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

Background: The robustness is a measurement of an analytical chemical method and its ability to contain unaffected by little with deliberate variation of analytical chemical method parameters. The analytical chemical method variation parameters are based on pH variability of buffer solution of mobile phase, organic ratio composition changes, stationary phase (column) manufacture, brand name and lot number variation;flow rate variation and temperature variation of chromatographic system. The analytical chemical method for assay of Atropine Sulfate conducted for robustness evaluation. The typical variation considered for mobile phase organic ratio change, change of pH, change of temperature, change of flow rate, change of column etc. Purpose: The aim of this study is to develop a cost effective, short run time and robust analytical chemical method for the assay quantification of Atropine in Pharmaceutical Ophthalmic Solution. This will help to make analytical decisions quickly for research and development scientists as well as will help with quality control product release for patient consumption. This analytical method will help to meet the market demand through quick quality control test of Atropine Ophthalmic Solution and it is very easy for maintaining (GDP) good documentation practices within the shortest period of time. Method: HPLC method has been selected for developing superior method to Compendial method. Both the compendial HPLC method and developed HPLC method was run into the same HPLC system to prove the superiority of developed method. Sensitivity, precision, reproducibility, accuracy parameters were considered for superiority of method. Mobile phase ratio change, pH of buffer solution, change of stationary phase temperature, change of flow rate and change of column were taken into consideration for robustness study of the developed method. Results: The limit of quantitation (LOQ) of developed method was much low than the compendial method. The % RSD for the six sample assay of developed method was 0.4% where the % RSD of the compendial method was 1.2%. The reproducibility between two analysts was 100.4% for developed method on the contrary the compendial method was 98.4%.

Development and Validation of a UPLC Method for the Determination of Docetaxel and Its Related Substances in Pharmaceutical Dosage Forms, an Antineoplastic Agent

A novel, simple, and sensitive Ultra Performance Liquid Chromatography (UPLC) method was developed and validated for the quantification of process-related impurities and degradants, as well as the assay of Docetaxel. The stability-indicating capability of the method was demonstrated through forced degradation studies and a comprehensive mass balance evaluation. Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 µm), with gradient elution. The mobile phase A comprised a mixture of water, methanol, and acetonitrile (500:300:200, v/v/v), while mobile phase B was acetonitrile and water (800:200, v/v). The flow rate was set at 0.4 mL/min, with detection at 232 nm using a photodiode array detector. The method exhibited excellent performance, with a tailing factor of 1.10 for Docetaxel. The method was rigorously validated for precision, accuracy, linearity, LOD, LOQ, ruggedness, specificity, and robustness. Forced degradation studies confirmed the method’s suitability for stability analysis. Stability testing on the drug substance was conducted following ICH guidelines.

Methodology Validation of Microbial Limit Test of Bupi Qiangli Ointment

[Objective] This study was conducted to establish a microbial limit test method for Bupi Qiangli Ointment. [Method] The conventional method and medium dilution method were used for bacterial, mold and yeast counting in sample recovery test. [Result] The medium dilution method （1：10 test solution, 0.5 ml/plate） could effectively eliminate the inhibition effect of the Bupi Qiangli Ointment, and the recovery of Staphylococcus aureus was greater than 70% in the 3 batches of samples; and the conventional method exhibited the recoveries of E. coil, Bacillus subtilis, Candida albicans and Aspergillus greater than 70% in the 3 batches of samples. [Conclusion] Due to Bupi Qiangli Ointment has strongly antibacterial effect on Staphylococcus au- reus, the medium dilution method was used for bacterial counting, and the conventional method was used for mold and yeast counting; and the conventional method was used for controlled bacterium examination of E. coll.

Comparison of Crop Model Validation Methods

In this paper, the many indices used in validation of crop models, such as RMSE （root mean square errors）, Sd （standard error of absolute difference）, da （mean absolute difference）, dap （ratio of da to the mean observation）, r （correlation）, and R2 （determination coefficient）, are compared for the same rice architectural parameter model, and their advantages and disadvantages are analyzed. A new index for validation of crop models, dap between the observed and the simulated values, is proposed, with dap〈5% as the suggested standard for precision of crop models. The different kinds of validation methods in crop models should be combined in the following aspects：（1） calculating da and dap; （2） calculating the RMSE or Sd; （3） calculating r and R2, at the same time, plotting 1：1 diagram.

Experimental validation method of elastic thin rod model for simulating the motional cable harness

To analyze the spring disturbance torque caused by motionai cable harness in a stabilized platform, the Kirchhoff theory based cable harness model has been previously developed to dynamically simulate the motional cable harness. In this paper, this model was validated by comparing the simulation results with the experiment results （ both the spring force and the deformed profile of the motional cable harness）. In the experiment, a special optical measuring instrument based on binocular vision was developed and the motion and deformation of cable harness were measured. A simpli- fied stabilized platform system was constructed, and the absolute value of spring disturbance force during the motion of this simplified frame was obtained by using a force gauge （0. 02 N precision）. The physical parameters of experimental specimen were also measured. The experimental and simulated results showed good agreement. These results should be useful for better motional cable harness layout design and reliable evaluation of the spring disturbance torque.

New Stability Indicating Method for Quantification of Impurities in Amlodipine and Benazepril Capsules by Validated HPLC

A stability indicating LC method was developed for the simultaneous determination of Amlodipine and Benazepril capsules in pharmaceutical dosage form. Efficient chromatographic separation was achieved on Symmetry C18 stationary phase with simple combination of amobile phase containing 750 mL of DI Water, 250 mL of Acetonitrile and 2 mL of Octylamine into suitable container with adjusted pH to 2.50 ± 0.05 with the aid of Ortho phosphoric acid delivered in an isocratic mode and quantification was carried out using UV detection at 240 nm at a flow rate of 1.0 mL·min-1 with an injection volume of 20 μl and ambient column temperature. This method is capable to detect both the drug components of Amlodipine and Benazepril in presence of their degradation products (Amlodipine Imp-A and Benazepril Impurity-C) with a detection level of 0.05%. Amlodipine/Benazepril in their combination drug product were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the samples were analysed. Peak homogeneity data of Amlodipine and Benazeprilis were obtained using PDA detector, in the stressed sample chromatograms, demonstrating the specificity. The method shows excellent linearity over a range of 0.05%-2.0% for Amlodipine, Amlodipine Impurity-A and 0.05%-5.0% for Benazepril and Benazepril Impurity-C. The correlation coefficient for Amlodipine and Benazepril is 1. The relative standard deviation was always less than 2%. The proposed method was found to be suitable and accurate for quantitative determination and the stability study of Amlodipine and Benazepril in pharmaceutical preparations. The developed HPLC method was validated with respect to linearity & range, accuracy, precision and robustness.

Quality Control of Tramadol in Kisangani: Development, Validation, and Application of a UV-Vis Spectroscopic Method

Context: Substandard and falsified medicines are circulating in low-income countries mostly without any control. We availed a simple and not expensive UV-Vis spectroscopic method to evaluate the quality of tramadol in Kisangani before and during the Covid-19 period. Methods: For the analytical quantitative method, an experimental design was applied to set up the optimal levels of the selected factors, namely, pH of dissolution medium, type of cuvette, and wavelength. Taking into account the capsule pharmaceutical formulation within 80 - 120 μg·mL-1 concentration range, we analyzed 89 tramadol samples from pharmacies and hospitals of the six Kisangani municipalities. Results: pH showed a significant effect on absorbance, whereas quartz cuvette and wavelength did not. A typical 100 μg·mL-1 tramadol solution gave an absorbance of 0.64 at 272 nm. Validation highlighted a matrix effect observed with a 6% bias. A correction factor of 0.9372 allowed to improve the accuracy profile, which were then totally included within the 10% acceptance limits. Quality control revealed that 25 samples out of 89 were not compliant in terms of manufacturing license, registration status in DRC and content as well. Conclusion: This study showed that the strengthening of analytical strategy in Kisangani is a need.

Solriamfetol impurities:Synthesis,characterization,and analytical method(UPLC-UV)validation

Given that impurities may affect the quality and safety of drug products,impurity identification and profiling is an integral part of drug quality control and is particularly important for newly developed medications such as solriamfetol,which is used to treat excessive daytime sleepiness.Although the highperformance liquid chromatography analysis of commercial solriamfetol has revealed the presence of several impurities,their synthesis,structure elucidation,and chromatographic determination have not been reported yet.To bridge this gap,we herein identified,synthesized,and isolated eight processrelated solriamfetol impurities,characterized them using spectroscopic and chromatographic techniques,and proposed plausible mechanisms of their formation.Moreover,we developed and validated a prompt impurity analysis method based on ultrahigh-performance liquid chromatography with UV detection,revealing that its selectivity,linearity,accuracy,precision,and quantitation limit meet the acceptance criteria of method validation stipulated by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use.Thus,the developed method was concluded to be suitable for the routine analysis of solriamfetol substances.

Validation method for simulation models with cross iteration

Cross iteration often exists in the computational process of the simulation models, especially for control models. There is a credibility defect tracing problem in the validation of models with cross iteration. In order to resolve this problem, after the problem formulation, a validation theorem on the cross iteration is proposed, and the proof of the theorem is given under the cross iteration circumstance. Meanwhile, applying the proposed theorem, the credibility calculation algorithm is provided, and the solvent of the defect tracing is explained. Further, based on the validation theorem on the cross iteration, a validation method for simulation models with the cross iteration is proposed, which is illustrated by a flowchart step by step. Finally, a validation example of a sixdegree of freedom (DOF) flight vehicle model is provided, and the validation process is performed by using the validation method. The result analysis shows that the method is effective to obtain the credibility of the model and accomplish the defect tracing of the validation.

Validation methodology for distribution-based degradation model

Distribution-based degradation models （or graphical approach in some literature） occur in a wide range of applications. However, few of existing methods have taken the validation of the built model into consideration. A validation methodology for distribution-based models is proposed in this paper. Since the model can be expressed as consisting of assumptions of model structures and embedded model parameters, the proposed methodology carries out the validation from these two aspects. By using appropriate statistical techniques, the rationality of degradation distributions, suitability of fitted models and validity of degradation models are validated respectively. A new statistical technique based on control limits is also proposed, which can be implemented in the validation of degradation models＇ validity. The case study on degradation modeling of an actual accelerometer shows that the proposed methodology is an effective solution to the validation problem of distribution-based de qradation models.

Validation of a HPLC Method for Quantification of Thiamine and Its Phosphate Esters in Rat Brain Tissue

The present data show a fast and efficient biological sample processing method for the extraction of thiamine (vitamin B1) and its mono-(TMP) and di-(TDP) phosphate esters from hippocampus, thalamus and prefrontal cortex (PFC) and blood sample of the rodents. In addition, using the hippocampus and standards of these three compounds we validated an isocratic fluorescence HPLC procedure for a simultaneous detection of them in a single chromatogram within a total run time of about 12 min. Reproducibility for TDP, TMP and B1 was 2.66%, 4.50% and 7.43% (intraday) and 37.54%, 25.39% and 25.87% (interday), respectively. Recovery assays were between 96.0% and 101.7%. The calibration curves were linear and the concentrations of the three compounds, all in nanomolar range, were determined in the brain areas and in the blood samples. When compared to the current methods in the literature, this new method provides information on essential variables, such as linearity range and limit of detection, reproducibility and stability of thiamine, TMP and TDP in rat brain samples. The present data on sample processing and B1 and its phosphate ester level determinations are the first to be validated using hippocampus samples of rats.

A Stability Indicating Reverse Phase-HPLC Method Development and Validation for the Estimation of Rucaparib in Bulk and Pharmaceutical Dosage Form

The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min-1 at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.

Titration Method Validation of Live Vaccines against Infectious Bursal Disease

Validating a method of analysis goes through different steps, which aims at testing the normality of measurements distribution, estimating the uncertainty of the components of a measurement （i.e., accuracy and correctness）, and finally, define the control tests of non degradation of the method performances. This paper outlines the steps for validating a biological method of analysis. It involves the construction of an experimental design, a statistical model, and the preparation of an interne laboratory reference material （pilot vaccine）. The latter is used to study the impact of deviation and variation factors, in order to, optimize the analytical method, to evaluate the bias （random error）, and to calculate the uncertainty of measurement, and make the control charts. This method is applied in the titration of live viral vaccines of Gumboro disease on chicken＇s embryos fibroblasts. The experimental results show that potential influence factors related to the titration method had no significant influence on the obtained results. Taking into account these results, an operating mode has been elaborated. The finalized method proved to be faithful to standard deviation of repeatability and reproducibility of 0.21 and 0.22, respectively, with a confidence level of 95%. The calculated uncertainty of measurement is equal to 0.2, which represents the average error level of a titer. A homogeneous stock of interne laboratory reference vaccine （MRIL）, with an average titer of 5.9 log DIT 50, was produced and the control chart set in away to provide the laboratory with an important tool of control and monitoring of the viral titers evolution in time, as well as, the mastery of the validated titration method performances.

Nurses’ movements within and between various paths when improving their communication skills—An evaluation of validation method training

Aims and objectives: To explore any changes in nurses’ skills at communicating with residents with dementia disease when using the validation method, as observed in one-to-one videotaped conversations. Background: Communication difficulties due to cognitive impairment among residents with dementia disease may complicate care situations. Training can improve nurses’ communication skills and increase care quality. The validation method aims to facilitate communication with residents with dementia disease through empathic and confirmatory approaches. Evaluations of the validation method have primarily focused on the residents’ perspective, and reports on nurses’ experiences are sparse. Improved communication and relationships with residents after validation method training have been described previously. Videotaped data could provide additional information about these earlier results. Design: A descriptive qualitative design. Methods: Eight nurses participated in a year of validation method training, including videotaped conversations with eleven residents. Videotapes with at least five months between the first and last recording were analysed and compared qualitatively. Results: The analysis revealed an overall pattern: nurses’ movements within and between various paths when improving their communication skills. This was based on three sub-patterns: from controlling communication towards developing attentiveness in communication, from ambiguous communication towards developing coherence in communication, and from being open and attentive towards having a refined attuned communication. Conclusions: All nurses developed their communication skills during the programme, albeit to different degrees. The findings are in congruence with the experiences described by nurses, and so it is reasonable to believe that the programme helped to improve the nurses’ skills in communicating with residents with dementia disease. Relevance to clinical practice: A validation method training programme could give nurses the possibility to develop their skills in communicating with residents with dementia disease.

Synthesis of an IS and Steviol Glycoside Analysis by a Validated Internal Standard Method

The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-steviol as IS. This is the 19-galactosyl ester of steviolmonoside (13-O-β-D-glucopyranosyl-steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 μm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of steviol glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy.

Determination and Validation of HPTLC Method for Cinacalcet Hydrochloride

Cinacalcet Hydrochloride (CIN) is a new calcimimetic agent indicated for the use in hypercalcemia. The present work is aimed at development and validation of a novel and simple high-performance thin-layer chromatographic (HPTLC) method for the analysis of Cinacalcet Hydrochloride (active pharmaceutical ingredient, API. In the method, Aluminum-backed silica gel 60 F254 plates (10 × 10 cm) were used as stationary phase and chloroform: acetonitrile (6:4, v/v) as the mobile phase, which showed compact bands of Cinacalcet HCl (RF 0.30 ± 0.02). Quantitative analysis was carried out by densitometry at a wavelength of 282 nm. Linear regression analysis for the calibration spots showed good correlation ship with regression co-efficient r2 = 0.9994 in the range of 40 - 160 ng/band. The developed method suitability for quantification of CIN was learned by validating it as per the ICH guidelines. CIN detection limit was 0.48 ng/band and the quantification limit was 1.59 ng/band. The proposed method was found to be linear (r2 = 0.999), precise (%RSD < 2% for intraday and intermediate precision), accurate, specific, and robust. Further, the developed method was validated and found suitable for stress induced studies, since presence of degradants has no effect on CIN estimation. The proposed method was found to be simple, sensitive, precise, accurate and reproducible for the estimation of CIN.

Analytical Method Development and Validation of Some Biosimilar Drugs by High Performance Thin Layer Chromatography

A simple and rapid HPTLC analytical method has been developed and validated for the determination of Etanercept and Filgrastim in pure form and in marketed formulation. Both the drugs were chromatographed on silica gel 60 F254s HPTLC plates, as stationary phase. The mobile phase optimized for Filgrastim and Etanercept was Water: n-butanol (7.5:2.5 v/v) and Isopropyl alcohol: water (6.5:4.5 v/v), respectively. The chromatogram obtained was scanned at 225 nm and 222 nm for filgrastim and etanercept which resulted in a retention factor of 0.45 ± 0.07 and 0.32 ± 0.03, respectively. The method was validated for parameters like linearity, accuracy, precision, specificity and robustness. Recovery studies were performed at three concentration levels, to demonstrate suitability, accuracy and precision of proposed method. Statistical analysis proved that the proposed method is accurate and reproducible with linearity in the range of 500 to 3000 ng/band for filgrastim and 200 to 1200 ng/band for etanercept. The limit of detection and limit of quantification for filgrastim was found to be 63.418 ng/band and 192.177 ng/band. For etanercept, LOD and LOQ were found to be 33.381 ng/band and 101.153 ng/band, respectively. The proposed method can be employed for the routine analysis of selected biosimilars.

Oocyte vitrification:A local validation of the method

Is there a really need to validate oocyte vitrification technique in an ART laboratory before establishing it in daily practice? Validation of micromanipulationbased technique, in this case oocyte vitrification, is essential prior to enlarging its use to routine practice. Oocyte vitrification is a new worldwide used technique and legal recently inFrance. This micromanipulation needs to be performed by a skilled and experienced embryologist and requires an internal assessment in each ART unit before any wide use. We designed a prospective study, from September 2011 to July 2012, using sibling oocytes from women who recovered more than 12 Metaphase II oocytes. A part of freshly recovered oocytes underwent immediate ICSI while the remaining oocytes were vitrified. 87 couples undergoing ICSI were selected based on number of mature oocytes available on the recovery day after denudation. A part of fresh MII oocytes were microinjected and the others were vitrified using an open system (Cryotop?). The major criterion of interest was the number of embryo transferred/ number of Metaphase II ratio for after ICSI on fresh oocytes (42/211) versus vitrified/warmed oocytes (51/204) (p > 0.05). Secondary studied criteria were survival rate (80.5% ± 26.3%), fertilization rate (68.9 ± 33.5) and finally, cumulative pregnancy rate obtained in this study is 40.2%. One of the benefits of such practice is the limitation of embryo freezing. However, the study design delays oocytes warming cycles, due to pregnancies triggered by the transfer of fresh derived oocyte embryos and to the priority to transfer all the frozen embryos before starting oocytes warming. Moreover, no data is available about children’ health. Oocyte vitrification represents not only a change in our daily practice to improve cumulative pregnancy rate but also a promising tool to develop egg banking and donation. Clinical Trials Registration number: 209 R02.

Comparative Analysis and Validation Methodologies of GC and HPLC for Analysis of Cholesterol in Meat Products

This study validated different extraction methodologies and compared the quantification of cholesterol by gas chromatography (GC) and high performance liquid chromatography (HPLC) in mg per 100 g of Italian-type salami and traditional bologna. The GC method used was direct saponification of the samples without derivatizations and the HPLC method was used to extract of the lipid samples. The GC limits of detection and quantification obtained for cholesterol were, respectively, 0.001 and 0.003 mg.g–1. The HPLC values were 0.005 mg.g–1 and 0.016 mg.g–1. The GC recovery rate was 97.10 ± 0.13 and that of HPLC was 93.33 ± 0.22. Comparison of the cholesterol quantity found using the two chromatographic techniques shows that both are capable of quantifying cholesterol in the foods. With regard to costs, analysis time, the cost/benefit relationship was better with gas chromatography than that obtained with high performance liquid chromatography.

Development and Validation of Stability Indicating RP-HPLC-PDA Method for Tenatoprazole and Its Application for Formulation Analysis and Dissolution Study

In the present study, comprehensive stress testing of tenatoprazole was carried out according to ICH guide-line Q1A (R2). Tenatoprazole was subjected to stress conditions of hydrolysis, oxidation, photolysis and neutral decomposition. Extensive degradation was found to occur in acidic, neutral and oxidative conditions. Mild degradation was observed in basic conditions. The drug is relatively stable in the solid-state. Successful separation of drug from degradation products formed under stress conditions was achieved on a Kromasil C18 column (250 mm × 4.6 mm, 5.0 μ particle size) using methanol: THF: acetate buffer (68:12:20 v/v) pH adjusted to 6.0 with acetic acid as mobile phase, flow rate was 1.0 mL●min–1 and column was maintained at 45°C. Quantification and linearity was achieved at 307 nm over the concentration range of 0.5 - 160 μg●mL–1 for tenatoprazole. The method was validated for specificity, linearity, accuracy, precision, LOD, LOQ and robustness.