Validation Of Method For Determination Research Articles

Validation of methods for determination of lead and cadmium content in butterfly pea flowers (Clitoria ternatea) in the Cai Rang district, Can Tho city by atomic absorption Spectrophotometer AAS

The content of cadmium (Cd), lead (Pb) was determined in butterfly pea flower samples by atomic absorption Spectrophotometer (AAS). Method validation was evaluated through of parameters such as linear range, the limit of detection (LOD), the limit of quantitation (LOQ), recovery, repeatability, and intermediate precision. The validation results showed that the LOD of Cd, Pb were 1.20 and 6.20 μg/kg, respectively, while the LOQ of Cd and Pb were 4.01 and 20.5 μg/kg, respectively. The recovery of Cd is 96 - 98% and Pb is 98 - 100%. The relative repeat standard deviation (RSDr) of Cd, Pb is in the range of 1.50 - 2.50% and 1.90 - 2.13%, respectively. Meanwhile, the intermediate precision of Cd, Pb is 2.02% and 1.97%, respectively. The working range of the method is 0.2 - 4 μg/L (Cd) and 3 - 30 μg/L (Pb) with the standard curve equations having the correlation coefficient R2 ≥ 0.995. The quantitative method of Cd, Pb by atomic absorption Spectrophotometer is accurate and highly reliable, used to analyze butterfly pea flower samples collected in Cai Rang district, Can Tho city.  

Development and validation of a high performance liquid chromatography-MS/MS method for determination of SOMCL-15-290 in a first-in-human study.

Background: SOMCL-15-290 is a novel inhibitor that targets FGF receptor, CSF1 receptor and VEGF receptor (kinase insert domain receptor). Aim: This study was aiming at developing a specific high performance liquid chromatography-MS/MS method for quantifying SOMCL-15-290 in human plasma and supporting the first-in-human study. Methods: Plasma samples were prepared using the protein precipitation method and separated on a C18 110A column with acetonitrile and 0.2% formic acid solution as mobile phases. Quantification of SOMCL-15-290 was operated on an Xevo-TQS triple quadrupole tandem mass spectrometer in electrospray ionization positive mode. Results & conclusion: The validated determination method of SOMCL-15-290 has proved feasible and was successfully utilized in the first-in-human study of SOMCL-15-290 in advanced solid tumor patients.

Development and Validation of UV Spectrophotometric Method for Determination of Chrysin and Its Solubility Studies

Development and Validation of UV Spectrophotometric Method for Determination of Chrysin and Its Solubility Studies

Validation of RP-HPLC Method for Determination of Omeprazole in Dissolution Media and Application to Study in-vitro Release from Solid- SNEDDS

Background: Omeprazole has poor water solubility, is unstable in acidic solutions, and undergoes first pass metabolism which results in lowering its bioavailability. A solid Self-Nano Emulsifying Drug Delivery System (SNEDDS) was previously prepared to enhance its dissolution. Objective: Development and validation of a RP-HPLC method with UV detection for the determination of omeprazole in 0.1N HCl and in 0.01 M phosphate buffer (pH 7.4). Methods: Validation was according to the ICH Q2 (R1) guidelines in terms of linearity, accuracy and precision, lower limit of quantification, sensitivity, specificity, and robustness. The developed and validated method was used to study the in-vitro dissolution of the drug from the solid-SNEDDS, commercial products and of the unprocessed drug. The dissolution was studied in 500 ml of 0.1N HCl during the first 2 hours, and 900 mL of 0.01 M phosphate buffer (pH 7.4) during the last hour (37 ± 0.5 oC and 100 rpm). Results: The method was linear in the range 1-50 μg/ml, accurate and precise as indicated by the ANOVA test. It was specific to the drug and the pharmaceutical excipients did not affect the determination of its concentration. The method was robust to small changes in pH, composition, and flow rate of the mobile phase. The dissolution rate of omeprazole from the Solid-SNEDDS was faster than that from two commercial dosage forms and than the dissolution rate of the unprocessed drug. Conclusion: The method met the acceptance criteria and was applied successfully in studying the rate of dissolution of the drug.

Management of validation of HPLC method for determination of acetylsalicylic acid impurities in a new pharmaceutical product

The work mainly focused on a validation of the method for determining the content of salicylic acid and individual unknown impurities in new pharmaceutical product—tablets containing: 75, 100 or 150 mg of acetylsalicylic acid and glycine in the amount of 40 mg for each dosage. The separation of the components was carried out by means of HPLC, using a Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) as the stationary phase. The mobile phase consisted of a mixture of 85% orthophosphoric acid, acetonitrile and purified water (2:400:600 V/V/V). Detection was carried out at a wavelength of 237 nm, with a constant flow rate of 1.0 ml min−1. In order to verify the method, linearity, precision (repeatability and reproducibility), accuracy, specificity, range, robustness, system precision, stability of the test and standard solution, limit of quantification and forced degradation were determined. Validation tests were performed in accordance with ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines. The method was validated successfully. It was confirmed that the method in a tested range of 0.005–0.40% salicylic acid with respect to acetylsalicylic acid content is linear, precise and accurate.

Development and validation of a LC-PDA method for methylphenidate analysis in sewage

Methylphenidate (MPH) is an important emerging pollutant found in effluents and wastewater. Thus, we aimed to develop and validate a method for detection and quantitation of MPH residues in sewage through high performance liquid chromatography coupled with photodiode array detector (LC-PDA). Here we describe a selective, accurate, precise, and valid method for determination of MPH in sewage with a total running time of 10 min, with limits of detection and quantification of 0.27 and 0.92 µg/mL, respectively. MPH retention peak was observed at 5 min. The method was applied to MPH analysis in a sewage sample pretreated with solid phase extraction, obtaining a result of 2.8 µg/L of MPH. Thus, the developed method can be considered feasible to be applied to MPH residual contamination analysis in sewage using a widely available apparatus.

Validation of the ICP-MS Method for Determination of Trace Elements Provided by ICH Q3D Guideline in Fosamprenavir Calcium

Validation of the ICP-MS Method for Determination of Trace Elements Provided by ICH Q3D Guideline in Fosamprenavir Calcium

Development and Validation of an Analytical Method for Determination of Al, Ca, Cd, Fe, Mg and P in Calcium-Rich Materials by ICP OES.

Four procedures based on closed-vessel microwave-assisted wet digestion with different oxidative reagents, including HNO3 (P1), HNO3 + H2O2 (P2), aqua regia (P3) and Lefort aqua regia (P4), for preparation of calcium (Ca)-rich materials prior to determination of total concentrations of Al, Ca, Cd, Fe, Mg and P by inductively coupled optical emission spectrometry (ICP OES) were compared. It was found that digestion with Lefort aqua regia (P4) provided the best results for all examined elements, i.e., precision of 0.30–4.4%, trueness better than 2%, recoveries of added elements between 99.5–101.9%, and limits of detection within 0.08–1.8 ng g−1. Reliability of this procedure was verified by analysis of relevant certified reference materials (CRMs), i.e., Natural Moroccan Phosphate Rock—Phosphorite (BCR-O32). Additionally, selection of appropriate analytical lines for measurements of element concentrations, linear dynamic ranges of calibration curves and matrix effects on the analyte response were extensively investigated. Finally, the selected procedure was successfully applied for routine analysis of other Ca-rich materials, i.e., CRMs such as NIST 1400 (Bone Ash), CTA-AC-1 (Apatite Concentrate Kola Peninsula) and NCS DC70308 (Carbonate Rock), and six natural samples, such as a dolomite, a phosphate rock, an enriched superphosphate fertilizer, pork bones, pork bones after incineration, and after steam gasification.

Bioanalytical Method Validation of an RP-HPLC Method for Determination of Rifampicin in Liver Perfusion Studies

Background: The number of validated quantification methods for rifampicin, a prototypical Oatp inhibitor, in biological rat samples was limited. Objective: This study was conducted to validate a modified reversed-phase liquid chromatographic method for the determination of rifampicin in rat liver tissue according to the current ICH M10 Bioanalytical Method Validation Draft Guideline (2019) for application to samples of in situ rat liver perfusion studies. Methods: Liver tissue samples were obtained from recirculatory in situ rat liver perfusion studies. The analysis was performed on a C18 column with a mobile phase composed of 0.05 M phosphate buffer (pH 4.58): acetonitrile (55:45, v/v). The assay was validated for selectivity, calibration curve and range, matrix effect, carry-over, accuracy and precision, reinjection reproducibility, and stability. Results: he method was considered selective and stable, without having carry-over and matrix effects. The calibration curve was linear (R2: 0.9983) within the calibration range (0.5-60 ppm). Accuracy and precision values fulfilled the required limits. Liver concentrations of rifampicin in liver tissue, obtained after 60 min perfusion with 10 μM and 50 μM of rifampicin, were 45.1 ± 11.2 and 313.4 ± 84.4 μM, respectively. Conclusion: The bioanalytical method validation was completed and the method was successfully applied for the determination of rifampicin in rat liver tissue.

Development and Validation of RP-HPLC Method for Determination of Amlodipine Besylate, Telmisartan and Rosuvastatin Calcium from Synthetic Mixture

Development and Validation of RP-HPLC Method for Determination of Amlodipine Besylate, Telmisartan and Rosuvastatin Calcium from Synthetic Mixture

Development and validation of method for determination of organophosphorus pesticides traces in liver sample by GC-MS/MS-ion trap

AbstractA method for simultaneous determination of trace of four organophosphorus pesticides residues in animal liver samples has been developed and validated. This method is based on the preliminary sample preparation using extraction of target compound with a mixture of toluene-cyclohexane by means of up-to-date accelerated solvent extraction (ASE), liquid-liquid partitioning with acetonitrile and hexane, additional clean up step using QuEChERS method. Further the obtained analytes are determined by gas chromatography with ion-trap detector. The validation of the method is performed in accordance with the recommendations in Document SANTE/11945/2015 and it meets the acceptability criteria for precision, mean recovery and limits of quantification. The samples were investigated by analysing blank liver samples and samples spiked with the target analytes chlorpyrifos-methyl, parathion and pirimiphos-methyl at levels of 25, 50, and 75 ng/g and with diazinon at levels of 15, 30, and 45 ng/g. The recovery for all compounds were in the range from 73 to 104% which perfectly fit with requirements of documents and European legislations. The repeatability and within-laboratory reproducibility also reveal acceptable in documents coefficient of variation and uncertainty less than 20 and 18%, respectively. The limits of quantification were less than 3 ng/g for all compounds and allowed determination of residues below the maximum residue levels (MRLs) set in Regulation (EC) Nº 396/2005.

Development and Validation of a Biolayer Interferometry Method for Determination of Human Anti-PD-1 Monoclonal Antibody Concentration in Cynomolgus Serum

Some types of immunotherapy of malignant tumours are aimed at restoration of T-cells’ ability to recognize and eliminate cancer. Programmed cell death ligand-1 (PD-L1) overexpression is characteristic of many human tumours and is associated with poor prognosis for patients. The development of monoclonal antibodies (mAbs) specific for PD-L1 or PD-1 is a promising area of immunotherapy of malignant tumours. However, before a therapeutic antibody-based product enters the market, it is necessary to ensure its safety and efficacy, i.e. perform a full scope of preclinical and clinical studies.The aim of the study was to develop and validate a bioanalytical method that does not require additional labeling and that could be used for determination of mAbs specific for human PD-L1 in the blood serum of a biological test system during preclinical studies.Materials and methods: an antigen in the form of a dimer of PD-1 extra-cellular domain covalently bonded to the Fc-fragment of human IgG (R&D Systems, USA) was used in the study. The antigen was immobilised on Dip and Read™ Protein A biosensors (Fortebio, USA). The therapeutic anti-PD-1 antibody GNR-051 was developed and produced by IBC “Generium” (Russia). The healthy cynomolgus monkey serum samples used as matrix were obtained from the Research Institute of Medical Primatology (Sochi, Russia). The assessment of binding was performed using Octet® QKe interferometer (Fortebio, USA) by real-time analysis of the dose-dependent rate of the antigen-antibody complex formation.Results: the paper presents experimental data on the development and validation of the test method for determination of the therapeutic PD-1-binding mAb concentration in cynomolgus monkey serum in the antibody concentration range from 2 to 2500 µg/mL. The authors assessed the calibration curve reliability, between-run and within-run precision and accuracy, dilution linearity, specificity and selectivity of the test method.Conclusions: the authors developed and validated the biolayer interferometry-based method for determination of therapeutic mAbs concentration. The method was shown to comply with the Eurasian Economic Union’s regulatory requirements in terms of the main validation parameters: analytical range, accuracy, precision, and selectivity.

Experience in Validation of Methods for Determination of Radiochemical Impurities in Radiopharmaceuticals

Most important quality attributes of any radiopharmaceutical (RPh) are its radiochemical purity (RCP) or content of radiochemical impurities (RCIs) that have to comply with respective norms and limits. However, at present, there is no unified approach to validation of analytical methods in the context of highly radioactive samples.The aim of the study was to develop an approach to validation of methods for determination of RCI content in RPhs.Materials and methods: the authors determined the content of RCIs in a radiopharmaceutical formulation containing a complex of technetium-99m and methylenediphosphonic acid by the radiometric method after isolation of impurities from the main compound by thin-layer chromatography using silica gel and methyl ethyl ketone (for sodium pertechnetate determination) and silica gel and 13.6% sodium acetate solution (for determination of hydrolysed reduced technetium-99m). The radioactivity was registered by a chromatogram scanner with a detector of gamma-rays with energies from 0.05 to 1.5 MeV.Results: the paper analyses existing official approaches to validation of analytical procedures and compares them with the results of experimental studies described in available publications. It assesses the validation parameters for compliance with the acceptance criteria set forth in the current regulations and substantiates selectivity of chromatographic determination of impurities under the selected test conditions. Coefficients of variation for repeatability, reproducibility, and accuracy did not exceed 4.5, 2.8, and 8.9%, respectively, given the relative error of not more than 10.5%. The study demonstrated signal linearity for the 10-fold dilution of the standardised sodium pertechnetate solution, it also demonstrated correspondence between the applied and detected radioactivity when performing the test in the impurity content range of 0.5–5%. The validation procedure was associated with significant radiation burden for the personnel of the quality control laboratory.Conclusions: the authors suggested a methodological approach to validation of methods for determination of RCI content in technetium-99m-based RPhs. This approach may be used in the development of a guideline on validation of analytical methods for RCP/RCI determination in RPhs, or for introduction of relevant sections into existing documents.

Optimization and validation of HPLC method for determination of related and degradation products in Paracetamol tablets 500 mg

Optimization and validation of HPLC method for determination of related and degradation products in Paracetamol tablets 500 mg

Validation of Liquid Chromatographic Analytical Method for Determination of Cephalexin and Aspirin in Pure and Pharmaceutical Preparations

Validation of Liquid Chromatographic Analytical Method for Determination of Cephalexin and Aspirin in Pure and Pharmaceutical Preparations

Validation of an analytical method for determination of eight food dyes in beverage and fruit roll-ups by ion-pair HPLC-DAD

Background and aims: Synthetic dyes are widely used as food additives to avoid the loss of original dye in processed foods and to make the foods more attractive to consumers. For simultaneous determination of 8 most commonly used synthetic colors in beverage and foodstuff, an efficient, selective, and sensitive method is suggested. Methods: To analyze food colors in different beverages and fruit roll-ups, a method using Ion-pair high-performance liquid chromatography with diode array detector was suggested and validated. The separation of dyes from beverage and foodstuff was done by solid-phase extraction (SPE). The ultrasound-assisted solvent extraction method was used to extract dye from fruit roll-ups. Results: The limit of detection and the limit of quantification ranged from 0.036 to 0.07 and 0.098 to 0.2 µg/mL, respectively. IP-HPLC-DAD method was validated using precision (RSD %) and accuracy (Recovery %) of two concentrations of 0.5 and 1 µg/mL in terms of intra- and inter-day. SPE method was also validated using intra- and inter-day precision and accuracy. The most commonly detected dye in the tested samples was carmoisine with a concentration of 386 µg/g. Additionally, the concentration of dye was higher than the permitted level in 28.6% of beverages, 40% of edible ice products, and 100% of fruit roll-ups (Lavashak). Conclusion: This method is an effective, appropriate, accessible, reliable and safe analytic method to analyze eight food dyes

Development and validation of an HPLC method for determination of rofecoxib in bovine serum albumin microspheres.

A simple and reliable HPLC method was developed and validated for determination of rofecoxib in bovine serum albumin microsphere. The analyses were performed on a C18 column (150 x 4.6 mm, 5 μm particle size) at room temperature with UV detection at 272 nm. The mobile phase was composed of acetonitrile-0.1% o-phosphoric acid solution in water (1:1, v/v) mixture, and flow rate was set to 1 mL/min. The method was validated according to the international guidelines with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity, and robustness. The detection and quantification limit of the method were 1.0 μg/mL and 2.5 μg/mL, respectively. The method was linear in the range of 2.5–25 μg/mL with excellent determination coefficients (R2 >0.99). Intra-day and inter-day precision (<1.76% RSD) and accuracy (<0.55 % Bias) values of the method also fulfilled the required limits. It was concluded that the developed method was accurate, sensitive, precise, and reproducible according to the evaluation of the validation parameters. The applicability of the method was confirmed for in vitro quantification of rofecoxib in bovine serum albumin microspheres.

A new approach of specific determination for 6-chlorobenzo[a]pyrene and 7-chlorobenzo[a]anthracene in six different oils

This study developed a new method for analysing 6-ClBaP and 7-ClBaA in six oil samples using liquid-liquid extraction and solid-phase extraction followed by GC-QQQ-MS. The recoveries of 6-ClBaP and 7-ClBaA were 44.26 ± 2.81% and 92.61 ± 4.09%, respectively. Both the instrument and method LODs were lower than 0.2 μg kg−1, while the intra-day and inter-day RSD were less than 5%. Five waste frying oils and one peanut oil were tested with the validated determination method. The concentrations of 6-ClBaP and 7-ClBaA in the tested samples ranged from no detection to 0.29 μg kg−1 and no detection to 0.20 μg kg−1, respectively. The data of 6-ClBaP and 7-ClBaA detected in this study suggest that a large scale of investigation on Cl-PAHs in foods is necessary.

Development and Validation of Spectrophotometry UV-Vis Method for Determination of Primaquine and Chloroquine in Liposome Dosage Form

Development and validation of UV-Vis Spectrophotometry method for simultaneous determination of primaquine and chloroquine in liposome dosage form has been carried out. The method was tested for selectivity, linearity, accuracy, and precision. A phosphate buffer solution pH 7.4 was used as a solvent and observations were made at wavelengths of 220 and 260 nm for simultaneous equations method. The selectivity results of primaquine and chloroquine showed no interference from liposome. Linearity result (r value) of the simultaneous equation method was 0.9998 with the value of Vxo less than 0.5% for both primaquine and chloroquine in the concentration range of 2–10 mg/L. Accuracy was done using spiked placebo method and obtained data analyzed using simultaneous equation method. Persentage recovery of primaquine was 89–97% and 79–108% for chloroquine. The intra-and interday precision of primaquine were 1.72 and 2.57%, respectively. Whereas the intra-and interday precision of chloroquine were 6.93 and 8.77, respectively. Further observation using chromatography method need to be done to have better accuracy results for both substances.

Development and validation of hplc method for determination of andrographolide in raw material and tablet of ethyl acetate fractions of Andrographis paniculata

A simple and selective HPLC method for the determination of andrographolide in raw material and tablet formulation of Andrographis paniculata was developed and validated. The Method was performed using a Poroshell 120 EC-C18 column (3.0 x 50 mm i.d., 2.7 μm particle size), column temperature was maintained at 30°C, isocratic elution with mobile phase of methanol: water (pH 3.05 with phosphoric acid) (50:50 v/v) with flow rate of 0.3 ml/min., 0.5 μl injection volume and detected at 228 nm. The proposed method is selective to separate the peak of andrographolide from other components, generating symmetry peak and good resolution with the retention time of andrographolide of 2.6 min. The calibration plots showed a good linear relationship with r2 = 0.9996 in the concentration range from 50 to 1000 ppm. The LOD and LOQ were 4.89 ppm and 16.19 ppm, respectively. The recovery of the method was found between 93.76 and 101.72% and the relative standard deviations of method was found between 1.60% and 2.39%. The proposed method is suitable for quality control of raw material and tablet formulation of Andrographis paniculata.