Analysis Of Related Substances Research Articles

Development of an extemporaneous preparation formulation using a simple and non-solubilizing matrix for first in human clinical study

Extemporaneous preparation (EP) formulation is an attractive strategy to accelerate the formulation development of new chemical entities for first entry into human study. In this work, an EP suspension formulation for a development drug candidate GDC-6599 was successfully developed. The formulation spanned a wide concentration range from 0.1 to 2.0 mg/mL. A non-solubilizing vehicle, 0.6 % (w/v) methylcellulose solution was used to suspend GDC-6599. An aversive agent denatonium benzoate at an extremely low level (6 ppm) was applied as a taste masking agent. This enabled a simple matrix for the analysis of related substances from GDC-6599 during all stability studies. Microcrystalline cellulose at 10 mg/mL concentration was added to the EP formulation to generate a suspension appearance, leading to the success of using a single placebo for matching active formulation at all concentrations. The developed formulation demonstrated excellent homogeneity, sufficient stability and passed microbiological enumeration test. Rinsing performance test demonstrated that greater than 99.8 % amount of drug was successfully recovered by rinsing with water twice, providing guidance for clinical dosing. Biopharmaceutical assessment was conducted by both in silico simulation and in vitro tests. Greater than 90 % bioaccessibility of the EP suspension formulation was obtained via an in vitro system mimicking the human gastrointestinal absorption, consistent with the result from the in silico modeling. The developed EP formulation was successfully used to support the early single ascending dose (SAD) cohorts of GDC-6599 Phase I clinical study. The formulation matrix and assessment workflow developed in this work are generalizable as a platform for EP formulation development of new chemical entities for early phase clinical studies.

Development and validation of stability indicating UPLC methods for related substances and assay analyses of ricobendazole hydrochloride

Ricobendazole hydrochloride is an active ingredient of a veterinary antiparasitic drug. The aim of this study was to investigate the degradation of ricobendazole hydrochloride under stress and stability testing conditions, for which we developed and validated the first stability indicating, specific, precise, accurate, and robust assay and related substances UPLC methods. The Acquity UPLC BEH C18 column was used for the related substances and assay analyses of ricobendazole hydrochloride, and the analyses were performed at 25 °C sample and 30 °C column temperatures with a 2 µL injection volume. In both methods, a mixture of water and methanol (60:40, v/v) was used as the diluent, mobile phase A was a phosphate buffer (50 mM potassium dihydrogen phosphate solution, pH 3.2 ± 0.05, adjusted with 10% o-phosphoric acid), and mobile phase B was a mixture of mobile phase A and acetonitrile (50:50, v/v). For the analysis of related substances, a gradient elution system was used at a flow rate of 0.4 mL/min for 35 min with a detection wavelength of 220 nm, while for the assay analysis; a gradient elution system was used at a flow rate of 0.3 mL/min for 15 min with a detection wavelength of 290 nm. The calibration curves showed excellent linearity with high R-squared (R2) values for each compound, ricobendazole (0.9998, 0.249 – 3.740 µg/mL), ricobendazole amine (0.9998, 0.255 – 3.819 µg/mL), albendazole (0.9998, 0.255 – 7.646 µg/mL), and albendazole sulfone (1.0000, 0.251 – 15.090 µg/mL) indicating a strong correlation between the concentrations of the compounds and their respective peak areas in the UPLC analysis. The method showed excellent accuracy with relative standard deviation values of less than 2.5%. The stress and photostability studies showed that ricobendazole hydrochloride was insensitive to daylight and UV radiation and showed significant degradation at elevated temperature (85 °C, 9 days) and under all hydrolysis and oxidation conditions. The major impurity was ricobendazole amine under thermal and hydrolysis conditions, while albendazole sulfone was the major oxidative impurity.

A full-scale tracing study of "ghost peaks" encountered in impurity analysis of budesonide based on experimental operation inspection-liquid chromatography/mass spectrometry fingerprint-mechanism based stress studies integrated strategy.

Budesonide is an active pharmaceutical ingredient used in various dosage forms of finished products for the treatment of asthma. During the process of drug development, unbiased analysis of related substances is of utmost significance for both pharmaceutical research and quality control purposes. In this work, the official method documented in the United States Pharmacopoeia was selected to determine the related substances of budesonide considering the pros and cons of critical chromatographic parameters, compared to the European Pharmacopoeia. In doing so, several unpredictable interference peaks, namely "ghost peaks", were observed occasionally during analysis. A strategy that integrated information derived from experimental operation inspection, liquid chromatography/mass spectrometry fingerprint analysis, and mechanism-based stress studies was then proposed for comprehensively and quickly exploring those non-degradable and degradable peaks. Some ghost peaks were found to originate from nylon syringe filter, illumination, and alkali borosilicate glass high-performance liquid chromatography vials. Besides, degradation pathways under alkaline conditions were also unraveled through liquid chromatography-mass spectrometry qualitative analysis. Overall, an optimization of the analytical methodology based on the United States Pharmacopoeia for its application in impurity analysis of budesonide and corresponding formulations was carried out with the design of experiments, by which "ghost peaks" could be suppressed or prevented. The results obtained herein are not only crucial to studies on budesonide's stability or degradation kinetics but also contribute to clarifying the impurity research of other drugs.

Stability of pemetrexed disodium in sodium chloride 0.9% w/v intravenous Viaflo infusion bags

ObjectivesThe aim of this study was to assess the stability of pemetrexed disodium (Alimta), reconstituted in 100 mL sodium chloride 0.9% w/v intravenous infusion bags (Baxter Viaflo) at two target...

Synthesis and Characterization of Potential and Degraded Impurities of Regadenoson

Background: Regadenoson is an A2A adenosine receptor agonist that is a coronary vasodilator and commonly used as a pharmacologic cardiac stressing agents. Methods: HPLC method was used for the analysis of related substances. The degraded impurities during the process were isolated and characterized by IR, Mass and NMR spectral analysis. Results: Forced degradation study of regadenoson under conditions of hydrolysis (neutral, acidic and alkaline) and oxidations suggested in the ICH Q1A(R2) was accomplished. The drug showed significant degradation under all the above conditions. On the whole, five novel degradation products were found under diverse conditions along with process related impurities which were not reported earlier. Conclusion: All the degradation products were well characterized by using advanced spectroscopic techniques like IR, 1H NMR, 13C NMR and Mass spectra. The identification of these impurities will be productive for the quality control during the production and stability behavior of the regadenoson drug substance.

Optimization of a liquid chromatography method for the analysis of related substances in daclatasvir tablets using design of experiments integrated with the steepest ascent method and Monte Carlo simulation

Analytical method for the determination of related substances (RS) in Daclatasvir tablets was optimised using quality by design (QbD) approach. Seven degradants (each more than 1.0%) generated during oxidation study, adversely affected the selectivity of the method. Coelution of the degradant peaks with API and known impurities, suggested failure in developing a stability indicating method. To overcome the shortcomings and develop a robust method, QbD principles were incorporated. Resolution was the critical quality attribute (CQA) and buffer pH, column oven temperature, gradient slope and flow rate were the critical method variables (CMVs) studied through design of experiments (DoE). Discovery of an unknown impurity (named as impurity D, about1.0%) was a key finding from this DoE study. The most crucial responses viz. Resolution between impurity D and the main peak and resolution between the main peak and impurity E demanded contradictory pH requirements. To select the right pH, responses were prioritised and eventually to attain the desired resolution between Daclatasvir and impurity E the value for pH was fixed to 3.0. Next, to improve resolution between impurity D and Daclatasvir, method of steepest ascent was applied to locate an apt value for column oven temperature. Accordingly, experiments were performed at different temperatures along the path of rapid increase in response. Finally, at 45 °C (pH :3.0), both the critical pairs were well resolved. The global optimum was determined through a Response surface methodology (RSM) design with pH and column oven temperature as critical factors. pH 3.0, column oven temperature 44 °C, % MP. B 45% and flow rate 1.0 mL min−1 was found to be the optimum condition. Further, the design space was complimented by establishment of a robust zone through Monte Carlo simulation and capability analysis. An analytical control strategy (ACS) was set up to ensure that the method repeatedly meets its acceptance criteria. The optimised method was successfully validated within the factor ranges mentioned in the ACS. Despite various intricacies, the QbD approach facilitated systematic optimisation of a stability indicating robust method.

Qualitative and quantitative assessment of related substances in the Compound Ketoconazole and Clobetasol Propionate Cream by HPLC-TOF-MS and HPLC

Related substances in pharmaceutical formulations are associated with their safety, efficacy and stability. However, there is no overall study already published on the assessment of related substances in the Compound Ketoconazole and Clobetasol Propionate Cream. In this work, a reliable HPLC-TOF-MS qualitative method was developed for the analysis of related substances in this preparation with a quick and easy extraction procedure. Besides the active pharmaceutical ingredients, two compounds named ketoconazole impurity B′ optical isomer and ketoconazole impurity E were identified. Furthermore, a new HPLC method for qualitative and quantitative assessment on related substances and degradation products, which were found in the stability test, was established and validated. The single standard to determine multi-components method was applied in the quantitative analysis, which was an effective way for reducing cost and improving accuracy. This study can provide a creative idea for routine analysis of quality control of the Compound Ketoconazole and Clobetasol Propionate Cream.

Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC–MS/MS

Ten related substances in coenzyme A (CoA) were detected using a newly developed gradient reverse phase high performance liquid chromatographic (HPLC) method. A highly specific and efficient LC–MS/MS method was developed to characterize process-related substances and the major degradation products, and three unknown related substances were further synthesized and characterized by H-NMR and C-NMR spectroscopy. Synthesized samples of the related substances were used for quantitative analysis by HPLC. The method was validated according to ICH guidelines with respect to specificity, precision, accuracy and linearity. The forced degradation studies included acidic, alkaline, oxidative, photolytic and thermal stress conditions. Furthermore, we depicted and speculated the probable mechanism of formation of related substances and the plausible fragmentation mechanism showed that all the related substances came from the manufacturing process. Characterization, synthesis and quantitative analysis of related substances were discussed in detail, and were critical for quality control, manufacturing process optimization and CoA monitoring, all of which were important to ensure the security of CoA.

SENSITIVE DETERMINATION OF RELATED SUBSTANCES IN PIOGLITAZONE HYDROCHLORIDE BY HPLC

Objective: An efficient, high performance liquid chromatographic method has been developed and validated for the quantification of related substances in pioglitazone hydrochloride drug substance.Methods: This method includes the determination of three related substances in pioglitazone hydrochloride. The mobile phase A is 0.1% w/v triethylamine in water with pH 2.5 adjusted by dilute phosphoric acid. The mobile phase B is premixed and degassed mixtures of acetonitrile and methanol. The flow rate was 1 ml/min. The elution used was gradient mode. The HPLC column used for the analysis was symmetry C18 with a length of 250 mm, the internal diameter of 4.6 mm and particle size of 5.0 microns.Results: The developed method was found to be linear with the range of 0.006-250% with a coefficient of correlation 0.99. The precision study revealed that the percentage relative standard deviation was within the acceptable limit. The limit of detection and limit of quantitation of the impurities was less than 0.002%and 0.006% with respect to pioglitazone hydrochloride test concentration of 2000 µg/ml respectively. This method has been validated as per ICH guidelines Q2 (R1).Conclusion: A reliable, economical HPLC method was magnificently established for quantitative analysis of related substances of pioglitazone hydrochloride drug substance.

A simultaneous determination of related substances by high performance liquid chromatography in a drug product using quality by design approach

The combination of Abacavir, Lamivudine and Dolutegravir is an anti-retroviral formulation that displays high efficacy and superiority in comparison to other anti-retroviral combinations. Analysis of related substances in this combination drug product was very challenging due to the presence of nearly thirty peaks including the three active pharmaceutical ingredients (APIs), eleven known impurities and other pharmaceutical excipients. Objective of this study was to develop a single, selective, and robust high performance liquid chromatography method for the efficient separation of all peaks. Initially, one-factor-at-a-time (OFAT) approach was adopted to develop the method. But, it could not resolve all the critical peaks in such complex matrix. This led to the advent of two different HPLC methods for the determination of related substances, one for Abacavir and Lamivudine and the other for Dolutegravir. But, since analysis of a single sample using two methods instead of one is time and resource consuming and thus expensive, an attempt was made to develop a single and robust method by adopting quality by design (QbD) principles. Design of Experiments (DoE) was applied as a tool to achieve the optimum conditions through Response surface methodology with three method variables, pH, temperature, and mobile phase composition. As the study progressed, it was discovered that establishment of the design space was not viable due to the completely distant pH requirements of the two responses, i.e. (i) retention time for Lamivudine carboxylic acid and (ii) resolution between Abacavir impurity B and unknown impurity. Eventually, neglecting one of these two responses each time, two distinguished design spaces have been established and verified. Edge of failures at both design spaces indicate high probability of failure. It therefore, becomes very important to identify the most robust zone or normal operating range (NOR) within the design space with low risk of failure and high quality assurance. For NOR establishment, Monte Carlo simulation was performed on the basis of which process capability index (Cpk) was derived. Finally, the selectivity issue problem faced due to the pH dependency and the dissimilar pH needs of the two critical responses was resolved by introducing pH gradient into the program. This new ternary gradient program has provided a single robust method. Thus, two HPLC methods for the analysis of the combination drug product have been replaced with a selective, robust, and cost effective single method.

Comprehensive supercritical fluid chromatography combined with mass spectrometric detection for the analysis of related substances in polyene phosphatidylcholine extracted from soybean and its commercial capsule

Comprehensive supercritical fluid chromatography combined with mass spectrometric detection for the analysis of related substances in polyene phosphatidylcholine extracted from soybean and its commercial capsule

Chemical stability of drugs: Influence of light and temperature on montelukast stability in solution

In this paper, influence of temperature and light on stability of montelukast in solutions (standard substance, chewable tablets and film coated tablets) which were prepared during routine analysis of related substances was investigated. The content of montelukast and degradation products was determined by HPLC method at defined time intervals (influence of temperature was tested at the beginning, after 24 hours, and after 48 hours, and influence of light was tested at the beginning of the experiment and after 12 hours). Stability of montelukast in tested solutions under influence of temperature was acceptable. The content of montelukast ranged from 98 % to100 %, which meets the pharmacopoeia requirements and specifications. Individual and total impurities were within acceptable limits. Presence of photosensitive functional groups in structure of montelukast led to significant photodegradation. Individual and total impurities in samples which were under influence of light were above acceptable limits. Photodegradation occured during first 12 hours. Content of MOK-3 sulphoxide and unknown impurity on RRT 0.76 was above limits in all examined samples. Over a period of 12 h the content of those impurities grew 8-40 fold compared to time zero values.

Analysis of related substances in synthetical arbutin and its intermediates by HPLC–UV and LC–ESI–MS

A high performance liquid chromatography with ultraviolet detection was proposed for the separation and determination of relative substances in industrial arbutin and its synthetical intermediates 4-acetoxyphenol (ATP) and arbutin pentaacetate (ABPA). Liquid chromatography–mass spectrometry was used for further confirmation of these relative substances. As relative impurities, hydroquione (HQ) and 1,4-diacetoxybenzene (DATB) in industrial ATP, HQ, ATP, and DATB in industrial ABPA, and HQ, ATP, DATB, and ABPA in industrial arbutin were, respectively, quantified. As the results showed, the linearity of standard curves was excellent with relative coefficients of over 0.999 for all the detected components, and the intra- and inter-day precisions of impurities determination were satisfactory, with relative standard deviations of not more than 4.0 %. The distribution and transfer of related substances were elucidated in the synthesis process of arbutin. The established HPLC method not only has great potential in quality control of arbutin but can also be used to instruct the optimization of the synthesis procedure.

Development of a RP-LC method for a diastereomeric drug valganciclovir hydrochloride by enhanced approach

Two methods, prescribed in USP, for the analysis of related substances of valganciclovir hydrochloride drug substance, were evaluated in terms of selectivity and ease of use. A new, simple, selective, stability indicating and user friendly RP-LC method was developed for related substances analysis. The developed single method is capable of separating all known impurities, which are quantified by two methods of USP. A central composite design was applied to optimize the critical chromatographic parameters. A multi step gradient program was strategically designed and a part of the program was optimized through Design of Experiments. Separation was achieved with a Zorbax SB C18 column with 0.1% trifluoro acetic acid and methanol in gradient elution. Design space is proposed graphically for the robust operation of the method. The method is linear, precise and accurate from LOQ level to 150% level of specification limit of impurities. Simple modification in the gradient program with reduced run time is also proposed for assay and diastereomer ratio.

Analysis of related substances in bisoprolol fumarate on sub-2-μm adsorbents

Summary The new analytical method of very high pressure liquid chromatography (VHPLC) has been designed for the estimation of related substances in bisoprolol fumarate. Several columns filled with sub-2-μm totally porous and shell adsorbent have been tested and compared during the method development. The columns' resolution was investigated. The shell adsorbent appears to have smaller resolution than the totally porous adsorbent. The tips and recommendations for the HPLC-VHPLC method transfer are presented. Significant reduction in analysis run time and solvent consumption has been obtained.

Identification, preparation and UHPLC determination of process-related impurity in zolmitriptan

A new impurity was detected and determined using gradient ion-pair UHPLC method with UV detection in zolmitriptan (ZOL). Using MS, NMR and IR study the impurity was identified as (4 S,4′ S)-4,4′-(2,2′-(4-(dimethylamino)butane-1,1-diyl)bis(3-(2-(dimethylamino) ethyl)-1 H-indole-5,2-diyl))bis(methylene)di(oxazolidin-2-one) (ZOL-dimer). The standard of ZOL-dimer was consequently prepared via organic synthesis followed by semipreparative HPLC purification. The UHPLC method was optimized in order to selectively detect and quantify other known and unknown process-related impurities and degradation products of ZOL as well. The presented method which was validated with respect to linearity, accuracy, precision and selectivity has an advantage of a very quick UHPLC chromatographic separation (less than 7 min including re-equilibration time) and therefore is highly suitable for routine analysis of related substances and stability studies of ZOL.

Substitute Technology for Reference Substances in the Analysis of Impurities in Cefonicid for Injection with HPLC Using a Diode Array Detector

With HPLC using a diode array detector (DAD), a method of substitution for reference substances in impurity profiling control was developed that combined peak tracking by the correlation of spectra with application of correction factors for determination of each impurity. For qualitative analysis, two-dimensional (2D) standard spectrochromatographic data produced by HPLC-DAD were compared with sample data to develop 2D chromatographic spectral correlative maps so that the three target impurities were recognized without preparation and injection of reference solutions. For quantitative analysis, correction factors among cefonicid and the three impurities were established. The correction factors were 1.06 for 5-mercapto-1,2,3,4-tetrazole 1-methyl sulfonic acid detected at 255 nm, 0.77 for 7-aminocephalosporanic acid detected at 265 nm, and 0.97 for methoxycefonicid detected at 268 nm. The method could be used in analysis of related substances in cefonicid for injection without recourse to chemical reference standards of the three impurities.

Analysis of Duloxetine Hydrochloride and Its Related Compounds in Pharmaceutical Dosage Forms and In Vitro Dissolution Studies by Stability Indicating UPLC

A reproducible gradient reversed-phase ultra-performance liquid chromatographic method is developed for quantitative determination of duloxetine hydrochloride in pharmaceutical dosage forms. The method is also applicable for analysis of related substances and for study of in vitro dissolution profiles. Chromatographic separation is achieved on a 50 mm × 4.6 mm, 1.8 μm C-18 column. Mobile phase A contains a mixture of 0.01 M KH(2)PO(4) (pH 4.0) buffer, tetrahydro furan, and methanol in the ratio 67:23:10 (v/v/v), respectively, and mobile phase B contains a mixture of 0.01 M KH(2)PO(4), (pH 4.0) buffer, and acetonitrile in the ratio 60:40 (v/v), respectively. The flow rate is 0.6 mL/min, and the detection wavelength is monitored at 236 nm. Resolution of duloxetine hydrochloride and three potential impurities is greater than 2.0 for all pairs of components. The drug was subjected to ICH prescribed hydrolytic, oxidative, photolytic, and thermal stress conditions. Method is validated for linearity, specificity, accuracy, precision, ruggedness, and robustness.

Influence of substituent groups at the 3‐position on the mass spectral fragmentation pathways of cephalosporins

The structural fragment ions of nine cephalosporins were studied by electrospray ionization quadrapole trap mass spectrometry (Q-Trap MS(n)) in positive mode. The influence of substituent groups in the 3-position on fragmentation pathway B, an alpha-cleavage between the C7-C8 single bond, coupled with a [2,4]-trans-Diels-Alder cleavage simultaneously within the six-membered heterocyclic ring, was also investigated. It was found that when the substituent groups were methyl, chloride, vinyl, or propenyl, fragmentations belonging to pathway B were detected; however, when the substituents were heteroatoms such as O, N, or S, pathway B fragmentation was not detected. This suggested that the [M-R(3)](+) ion, which was produced by the bond cleavage within the substituent group at the 3-position, had a key influence on fragmentation pathway B. This could be attributed to the strong electronegativity of the heteroatoms (O, N, S) that favors the production of the [M-R(3)](+) ion. Moreover, having the positive charge of the [M-R(3)](+) ion localized on the nitrogen atom in the 1-position changed the electron density distribution of the heterocyclic structure, which prohibits a [2,4]-reverse-Diels-Alder fragmentation and as a result fragmentation pathway B could not occur. The influence of the substituent group in the 3-position was determined by the intensity ratio (e/d) of ions produced by fragmentation pathway A, a [2,2]-trans-Diels-Alder cleavage within the quaternary lactam ring, including the breaking of the amide bond and the C6-C7 single bond (ion d), and fragmentation pathway B (ion e). The results indicate that the electronegativity of the substituent group was a key influencing factor of pathway B fragmentation intensity, because the intensity ratio (e/d) is higher for a chlorine atom, a vinyl, or a propenyl group than that of a methyl group. This study provided some theoretical basis for the identification of cephalosporin antibiotics and structural analysis of related substances in drugs.

A new stability indicating HPLC method for related substances in zolmitriptan

A sensitive, precise, specific, linear and stability indicating isocratic HPLC method was developed for the analysis of related substances in zolmitriptan. The potential known related substances are (S)-4-(4-aminobenzyl)-1,3-oxazolidin-2-one (impurity I) and (S)-4-(4-hydrazinobenzyl)-1,3-oxazolidin-2-one (impurity II). The method can be used for the detection and quantification of known and unknown impurities and degradants in the drug substance zolmitriptan during routine analysis and also for stability studies in view of its capability to separate degradation products.