Chromatographic Assay Method Research Articles

A Comparative Study of Cystatin C and Creatinine as a Preliminary Marker of Nephropathy in Type 2 Diabetic Patients

Background: Type 2 diabetes disorder (T2DD) is one of the most common disorder in societies, and its causes are genetic and acquired. This disorder is characterized by an increase in the concentration of glucose in the blood. High concentrations of glucose in the blood for long periods lead to complications, the most important of which is diabetic nephropathy (DNP), which is characterized by the gradual loss of the kidney's filtration function.Material and methods: The present study was designed based on collecting twenty patients with newly DNP (as first group) and twenty healthy individuals (as second group). The levels of HbA1c, Creatinine and Cystatin C of all study individuals were measured using Chromatographic assay method, Kinetic colorimetric method and Immunoturbidimetric assay techniques respectively.Results: This study used the t-test statistical method to compare groups as well as sensitivity and specificity based on HbA1c, creatinine and Cystatin C biomarkers. The current study showed a higher the HbA1c percentage, creatinine level and Cystatin C level in the first group compared to the second group. On the other hand, the current study showed that Cystatin C has greater sensitivity and specificity than creatinine towards the DNP disease.Conclusion: The current study concluded by stating the importance of the role of Cystatin C as an early indicator of DNP disease, as the current study proved that Cystatin C has greater sensitivity and specificity than creatinine towards DNP disease

A practical approach to estimate analytical method variability from routine testing

The performance of analytical test methods is critical to ensure decisions that affect efficacy and quality of pharmaceutical products are based on accurate and reliable results. As described in USP <1220> and advocated for in ICH Q14, continued verification of critical method attributes linked to bias and precision is essential to ensure method performance throughout the lifecycle of an analytical test method. As continued verification programs for analytical methods within the pharmaceutical industry mature, additional monitoring tools are required to deliver robust and cost-effective verification programs. Herein, a novel methodology is presented to evaluated analytical method variability directly from results generated during routine method execution. The implementation of the methodology is demonstrated for a small molecule liquid chromatographic assay method utilizing a single-point external reference calibration. Approaches to reduce the required data to be collected and broaden the applicability of the methodology to a wide range of analytical methods is described. Finally, the application of the methodology to method development activities is discussed to aid in the identification of variability sources and effectively select replication strategies, thus allowing a holistic understanding of method variability throughout the entirety of the method lifecycle.

Development and validation of stability indicating ultra‐high‐performance liquid chromatographic method for assay and impurities of mexiletine hydrochloride and mexiletine hydrochloride capsules

AbstractMexiletine (MEX) is an antiarrhythmic drug used to treat acute and chronic ventricular arrhythmias. A simple isocratic, rapid, specific, stability‐indicating, and sensitive reverse‐phase ultra‐high‐performance liquid chromatography (UHPLC) method was developed and successfully validated to quantify the assay and impurities of MEX hydrochloride. The new UHPLC method showed the optimum separation of MEX and its seven impurities in 20 min by using sodium acetate buffer (0.14 mM) with 0.3% of glacial acetic acid (pH 4.8) and methanol in the ratio of 40:60 (v/v) as mobile phase. The method used an Acquity HSS T3 (100 × 3.0 mm, 1.8 µm) chromatographic column at a 0.2 mL/min flow rate. The injection volume is 2 µL, and 254 nm is the detection wavelength. Note that, 25°C is the column oven temperature. The method is linear over the concentration range of the quantification limit (0.03%) to 150% (0.3%) of the specification level (0.2%) for the impurities and 50%–150% for the assay method. The quality control lab of bulk drugs and finished dosage form successfully applied the new UHPLC method for quantification of assay and impurities of MEX hydrochloride.

Paper-based radial chromatographic assay for rapid in situ measurement of gelatinized starch concentration

Starch gelatinization is pivotal in food processing, impacting viscosity, texture, and functionality. However, current methods for determining concentration and viscosity of gelatinized starch (GS), such as falling ball and rotational viscometers, are impractical for in situ measurements due to bulky equipment. Here, we introduce a paper-based radial chromatographic assay (PRCA) method for rapidly measuring the concentration of gelatinized starch (GS). We utilized Evans Blue (EB) as a chromatic agent to label GS and generate color-ring patterns on a nitrocellulose membrane based on the “Coffee-ring” effect. The resulting PRCA patterns varied depending on the GS concentrations, yielding a good linear correlation (R2 > 0.967) between grayscale value and viscosity. This method enables rapid measurement of GS concentration within 10 s, even with varying ratios of amylose to amylopectin. PRCA demonstrates a detection limit for GS concentration between 0.027% and 0.051%, which is 10-20 fold lower than that of traditional rotational-viscometer. The intensity of PRCA patterns at pH 3 (45.8) was significantly lower than those at higher pH levels (5 to 9), consistent with the decreased viscosity measured by the viscosimeter. The intensity of PRCA patterns did not change with increasing temperature from 25°C to 90°C, despite the significant decrease in viscosity from 345 mPa•s to 195 mPa•s. Additionally, PRCA can monitor changes in starch viscosity during the hydrolysis process, which decreases from 744 mPa•s to 6 mPa•s. This capability provides an efficient means of measuring the concentration and viscosity of GS during food processing.

Development and validation of RP-HPLC method for estimation of anti- hypothyroidism drug in capsule dosage form

A simple, accurate, sensitive and precise Reverse Phase-High Performance Liquid Chromatographic assay method for estimation of Levothyroxine sodium in dosage form was successfully developed. The chromatographic separation was performed on Welch Ultisil XB-CN, 150 x 4.6 mm, 5µm. column. The mobile phase consists of 0.05% OPA Buffer solution, and Acetonitrile in the ratio of 72:28, v/v. Mobile phase was delivered at a flow rate of 1.0 ml/min. Samples were injected 50 μL the column temperature was kept at 40 0C and sample temperature 150C. The wavelength 221 nm were selected for the evaluation of the chromatogram. The retention time of the drug was found to be 3.7 min. The developed method was found to be linear in a concentration range of 5-15 μg/ml of the drug (r2= 0.999). The low value of % RSD indicates reproducibility of the method. Thus this method can be used for routine analysis of pomalidomide in formulation and to check the stability of bulk samples.

Development and Validation of Stability Indicating RP-HPLC Method for Quantitative Estimation of Enzalutamide in Enzalutamide Capsules Dosage Form

Objectives: A precise, accurate and selective stability-indicating reverse phase high performance liquid chromatographic assay method has been developed for the quantitative estimation of Enzalutamide in Enzalutamide capsules dosage form. Materials and Methods: The separation was achieved by using a stationary phase Waters X-Bridge Shield RP18 (150 x 4.6 mm, 3.5μ) and the mobile phase consisted of perchloric acid buffer and acetonitrile in the proportion of (20:80 volume/volume). The run velocity was 1.2 mL/min. Enzalutamide was identified using UV detector at the wavelength of 210 nm. Column oven temperature 25°C and sample cooler temperature 25°C and infused quantity 20 μL, run time was 15 min. Results: As there is no meddling flanked by blank and placebo at the retention time of Enzalutamide. Degradation study results were shown significant degradation was observed in acid and oxidation (peroxide) stress condition. Hence it can be concluded that Enzalutamide is sensitive to acid and oxidation. To obtain system precision, a study was conducted with six replicate injections. %RSD was estimated from the peak areas of Enzalutamide establish to be 0.55% correspondingly. The relative standard deviation for method exactitude was establish to be 0.55%. The suggested HPLC technique was linear over the range of 100.6-301.8 μg/mL, the correlation coefficient was establish to be 0.9999. The accuracy studies were shown as % recovery for Enzalutamide 50% to150% level. The limit of % recovered revealed is in the assortment of 98 and 102% and the consequences obtained were establish to be within the limits. Hence the technique was establish to be accurate. The solution steadiness of the standard and samples are stable upto 48 hr on a bench top and refrigerator (2-8°C). The method is robust for changes like flow rate and column oven temperature. Performed the filter validation for sample solution 0.45 μm PVDF and 0.45 μm Nylon filterers are suitable for filtration. The method has validated as per ICH guidelines and all the validation parameters are satisfy the ICH Q2 specification acceptance limits. Conclusion: The developed method was validated for an assortment of parameters as per ICH guidelines like accuracy, precision, linearity, specificity, system suitability, solution stability and robustness. The consequences obtained were within the acceptance criteria. So, it can be concluded that the urbanized technique is simple, precise, cost-effective, eco-friendly, and safe and can be successfully employed for the routine analysis of Enzalutamide in bulk and pharmaceutical dosage forms.

Development and Validation of New RP-HPLC Method for Estimation of Pramipexole Dihydrochloride in Bulk and Pharmaceutical Formulation

A novel high-performance liquid chromatographic assay method was developed and validated for the quantitative determination of the anti-Parkinson agent pramipexole dihydrochloride monohydrate in bulk and its tablet dosage form. In this perspective, the chromatographic separation was accomplished on Eclipse XDB-12 C18 (150 mm x 4.6 mm, 5 μm particle size) column using UV detection at 263 nm. The mobile phase consisted of distilled water: acetonitrile (10: 90 v/v), run at a flow rate of 1.0 mL/min with isocratic elution. The method was validated in accordance with ICH guidelines by evaluating the system suitability, linearity, limits of detection (LOD) and quantitation (LOQ), precision, accuracy, specificity, selectivity and short-term stability. Our findings revealed that retention time for pramipexole dihydrochloride was found to be 5.2 minutes. The linearity range was established between 6.25-225.0 μg/mL with a mean recovery of 101.26 % ± 0.56. The limits of detection and quantification were determined to be 4.18 μg/mL and 12.66 μg/mL, respectively, indicating that the method is very sensitive. Intra and inter-day precision were within acceptable limits (RSD

Development and Validation of Fast and Robust Stability Indicating RP-HPLC Method for Simultaneous Estimation of Tolperisone Hydrochloride and Etoricoxib in Pharmaceutical Dosage Form

Objectives: A precise, accurate and selective stability-indicating reverse phase high performance liquid chromatographic assay method has been developed for the simultaneous quantitative determination of Tolperisone hydrochloride and Etoricoxib in tablets. Methods: The chromatographic separation of drugs was attained by using Eclipse plus C18 (150 mm × 4.6 mm, Agilent 5µm) column at room temperature. The composition of mobile phase was mixture of 0.035M triethylamine (pH 3.0 adjusted with orthophosphoric acid) and acetonitrile in ratio of 70:30 v/v and flow rate of mobile phase 1.0 ml/min with isocratic elution. The signal of eluents was observed at 290 nm by using diode array detector. Results: The retention time of Tolperisone hydrochloride and Etoricoxib were found to be 2.826 min and 7.566 min, respectively. The linearity ranges for both drugs were found to be 5-15 μg/ml and the percent recoveries were found to be 99.39% and 99.15% for Tolperisone hydrochloride and Etoricoxib respectively. Various forced degradation conditions, such as alkali hydrolysis, acid hydrolysis, thermal degradation, photolytic degradation, and oxidative degradation, were applied to the drug ingredients and drug formulation. The degradants were efficiently separated from the drugs by using optimized chromatographic conditions. The developed method was validated as per recommendation parameters of International council on harmonization guidelines Q2(R1). Conclusion: The validation parameters indicate that the drug substances were efficiently separated from its degradants and developed method can be routinely applied for the simultaneous quantitative determination of Tolperisone hydrochloride and Etoricoxib in combined tablet formulation in the quality control laboratory.

Chromatographic method development for the simultaneous assay of pseudoephedrine hydrochloride and chlorphenamine maleate in oral dosage formulations

Common cold medicines usually present as multi-component products and constitute one of the largest groups of ‘over-the-counter’ medicines marketed in Ghana. Due to their wide patronage and easy accessibility, their quality control is of utmost importance to medicine consumers. In this study, the Design of Experiment (DOE) concept is adopted to develop a chromatographic method for the simultaneous assay of pseudoephedrine hydrochloride and chlorphenamine maleate in commercially available products. The separation of the drugs was achieved on an Agilent Eclipse Plus C18 analytical column (4.6 × 150 mm; 5 µm) and detected at 252 nm, while maintaining column temperature at 30 oC. The method parameters, including acetonitrile concentration in the mobile phase, mobile phase pH and flow rate were optimized using a Central Composite Design study, while monitoring the method attributes, asymmetric factors, selectivity, retention times, and resolution. The outcome showed a good correlation between experimental and predictive values throughout the modeled Design Space, with a Desirability of 1.000. The conditions optimized included a mobile phase containing acetonitrile, 50 mM aqueous acetate buffer solution (pH = 3.19) and triethylamine (15.34:84.65:0.01 v/v/v), and a flow rate of 1.437 mL/min. The method was then validated following the International Council for Harmonization Q2(R) guidelines to establish linearity and range, accuracy, precision, robustness, and stability of the test solution. The developed method was successfully applied to estimate pseudoephedrine hydrochloride and chlorphenamine maleate contents in single and fixed-dose formulations commercially available (N = 38).

A NOVEL VALIDATED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC STABILITY-INDICATING ASSAY METHOD IN REVERSE PHASE FOR ESTIMATION OF HYDROXYPROGESTERONE CAPROATE IN AN INJECTABLE FORMULATION.

A high-performance liquid chromatographic stability-indicating assay method in reverse-phase has been developed and validated for the quantitative measurement of Hydroxyprogesterone corporate in injectable formulation prepared in castor oil base containing excipient benzyl benzoate as preservative. The reverse-phase method was developed by using Sunfire C18 column having dimensions (150 x 4.6 mm x 5μm) with a mobile phase[A] comprised of water, acetonitrile, and Methanol mixed in 40:30:30 v/v ratio and mobile phase [B] comprised of water, acetonitrile, and Methanol mixed in 20:40:40 v/v ratio pumped gradient program at a flow rate of 1.0 ml/min where the thermostat of the column oven controlled at 30°C and sample compartment at 5°C. The injection volume is set at 20μL. Hydroxyprogesterone corporate has a retention time of 10 minutes with UV detection at 240 nm. The detector was showing linear response in a range of 0.25 μg/ml to 150 μg/ml for Hydroxyprogesterone corporate. The guidelines of the International Conference on Harmonization on guidance for industry (2005) validation of analytical procedures, text, and methodology Q2(R1) were followed for the validation of this novel method. For quantification of Hydroxyprogesterone corporate the developed method was found to be genuinely exact precise, accurate, linear, robust, rugged, stable. and cost-effective.

Development and Validation of Q-Absorbance Ratio by UV-Spectrophotometric Method for Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Combined Dosage Form

Advantages of simultaneous stability studies are the identification of new degradation products, to understand mutual induction and/or inhibition of rates of degradation and to analyze the degradation products of both drugs. Various ultraviolet spectroscopic and high performance liquid chromatographic assay methods were reported for the estimation of metformin, sitagliptin, pioglitazone, glimepiride and simvastatin individually and in combination with other drugs. All the above reported methods were based on the estimation of metformin, sitagliptin, pioglitazone, glimepiride and simvastatin alone or in combination with other drugs. The degradation products were generated and successfully separated by the developed and validated high performance liquid chromatographic methods for the estimation of the selected anti-diabetic drug combinations. The aim of the study was to develop and validate of Q-Absorbance Ratio UV-Spectrophotometric Method for Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Combined Dosage Form.&#x0D; Keywords: Metformin, Method Development, Validation, Empagliflozin, UV-Spectrophotometer.

Approved stability indicating reversed-phase-high performance liquid chromatographic assay method for Palonosetron HCl in formulations

Approved stability indicating reversed-phase-high performance liquid chromatographic assay method for Palonosetron HCl in formulations

Development of Chromatographic Methods for Assay of Contaminants of Russian-Made Tramadol Hydrochloride Substance

Methods for identifying contaminants in Russian-made tramadol hydrochloride substance were developed. Detection of the RS,SR-isomer in tramadol hydrochloride substance was by HPLC with UV detection. Identification of (2RS)[(dimethylamino)methyl]cyclohexanone was by chromatography in a thin layer of sorbent on Sorbfil F254 plates. The HPLC method was validated in terms of efficiency, linearity, and reproducibility. Detection sensitivity for contaminants was 0.0001 mg/cm3 (0.02%).

Stability-indicating high-performance liquid chromatographic assay methods for drugs in pharmaceutical dosage forms: Part I

A review of applications of stability-indicating high-performance liquid chromatographic (HPLC) assay methods for drugs in various pharmaceutical dosage forms is presented. A survey of articles primarily on the significant developments in this field is covered in this paper. Selected substances are grouped to integrate drugs that share similar uses or actions. Papers selected by the authors are briefly summarized, and HPLC systems developed in these papers for stability testing of pharmaceuticals are tabulated to assist readers in selecting an appropriate system for a given pharmaceutical. The information given in the table includes the drug names, materials, column types, mobile-phase compositions, detections, internal standards used and the literature references.

Stability-indicating High-Performance Liquid Chromatographic assay methods for drugs in pharmaceutical dosage forms: Part II

Stability-indicating High-Performance Liquid Chromatographic assay methods for drugs in pharmaceutical dosage forms: Part II

Separation and Simultaneous Quantitation of Montelukast Sodium and Ebastine in Tablets by using Stability-Indicating Liquid Chromatographic Method

Objectives: A precise, accurate and selective stability-indicating reverse&nbsp;phase high performance liquid chromatographic assay method has been&nbsp;developed for the simultaneous quantitative determination of Montelukast&nbsp;sodium and Ebastine in tablets. Methods: The chromatographic separation&nbsp;of drugs was attained by using Hypersil octadecyl silane C18 (250 x 4.6&nbsp;mm, 5μm) column at room temperature. The composition of mobile phase&nbsp;was methanol, acetonitrile and 0.02M ammonium acetate buffer (pH 5.5&nbsp;adjusted with dilute acetic acid) in the ratio of 80:15:05 v/v/v and flow rate&nbsp;of mobile phase 1.0 ml/min with isocratic elution. The signal of eluents&nbsp;was observed at 244 nm by using diode array detector. Results: The retention&nbsp;time of Montelukast sodium and Ebastine were found to be 4.28 min&nbsp;and 6.63 min, respectively. The linearity ranges for both drugs were found&nbsp;to be 10-50 μg/ml and the percent recoveries were found to be 99.16%&nbsp;and 100.12% for Montelukast sodium and Ebastine respectively. The drug&nbsp;substances and drug products were treated with various forced degradation&nbsp;conditions like acid hydrolysis, alkali hydrolysis, oxidative degradation,&nbsp;thermal degradation and photolytic degradation. The degradants were efficiently&nbsp;separated from the drugs by using optimized chromatographic conditions.&nbsp;The developed method was validated as per recommendation parameters&nbsp;of International conference on harmonization guidelines Q2(R1).&nbsp;Conclusion: The validation parameters indicate that the drug substances&nbsp;were efficiently separated from its degradants and developed method can&nbsp;be routinely applied for the simultaneous quantitative determination of&nbsp;Montelukast sodium and Ebastine in combined tablet formulation in the&nbsp;quality control laboratory.

European Bioanalysis Forum Recommendation on Singlicate Analysis for Ligand Binding Assays: Time for a New Mindset

It is well accepted that chromatographic assay methods employ singlicate analysis for toxicokinetic and pharmacokinetic analysis. While conversely, it has been the norm for ligand-binding assays to be run in at least duplicate analyses, stemming mainly from concerns over inherent assay variability and reagent quality. Regulatory guidelines and guidance on bioanalytical method validation has, in the most part, recommended multiple replicates for immunoassays and this has led to the industry being comfortable and familiar with duplicate analysis. Over the last few years, the discussion on whether singlicate analysis is acceptable for ligand-binding assays has grown and the status quo is being challenged for regulated bioanalysis performed using immunoassays. Through interrogation of preclinical and clinical pharmacokinetic assay data from the European Bioanalysis Forum community, the application of a singlicate analysis strategy has shown to have no impact on toxicokinetic and pharmacokinetic parameters when compared with duplicate analysis from the same studies. Therefore, now is the time to adopt a new mindset when it comes to sample analysis for toxicokinetic and pharmacokinetic ligand-binding assays and embrace singlicate analysis in the regulated environment.

Stability Indicating RP-UPLC Photo-Diode Array based Method for Determination of Edoxaban Tosylate

The objective of the current study was to develop a specific, precise, accurate and robust gradient stability indicating reversed-phase ultra performance liquid chromatography (RP-UPLC-PDA) assay method and validated for determination of edoxaban tosylate in API. Gradient separation was achieved on an acquity UPLC BEH C18 column (50 mm, 2.1 mm and 1.7 μm) column using mobile phase of acetoitrile:20 mM potassium dihydrogen phosphate, pH 3.0 ± 0.05 adjust with OPA at flow rate of 0.6 mL/min, the injection volume was 1 μL and the detection was carried out of 289 nm by using photo-diode array detector. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress condition. The method was linear in the drug concentration range of 100-300 μg/mL with correlation coefficient of 0.999. Degradation products produced as a result of stress studies did not interfere with detection of edoxaban tosylate and the assay, thus developed stability indicating method can be used for routine analysis in pharmaceutical industry.

Detection of Tadalafil and Its Analogues in Health Wine and Oral Liquid by Microwell Lateral Flow Immunochromatography Assay

To monitor illegal addition of tadalafil and its analogues in health food, a new polyclonal antibody was prepared by immunizing New Zealand white rabbits with a common skeleton structure hapten combined with an unsaturated linear alkyl arm hapten strategy, which could simultaneously recognize tadalafil, amino tadalafil and N -Desmethyl tadalafil. A microwell lateral flow immune- chromatographic assay ( mw LFIA) method combined with simple sample pretreatment was developed. The mw LFIA showed that the LOD for three tadalafil drugs was between 0.05 ng mL −1 and 0.30 ng mL −1 , the cut-off values were between 50 ng mL −1 and 100 ng mL −1 . Cross-reactivity (CR) results showed no interference from substances with similar structures, indicating a good specificity against tadalafil, amino tadalafil and N -Desmethyl tadalafil. The average recoveries of three drugs in spiked health wine and oral liquid ranged from 78.7% to 117.8% with an RSD of less than 15%, showing a good aggreement with the results of HPLC-MS/MS method. The developed mw LFIA strip was suitable for the rapid detection of tadalafil and its analogues in health wine and oral liquid samples. A microwell lateral flow immunochromatographic assay ( mw LFIA) for simultaneous determination of tadalafil, amino Tadalafil and N -desmethyl Tadalafil was developed with high accuracy and sensitivity. The mw LFIA method was successfully applied to the detection of Tadalafil and its analogues residue in analogues health wine and oral liquid samples.

SIMULTANEOUS QUANTIFICATION OF PENTAZOCINE AND NALOXONE BY STABILITY INDICATING REVERSE-PHASE-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD

Objective: The objective of the study was to develope a stability indicating high-performance liquid chromatographic (HPLC) method for simultaneous assay of pentazocine and naloxone in bulk and tablets.&#x0D; Methods: Pentazocine and naloxone were analyzed on Dionex C18 column using 0.1M K2HPO4 buffer (pH 4.0) and methanol (60:40, v/v) as the mobile phase. The concentration of pentazocine and naloxone was quantified by photodiode array detector set at 248 nm. The method was validated in compliance with ICH rules. Pentazocine and naloxone tablet formulation was subjected to forced degradation such as acid, neutral and alkali hydrolysis, oxidation, photo, and thermal degradation.&#x0D; Results: The method was linear, with R2=0.9999 in the concentration range 100–300 μg/ml for pentazocine and R2=0.9995 in the concentration range 1–3 μg/ml for naloxone. The level of detection and quantification was 0.097 μg/ml and 0.322 μg/ml for pentazocine and 0.0073 μg/ml and 0.0243 μg/ml for naloxone, respectively. The degraded products are resolved well from pentazocine and naloxone with significantly different retention time values. From validation results, it was proved that the method is selective, precise, robust, and accurate for the estimation of pentazocine and naloxone simultaneously.&#x0D; Conclusion: The developed stability-indicating HPLC method can be applied for quantitative determination of pentazocine and naloxone in tablets.