Isocratic HPLC Method Research Articles

Determination of Mesalamine in Bulk and Suppository Dosage Forms through the Development and Validation of Stability- indicating RP-HPLC Method

Background: In the current study, a simple and cost-effective stability-indicating RP-HPLC method was developed and validated to estimate the mesalamine from both bulk and pharmaceutical dosage forms. Methods: An isocratic HPLC method using a reverse phase HiQSilC18 column (250 x 4.6 mm, 5μm) and a mobile phase methanol: ammonium acetate buffer (90:10 v/v) were employed as the mobile phase with a flow rate of 1 mL/min at 25°C. Detection was carried out at 305 nm, and the injection volume was 20μl. The developed method was validated as per ICH Q2 guidelines. Mesalamine has been subjected to various stress testing conditions, such as hydrolysis of acid and base, thermal degradation, oxidation, and photolysis. Also, methods have been validated with regard to linearity, accuracy, precision, and robustness. Results: The RT of mesalamine was determined to be 3.550 min ± 0.024 minutes, providing a reliable marker for its identification. The method was found to be linear between 5-30 μg/mL concentration with (R²) of 0.994. This demonstrated the method's ability to measure varying concentrations of mesalamine accurately. Additionally, the percentage recovery of mesalamine was approximately 100%, confirming the accuracy of the developed method. The parameters for system suitability have also been found to be within acceptable limits. Force degradation studies reinforced the method's selectivity and sensitivity in detecting mesalamine under various degradation scenarios. Notably, mesalamine significantly degraded in an acidic environment. Conclusion: In conclusion, our proposed RP-HPLC method provides a sensitive, accurate, and precise means of analyzing mesalamine in both bulk and pharmaceutical dosage forms.

Validation of an Isocratic HPLC Method for Simultaneous Estimation of Major Phytosterols in Prunus spinosa L. extracts.

This study aimed to develop a rapid method for separation of stigmasterol, campesterol and β-sitosterol in Prunus spinosa L. (sloe) fruit extracts by High Performance Liquid Chromatography system. Samples were prepared by Soxhlet extraction method and separated on a high strength silica C18 column using acetonitrile-methanol mobile phase and Photodiode Array Detector. The optimized method resulted in a linear calibration curve ranging from 1.7 ng mL-1 to 130 ng mL-1 for all three phytosterols. Analyses of internal and external phytosterol standards showed good linearity (R2 of 0.998 to 0.999); LOD and LOQ were determined to be 2.33×10-7-2.18×10-4 and 7.07×10-7-6.60×10-4 mg mL-1, respectively. Repeatability and reproducibility precision analyses showed acceptable values of RSD %. β-sitosterol was the predominant phytosterol (51.53-81.03 % of total) among all samples. Method validation parameters indicated that this analytical method can be applied for accurate and precise determination of campesterol, stigmasterol and β-sitosterol, in selected extracts.

Two chromatographic methods for determination of cytarabine and dexamethazone; two co-administered drugs for the treatment of leukemia: green analytical perspective

Two sensitive, straightforward and repeatable chromatographic techniques were developed for the determination of Cytarabine HCl and Dexamethazone in their pure form and spiked human plasma without prior separation. The drugs are used co-administered for the treatment of Leukemia, a certain type of blood cancer. Method (A) is an isocratic chromatographic HPLC method; separation was accomplished on C18 column using the eluting mixture of 6.9 g/L Monobasic Sodium Phosphate pH 3: methanol (70:30, v/v) and detection was at 275 nm. Concentrations were in the range of 0.2–15 μg/mL for both CYT and DEX. Method (B) is a HPTLC method in which separation was attained on HPTLC F254 plates using methanol: ethyl acetate: ammonia, (7.8:2:0.2, by volume) as eluting solvents and detection was at 275 nm. Concentrations were in the range of 0.1–4 μg/band for both CYT and DEX. The parameters for system suitability testing were evaluated to determine the effectiveness of the developed chromatographic procedures in terms of performance. The recently developed techniques were applied for the determination of the drugs under investigation in spiked human plasma. Validation parameters were examined in accordance with US-FDA criteria. All results were found to be within the acceptable ranges. To evaluate the greenness characters of the proposed methods to the environment; three greenness assessment tools including eco-scale assessments (ESA), green analytical procedure index (GAPI), and Analytical Greenness calculator (AGREE) were used. Acceptable and satisfying results that demonstrated the greenness characteristics of the suggested methods were attained.

Development and Validation of Novel HPLC Methods for Quantitative Determination of Vitamin D3 in Tablet Dosage Form.

In the present work, an efficient isocratic HPLC method was developed for the precise and accurate estimation of vitamin D3 in tablet form. The chromatographic conditions comprised an L3 silica column (5 µm in particle size, 4.6 mm × 250 mm) with a mobile phase n-hexane/ethyl acetate (85:15 v/v) with a flow rate of 2.0 mL/min and a detection wavelength of 292 nm. The new methodology was validated for accuracy, precision, specificity, robustness, and quantification limits according to an official monograph of USP/BP and ICH guidelines. The peak areas of the six replicates of the homogeneous sample were recorded. The mean value obtained was 67,301, and the relative standard deviation (RSD) was 0.1741. The linearity and range were in the acceptable bounds, i.e., 0.999, which was calculated using regression line analysis. The results show that the method is truly acceptable as the RSD, as the flow rate was 0.81%, while for the mobile phase composition, it was 0.72%, which lies in the acceptable range. The limit of detection (LOD) and the limit of quantification (LOQ) values were 0.0539 µg/mL and 0.1633 µg/mL, respectively. The % RSD of the intra and inter-day precision of the method was deemed acceptable according to the international commission for harmonization guidelines. The developed method has potential to be used for the detection and quantification of vitamin D3 during routine analysis for tablets in dosage form.

Simultaneous determination of four non paraben preservatives (benzyl alcohol, phenoxyethanol, sodium benzoate, and salicylic acid) in shampoos by an isocratic HPLC method

Salicylic acid (SA), sodium benzoate (SB), phenoxyethanol (PO), and benzyl alcohol (BA) are “non paraben” preservatives commonly used in cosmetic products like shampoos, and often in combination of at least two compounds to assure their antimicrobial effectiveness at low concentrations. In this study, an HPLC-DAD method was developed and validated for simultaneous determination of SA, SB, PO, and BA in commercial shampoos. The method was carried out using an YMC Triart C18 (250 × 4.6 mm, 5 µm) column with a mixture of methanol and 0.02 M solution of phosphate buffer pH 2.7 (35 : 65, v/v) as mobile phase. The flow rate of mobile phase was maintained at 1.0 mL per minute, the injection volume was 20 µL and the UV detection was done at 208 nm for BA and at 230 nm for SA, SB and PO. The method was fully validated according to requirements on analytical method performance of AOAC International and validation results confirmed its reliability and suitability for intended application. The method was employed to determine the four “non paraben” preservatives in 18 commercial shampoos, finding the actual content of these substances in all samples conformed to their regulatory limits.

Development and Validation of a Method for Trace Level Zinc Quantification in Pharmaceutical Zinc Supplements Using a Carboxyl Functional Group Packed Column and Refractive Index Detector.

Zinc helps with cell division, growth, wound healing, and carbohydrate breakdown. Humanbeings have to obtain zinc from food or supplements because our bodies do not produce it naturally. In view of the greater advantages (such as low cost, time of analysis, and stability-indicating) compared to other quantification methods (titration, ion chromatography, Atomic absorption spectroscopy) proposed in the literature, a refractive index detector coupled with HPLC has been used in quantification of zinc. The goal of this research is to develop and validate a sensitive, low-cost, high-resolution, and stability- indicating method for detecting and quantifying zinc levels in zinc supplement pharmaceutical products (injectables, tablets, and capsules). A novel isocratic reverse-phase HPLC method with a refractive index detector (with sensitivity 64, detector temperature 35°C, and positive polarity) was developed using a carboxyl functional group packed column and 0.8% (v/v) formic acid as the mobile phase to detect and quantify zinc content. The reported method has a good optimal sensitivity (LOQ: 0.006 mg/mL and LOD: 0.002 mg/mL). The correlation coefficient (r) obtained from the zinc calibration plot was greater than 0.998, indicating that the method was linear and that there was a strong correlation between zinc concentration (0.006 mg/mL to 0.375 mg/mL) and peak response. The accuracy at LOQ level was found to be 95-105% and 97-103% at the remaining levels (50, 100, and 150%). The proposed method was successfully developed and validated as per International council for harmonisation (ICH) guidelines. Therefore, this method can be used for the quantitative testing of zinc in the QC laboratory. A novel method was developed for zinc levels determination in pharmaceutical products using HPLC with a refractive index detector. The present approach has a quick run time of 10 min and is inexpensive.

QUANTITATIVE AND QUALITATIVE ANALYSIS OF STEVIOL GLYCOSIDE IN STEVIA BY VARIOUS ANALYTICAL METHODS: A REVIEW

Stevia rebaudiana, a perennial herb in the family Asteraceae, is a popular natural sweetener with sensory and functional attributes that outperform those of conventional high-potency sweeteners. Stevia rebaudiana leaves contain sweet glycosides that have biological effects. The various glycosides included in extracts must be measured since, in addition to the major glycosides, they may also contribute to activity. The isocratic HPLC method was validated for the identification of dulcoside A, steviolbioside, rebaudioside C, and rebaudioside B. The development and validation of the HPTLC technique for the simultaneous measurement of three steviol glycosides in Stevia rebaudiana leaves-steviolbioside, stevioside, and rebaudioside-A-also employed the ICH (International Conferences on Harmonisation) guidelines.

Application of Box-Behnken Design and Response Surface Methodology for Selecting the Optimum RP-HPLC Conditions for the Simultaneous Determination of Paracetamol and Diclofenac Sodium Along With Three Skeletal Muscle Relaxants in Three Different Pharmaceutical Dosage Forms.

An isocratic HPLC method has been developed and validated for estimating paracetamol and diclofenac sodium simultaneously with three skeletal muscle relaxants, namely, methocarbamol, tizanidine hydrochloride and chlorzoxazone in their pure standard mixtures and in different multi-component dosage forms in a single chromatographic run. HPLC separation was achieved on a C18 Inertsil ODS-3V 5μm column (250 × 4.6mm) using a mobile phase mixture containing acetonitrile and 25mM phosphate buffer (pH7.4 adjusted with NaOH) in the proportion of (39.7:60.3, v/v) pumped at 1.2mL/min flow rate with UV detection at 220nm. An experimental design was used by applying Plackett-Burman design for screening the most critical predictors affecting the chromatographic separation and Box-Behnken design for optimizing the selected predictors and creating the response surface between the selected predictors and the interested responses. ICH recommendations were applied for validating the proposed method with regard to linearity, precision, accuracy, selectivity, limits of detection and quantitation, and robustness. There are many applications for the optimized method that can be applied for routine estimation of the cited drugs in laboratories of quality control and pharmaceutical industries to save money and time and to reduce material waste and effort.

A simple and reliable isocratic high-performance chromatographic assay for the simultaneous determination of hydrophilic benzophenone-4 and lipophilic octocrylene in sunscreens.

This work aimed to develop a simple HPLC method for the simultaneous quantitative determination of the ultraviolet (UV) filters, hydrophilic benzophenone-4 and lipophilic octocrylene, in the presence of three other commonly used UV filters, avobenzone, octisalate and homosalate. Reverse-phased HPLC was performed on a C18 column. A scouting gradient was initially used to determine the approximate mobile phase composition required for efficient analyte elution and separation before further optimization. The assay was validated with regard to specificity, linearity, intra- and inter-day accuracy and precision, limits of detection and limits of quantification. An ultrasound dispersion extraction method for the UV filters from a commercial sunscreen was developed, and the extraction efficiencies from spiked samples were calculated. An acetonitrile-methanol-water mixture (20:67:13, v/v/v), where the water component contained 0.2% trifluoroacetic acid (v/v), was found to be the optimal mobile phase at a flow rate of 1.0 mL/min. The assay was linear between 1.0-100 μg/mL for both benzophenone-4 and octocrylene (both correlation coefficients were above 0.999). There was no interference from the excipients of the sunscreen nor from the three other UV filters. The intra- and inter-day accuracy was between 90.0-104.6% for both analytes. Extraction recoveries from a spiked commercial sunscreen were between 95.4 ± 2.1% to 98.5 ± 2.1% for benzophenone-4, and between 87.3 ± 2.3% and 98.9 ± 3.1% for octocrylene. All validation parameters were within the acceptance criteria set out in the International Council for Harmonization (ICH) guidelines. The HPLC assay showed the extracted quantities of benzophenone-4 and octocrylene from the commercial sunscreen closely matched claimed quantities. The developed isocratic HPLC method was suitable for simultaneously determining the hydrophilic benzophenone-4 and lipophilic octocrylene in the presence of other commonly used UV filters. Additionally, the extraction method was simple and effective for accurately quantifying the UV filters in a commercial sunscreen.

A new simple liquid chromatographic assay for gentamicin in presence of methylparaben and propylparaben

AbstractGentamicin sulfate is a potent broad spectrum aminoglycoside antibiotic which is used against Gram-positive and Gram-negative bacteria. A simple, isocratic HPLC method for separation, identification and determination of gentamicin and parabens (methylparaben and propylparaben) was developed and validated. To our knowledge there is no report about simultaneous determination of those three analytes in pharmaceutical products. The optimum chromatographic conditions were achieved on CN column with a mobile phase consisting of 0.15% triethylamine in 10 mM KH2PO4 aqueous solution (final pH 3.0 adjusted with H3PO4) and methanol in the ratio 70:30 (v/v), providing selective quantification of analytes within 5 min. The method was successfully validated according to ICH guidelines acceptance criteria in terms of selectivity, linearity, accuracy, precision and robustness. The linearity of the method was proved in defined concentrations ranges for gentamicin (0.32–1.04 mg mL−1), methylparaben (0.0072–0.0234 mg mL−1) and propylparaben (0.0008–0.0026 mg mL−1). Relative standard deviations calculated for all analytes in precision testing were <2% (analysis repeatability) and <3% (intermediate precision). Recovery values were between 98.87% and 101.67%. Chromatographic parameters are not significantly influenced by small variations of column temperature, pH and molarity of KH2PO4. Finally, the method was successfully applied for quantitative determination of gentamicin and parabens in commercially available solution for injection. Proposed HPLC method is found to be promising in terms of simplicity, analysis times and non-use of derivatization and ion-pair agents.

Stability Indicating Isocratic HPLC Method for Bilastine and Characterization of Forced Degradation Products by LC-MS/MS

Abstract: The present study aims to develop and validate a simple, precise, accurate, stability indicating and isocratic reversephase high-performance liquid chromatography (RP-HPLC) method for estimating Bilastine in bulk and synthetic mixture. Bilastine from its degradation products were well separated and estimated on Discovery C8 column (250 mm x 4.6 mm, 5μm) using methanol: 0.1% ortho-phosphoric acid (55:45 %v/v) as a mobile phase and detection was performed at 276 nm by PDA detector. The degradation of Bilastine was studied under different ICH recommended stress conditions. The developed stability-indicating method was validated for system suitability, linearity, accuracy, precision, robustness, detection limit and quantitation as per ICH guidelines. The method was linear over 25-150 μg/ml. Mathematically computed Limit of Detection and Limit of Quantitation were found to be 0.19μg/ml and 0.57μg/ml, respectively. It was discovered that Bilastine degrades under acid and oxidation conditions. By using LC-MS/MS analysis, the structure of Bilastine breakdown products created amid acidic and oxidative settings was elucidated. A complete fragmentation pathway of Bilastine was established to elucidate the degradation products' structures using LC–MS/MS. The elemental concentrations of oxidation products and their fragmentation products were studied using the acquired mass values. The overall data was utilized to describe the degradation of products and their fragmentation process. The developed method can be used for routine analysis of Bilastine in dosage form as it can estimate of Bilastine in presence of excipients and degradation products.

Dietary supplement mislabelling: case study on selected slimming products by developing a green isocratic HPLC method for their quality control

Nowadays, a huge population consumes Dietary supplements for losing weight. Products are often claimed as botanical blends, yet they aren't necessarily safe. Misleading labels are also very common. Thus, validated analytical methods for a wide range of slimming compounds are highly needed. Herein, we present a simple HPLC/PDA method for the quantitation of seven popular slimming ingredients. Studied compounds were Caffeine, Raspberry Ketone, trans-Resveratrol, p-Synephrine, p-Octopamine, p-Hordenine and 2-phenethylamine. After optimization, separation was carried out on a C18 column and mobile phase was a mixture of Acetonitrile:Water containing 0.1% phosphoric acid (50:50, %v/v). The last compound was eluted at 9.76 min. Separation was efficient showing baseline- separated symmetric peaks, without using any gradient programs, organic mobile phase modifiers or modified stationary phases. Method validation was done following ICH guidelines. Calibration curves were linear over wide concentration ranges and calculated LOD values were in the range 0.02–0.09 µg/mL. Method greenness was assessed using Analytical Eco-scale, GAPI and AGREE metric tools. Further, four random sample products purchased from online supplement stores were assayed. Results proved some mislabeling actions. To support our findings, standard addition was carried out and average % recoveries were 96.67 – 101.44% with standard deviation ≤ 2.83 between measurements.

A Rapid and Sensitive stability indicating Rp-HPLC method development for the quantitative analysis of empagliflozin & linagliptin in bulk & synthetic mixture

An isocratic HPLC method was developed using, Shimadzu C18 column (250 mm × 4.6 mm, 5 µm) with an isocratic binary mobile phase consisting of Acetonitrile: Buffer in a ratio (80:20 v/v) pH 3.0 adjusted with Orthophosphoric acid and flow rate monitored at 0.80 ml/min. The UV detector was used for simultaneous analysis of two drugs at a common wavelength of 226 nm and each injection volume was 20 µl. The retention time for Empagliflozin and Linagliptin was found to be 3.714 min and 3.064 min, respectively. Empagliflozin and Linagliptin produce degradation products in acidic, alkaline, thermal, and UV stress. The result of the assay of Empagliflozin and Linagliptin shows that the degradation product does not interfere with the analytical procedure quantitively when these drugs are analyzed.

Box–Behnken experimental design for optimizing the HPLC method to determine hydrochlorothiazide in pharmaceutical formulations and biological fluid

An isocratic HPLC method was developed to quantify hydrochlorothiazide (HCT) and its impurities (A, B, C). Response surface methodology combined Box-Behnken design was applied to optimize the effects of four independent factors; pH (2–4), acetonitrile (2–15%), methanol (2–15%) and flow rate (0.7–1.3 mL min−1) against the response. The current investigation aimed to choose the utmost significant factors with the maximum influences of chromatographic separation: resolution, tailing factor, and the number of theoretical plates. Statistical analysis of variance (ANOVA) indicated the best conditions for separating the compounds and the most influential parameters. The plots (contour, 3D surface and perturbation) described the statistically significant parameters quantitatively and their interactions. The experimentally derived RSM model was validated using a t-test with observed R2 value, adjusted R2, predicted R2 and F-values, indicating that the developed model was significant. The proposed LC was combined with a PDA detector and detected at 210 nm. The HCT highest separation was achieved on the ODS Hypersil C18 (250 mm × 4.6 mm, 5 μm) column using the following conditions: pH (3.497), acetonitrile (10.6%), methanol (16.2%), flow rate 1 mL/min. The method validated was precise and linear in 1.25–12.75 µg mL−1. The validated method's value of LOD & LOQ was 1.09 and 3.33 µg mL−1, respectively. Youden and Steiner’s factorial design with typical and alternative conditions were performed to verify its robustness study. The % RSD values were within the accepted range of ICH guidelines. Therefore, the proposed method can be applied for routine analysis of HCT and its impurities in pharmaceutical formulation and biological fluid for routine quality control sample analysis.

Development and Validation of a Stability-indicating HPLC Method for Determination of Insulin Detemir and Preservatives in their Injection Products

Insulin detemir (ID) is a long-acting form of insulin that is characterized by the covalent attachment of a lipophilic tail of myristate, and commercially available as Levemir®. No satisfactory simple isocratic stability-indicating HPLC method has been reported for its quality control. A novel simple and isocratic reversed-phase HPLC method was developed and validated for the simultaneous determination of ID along with its dosage form additives in the available commercial preparations. The method employed C4 column (5μm, 250 × 4.6 mm), a mobile phase consisting of 50 mM phosphate buffer pH 2.7, acetonitrile, triethylamine (62:37:1) and 0.02 g/ml sodium sulfate, that was delivered isocraticaly at a flow rate of 1.5 ml/min, and detection performed at 214nm. The method was properly validated and was shown linear over the range 80–120% of the assay concentration for ID, phenol, and m-cresol. The method was also selective, specific, precise, and accurate. Furthermore, the validated method was applied to separate the major degradation products in those preparations. Forced degradation studies in different pH values, which are the first to be reported for ID, showed that the degradation products were baseline separated from ID itself and/or other formulation additives. Thus, the method has been demonstrated to be a stability-indicating assay.

Quantitation of Ciprofloxacin by RP-HPLC from Active and Dosage Formulations

A simple, sensitive isocratic reversed-phase HPLC method for detecting and quantifying ciprofloxacin in pharmaceutical dosage forms has been developed and validated according to ICH guidelines. In the method, the separation was achieved on a C18 reversed-phase column with a mobile phase acetonitrile-water at a ratio of (80:20). The pH was adjusted to 2.7 using 85% of phosphoric acid with ultraviolet detection at 275.0 nm. A flow rate of 1.7 mL/minute was used at room temperature. The method'

Effect of experimental hyperglycemia on intestinal elimination and biliary excretion of ibuprofen enantiomers in hyperglycemic rats

Diabetic complications are mostly due to hyperglycemia. Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the activities of drug-metabolizing enzymes and membrane-integrated transporters, which can modify the fate of drugs and other xenobiotics. An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion of ibuprofen enantiomers in the rat. Experimental diabetes was induced by intravenous (i.v.) administration of streptozotocin. The intestinal perfusion medium contained 250 µM racemic ibuprofen. A validated isocratic chiral HPLC method with UV detection was developed to determine the amount of the two enantiomers in the intestinal perfusate and the bile. The results indicated that experimental diabetes doesn’t cause a statistically significant difference in the disappearance of ibuprofen enantiomers from the small intestine. Analysis of the bile samples detected only the (S)-IBP enantiomer. Excretion of the ibuprofen enantiomer to the bile decreased in experimental diabetes. The observed changes can affect the pharmacokinetics of drugs administered in hyperglycemic individuals.

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cyclosporine A in Combination with α-Linolenic Acid

A suitable isocratic reverse-phase HPLC method was developed for the simultaneous estimation of cyclosporine A and α-linolenic acid (ALA) in their synthetic mixture formulated to evaluate the synergistic therapeutic effect. The reversed phase HPLC chromatographic method was developed to obtain two separate peaks within short retention time period (30 min run time), without any interference from excipients peaks. The chromatographic separation was achieved on C18 column (5 μm, 4.6 × 250 mm) at 50 ± 0.3 ºC. The analytes were eluted at a flow rate of 0.8 mL/min using ultraviolet detector at 210 nm with mobile phase of 1% v/v orthophosphoric acid in water: acetonitrile:2-propanol (25:60:25 v/v/v). The proposed method was validated for specificity, precision, linearity, accuracy and system suitability. All validation parameters were within the acceptable range.

Dietary Supplement Mislabelling: A Case Study on Selected Slimming Products by Developing a Green Isocratic Hplc Method for Their Quality Control

Dietary Supplement Mislabelling: A Case Study on Selected Slimming Products by Developing a Green Isocratic Hplc Method for Their Quality Control

Monitoring of Gold Biodistribution from Nanoparticles Using a HPLC-Visible Method

There is intensive research using gold nanoparticles for biomedical purposes, which have many advantages such as ease of synthesis and high reactivity. Their possible small size (<10 nm) can lead to the crossing of biological membranes and then to problematic dissemination and storage in organs that must be controlled and evaluated. In this work, a simple isocratic HPLC method was developed and validated to quantify the gold coming from nanoparticles in different biological samples. After a first carbonization step at 900 °C, the nanoparticles were oxidized by dibroma under acidic conditions, leading to tetrachloroaurate ions that could form ion pairs when adding rhodamine B. Finally, ion pairs were extracted and rhodamine B was evaluated to quantify the corresponding gold concentration by reversed-phase HPLC with visible detection. The method was validated for different organs (liver, spleen, lungs, kidneys, or brain) and fluids (plasma and urine) from rats and mice. Lastly, the developed method was used to evaluate the content of gold in organs and fluids after intravenous (IV) injection of nanoparticles.