Validated HPLC Method Research Articles

Development and validation of HPLC method for simultaneous determination of Leflunomide and folic acid in the nanoparticulate system by reversed-phase HPLC

Objective The main objective of this study was to develop a highly sensitive, accurate, and reproducible analytical method for the simultaneous detection of LEF and FA in polymeric nanocarriers. Significance Leflunomide (LEF), is widely employed in the treatment of rheumatoid arthritis (RA). However, long-term delivery of the drug is associated with systemic side effects. Therefore, folate (FA) conjugated LEF nanocarriers were fabricated for targeting the nanocarriers toward activated macrophages. HPLC is considered one of the most sensitive and precise analytical techniques for the simultaneous detection and estimation of different components in a particular sample. Methods Analysis was performed on HPLC (Shimadzu 10 A), having a reversed-phase C-18 column (Beckmen, 250 X 4.6 mm, 5 µm) equipped with a photodiode detector set at a wavelength of 260 nm (LEF) and 285 nm (Folic acid). The isocratic mobile phase was composed of acetonitrile, water, and trimethylamine in a ratio of 65:35:0.5 at pH 4. Rapid analysis of both agents was performed, with a total run time of 10 min (FA = 2.1 ± 0.1 min, LEF = 5.9 ± 1 min) at a 1 mL/min flow rate. Results The assay demonstrated good linearity of 0.9989 of 0.9997 for LEF and FA respectively with a recovery in the range of 95–100%. The method also depicted good specificity, and intra and inter-day precision based on relative standard deviation (RSD) values. Conclusions The study concludes, that the developed method was helpful in the detection and quantitation of lower values of both agents from polymeric nanocarriers.

Evaluation of Bioenhancing Effect of Piperine and its Analogs on the Pharmacokinetics of Verapamil

A present study was undertaken to understand the impact of piperine and some of its derivatives on the bioavailability of verapamil used in the treatment of cardiac disorders. On oral administration, it undergoes fast biotransformation resulting in its low bioavailability. Its bioavailability varies between 20 and 35%. Piperine (a well-known bioenhancer) has been reported to improve the bioavailability of a wide range of structurally and therapeutically varied medicines. Hence some derivatives of piperine were synthesized and evaluated for their bioenhancing effect on verapamil. The bioenhancing effect of piperine and synthesized derivatives was investigated and compared using a validated HPLC method per USFDA guidelines. The pharmacokinetic characteristics of verapamil alone and when combined with piperine and its synthetic analogs were also investigated. When administered to wistar rats, it was discovered that verapamil bioavailability was 1.54 times higher when given with piperine and 2.42 times higher when given with morpholine derivative. The study undertaken indicates that some synthetic derivatives of piperine can be successfully used to enhance the bioavailability of verapamil to a considerable extent

Kinetic studies of fluorocurcumin derivatives with anticancer activity

Two novel fluorocurcumin adducts with a difluoroboryl group exhibited activity in vitro against two bladder cancer cell lines, namely 5637 and SCaBER. The stability was evaluated under physiologically relevant conditions to mimic both the digestive tract and the bladder environment. Therefore, experiments were performed in hydrochloric acid at pH 1.2, and in buffer solutions with pH from 4.5 to 6.8 at 37°C. A validated HPLC method, allowing the monitoring of changes in substrates and products concentrations over time, was applied. Notably, the main decomposition product was the fluorocurcumin derivative without the difluoroboryl group. The decomposition of the fluorocurcumin adducts to the major products undergoes the pseudo-first-order kinetics reaction.

Development and validation of HPLC method for simultaneous determination of amlodipine besylate, telmisartan and hydrochlorothiazide in combined tablets

A combination of three active ingredients amlodipine besylate, telmisartan and hydrochlorothiazide has been used and proven effective in the treatment of hypertension in patients with moderate to severe hypertension, while it contributes to a reduction in product costs and increases patient compliance. However, there are no monographs in pharmacopoeias for the simultaneous quantification of these three active ingredients. Therefore, in order to contribute to the establishment of an in-house specification for domestic generic drugs, shorten the time of quantifying each ingredient separately, the study was carried out with the aim of developing and validating procedure for the simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide by high performance liquid chromatography. In this study, we conducted investigation into different chromatographic conditions, developed an analytical procedure for the simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide in tablets preparation. The procedure was validated in compliance with ICH guidelines. Suitable chromatographic conditions for simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide are: Xterra C18 column (250 x 4.6 mm, 5 μm), detection wavelength 230 nm, gradient program of mobile phase: acetonitrile and phosphate buffer pH 3.0, flow rate 1.0 ml/min, sample injection volume 10 μl, column temperature 30°C. The procedure was validated for systematic suitability, accuracy, specificity, linearity, precision, working range and robustness. A procedure for the simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide in tablets by high performance liquid chromatography has been successfully developed and validated.

Development and Validation of HPLC Method for Estimation of Some Active Analytes in Combined Dosage form an Application to In-vitro Dissolution Studies

This paper describes a new, simple, accurate and economical method of development and validation of HPLC method for the assessment of dicyclomine hydrochloride with omeprazol magnesium combine dosage form. The method development trial was carried out initially using C8 (100×0.46×3.5 μm) and C18 GraceSmart (250×4.6×5 μm) as stationary phase and acetate buffer (pH 4.5) and methanol as mobile phase in proportion 60:40v/v. The optimized conditions of factors were sample size 50 μL and wavelength (λmax) 215 nm. The developed method was then applied to in-vitro dissolution studies for the dicyclomine hydrochloride and omeprazole magnesium. As per ICH guidelines, stability testing was done. The estimated method can be used to analyze the pharma industries’ products.

HPLC Method Validation for Quantification of Lisinopril

This study aimed to develop a straightforward, sensitive, exact, quick, and accurate reverse phase high performance liquid chromatography (RP-HPLC) method for figuring out how much lisinopril is in pharmaceutical gels and other large amounts of medication. Agilent Zorbax Bonus-RP column (250 x 4.6 mm, 5μ) was used for the chromatographic separation. “A mobile phase composed of methanol and trifluoroacetic acid (50:50 v/v) was used to develop the analytical procedure. The flow was found to be occurring at a rate of 1-mL/min and with a wavelength of 215 nm. The retention time was 2.28 min. In a concentration range from 3–7 μg/mL (r2=0.998), the drug’s response was determined to be linear. The LoQ was 1.11 μg/mL, while the LoD was 0.36 μg/mL. Lisinopril’s %assay was determined to be 98.22%, while assays for the other medicines in the commercial formulation showed no interference from the excipients. This method functions well and can be applied to routine analysis.

A Different Perspective on the Characterization of a New Degradation Product of Flibanserin with HPLC-DAD-ESI-IT-TOF-MSn and its Pharmaceutical Formulation Analysis with Inter-Laboratory Comparison.

Flibanserin (FLB) was firstly synthesized as an antidepressant drug; however, due to its enhancing effects on sexual activity, it was approved for treatment of hypoactive sexual desire disorder in women in 2015. The aim of this study was to develop a new and fully validated HPLC method for analysis of FLB in pharmaceutical formulations besides its degradation products, and identification of possible formation mechanisms by using HPLC-DAD-ESI-IT-TOF-MSn. The HPLC separation was achieved in an Supelco Ascentis® Express series phenyl hexyl column (100 × 4.6 mm, ID 2.7µm). The mobile phase was acetonitrile-ammonium acetate solution (50:50, v/v, 10 mM, pH 5.4) mixture, which was pumped at the rate of 0.5 mL/min. Chromatography, detection and structural identification was performed by using LCMS-IT-TOF instrument (Shimadzu, Japan). 1-(2-(4-(3-hydroxy-5-(trifluoromethyl)phenyl)piperazine-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one is proposed as a novel degradation product, with a mass of 407.1695, and a formula of C20H21F3N4O2 with a margin of error about 0.001 ppm. The developed method is applicable with 98% accuracy within 2.5-50.0 µg/mL range. The LOD and LOQ was about 500 ng/mL and 1.50 µg/mL, respectively. The transferability and variation between laboratories were tested by inter-laboratory comparison and evaluated with one-way analysis of variance. A novel FLB degradation product, which was produced under oxidative forced degradation condition was observed and identified for the first time; in addition, the formation kinetics of the degradation product, besides decomposition of FLB was studied. Furthermore, an inter-laboratory comparison was carried out and application of the proposed method on pseudo Addyi® sample was tested using both instrument configurations. A novel stability indicating assay method was developed and fully validated according to ICH (Q2)R1 for the analysis of FLB in the pharmaceutical preparations. A new degradation product was identified in the oxidative forced degradation condition and characterized using HPLC-DAD-ESI-IT-TOF-MS3. Moreover, the possible mechanism and the formation kinetic of the degradation product were revealed. In addition, the developed method was transferred to another LC-PDA instrument for inter-laboratory comparison. Finally, the current method was applied to pseudo formulation of Addy® in both instruments and ANOVA was applied for evaluation.

A Comprehensive Look at the -13910 C>T LCT Gene Polymorphism as a Molecular Marker for Vitamin D and Calcium Levels in Young Adults in Central and Eastern Europe: A Preliminary Study.

Intolerance to dairy products resulting from the abnormal digestion of milk sugar (lactose) is a common cause of human gastrointestinal disorders. The aim of this study was to show that the -13910 C>T LCT gene polymorphism, together with genotypes of selected VDR gene polymorphisms and diet and nutritional status parameters, can impact the prevalence of vitamin D and calcium deficiency in young adults. This study was conducted on a group of 63 people, which comprised 21 individuals with primary adult lactase deficiency, and a control group of 42 individuals with no hypolactasia. The LCT and VDR gene genotypes were assessed using PCR restriction fragment length polymorphism (PCR-RFLP) analysis. A validated HPLC method was used to determine serum concentrations of 25(OH)D2 and 25(OH)D3. Atomic absorption spectrometry was used to determine calcium levels. Their diets (self-reported 7-day estimated food record), estimated calcium intakes based on the ADOS-Ca questionnaire and basic anthropometric parameters were assessed. The CC genotype associated with hypolactasia was found in 33.3% of the subjects. The presence of the CC variant of the LCT gene polymorphism in the study group of young Polish adults was found to be associated with significantly lower milk (134.7 ± 66.7 g/d vs. 342.5 ± 176 g/d; p = 0.012) and dairy product consumption (78.50 ± 36.2 g/d vs. 216.3 ± 102 g/d; p = 0.008) compared with lactase persistence. At the same time, people with adult-type primary intolerance were found to have statistically significant lower serum levels of vitamin D and calcium (p < 0.05). There was a higher chance of vitamin D and calcium deficiency and a lower intake in the group exhibiting lactase non-persistence (OR > 1). The AA variant of the VDR gene's BsmI polymorphism present in people with hypolactasia may further contribute to an increased risk of vitamin D deficiency. Exclusion of lactose from the diet, combined with impaired vitamin D metabolism, may also lead to inhibited calcium absorption by the body. Further research should be carried out on a larger group of subjects to clarify the relationship between lactase activity and vitamin D and calcium levels in young adults.

Development and Validation of RP HPLC Method for Estimation of Deferiprone and Its Related Impurity in Pharmaceutical Dosage Form

Development and Validation of RP HPLC Method for Estimation of Deferiprone and Its Related Impurity in Pharmaceutical Dosage Form

A Review of HPLC Method Development and Validation as per ICH Guidelines

Method development and validation are continuous processes that progress in parallel with the evolution of drug products. Changes encountered during drug development may require modifications to existing analytical methods. These modifications to the methods, in turn, may require additional validation. The advent of new techniques and improved instrumentation in the field of analysis may give way to more sensitive, precise, and accurate methods if the existing methods are erratic or unreliable; time-consuming, or too expensive. Thus, continuous new analytical method development and validation activities are essential for the growing drug development programs.

HPLC Method Development, Validation and Application to Determining In-Vitro Effect of Levofloxacin on the Availability of Gliclazide

HPLC Method Development, Validation and Application to Determining In-Vitro Effect of Levofloxacin on the Availability of Gliclazide

Development, Optimization, and Validation of HPLC Method for Quantification of Enzalutamide in Bulk and Pharmaceuticals

Development, Optimization, and Validation of HPLC Method for Quantification of Enzalutamide in Bulk and Pharmaceuticals

Phytochemical profile analysis by LC-ESI-MSn and LC-HR-ESI-MS and validated HPLC method for quantification of rutin in herbal drug and products from leaves of Croton blanchetianus.

Phytochemical analysis of Croton blanchetianus leaves was performed by. After that, a high performance liquid chromatography method was developed and validated for the determination of rutin in herbal drug and products of C. blanchetianus. The separation was achieved on a C18 column, and the mobile phase was composed of ultrapure water and methanol (acidified with trifluoroacetic acid) with a gradient of 0.8ml/min. The method was validated following international guidelines. The chemical analysis revealed the presence of flavonoids. Among them rutin was used as the standard for validation. In the HPLC the presence of rutin was observed at 24.7min. The method was robust, with no significant variations, and linear in the range evaluated with R2 > 0.99. Regarding the matrix effect, it was possible to prove the absence of interference of the constituents in the herbal drug. The precision was determined with a relative standard deviation of <1.34%. The recovery results were achieved between 89.29 and 101.21%. Furthermore, with partial validation, the method was proved to be suitable for the liquid extract, dry extract and effervescent granules. Therefore, this study demonstrated that the method is effective for the quality control analysis of C. blanchetianus leaves and products.

Determination of scums in medicine materials

The current study is describing the main degrading scums in bromazepam, diazepam, Cefixime trihydrate, cefazolin sodium, simvastatin, and lovastatin, among other materials and manufactured items. HPLC was used to characterize pharmaceutical substance scums by comparing their retention lengths to reference standards. Before deciding, they sorted all the material from its main scums. Scums fell between 1.23 to 20.57 min, and parent compounds had try values between 2.71 and 9.22. Thus, isolating and identifying these compounds takes 20 minutes. This study identified parent compounds and degradation products using HPLC after confirming the European Pharmacopeial methodology. HPLC method validation for parent pharmaceuticals and degradation scums examined specificity, linearity, precision, and accuracy. The process can be applied to commodities and waste products using each compound's values for these qualities. 98%–102% of parent and scum materials are recovered. The experimental technique repeats within 3%. In compliance with pharmacopeial and regulatory norms, scums above 0.1% must be determined for pharmaceutical safety, efficacy, and quality. It's vital due to the effects of packaging, transit, storage, and manufacturer synthetic pathways. Adjuvants may cause degradation scums. The selected drugs' raw material and finished product levels of their main degrading scums were examined, and a concentration range was provided.

Analytical quality-by-design approach for development and validation of HPLC method for the simultaneous estimation of omarigliptin, metformin, and ezetimibe: application to human plasma and dosage forms

A simple, selective, and sensitive RP-HPLC method was proposed for the simultaneous determination of two co-administered antidiabetic drugs (omarigliptin and metformin) with an anti-hyperlipidemic drug (ezetimibe) in a medicinally-recommended ratio of 2.5:50:1, respectively. The proposed procedure was optimized by adopting a quality-by-design approach. The influence of different factors on chromatographic responses was optimized by applying the two-level full factorial design (25). The optimum chromatographic separation was achieved using Hypersil BDS C18 column at 45 °C, and the mobile phase pumped isocratically composed of methanol: potassium dihydrogen phosphate buffer (6.6 mM; pH 7, 67:33% v/v) at a flow rate of 0.814 mL/min using 235 nm as a detection wavelength. The developed method was capable of separating this novel mixture in less than 8 min. The calibration plots of omarigliptin, metformin, and ezetimibe showed acceptable linearity over the ranges of 0.2-2.0, 0.5–25.0, and 0.1-2.0 µg/mL with quantitation limits of 0.06, 0.50, and 0.06 µg/mL, respectively. The proposed method was successfully applied to determine the studied drugs in their commercial tablets with high % recoveries (96.8-102.92%) and low % RSD values (less than 2%). The applicability of the method was extended to the in-vitro assay of the drugs in spiked human plasma samples with high % recoveries (94.3-105.7%). The suggested method was validated in accordance with ICH guidelines.

Validation of HPLC method for quantitative determination of active compounds in Thai traditional herbal medicine to treat gastrointestinal disease

Quality control is essential for determining the quality and consistency of medicinal plant products before their use. Thatbunjob is a Thai herbal formulation included in the National List of Essential Medicines for treating gastrointestinal diseases. This study aimed to develop and validate a high-performance liquid chromatography method for determining active compounds in Thatbunjob. The main compounds, gallic acid, chebulagic acid, rutin, and eugenol, were determined following the International Conference on Harmonization (ICH) guidelines. The method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ), and found to be linear, with a correlation coefficient (R2) ranging from 0.9995 to 0.9998. The LOD and LOQ values calculated from the calibration curve ranged from 7.29 to 20.29 and 22.09 to 61.48 µg/mL, respectively. The method also exhibited acceptable precision, with relative standard deviation values lower than 2%. The accuracy of the method was evaluated based on the recovery percentages, and found to be within an acceptable range (90.12–105.39%). The validated protocol developed in this study was employed to analyze the concentration levels of gallic acid, chebulagic acid, rutin, and eugenol in eight different Thatbunjob products commercially available in Thailand. The performance parameters show that the developed and validated method is appropriate for the quantitative determination of gallic acid, chebulagic acid, rutin, and eugenol in Thatbunjob.

Rapid and Efficient SpPRE Approach: A Step Towards Meeting High Demand of Bioactive Swertiamarin

ABSTRACT Swertiamarin has a potential for treating metabolic illnesses, Parkinson’s disease, and its hepatoprotective and antiaging effects are evident. In spite of high yield in herb Enicostemma littorale Blume, the complexity of phytochemical makeup of extract presents difficulties in purification of swertiamarin due to a closely eluting flavonoid glycoside(s) and has an impact on the final output. At the laboratory scale, advanced techniques such as centrifugal partition and counter-current chromatography have been examined. However, feasibility of scaling up these strategies is limited. We developed a four-step SpPRE technique for enriching swertiamarin in the extract which includes optimization of Solvent for extraction, liquid-liquid partitioning, Precipitation, and Resin Enrichment step. This technique enabled enrichment of swertiamarin from 10.9% to 81.7% with reduction in extract weight by 91%. Subsequently, a single-step silica gel column chromatography resulted into 97% pure swertiamarin. The SpPRE methodology has resolved the problem of repeated column chromatography as well as the sample loading over to silica gel. To determine the concentration of the compound at each step, a validated HPLC method was employed. Furthermore, the current approach’s ability to isolate the target component in a high yield will increase swertiamarin’s market value and research prospects.

A Validated HPLC Method for the Simultaneous Quantification of Curcumin, Silybin, and Psoralen: Application to Phytopharmaceuticals

A Validated HPLC Method for the Simultaneous Quantification of Curcumin, Silybin, and Psoralen: Application to Phytopharmaceuticals

Removal of fenamiphos, imidacloprid, and oxamyl pesticides from water by microalgal Nannochloropsis oculata biomass and their determination by validated HPLC method

The present study assessed the removal of fenamiphos, imidacloprid, and oxamyl pesticides from water using algal Nannochloropsis oculata biomass. Several factors, such as algal biomass concentration, incubation time, and pesticide concentration, were studied for their impact on pesticide removal. Analysis and quantification of pesticides by rapid HPLC have been developed and validated. The optimum conditions were obtained at 15 min, 50 mg/L of pesticide concentration, and 4,500 mg/L of the algal biomass with 92.24% and 90.43% removal for fenamiphos and imidacloprid, respectively. While optimum parameters of 10 min incubation, 250 mg/L of pesticide concentration, and 2,750 mg/L of the algal biomass exhibited 67.34% removal for oxamyl. N. oculata, marine microalgae, successively removed different concentrations of the tested pesticides from water, and the algal biomass showed a potential reduction of pesticides in polluted water samples.

Validated HPLC method for ceftriaxone from dried blood spots for pharmacokinetic studies and therapeutic drug monitoring in neonatal population.

Background: Pharmacokinetic evaluation is essential for the precise dosing of ceftriaxone in neonates. There is a need for developing a sensitive, affordable and convenient analytical method that can estimate ceftriaxone from dried blood spot (DBS) samples of neonates. Method: An HPLC-UV method was developed and validated as per ICH M10 for ceftriaxone from DBS and plasma using an Inertsil-ODS-3V column with gradient elution. DBS samples were extracted with methanol. Clinical validation was performed using neonatal samples. Results: The developed plasma- and DBS-based-HPLC method were linear from 2-700μg/ml and 2-500μg/ml, respectively, for ceftriaxone. Bland-Altman analysis indicated a strong interconvertibility between the plasma and DBS assays. Conclusion: Observed concentrations in clinical samples were comparable to the predicted concentrations, proving the clinical validity of the method.