Analytical Methods For Assay Research Articles

A rapid and highly sensitive HILIC-HPLC based analytical assay method for quantification of azacitidine and venetoclax in a single run

Venetoclax and Azacitidine combination has recently been approved by the USFDA to treat old people with acute myeloid leukemia. Pharmaceutical formulations of the combination of these two drugs are expected to be launched soon. Till date, an analytical method of any type has not been reported in literature for quantification of Venetoclax and Azacitidine in a single run. The current work set out to establish an HILIC-HPLC based analytical assay method for simultaneous quantification of Azacitidine and Venetoclax from the mixed sample. The method has been developed employing Poroshell HILIC column. Ammonium formate (10 mM, pH 5.0) and acetonitrile were used as a mobile phase in gradient programme. A variety of parameters, including linearity, accuracy, precision, specificity, robustness, and ruggedness were evaluated to validate the method. Azacitidine and Venetoclax were eluted at 8.8 min and 4.0 min, respectively. Method was linear over the range of 1-100 µg/mL. Recovery was found to be 98.29-99.28% and 101.84-101.95% for Azacitidine and Venetoclax, respectively. The method was reproducible with a relative standard deviation of less than two in precision studies. The developed method can suitably be employed for quantifying Venetoclax and Azacitidine in a single run from their mixed samples.

Robustness Study and Superior Method Development and Validation for Analytical Assay Method of Atropine Sulfate in Pharmaceutical Ophthalmic Solution

Robustness Study and Superior Method Development and Validation for Analytical Assay Method of Atropine Sulfate in Pharmaceutical Ophthalmic Solution

Advances in the Diagnosis, Monitoring, and Progression of Oral Cancer through Saliva: An Update.

The early detection of cancer, and in particular oral cancer, has been a priority objective of study in recent years. Saliva has been proposed as an easy-to-obtain means of providing the necessary information to diagnose malignant lesions in the oral cavity, since it can be obtained very easily and completely noninvasively. There are a number of molecules, known as biomarkers, which may be involved in the malignant transformation of oral lesions, and which have different natures. The involvement of proteins ("proteomics"), metabolites ("metabolomics"), and even certain genes in the structural changes of altered tissue has been investigated in order to establish validated parameters for the early diagnosis of oral cancer. In addition, the development of new analytical assay methods that can reduce costs and obtain better results in terms of sensitivity and specificity has been a key point in recent research in this field. Even though there are numerous biomarkers with results showing high sensitivity and specificity, there is still a need for more studies, with a larger sample and with analytical methods that can constitute a real advance in time and cost. Although salivary biomarkers are a promising new diagnostic tool for oral cancer, for the moment they do not replace biopsy as the "gold standard".

Establishment of a Rapid and Highly Sensitive Reverse-phase High-performance Liquid Chromatography Based Analytical Assay Method for Duvelisib

Establishment of a Rapid and Highly Sensitive Reverse-phase High-performance Liquid Chromatography Based Analytical Assay Method for Duvelisib

A Validated High-Pressure Liquid Chromatography (HPLC) Method for Molnupiravir

Molnupiravir is an approved antiviral drug that inhibits RNA replication of viruses. It is approved recently (November 2021) for the treatment of infectious diseases caused by SARS-CoV-2. However, it is reported that Molnupiravir is a nucleoside analogue and shown that nucleoside analogues are reagents that resemble the structure of natural nucleosides. It is widely applied in antiviral and anticancer therapy. Therefore, there is a general need appeared for an efficient, fast, simple, and validated analytical assay method for the quantification of this new product in any samples. We report here that an HPLC method was developed, optimized, and validated for molnupravir. The developed method was found to be easy to apply, cheap, and time-saving. Isocratic elution with 10mM Phosphate Buffer pH:7 / Acetonitrile mixture (80:20) with a Phenomenex C18 column was performed and a photodiode array detector was used for the detection. The total time for the analysis was only 10 minutes. The method was found to be selective, linear, accurate, reproducible, and robust. This proposed method can be easily adapted for molnupravir analyses in pharmaceutical products or in any other biological or non-biological samples.

Analytical Method Development of Content and Dissolution Assay of Ursodeoxycholic Acid Capsule

Ursodeoxycholic acid is an active compound to treat cholesterol gallstones which is included in Biopharmaceutical Classification System class II. Ursodeoxycholic acid capsule formulations have been developed by the pharmaceutical industries. Due to requirement, active ingredient testing and dissolution tests need to be routinely carried out to control quality of the drug. Therefore, aim of this study was to develop a simultaneously analytical method for assay and dissolution test. Optimum condition of analysis were using HPLC-RID, C 18 column (5 µm; 3.9 x 300 mm), flow rate 1 mL/min, 50 µL volume injection, acetonitrile:buffer phosphate pH 3.0 (55:45 v/v) as mobile phase, column, detector, and sample temperature were 40 o C. This method provides good linearity in a range of 50-130 % concentration for assay and dissolution test with correlation coefficient values of 1 and 0.9995, respectively. LOD and LOQ for assay were in value of 4.892 µg/mL and 14.824 µg/mL whereas for dissolution tests in a value of 6,501 µg/mL and 19,701 µg/mL. The average percent recovery for assay and dissolution test were in a range of 100.018±0.888% and 98.936±2.124 %. Repeatability and intermediate precision of assay were obtained by determining RSD values of 0.466% and 0.279-0.483 %, while the dissolution test were 1.137% and 0.032-0.289 %. The validation show this method are linear, selective, accurate, and precise. Keywords: Assay, dissolution test, Ursodeoxycholic acid, validation

Comparative study of four greenness assessment tools for selection of greenest analytical method for assay of hyoscine N-butyl bromide

Several assessment tools were recently introduced for the evaluation of the greenness of analytical methods. Each tool has advantages, disadvantages, and a unique assessment protocol. The final results obtained from each assessment tool may lead to a dissimilar conclusion about the selection of the greenest method, which makes the decision confusing as to which an assessment tool relies on. Accordingly, in this comparative case study, four greenness assessment tools-National Environmental Methods Index (NEMI), Eco-Scale Assessment (ESA), Green Analytical Procedure Index (GAPI), and Analytical GREEnness metric (AGREE)-were tested to evaluate 16 chromatographic methods described in the literature for the assessment of the commonly used antispasmodic drug Hyoscine N-butyl bromide (HNBB). The importance of applying more than one assessment tool when evaluating the greenness of analytical methods is explained in this study. Despite NEMI tool simplicity, it was the least effective in providing information about the analytical method as 14 out of 16 methods had the same NEMI pictogram. ESA and AGREE provided reliable numerical assessments that differed in their total scores whereas the total scores were out of 100 and 1 for each, respectively. AGREE has the merits over ESA with respect to automation and highlighting the weakest points in analytical techniques that need further improvements in terms of greenness. GAPI and AGREE provide fully descriptive three-colored pictograms. The main disadvantage of GAPI is complexity compared to NEMI and ESA. AGREE has the merits of simplicity and automation over GAPI. Based on the results, recommendations are made for ESA, GAPI, and AGREE tools, which provide reliable and precise results about the greenness of the method. Planning for the greenness of analytical methods should be assured before practical trials in a laboratory for reduction of chemical hazards released into the environment. Moreover, inclusion of the evaluation of greenness of analytical methods in method validation protocols is strongly recommended.

LC-Q-TOF-MS driven identification of potential degradation impurities of venetoclax, mechanistic explanation on degradation pathway and establishment of a quantitative analytical assay method

Venetoclax is a selective orally active Bcl-2 protein inhibitor very recently approved by USFDA to treat chronic lymphocytic leukemia and other hematological malignancies. Postmarketing surveillance of any drug depends on its acceptability based on risk to benefit ratio. When risk outweighs the benefits, withdrawal of an already marketed drug is warranted. Presence of impurity is the primary cause of increased risk in a drug substance or drug product. With the discovery of newer molecules, it is of great importance to establish advanced analytical techniques for quantification of the drugs as well as their related impurities to address the prospective regulatory queries even if it is already in the market. In this study, a quantitative analytical assay method has been developed and validated for quantification of venetoclax in presence of its degradation impurities. A stress study was performed to examine the stability of the drug in hydrolytic, oxidative, thermolytic and photolytic environments. Venetoclax was found to be prone to degradation in acidic hydrolytic and oxidative stress conditions. Three new degradation impurities have been identified and characterized with the help of LC-Q-TOF-MS with accurate mass measurement and their putative structures have been proposed. Furthermore, for the first time, a possible degradation pathway has been established with mechanistic explanation. Moreover, the analytical method developed in this study will be of immense help for routine analysis of quality control and stability study samples of venetoclax in industry and research laboratories.

Xanthene based spectroscopic probe for the analysis of HCV antiviral, daclatasvir dihydrochloride, through feasible complexation reaction

Two simple and rapid analytical assay methods were developed and validated utilizing spectroscopic techniques for sensitive and selective estimation of daclatasvir dihydrochloride in pharmaceutical preparations based on anion pair complex formation with eosin Y. The maximum absorbance of the formed complex was achieved at 545 nm in 0.1 M acetate buffer at pH 3.2 and Beer's law for absorbance against daclatasvir dihydrochloride concentration obeyed in the range of 1.0–10.0 μg mL−1 for the spectrophotometric method (method I). The technicality of spectrofluorimetric method (method II) based on measuring the quenching effect of daclatasvir dihydrochloride on the native fluorescence intensity of eosin Y at the same pH and the measurements were accomplished at 543 nm after excitation at 301 nm. The rectilinear calibration graph between daclatasvir dihydrochloride concentration and the fluorescence quenching values (ΔFI) was achieved over the range of 0.1–2.0 μg mL−1. The formation constant for the binary complex was 4.4 × 104 and Gibb's free energy change was −2.7 × 104 J mol−1. Both methods were validated in agreement with ICH guidelines and a successfully applied for the analysis dosage forms with good % recovery. A suggestion for the reaction pathway was supposed.

Inhibition of free radical activity by dual PPAR α and PPAR γ agonist using analytical assay methods

Background: Overproduction of free radicals involved in the pathology of a wide variety of clinical disorders. Poor glycaemic control in diabetic people leads to free radical production responsible for diabetic related complications. Antioxidants produces resistance against the oxidative stress by scavenging free radicals may useful in treating diabetic related complications. Saroglitazar is a newer antidiabetic drug act on dual Peroxisome Proliferator Receptor Agonist α (PPAR α) and PPAR γ agonist with protection effect on Diabetes mellitus induced lipid dystrophy. Our study was done to evaluate the in vitro antioxidant effect Saroglitazar by 1, 1 Diphenyl 2 Picryl hydrazide (DPPH) and Nitric Oxide (NO) method.Methods: In this study, we demonstrated invitro antioxidant activity by using 10 mg/dl stock solutions of Saroglitazar. DPPH and NO free radical scavenging test were done for different concentration of Saroglitazar.Results: Saroglitazar showed concentration dependent free radical scavenging activity in DPPH assay. In DPPH assay at higher concentration 1000ug concentration showed 49.18% free radical scavenging activity. At lower concentration 10ug showed 17.18% free radical scavenging activity. NO scavenging activity at lower concentration 100ug showed 55.15% activity. But the higher concentration (1000ug) only slight increase in 60.15% activity.Conclusions: Thus Saroglitazar invitro antioxidant analysis proved that it is a potent antioxidant.

Characterization of degradation products of ivabradine by LC-HR-MS/MS: a typical case of exhibition of different degradation behaviour in HCl and H2SO4 acid hydrolysis.

A validated stability-indicating HPLC method was established, and comprehensive stress testing of ivabradine, a cardiotonic drug, was carried out as per ICH guidelines. Ivabradine was subjected to acidic, basic and neutral hydrolysis, oxidation, photolysis and thermal stress conditions, and the resulting degradation products were investigated by LC-PDA and LC-HR-MS/MS. The drug was found to degrade in acid and base hydrolysis. An efficient and selective stability assay method was developed on Phenomenex Luna C18 (250 × 4.6 mm, 5.0 µm) column using ammonium formate (10 mM, pH 3.0) and acetonitrile as mobile phase at 30 °C in gradient elution mode. The flow rate was 0.7 ml/min and detection wavelength was 286 nm. A total of five degradation products (I-1 to I-5) were identified and characterized by LC-HR-MS/MS in combination with accurate mass measurements. The drug exhibited different degradation behaviour in HCl and H2SO4 hydrolysis conditions. It is a unique example where two of the five degradation products in HCl hydrolysis were absent in H2SO4 acid hydrolysis. The present study provides guidance to revise the stress test for the determination of inherent stability of drugs containing lactam moiety under hydrolytic conditions. Most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation pattern of the drug and its degradation products. In silico toxicity revealed that the degradation products (I-2 to I-5) were found to be severe irritants in case of ocular irritancy. The analytical assay method was validated with respect to specificity, linearity, range, precision, accuracy and robustness.

Sensitive and Selective Reversed-Phase High Performance Liquid Chromatographic-UV Spectrophotometric Determination of Dextromethorphan and its CYP2D6 Mediated Metabolite, Dextrorphan in Human Urine

Purpose: To develop a simple, sensitive and selective method for the determination of dextromethorphan and its metabolite, dextrophan in human urine using reversed-phase high performance liquid chromatography with UV-spectrophotometric detection (RP-HPLC-UV). Methods: Pre-column sample clean-up was carried out by liquid-liquid extraction of the analytes with chloroform: isopropanol (70:30) solution after alkalization of 1000 µL sample and spiking of internal standard, morphine. The samples were chromatographed in a reversed-phase (C-18) ultra sphere silica (5µm particle size and 250 x 4.6 mm I.D). The mobile phase consisted of methanol: acetonitrile: 0.5% w/v ammonium acetate (10:10:80) adjusted to pH 2.8 with orthophosphoric acid and pumped through the column at 1ml/min flow rate. The analytical method was validated for accuracy and precision as well as the recovery of the analytes, dextromethorphan and its metabolite, dextrophan over the concentration range of 0.20 to 5.0µg/ml. Results: The standard curves were linear over the concentration range of 0.2 to 5.0µg/ml for dextromethorphan and dextrorphan. The regression coefficients (R 2 ) of the analytes were >0.99. The method was reproducible with coefficient of variation for the analytes being < 10 %. Dextromethorphan was well resolved from its metabolite, dextrorphan and the internal standard, morphine. The limits of detection of dextromethorphan and dextrorphan were 50ng/ml and the recoveries and accuracies were greater than 85 and 90 %, respectively. Conclusion: The analytical assay method exhibits good precision and selectivity and it was applied to the analysis of dextromethorphan and dextrorphan in urine for the assessment of CYP2D6 activity.

Validation of an Analytical Method for Assay of Immunoreactivity of 99mTechnetium‐radiolabelled Monoclonal Antibodies

A validation is described of the popular Lindmo assay which is widely used for the determination of immunoreactive fraction of radiolabeled monoclonal antibodies used in tumour targeting. 99mTechnetium-labelled PR1A3 anti-carcinoembryonic antigen (CEA) antibody was incubated with tumour cells bearing the appropriate CEA epitope over a range of concentrations and using optimized assay conditions. Cell-bound radioactivity was measured following centrifugation and the immunoreactive fraction determined by extrapolation of a Burke-Lineweaver plot to the y-axis. The specificity, repeatability, reproducibility, accuracy, sensitivity and linearity of the assay were explored and found to lie within acceptable limits.

Comparison of IFCC and NGSP methods for determination of glycated haemoglobin using advanced regression techniques

Abstract The objective of this work was a correct statistical comparison of two assay methods for determination of glycated haemoglobin HbA1c. The immunoturbidimetric determination of HbA1c was performed in two ways: using an automatic analyser Hitachi 912, calibrated according to the IFCC reference system (International Federation of Clinical Chemistry and Laboratory Medicine) and using an analyser Advia 1200 and the NGSP reference system (National Glycohaemoglobin Standardization Program). For statistical comparison of these two analytical methods several advanced regression methods were used, which respect random errors of both compared methods. Specifically, Deming regression with and without weights, orthogonal regression, and Passing-Bablok regression were employed. The results demonstrate that the investigated analytical assay methods do not correspond to each other. The summarized results indicate usefulness of better harmonisation of two existing reference systems.

Choice of analytical assay methods from a pharmacopoeial point of view

The purpose of the European Pharmacopoeia is to promote public health by the provision of recognised common standards for use by healthcare professionals and others concerned with the quality of medicines. Such standards are to be appropriate as a basis for the safe use of medicines by patients and consumers. Their existence:

Degradation studies of pentoxifylline: Isolation and characterization of a novel gem-dihydroperoxide derivative as major oxidative degradation product

Pentoxifylline was subjected to various stress conditions and degradation profile was studied with conventional LCMS. Interestingly, under oxidative stress conditions the drug substance underwent distinct transformation to give rise to a single major degradation product. The structure of this product was elucidated using 1D, 2D NMR spectroscopy, high resolution mass spectrometry (Q-TOF LC/MS) and found to be a novel gem-dihydroperoxide, namely 1-(5,5-Bis-hydroperoxy-hexyl)-3,7-dimethyl-3,7-dihydro-purine-2,6-dione. An efficient stability indicating liquid chromatographic separation method was developed for pentoxifylline and its three degradation products (including two from base hydrolysis) using 1.8 μm, C18 reverse phase column and UHPLC. Baseline separation was achieved with a run time of 4 min. The analytical assay method was validated with respect to system suitability, specificity, linearity, range, precision, accuracy and robustness.

Method development for HILIC assays

In this review, method development for hydrophilic interaction LC (HILIC) is highlighted. HILIC is a chromatographic technique that uses aqueous-organic solvent mobile phases with a high organic-solvent fraction, and a hydrophilic stationary phase. It is mainly applied for the separation of polar and hydrophilic compounds. Method development, in general, can be done uni- or multivariately. In the univariate approach, the factors that are expected to potentially affect the separation of the compounds will be examined sequentially and one-at-a-time. All HILIC methods found in the literature were developed in this way. For these methods, the analytes, the considered factors, the selected responses, and the finally chosen experimental conditions are discussed in this review. Where examined, the method validation and the comparison with other analytical assay methods is also described. For the multivariate method-development approach, which is based on the use of experimental designs, only a possible strategy is presented, because of the lack of relevant publications in the literature.

Analytical methods for assay of ellagic acid and its solubility studies

Ultra-violet (UV) spectrophotometric and high performance liquid chromatographic methods for quantitative determination of ellagic acid (EA), an antioxidant were developed. The analytical methods were validated for linearity, accuracy, intra- and inter-day variability, and precision. EA was eluted using a polyethylene glycol (PEG) column with mobile phase composed of acetonitrile and 5 mM potassium dihydrogen orthophosphate buffer pH 2.5 (80:20, v/v). The UV and high performance liquid chromatography (HPLC) methods have lower detection limits of 0.2 and 0.1 μg/ml, respectively. Because of the increasing pharmaceutical interest in phytochemicals and their solubility problems, solubility studies for EA were also carried out. Various organic solvents and surfactants were screened for assessing solubility of EA and the compound was found to be soluble in some pharmaceutically acceptable solvents like triethanolamine, polyethylene glycol 400 and N-methyl pyrrollidone (NMP). Aqueous and pH dependent solubility of EA were determined using UV and HPLC methods, respectively.

Is it the Method or the Process—Separating the Causes of Low Recovery

During the development of a tablet formulation, a solvent capable of extracting 100% of the drug from the tablet excipients must be identified as part of the analytical assay method. When a low drug recovery from a tablet is observed with the assay method, it must be determined whether a problem with the manufacturing process exists, or if the extraction of the drug was incomplete. A solvent screen study was conducted with CP‐122,721 prototype formulations to select a robust solvent for the assay method. However, low tablet assay values (ca. 95%) were routinely observed during tablet formulation development and process scale up. Drug‐excipient interactions in a variety of solvents were subsequently evaluated to confirm the selection of the extraction solvent as capable of 100% extraction. At this point the focus of the investigation was placed on process‐related sources of low recovery, such as loss of drug to manufacturing equipment and/or segregation during the tableting process. The results suggest that the low drug recovery observed for the CP‐122,721 tablets was due to segregation during the manufacture, while the selected extraction solvent was able to eliminate any interactions between CP‐122,721 and the tablet excipients.

Validation of a near-infrared transmission spectroscopic procedure: Part B: Application to alternate content uniformity and release assay methods for pharmaceutical solid dosage forms

NIR analytical methods can be validated to meet the requirement of demonstrating that it is suitable for the analysis of the materials for which it is being used. Applying previously described protocols for NIR methods to the analysis of two types of pharmaceutical products shows that for these products, NIR is suitable as an alternate analytical method for assay and for content uniformity.