European Pharmacopoeia Method Research Articles

Development, Validation and Application of a Fast Sequential Method for Na, K, Ca and Mg Determination in Hemodialysis Solutions by HR-CS F AAS.

Hemodialysis solutions are liquid concentrates used during hemodialysis sessions. They are commonly used as a renal replacement therapy. This study aimed to develop a method to determine Na, K, Ca and Mg in hemodialysis solutions by high-resolution continuum source flame atomic absorption spectrometry (HR-CS F AAS) using Design of Experiments (DOE). The combination of the Doehlert Matrix with the desirability function was used to establish a compromise condition in the optimization of the significant variables studied: 60 L/h for acetylene flow rate, 8 mm for burner height, 0.25% (w/v) and 0.60% (w/v) for Cs and La concentrations. The method was validated by following the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and parameters such as the selectivity, linearity, precision, accuracy and robustness were evaluated. Calibration curves with the ranges of 25-130 mg/L for Na, 1.0-5.0 mg/L for K and Ca, and 0.30-0.70 mg/L for Mg were employed. The method proved to be precise (RSD lower than 2.7%) and accurate (mean recovery data, contemplating the three levels added were: 103.0% for Na, 100.5% for K, 101.3% for Ca and 101.5% for Mg). The developed method enables the sequential multielement determination of Na, K, Ca and Mg in a single run. Requiring only one dilution step, this method significantly reduces analysis time for both sample and standard solutions preparation and measurement. This study presents the first method reported in the literature for multielement determination in hemodialysis solutions, offering an alternative approach to the current European Pharmacopeia method.

Evaluation of acellular pertussis vaccine: comparisons among different strains of mice

ABSTRACT The current study was designed to comparatively analyse the reactions of different mouse strains in response to acellular pertussis (aP) vaccine, with attempt to further provide a reference for aP vaccine evaluation. NIH mice, ICR mice, and BALB/c mice adopted from different pharmacopoeias and studies were utilized to measure the immune protection and immunogenicity of the same batch of aP vaccine according to the Modified intracerebral challenge assay (MICA) from some Asian pharmacopoeias and the pertussis serological potency test (PTST) method from European Pharmacopoeia. Based on our results, the aP vaccine detected by NIH mice had the best potency. So the NIH mice were more suitable for detecting the immune protection of aP vaccine by the MICA method. Given that the levels of PT-IgG and FHA-IgG antibodies in ICR mice were the highest, and the levels of Th1 and Th2 cells were significantly increased (P < .01), it was more suitable for the detection of immunogenicity of aP vaccine by PSPT method. Spleen lymphocytes were stimulated by PT and FHA. And the levels of IL-4 in ICR mice and NIH mice were significantly increased, so were the levels of IL-17, IL-23, IL-27, and TNF-α in BALB/c mice. NIH mice have stronger adaptive immunity and the weakest inflammatory response, and ICR mice have enhanced adaptive immunity and inflammatory responses, both of which can be thereby used for evaluation by different pharmacopoeia methods. NIH was more suitable for the MICA method of Chinese Pharmacopoeia, and ICR for the PSPT method of European Pharmacopoeia.

Development and validation of a stability-indicating RP-HPLC method for the determination of fifteen impurities in rivaroxaban

A simple and stability-indicating reverse phase high-performance liquid chromatographic (RP-HPLC) method for the determination of rivaroxaban (RIX) and its related substances was developed. Fifteen impurities of RIX, including three unreported isomers, were identified, synthesized, purified, and confirmed using MS, 1H NMR, 13C NMR, and HSQC spectral methods. This new method offered baseline separation for all monitored impurities, and was fast and reliable when compared to the European Pharmacopoeia method. Optimum separation for RIX and its related impurities was achieved on an octyldecyl silica column (YMC Core C18, 4.6 ×100 mm, 2.7 µm) by using a gradient HPLC method in 38 min. The final method was validated with respect to precision, LOD and LOQ, linearity, accuracy, and robustness. This developed method was suitable for routine quality control and drug analysis of RIX active substance.

Multi-Dose Formulation Development for a Quadrivalent Human Papillomavirus Virus-Like Particle-Based Vaccine: Part I - Screening of Preservative Combinations

The development of multi-dose, subunit vaccine formulations can be challenging since antimicrobial preservatives (APs) often destabilize protein antigens. In this work, we evaluated Human Papillomavirus (HPV) Virus-Like Particles (VLPs) to determine if combining different APs used in approved parenteral products, each at lower concentrations than used alone, would maintain both antimicrobial effectiveness and antigen stability. To identify promising AP combinations, two different screening strategies were utilized: (1) empirical one-factor-at-a-time (OFAT) and (2) statistical design-of-experiments (DOE). Seven different APs were employed to screen for two- and three-AP combinations using high-throughput methods for antimicrobial effectiveness (i.e., microbial growth inhibition assay and a modified European Pharmacopeia method) and antigen stability (i.e., serotype-specific mAb binding to conformational epitopes of HPV6, 11, 16 VLPs by ELISA). The OFAT and DOE approaches were complementary, such that initial OFAT results (and associated lessons learned) were subsequently employed to optimize the combinations using DOE. Additional validation experiments confirmed the final selection of top AP-combinations predicted by DOE modeling. Overall, 20 candidate multi-dose formulations containing two- or three-AP combinations were down-selected. As described in Part 2 (companion paper), long-term storage stability profiles of aluminum-adjuvanted, quadrivalent HPV VLP formulations containing these lead candidate AP combinations are compared to single APs.

In vitro Comparison of Pancreatic Enzyme Preparations Available in the Indian Market

PurposePancreatic enzyme replacement therapy (PERT) involves exogenous enzyme supplementation and is used in the treatment of pancreatic exocrine insufficiency. Clinical efficacy of PERT preparations is a function of physical properties and release kinetics that vary between commercially available products. In this study, we evaluated the physical properties, in vitro dissolution, and release kinetics of commercially available pancreatic enzyme preparations available in the Indian market.MethodsPhysical properties such as particle size distribution and water content of the capsules were measured by dynamic light scattering and Karl–Fischer titration method, respectively. An analytical procedure based on the European pharmacopoeia (EP) method was used to determine lipase activity, and a modified United States pharmacopoeia (USP)–based method was used for dissolution studies. Enzyme release was ascertained under gastroduodenal conditions in buffered media.ResultsConsiderable variations in physical properties such as particle size and water content were observed between pancreatic enzyme preparations. Some preparations failed to meet the labeled lipase content as per USP standards (>90% label claim) and showed inconsistent release behavior (>5% relative standard deviation).ConclusionDifferences exist between pancreatic enzyme preparations in terms of physical properties, dissolution, and release behavior that can affect their clinical efficacy. The present study suggests, therefore, that these preparations should not be used interchangeably.

Computer-assisted UHPLC method development and optimization for the determination of albendazole and its related substances

Computer-aided ultrahigh performance liquid chromatographic (UHPLC) method development and optimization was undertaken in order to replace an underperforming European Pharmacopoeia method for the determination of albendazole and its related substances. In the preliminary screening, a temperature-gradient time bidimensional model was chosen to aid selection of the proper stationary phase. Hereinafter temperature-gradient time-ternary composition and temperature-gradient time-pH tridimensional models were applied for the optimization of critical method parameters. The simulation and in silico robustness testing were realized using DryLab modeling software. The final method was validated for quantification of impurities and assay of the active substance according to the current ICH guidance. The validated methods were tested on a real, commercial tablet formulation. The experimental design-based and software-assisted method development proved to be a fast and reliable way of replacing a method with inadequate selectivity and long runtime with a robust UHPLC-based method, which offers baseline separation for all monitored impurities in 10 min. Results confirm that software-based chromatographic modelling can not only speed up the analytical method development process, but also improve the reliability of the developed method.

Development of a Novel, Fast, Simple HPLC Method for Determination of Atorvastatin and its Impurities in Tablets

Our main target and concept was to develop a method for the determination of the most prescribed antilipemic drug, atorvastatin, together with its related substances, with a single sample preparation and during a single chromatographic run, in the shortest possible period of time, with the lowest possible mobile phase consumption. A new rapid, simple chromatographic method for the determination of atorvastatin and its main specified impurities was developed, using different chromatographic columns. With this new concept of a mobile phase and a powerful core–shell, or a superficially porous silica-based column, satisfactory results for targeted parameters, such as critical peak resolution, run time length, and column backpressure, were achieved. The analysis is performed within a run duration of less than 15 min, which is about six times shorter than the official European Pharmacopoeia method. The chromatogram performances suggests that the method limit of quantification (LOQ) can be about 7 times lower, and the limit of detection (LOD) about 20 times lower, using an injection volume of only 2 µl. This was confirmed by the performed method validation in accordance with the International Conference on Harmonization (ICH) guideline for the validation of analytical procedures Q2(R1), where the selectivity, linearity, accuracy, precision, limit of quantification, and limit of detection were tested and confirmed.

Determination of Anthracene Derivatives in the Antihaemorrhoidal Medicinal Herb Mixture Proctophytol® Using the European Pharmacopoeia Method

Specialists in pharmacognosy are facing an important task of improving quality control methods for combination herbal medicinal products in order to enable reliable assessment of the content of active substances that are responsible for the drug’s pharmacological effect.The aim of the study was to investigate the possibility of using the European Pharmacopoeia method to determine the total content of anthracene derivatives in the antihaemorrhoidal medicinal herb mixture Proctophytol® and to propose limit values for the total content of anthracene derivatives.Materials and methods: individual mixture components containing anthracene derivatives, such as senna leaves and frangula bark, as well as model mixtures containing these individual components in a 1:1 ratio, and a model mixture imitating Proctophytol® were used in the study. The determination of the anthracene derivatives content was carried out using spectrophotometry.Results: it was demonstrated that the European Pharmacopoeia method could be used to assess the quality of Proctophytol® in terms of anthracene derivatives total content. The spectrophotometric method described in the European Pharmacopoeia has advantages over the methods described in manufacturer specifications for Russian products, because it allows for thorough extraction of the active substances and is standardized for anthracene derivatives.Conclusions: the European Pharmacopoeia method was adjusted to determine anthracene derivatives in the medicinal herb mixture Proctophytol®. It was proposed to use 70% ethanol instead of 70% methanol as extraction solvent. The authors identified optimum sample weights and test solution dilutions, and calculated and verified the limit for anthracene derivatives content—“Not less than 1.9%”. The adjusted method can be recommended for inclusion in the monographs of the State Pharmacopoeia of the Russian Federation for medicinal herb mixtures similar to Proctophytol®.

Rapid Capillary Zone Electrophoresis of Recombinant Erythropoietin by the Use of Dynamic Double Layer Coating

The determination of erythropoietin (EPO) isoforms by using capillary zone electrophoresis (CZE) has been a key study in the quality control of erythropoietin products. However, the current European Pharmacopeia method has been reported to provide poor reproducibility, tedious preconditioning, long separation times exceeding 60 min, and the employment of toxic reagents. In this study, a novel rapid CZE method, in which a double layer dynamic coating technique was developed for the determination of erythropoietin isoforms by the minimization of protein adsorption to the inner capillary walls and to accelerate the throughout of the protocol. The results obtained by the measured procedure were demonstrated to meet the European Pharmacopeia requirements. Real samples from different vendors were also analyzed. This rapid CZE method was shown to be suitable for the quality control (QC) erythropoietin with better reproducibility and higher sample throughput.

An Improved Reversed-Phase High-Performance Liquid Chromatography Method for the Analysis of Related Substances of Prednisolone in Active Ingredient.

Prednisolone, an important active pharmaceutical ingredient, is a synthetic glucocorticoid used for the preparation of various pharmaceutical products with anti-inflammatory and immunosuppressive properties. It is a challenge in high-performance liquid chromatography (HPLC) to separate the prednisolone peak and its structurally related substance (hydrocortisone), which only differs in a double bond at the C-1 position. Successful application of the HPLC method according to the European Pharmacopoeia monograph for related substances of prednisolone is very often limited to the chromatographic system available. This is due to the nonbaseline separation of the prednisolone and hydrocortisone peaks, which is strongly influenced by the instrument parameters and the chosen C18 column. First, an adjusted European Pharmacopoeia method for related substances of prednisolone was developed within the allowable adjustments. Next, an improved stability-indicating reversed-phase HPLC method for related substances of prednisolone was developed and validated for use in quality control laboratories for routine analysis. The optimized separation was performed on a Phenomenex Gemini C18 column (150 mm × 4.6 mm, 3 μm) using a gradient mobile-phase system consisting of acetonitrile/tetrahydrofuran/water (15:10:75 v/v/v), acetonitrile/water (80:20 v/v), and ultraviolet detection at 254 nm. A baseline separation was achieved, and stability indicating capability was demonstrated by a forced degradation study. A full validation procedure was performed in accordance with International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines.

Comparison of the NMR and the Acid Value Determination Methods for Quality Control of Input Polysorbates

Polysorbates (PS) are the most common non-ionic surfactants used in protein formulations. Their degradation has been studied intensively in recent years. Ester bond hydrolysis is one of many pathways of PS degradation that can lead to accumulation of free fatty acids (FFAs) and particle formation. The distribution and quantity of FFAs in PSs impacts directly on product quality. Characterization of input PS is highly relevant, because the initial content of FFAs differs greatly between manufacturers. The purpose of this study was to set up a quick and simple analytical method for the quantitative evaluation of FFAs in PS. The content of FFAs was measured for selected PS 20 and 80, using two methods, 1H nuclear magnetic resonance spectroscopy (1H NMR) and the European pharmacopoeia method for determining acid value (IA). These methods have been evaluated using the method of standard addition and, based on the results, they are interchangeable. It was concluded that 1H NMR is a useful tool for quality control of input PS and a rapid method for indicating the rate of PS degradation by hydrolysis and oxidation. Further, a newly discovered impurity in PS raw material, the long chain ketone 12-tricosanone, can be identified using 1H NMR.

Green alcoholic solvent and UAE extraction of oleuropein from the Olea europaea L.leaves: Experimental design, optimization, and comparison with Pharmacopoeia method

ABSTRACT Solvent extraction and UAE were studied for the extraction of the polyphenolic compounds from the olive leaf in MeOH/H2O and EtOH/H2O solvent systems. Box Wilson-CCRD design was used under RSM for the experimental design of alcoholic solvent extraction. Multi-response optimization was solved with the help of pareto areas and optimal input variable values were determined by the desirability function, optimization by fuzzy similar precedable ratio method. It is seen that the developed solvent extraction methods are more advantageous to the European Pharmacopoeia method for especially oleuropein and the other phenolics which have synergetic effect in olive leaf extract.

New possibilities for pharmaceutical excipients analysis: Combustion combined with pyrohydrolysis system for further total chlorine determination by ICP-OES

An alternative method for the determination of total chlorine content in hydroxypropyl cellulose (HPC) was applied, combining a recently developed system based on a combustion step followed by pyrohydrolysis reaction. Using this approach it the determination of total chlorine by inductively coupled plasma optical emission spectrometry (ICP-OES) without interferences was feasible. It overcame the limitations of European Pharmacopoeia (EP) method for HPC analysis regarding to the inability to determine total chlorine in HPC, once some chlorine compounds (e.g., chloroform) that can not be identified by the official method (EP). The following parameters of combustion and pyrohydrolysis were evaluated: absorbing solution, sample mass, the use of powdered silica as retardant of combustion, oxygen flow rate and reaction time. Reference values for total chlorine were obtained after digestion using microwave-induced combustion and determination by ion chromatography (IC). Microwave-assisted extraction (MAE) was also investigated for Cl extraction. The accuracy of the proposed method was also evaluated by analyte recovery tests (agreement of 95–103%), as well as by the analysis of certified reference materials (CRMs). The agreement with the certified values was higher than 95% and the limit of quantification (LOQ) was 50 µg g−1. Up to 500 mg of sample were efficiently digested by the proposed method in 5 min (dissolved carbon in digests was below 50 mg L−1). Total chlorine content in samples of modified cellulose ranged from 284 to 576 µg g−1. Despite the relatively high chlorine content in all samples, the concentration was lower than the maximum limit allowed by the EP for HPC (0.5%).

An alternative methodology for the determination of the radiochemical purity of 11C-methionine

Background[11C]Methionine ([11C]MET) acts as an oncological radiopharmaceutical for measuring increased protein synthesis in brain tumours while having low uptake in normal brain an. In several Latin American countries, the official pharmacopoeias are the United States of America Pharmacopoeia (USP) and the European Pharmacopoeia (EP). For determination of the radiochemical purity, we used the monograph of L-methionine injection ([11C] methyl-methionine) of the EP (01/2002: 1617) because it is not available in the USP.We present herein an alternative methodology for the determination of the radiochemical purity, which allows the assessment of the [11C]CH3I and of the degradation radioactive products.ResultsThe proposed method enabled a greater number of impurities to be determined than could be identified using the EP method. Under the conditions proposed by EP, it is not possible to determine the presence of [11C]CH3I or other lipophilic radiochemical impurities in the final product.For the determination of enantiomeric purity according to EP a chiral thin-layer chromatography (TLC) allows the determination of D and L methionine between the established limits. This is a slow method and presents an inadequate detection due to the decay of the sample. An alternative HPLC method was optimized.ConclusionThe proposed radiochemical purity method allowed the identification of a greater number of radiochemical impurities compared to the method proposed by the EP monograph in 16 min.The proposed enantiomeric purity method enables to perform the determination in a reasonable amount of time.A discussion concerning the limit of 95% radiochemical purity proposed by the EP is necessary. If more radiochemical impurities can be detected, it is more reasonable to propose a limit of 90% for this parameter to perform clinical examinations.

Increased sensitivity of microbiological testing of cornea organ culture medium by additional resin treatment

ObjectiveThis validation study investigates the treatment of cornea organ culture medium (Modified Eagle Medium, Biochrom GmbH, Berlin, Germany) with RESEP, a new medical device for antibiotics removal, before microbiological testing...

Validation of a major modification of the mobile phase in the European Pharmacopoeia LC-UV human insulin assay

To prevent the buildup of salt crystals inside the chromatograph tubing and pump that occurs with the high mobile phase sodium sulfate concentration (148 mM) used in the European Pharmacopoeia method for the quantification of human insulin, the use of a lower concentration was proposed. A minimal concentration of 37 mM sodium sulfate maintained the chromatographic performance while avoiding instrumental damage. This buffer concentration modification is considered as a major change by the European Pharmacopoeia and this improved method therefore had to be validated. Using a single-point calibration (600 µg.mL−1) yielded adequate precision (the highest relative standard deviations for repeatability and intermediate precision were <5% and <8%, respectively) and accuracy (bias values between −8% and +7%) for the quantification of human insulin in solutions of known concentration and in commercial insulin preparations. The limits of detection and quantification (1.6 and 4.5 µg.mL−1, respectively) were largely sufficient for the purpose of the method.

A fast and robust hydrophilic interaction liquid chromatography tandem mass spectrometry method for determining methylpentose, hexose, hexosamine and hexonic acid in pneumococcal polysaccharide vaccine hydrolysates

The conventional UV/Vis spectroscopy methods recommended by the European Pharmacopoeia (EP) for determining hexosamine, hexonic acid and methylpentose in pneumococcal polysaccharide vaccine (PPSV) hydrolates are time-consuming due to derivatization process (typically, an analysis cycle is more than 4 h) and improvements of selectivity and precision of the methods are in demand. In this study, a new approach based on hydrophilic interaction liquid chromatography and triple quadrupole mass spectrometry (HILIC-MS/MS) was optimized to overcome the drawbacks of the EP methods for simultaneous determination of methylpentose, hexose, hexosamine and hexonic acid in PPSV hydrolysates. The chromatographic, MS and sample hydrolysis conditions were systematically investigated. A zwitterionic column, Click Cys, using a gradient elution with a mobile phase of 10 mM ammonium formate (pH 4.3) in acetonitrile from 72% to 21% in 6 min was applied for separating the targets, which exhibited low column bleeding, easy equilibration and long-term stability. The HILIC-MS/MS method showed a high sensitivity (LOD = 0.98 μg L−1 for hexonic acid), a good repeatability (RSD of peak area less than 1.669%), accuracy (92.9%–104.2%), recovery (97.6%–99.3%) and a wide linear range. The RSD of retention time obtained from more than 3000 injections in three months was less than 1.64%. The new method was compared with the EP method for determining hexosamine in 23 serotypes of PPSV hydrolysates. The results indicated that the new HILIC-MS/MS method was highly selective, accurate, stable and extremely fast due to without need of derivatization, as compared to the conventional EP methods.

A new two-strip TLC method for the quality control of technetium-99m mercaptoacetyl-triglycine (99mTc-MAG3)

Background99mTc-mercaptoacetyl-triglycine (99mTc-MAG3) has been used for dynamic renal imaging since about 30 years. Free pertechnetate (99mTcO4), colloidal 99mTc ((99mTcO2)n), 99mTc-tartrate (precursor), precomplexes (99mTc-(MAG3)x) and lipophilic 99mTc-MAG2 are the main radiochemical impurities that may occur in the preparation. The total amount of these impurities has to be identified before release of the product for patient administration to guarantee patient safety and good image quality. The European Pharmacopoeia suggests a method based on high-pressure liquid chromatography analysis in combination with a paper chromatography. This analytical method is time consuming, expensive and requires specially trained technicians. As a consequence, it is not widely applied in nuclear medicine radiopharmacies.ResultsWe developed a simple method for radiochemical purity testing of 99mTc-MAG3. The method is based on thin layer chromatography with two strips to be developed in parallel. Method validation was carried out in comparison to the official methods of the companies and to the European Pharmacopoeia method. It was tested on specificity, accuracy, robustness and precision.ConclusionThe proposed method is able to identify and quantify the sum of all impurities occurring in the preparation, respecting the acceptance criteria for the radiochemical purity defined by the official methods. Hydrophilic and lipophilic compounds are identified separately and results are obtained within less than 20 minutes. Our method is simple, cost effective, fast and is suitable for employing dose calibrators or radiometric scanners.

Method for sterility testing of corneal storage and transport media after removal of interfering antimicrobials: prospective validation study in compliance with the European Pharmacopoeia

ObjectiveThis study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C, and the transport/deswelling medium, CARRY-C, according to the method suitability test, as defined by...

User-Friendly HPLC Method Development and Validation for Determination of Enalapril Maleate and Its Impurities in Enalapril Tablets.

The official method for the determination of Enalapril Maleate and its related substances in European Pharmacopoeia (EP) is a gradient liquid chromatographic method. The method used styrene-divinylbenzene copolymer column, mobile phase buffer pH 6.8 and column oven temperature 70°C. In this method, the separation between main component Enalapril and Ph. Eur. Imp-A was not completed hence the achieving system suitability requirement is a tough task and it requires quite often adjustment in chromatographic parameters. Moreover, column oven temperature 70°C is not user friendly to HPLC instruments and users. In this study, several changes were introduced to the method in order to improve the separation, peak shapes and to overcome the column oven temperature. A new user-friendly stability-indicating RP-HPLC method was developed for Enalapril related substances analysis. The developed method uses a ZORBAX Eclipse XDB-C18 column with column oven temperature at 55°C and mobile phase containing acetonitrile and a phosphate buffer at pH 3.0. The method is capable of separating all the known impurities with resolution more than 3.5, which is much better than that obtained with the existing monograph methods. The optimized method was validated and demonstrated to have acceptable specificity, sensitivity, linearity, accuracy, precision, robustness, solution stability and equivalency to the EP method. The developed method proved to be applicable to a wide number of C18 reversed-phase columns. In addition, the Enalapril assay method also presented with 20 min run time.