipse C18 column using a gradient mixture of acetonitrile–water as a mobile phase and scanning was performed at 260?nm (for SOF) and 330?nm (for LED). The two drugs were completely separated from each other and from plasma, where plasma peak appeared at 2.76?±?0.05?min, SOF at 4.25?±?0.05, and LED at 7.35?±?0.05. The developed method showed high sensitivity, the drugs showed linearity in the range of 1–45?µg/mL for both pure form and spiked human plasma. Three freeze–thaw cycles were performed separately at two different temperatures, ?8 and ?20°C. No significant loss of the studied drugs were observed during repeated thawing and freezing. Validation parameters such as accuracy, precision, robustness, and ruggedness were tested in compliance with USP recommendations, where acceptable results were obtained. Applying to pharmaceutical formulation showed no interference from tablet excipients.     

Development and validation of the stability-indicating LC-UV method for the determination of cefoselis sulphate

The stability-indicating LC assay method was developed and validated for quantitative determination of cefoselis sulphate in the presence of degradation products formed during the forced degradation studies. An isocratic, RP-HPLC method was developed with C-18 (250 × 4.6 mm, 5 μm) column and 12 mM ammonium acetate-acetonitrile (95:5 V/V) as a mobile phase. The flow rate of the mobile phase was 1.0 mL min⁻¹. Detection wavelength was 260 nm and temperature was 30°C. Cefoselis similarly to other cephalosporins was subjected to stress conditions of degradation in aqueous solutions including hydrolysis, oxidation, photolysis and thermal degradation. The developed method was validated with regard to linearity, accuracy, precision, selectivity and robustness. The method was applied successfully for identification and determination of cefoselis sulphate in pharmaceuticals and during kinetic studies.     

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 novel stability indicating RP-HPLC method for the quantitative determination of marbofloxacin in its tablets

A fast, feasible, isocratic, stability indicating reverse phase-high performance liquid chromatography (RP-HPLC) method has been developed and validated for the quantitative determination of marbofloxacin in marbofloxacin tablets. The method was developed using Zorbax SB C18 (150?mm?×?4.6?mm), 5-µm column thermostated at 30°C, mobile phase A (1?mL of trifluoroacetic acid in 1000?mL of water), mobile phase B (acetonitrile) in the ratio of 83:17?v/v at flow rate of 1.0?mL/min, and an injection volume of 10?µL. The analyte was monitored at a wavelength of 298?nm. The method was validated in accordance with the Food and Drug Administration (FDA) Veterinary International Conference on Harmonization guidelines. To demonstrate stability indicating ability of method, drug product was subjected to the stress condition of acidic, basic, humidity, thermal, oxidative, and photolytic degradation.     

Comparison of the response of four aerosol detectors used with ultra high pressure liquid chromatography

The responses of four different types of aerosol detectors have been evaluated and compared to establish their potential use as a universal detector in conjunction with ultra high pressure liquid chromatography (UHPLC). Two charged-aerosol detectors, namely Corona CAD and Corona Ultra, and also two different types of light-scattering detectors (an evaporative light scattering detector, and a nano-quantity analyte detector [NQAD]) were evaluated. The responses of these detectors were systematically investigated under changing experimental and instrumental parameters, such as the mobile phase flow-rate, analyte concentration, mobile phase composition, nebulizer temperature, evaporator temperature, evaporator gas flow-rate and instrumental signal filtering after detection. It was found that these parameters exerted non-linear effects on the responses of the aerosol detectors and must therefore be considered when designing analytical separation conditions, particularly when gradient elution is performed. Identical reversed-phase gradient separations were compared on all four aerosol detectors and further compared with UV detection at 200 nm. The aerosol detectors were able to detect all 11 analytes in a test set comprising species having a variety of physicochemical properties, whilst UV detection was applicable only to those analytes containing chromophores. The reproducibility of the detector response for 11 analytes over 10 consecutive separations was found to be approximately 5% for the charged-aerosol detectors and approximately 11% for the light-scattering detectors. The tested analytes included semi-volatile species which exhibited a more variable response on the aerosol detectors. Peak efficiencies were generally better on the aerosol detectors in comparison to UV detection and particularly so for the light-scattering detectors which exhibited efficiencies of around 110,000 plates per metre. Limits of detection were calculated using different mobile phase compositions and the NQAD detector was found to be the most sensitive (LOD of 10 ng/mL), followed by the Corona CAD (76 ng/mL), then UV detection at 200 nm (178 ng/mL) using an injection volume of 25 μL.     

Development and Validation of a High-Performance Liquid Chromatographic Determination of Ceftriaxone Sodium and Its Application to Drug Quality Control

Abstract

A reliable and sensitive RP-HPLC method was developed and validated for the quantitative estimation of ceftriaxone sodium (CFTZ) in pure drug and pharmaceutical dosage forms. The separation of ceftriaxone sodium was achieved on a Waters XTerra RP-18 (5 µm, 250 × 4.6 mm i.d.) column using photodiode array detector at 240 nm. The mobile phase consisted of 0.1 M triethylammoniumacetate–acetonitrile (60:40 v/v) mixture delivered at a flow rate of 1.0 ml/min. Accuracy, evaluated by means of the spike recovery method, was excellent, with percent recovery in the range 99.5–102% with precision in the range 0.3–1.2%.     

Simultaneous Determination of Maprotiline,Desipramine, and Moclobemide by Reversed-Phase High-Performance Liquid Chromatography and Statistical Optimization

Abstract

A method for separation of three antidepressants, maprotiline, desipramine, and moclobemide, by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated. To find optimal conditions and estimate the impact of individual parameters on the separation, a complete set of 2³ interdependent relationships of the mobile phase composition, temperature, and the volume flow rate were examined. Full separation of the investigated components from a laboratory mixture was achieved on a Supelcosil LC-18 (120 mm × 4.6 mm, 5 µm) column, using two solvent systems, 3% ammonium ion in water/ethanol and acetonitrile, and alternating isocratic gradient–isocratic elution modes. Relevance of the proposed method for therapeutic drug monitoring is anticipated.     

Identification of degradation impurities in aripiprazole oral solution using LC–MS and development of validated stability indicating method for assay and content of two preservatives by RP-HPLC

A simple and simultaneous reverse phase high-performance liquid chromatographic method was developed for the quantification of aripiprazole (ARI) and two preservatives, namely, methyl paraben and propyl paraben in ARI oral solution. The method was developed on ACE C18 (4.6?×?250?mm, 5?µm) column using gradient elution of 0.1% v/v trifluoroacetic acid in water and acetonitrile as mobile phase components. Flow rate of 1.0?mL/min and 30°C column temperature were used for the method at quantification wavelength of 254?nm. The developed method was validated in accordance with International Conference on Harmonization guideline for various parameters. Forced degradation study was conducted in acid, base, peroxide, heat, and light stress conditions. ARI was found to degrade in oxidation, acid hydrolysis, and heat while it was stable under the remaining conditions. Specificity of the method was verified using Photo Diode Array (PDA) detector by evaluating purity of peaks from degradation samples. Major degradation impurities formed during stress study were identified using liquid chromatography–mass spectrometry method. The present method was useful for determining the content of all the three main analytes present in the oral solution without interference from degradation impurities. The method was robust under the deliberately modified conditions.     

Development and application of a validated stability-indicating high-performance liquid chromatographic method using photodiode array detection for simultaneous determination of granisetron, methylparaben, propylparaben, sodium benzoate, and their main degradation products in oral pharmaceutical preparations

A simple, rapid, and sensitive RP-HPLC method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron), sodium benzoate, methylparaben, propylparaben, and 4-hydroxybenzoic acid (the main degradation product of parabens) in granisetron oral drops and solutions. The separation of the compounds was achieved within 8 min on a SymmetryShield RP18 column (100 x 4.6 mm id, 3.5 microm particle size) using the mobile phase acetonitrile--0.05 M KH2PO4 buffered to pH 3 using H3PO4 (3+7, v/v). The photodiode array detector was used to test the purity of the peaks, and the chromatograms were extracted at 240 nm. The method was validated, and validation acceptance criteria were met in all cases. The robust method was successfully applied to the determination of granisetron and preservatives, as well as their degradation products in different batches of granisetron oral drops and solutions. The method proved to be sensitive for determination down to 0.04% (w/w) of granisetron degradation product relative to granisetron and 0.03% (w/w) 4-hydroxybenzoic acid relative to total parabens.     

反相高效液相色谱法测定乳品及乳制品中的黄曲霉毒素M_1

采用反相高效液相色谱测定乳品及乳制品中黄曲霉毒素M_1(AFM_1)的含量。样品经氯仿提取,过硅胶固相萃取柱净化,用氯仿-丙酮(1+1)混合溶液将黄曲霉毒素M_1从固相萃取柱上洗脱下来。以ZORBAX SB C_(18)色谱柱为分离柱,水和乙腈为流动相梯度淋洗,用荧光检测器检测,外标法定量。黄曲霉毒素M_1在1.0～25μg·L~(-1)质量浓度范围内与其峰面积呈线性关系,检出限(3S/N)为0.05μg·kg~(-1)。应用此法测定了牛奶和乳粉中AFM_1的含量,并测得其平均回收率分别在76.0%～80.0%和76.7%～90.8%之间,相对标准偏差(n=6)均小于7.0%。     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

Determination of gemifloxacin on dried blood spots by hydrophilic interaction liquid chromatography with fluorescence detector: application to pharmacokinetics in rats

A highly selective, sensitive and rapid hydrophilic liquid interaction chromatographic method was developed and validated for determination of gemifloxacin on dried blood spots. The chromatographic separation was achieved on a reversed‐phase zwitterionic hydrophilic interaction liquid chromatographic ZIC®HILIC‐C₁₈ (4.6 × 100 mm; 5 µm) column using acetonitrile–10 mM ammonium acetate (pH 3.5; 80:20, v/v) as a mobile phase in an isocratic elution mode at a flow rate 0.6 mL/min at 27 °C. An on‐line fluorescence detector set at excitation and emission wavelengths of 269 and 393 nm, respectively was used for monitoring column eluents. Ciprofloxacin was used as an internal standard. The method was validated for accuracy, precision, linearity and selectivity by design of experiments following ICH guidelines. The assay exhibited a linear range of 25–5000 ng/mL for gemifloxacin on dried blood spots. The lower limit of detection was found to be 10 ng/mL. The intra‐ and inter‐assay coefficients of variation did not exceed 7.4% deviation of the nominal concentration. The recovery of GFX from dried blood spots was >95.0% and its stability was excellent with no evidence of degradation during sample processing for at least 3 months storage in a freezer at ?20 °C. Copyright © 2012 John Wiley & Sons, Ltd.     

Synchronized stability indicating RP-LC methods for determination of metolazone with losartan potassium or spironolactone in presence of their degradation products

Abstract

Two precise and selective stability-indicating RP-LC methods have been developed and validated for simultaneous determination of metolazone in its binary mixture with losartan potassium (method 1) and spironolactone (method 2) in the presence of their degradation products. For method 1, the chromatographic separation was achieved on Kromasil C₁₈ column, the mobile phase consisted of a mixture of 0.1% ortho-phosphoric acid in acetonitrile and 0.1% ortho-phosphoric acid in water (28:72, v/v) pumped at flow rate 2?mL/min and UV detection at 235?nm. Linearity was determined over the concentration range of 2–16µg/mL for metolazone and 40–320µg/mL for losartan potassium. For method 2, chromatographic separation of metolazone and spironolactone was achieved on a Symmetry C₈ column using a mobile phase that consisted of acetonitrile, methanol, and 0.1% ortho-phosphoric in water in gradient mode pumped at a flow rate 1.5?mL/min with programed wavelength detection. Linearity was determined over the concentration range of 2–16µg/mL for metolazone and 20–160µg/mL for spironolactone. The suggested methods were proved to be highly selective, precise and accurate for simultaneous determination of the cited drugs in their combined pharmaceutical dosage form in the presence of their degradation products. The proposed methods were validated in compliance with ICH guidelines.

1. Highlights

2. Synchronized determination of metolazone and co-formulated drugs in presence of their degradation products.

3. Act as a method for screening of metolazone and co-formulated drugs in quality control laboratories.

4. Validation of suggested methods according to ICH guidelines.

5. The pathway of degradation of metolazone under different stress conditions was proposed.

     

Isolation and characterization of process related impurities of olanzapine using HPLC and ESI-MS/MS

An RP-HPLC method for the simultaneous separation and determination of olanzapine (OLZ) and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on Inertsil ODS 3V (4.6 mm x 250 mm; particle size 5 microm) column using 0.2 M ammonium acetate (pH = 4.50) and ACN as mobile phase in gradient elution mode. The analytes were monitored by a photo diode array (PDA) detector set at 254 nm and the flow rate was kept at 1.0 mL/min. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. Four unknown process impurities observed consistently during the analysis of different batches of OLZ were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR. The proposed RP-HPLC method was successfully applied to the analysis of commercial formulations. The method could be of use not only for rapid and routine evaluation of the quality of OLZ in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations.     

Improving the universal response of evaporative light scattering detection by mobile phase compensation

Mobile phase compensation, first reported for the charged aerosol detector (CAD), was used as a suitable method to overcome problems related to the mobile phase-dependent response of the evaporative light scattering detector (ELSD). Mobile phase compensation was effectively performed both in the flow injection- and in gradient modes. Without compensation, the response factors of the ELSD for six sulfonamide drugs differed by a factor of two when varying the mobile phase composition between 10 and 90% acetonitrile. This change could be effectively eliminated using the technique of mobile phase compensation, where a secondary pump with a reversed gradient was used to provide the detector with a constant composition of the mobile phase. For identical experimental conditions, the ELSD showed a nearly constant, albeit somewhat reduced, response with compensation. This indicates that under such conditions, the ELSD behaved as a concentration-sensitive detector. The analysis of sulfonamides drugs at 0.05% level using gradient UPLC-ELSD separation with mobile phase compensation is demonstrated.