Impurities In Drug Substances Research Articles

RP-HPLC Method Development and Validation for the Estimation of Lansoprazole in Presence of Related Substances by QbD Approach

A rapid specific RP-HPLC method has been developed for the determination of Lansoprazole impurities in the drug substance. The control of pharmaceutical impurities is currently a critical issue in the pharmaceutical industry. The International Council for Harmonization (ICH) has formulated a workable guideline regarding the control of impurities. The objective of the recent study was to develop and validate a HPLC method for the quantitative determination of process-related impurities of Lansoprazole in pharmaceutical drug substance. Lansoprazole, 2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridinyl] methyl]-sulfinyl]- 1H-benzimidazole is an proton pump inhibitor used in the management of gastric ulcers. Chromatographic identification of the impurities was carried out by response surface methodology, applying a three-level Box Behnken design with three center points. Three factors selected were a mobile phase, flow rate, column temperature. Evaluation of the main factor, their interaction, and the quadric effect on peak resolution were done on Waters Symmetry C8, 250 x 4.6mm, 5µm column is used for the development of the method. The mobile phase consists of buffer and acetonitrile. The flow rate of the mobile phase was 1.0 ml/min with gradient elution. The column temperature is ambient and the detection wavelength is 235 nm. The injection volume was 10 µL. The method was validated as per ICH guidelines for linearity in the range of 50-150 µg/ml and the LOD & LOQ values obtained were 0.437×10-4 and 0.1325×10-3 µg/ml respectively which specifies the method's sensitivity. The proposed method was successfully used to determine the Lansoprazole impurities in drug substances.

Low-Level Determination of Mutagenic Nitrosamine Impurities in Drug Substances by LC–MS/MS

Since the detection of N-nitrosodimethylamine (NDMA) in a batch of valsartan in 2018, at levels exceeding ICH acceptable intake limits for mutagenic impurities, the analysis of nitrosamines has become an intense focus point for the pharmaceutical industry. The identification and low-level determination of nitrosamines in potentially affected materials is challenging and requires the application of highly sensitive analytical techniques. This article reviews the chronological development of the story and the regulatory landscape that has evolved. It will then discuss the development of analytical methods for the determination of a series of nitrosamines referenced by regulatory authorities, demonstrating separation of these compounds from the active pharmaceutical ingredient (API) and looking at how mass spectrometry (MS) can be applied to ensure that the required detection limits can be reached.

Stability-Indicating Method Development and Validation for Busulfan Drug Substance by Gas Chromatography-Flame Ionization Detector.

A new, simple and stability-indicating gas chromatography-flame ionization detection (GC-FID) method was developed and validated for the quantitative determination of busulfan and its organic impurities (OI) in drug substance without derivatization. The chromatographic attributes were achieved on a fused silica capillary column (0.53mm × 30m, 1.0μm, USP Phase G42), using hydrogen as a carrier gas with a split ratio of 1:1. Forced degradation studies were conducted to establish the stability-indicating capability and method specificity that showed the stressed busulfan peak was free from any co-elution. Robustness study demonstrated the chromatograms remained mostly unaffected under deliberate, but small variations of chromatographic parameters, establishing the reliability of the method during routine usage. The method was shown to be reliable, sensitive, specific, linear, accurate, precise and rugged in the 1,4-butanediol concentration range of 1-20μg/mL. The method, intended for compendial uses, is suitable for quantitative analysis of busulfan and its organic impurities in drug substances.

Chromatographic Separation of Synthetic Estrogen and Progesterone in Presence of Natural Congeners: Application to Saliva and Pharmaceutical Samples

Estradiol valerate (EST) and Norethisterone acetate (NOR) are co-formulated as a hormone replacement therapy. Naturally occurring steroidal hormones like estradiol, estrone and estriol are also considered as impurities in drug substance. Chromatographic separation of these five steroidal hormones was developed by HPLC and TLC. Reversed-phase chromatography was applied using either acetonitrile–methanol containing 0.05% β-cyclodextrin as a mobile phase for HPLC with gradient elution and chloroform–propanol–formic acid (5:3:2, by volume) as a developing system in TLC. The methods have been applied for determination of EST and NOR in presence of other natural congeners in biological samples as well as in drug products as official impurities. Solid-phase extraction was applied for saliva sample cleaning using testosterone as internal standard whereas ampoules and tablets formulations were used in pharmaceutical applications. The methods have been validated for bioanalytical and pharmaceutical analysis following the respective FDA and ICH guidelines. The obtained results were statistically compared with the official methods for both drugs.

A Validated Stability-Indicating Liquid Chromatographic Method for the Determination of Lorcaserin and Related Impurities in DRUG Substance Supported by Quality by Design.

Lorcaserin (LOR) is selective and potent antiobesity drug that targets the activation of the serotonin 5HT2C receptor. Here a novel, specific, sensitive stability indicating method was developed and validated for the quantitative determination of LOR and its process-related impurities using quality by design principles. By applying experimental design, the authors examine the multifactorial effect of parameters on the critical resolution pair and generated design space representing the robust design. LOR was subjected to stress condition and found stable at all condition, only found significant degradation at oxidative stress condition. The chromatographic separation of degradation product and its process-related impurities were achieved on a Phenomenox Luna phenyl-hexyl column (150×4.6mm×5μm), with mobile phase consisting of 10mM ammonium formate containing 0.1% ammonia solution; pH adjusted to 2.8 with trifluoroacetic acid as solvent A and methanol/acetonitrile (5/95) as solvent B delivered with gradient program at a flow rate of 1.0mL/min, column temperature was maintained at 25°C and analytes were monitored at 220nm. The injection volume was 5μL. The developed RP-LC method was validated and found linear, accurate, specific, selective, precise and robust. The structure of impurities was confirmed by direct mass analysis.

Detection of Two Genotoxic Impurities in Drug Substance and Preparation of Imatinib Mesylate by LC–MS/MS

As a potential DNA damaging substance, genotoxic impurities have been concerned by regulatory authorities in various countries. Two genotoxic impurities were found in imatinib mesylate which was a classical small molecule inhibitor of tyrosine kinase, and the analysis method has never been reported. A LC–MS/MS method was developed for the analysis of two genotoxic impurity materials: N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-aminopyrimidine (IMA) and N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine (IMN). The HPLC method utilized an ACQUITY UPLC HSS T3 C18 (150 × 2.1 mm, 1.7 μm) maintained at 40 °C. The mobile phase consisted of 0.02 M ammonium formate buffer (pH 3.4) and acetonitrile (containing 0.05% formic acid) in gradient elution mode. Detection was performed by triple quadrupole mass spectrometry fitted with ESI probe functioning in the positive ion mode and the following m/z 278/106 and 308/262 were used as qualifier and quantifier transitions. Flow rate was 0.4 mL min−1. The validation data demonstrated that the method had high sensitivity and selectivity. A linear calibration curve (correlation coefficient, r > 0.998) was obtained at the concentration range of 0.02–35.27 and 0.02–28.86 ng mL−1, respectively. The LOD of IMA in drug substances, tablets, and capsules were 0.0039, 0.0043, and 0.0044 ng mL−1, and LOD of IMN were 0.0034, 0.0035, and 0.0036 ng mL−1, respectively. Precision and accuracy were within the acceptable limit. The drug substances, tablets, and capsules from three manufacturers were used for inspection and all samples met the requirements. The developed LC–MS/MS method is robust and can be applied to detect the genotoxic impurities in imatinib mesylate. Mixed solution of Imatinib, IMA and IMN. A new LC-MS/MS method was developed for the analysis of two genotoxic impurities materials: N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-aminopyrimidine and N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine (IMN)

GENOTOXIC IMPURITIES: AN IMPORTANT REGULATORY ASPECT

Genotoxins are agents/carriers such as chemical or radiation that can cause the damage to DNA or chromosomal structure, thereby causing mutations and the process are called as genotoxicity. Identification and understanding of genotoxins at a primary stage of drug development would enable us to prevent the potential damage that can be caused by these genotoxic agents. Various regulatory agencies such as International Council for Harmonization and EMEA, USFDA, European Pharmacopeia guidance, guidance for oncology products provide guidelines to limits the level of impurities in drug substances and drug products. Nowadays, conventional protocol of isolation, various spectral analysis high-performance liquid chromatography (LC), Fourier transform infrared to on-line analysis using modern, sophisticated hyphenated tools, like gas chromatography-mass spectroscopy, LC-MS so on, as well as modern software based in silico drug designs are extensively used by industry, research, and development areas and also there is tremendous increase in publications in the literature involving their use. Our review article focused on the various regulatory guidelines, application of hyphenated tools, and in silico techniques in genotoxic impurity and degradation product profiling of small molecules. A brief explanation is made on possible pitfalls in the experimentation and data interpretation. From this review, it concluded that there are various countries having their own regulatory agencies and regulatory guidelines for drug approvals, which may be followed by applying new chemical entities the new drug application title (NDA) in new drug application as well as there are various conventional to modern software based techniques to quantification of genotoxic impurities.

New stability indicating RP-HPLC method for the determination of Abiraterone acetate, its related substances and degradation products in bulk and dosage form

A robust, specific, sensitive and precise reverse-phase HPLC method has been developed for the quantitative determination of an atypical anticancer drug Abiraterone acetate and its eighteen potential impurities in drug substances. Chromatographic separation was achieved on a Kinetex C18 (4.6 mm × 150 mm, 5µ) column, using buffer (1 g of KH2PO4 in 1000 ml water and adjusting the pH 3.1 with ortho phosphoric acid) and acetonitrile in gradient elution mode. The flow rate was kept at 0.8 ml/min and thermostated at 40 °C with runtime of 65 min. The optimized method was found to produce symmetric and sharp peaks with good separation between the processes related impurities and degradation impurities. Quantification is achieved with photodiode array detection at 205 nm over the concentration range of 0.2–3.0 mg/mL. Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability-indicating capability of the developed HPLC method. Developed method is validated as per ICH guidelines and found to be linear, accurate, specific, selective, precise, robust and useful during the process development as well as quality check in bulk drug manufacturing.

Nitrosamine Impurities in Drug Substances and Drug Products

Nitrosamine Impurities in Drug Substances and Drug Products

Design of Experimental Approach to Analytical Robustness Study for UHPLC Method Developed for Separation and Quantification of Spironolactone and its Impurities in Drug Substances

The present work demonstrates the development of an optimal, robust, validated UHPLC method for quantification of related impurities and assay determination of spironolactone. Design of experiment procedure, in combination with statistical evaluation of the data was used to test the robustness of developed method. A stability indicating method was established by forced degradation experiments. Analytical robustness was determined using design of experiment approach. The chromatographic separation was achieved with Agilent SB-C18 RRHD column using gradient elution with mobile phase-A consists of a mixture of 0.1 % each formic acid and ammonia in water and methanol as mobile phase-B respectively. The developed method is exhaustively validated for parameters like precision, accuracy, linearity, LOD, LOQ, ruggedness and robustness. The stability tests were also performed on drug substances as per ICH norms. Base line separation was achieved for all impurities, degradation products and the API. All impurities were eluted within 12 min, there was a remarkable 3.5-fold decrease in runtime and a clear baseline separation between all peaks in comparison with Ph.Eur monograph. A multi-dimensional design space was built to study the robustness of developed method using design expert software. Significant parameters such as effect of flow rate, buffer strength and mobile phase compositions were optimized at three levels. Plackett-Burman design was applied for screening of chromatographic conditions and factorial design was applied for optimization of essential factors in robustness studies.

HPTLC Method for Determination of R, R-Glycopyrronium Bromide and its Related Impurities.

An innovative high-performance thin layer chromatographic (HPTLC) method was designed, optimized and validated for the quantification of R, R-glycopyrronium bromide (GLY) and its related impurities in drug substance and drug product. Separation was performed on HPTLC plates pre-coated with silica gel 60F254 by dichloromethane:methanol:formic acid (10:0.5:0.5, v/v/v) as a developing system. GLY and its related impurities namely, glycopyrronium impurity G and glycopyrronium impurity J, were separated giving compact well-resolved spots with significant retardation factor (Rf) values of 0.17±0.02, 0.34±0.02 and 0.69±0.02, respectively. Quantification was done at 220nm in the ranges of 0.3-10 and 0.2-4.0μg/spot with limits of detection and quantification of 0.1, 0.3 and 0.05, 0.2μg/spot for GLY and its related impurities, respectively. Good accuracy was obtained with mean percentage recovery of 99.48±1.36, 100.04±1.32 and 99.61±0.80 and R2≥0.9968 for GLY and its impurities, consecutively. Validation parameters were presented according to the International Conference on Harmonization. The method was used to investigate impurity profile of GLY in drug substance and drug product and could be applied in routine analysis of the drug. Comparison between the developed method and the reported method revealed no statistical difference.

Understanding and Kinetic Modeling of Complex Degradation Pathways in the Solid Dosage Form: The Case of Saxagliptin.

Drug substance degradation kinetics in solid dosage forms is rarely mechanistically modeled due to several potential micro-environmental and manufacturing related effects that need to be integrated into rate laws. The aim of our work was to construct a model capable of predicting individual degradation product concentrations, taking into account also formulation composition parameters. A comprehensive study was done on active film-coated tablets, manufactured by layering of the drug substance, a primary amine compound saxagliptin, onto inert tablet cores. Formulation variables like polyethylene glycol (PEG) 6000 amount and film-coat polymer composition are incorporated into the model, and are connected to saxagliptin degradation, via formation of reactive impurities. Derived reaction equations are based on mechanisms supported by ab initio calculations of individual reaction activation energies. Alongside temperature, relative humidity, and reactant concentration, the drug substance impurity profile is dependent on micro-environmental pH, altered by formation of acidic PEG degradation products. A consequence of pH lowering, due to formation of formic acid, is lower formation of main saxagliptin degradation product epi-cyclic amidine, a better resistance of formulation to high relative humidity conditions, and satisfactory tablet appearance. Discovered insights enhance the understanding of degradational behavior of similarly composed solid dosage forms on overall drug product quality and may be adopted by pharmaceutical scientists for the design of a stable formulation.

Lesson Learnt from Recall of Valsartan and Other Angiotensin II Receptor Blocker Drugs Containing NDMA and NDEA Impurities

The presence of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) impurities in angiotensin II receptor blocker (ARB) drugs containing tetrazole ring has triggered worldwide product recalls. The purpose of this article is to identify the potential gap area in current pharmaceutical industry practice that might have led to the NMDA and NDEA impurities escaping the drug manufacturer's and FDA's attention. The impact of process change was not adequately assessed by the manufacturer of contaminated APIs (active pharmaceutical ingredients), and potential for generation of mutagenic or other toxic impurities was not considered. The safety and risk associated with a chemical synthetic process was also not evaluated. This is primarily due to current industry practice which focuses on controlling the impurities above reporting threshold. ICH Q3A and FDA guidance on genotoxic and carcinogenic impurities in drug substances and products need to be integrated so that the ICH Q3A decision tree (attachment 3) begins by checking whether the synthetic process has been evaluated for the potential to generate toxic impurities. The compliance with ICH Q3A limits should be carried out only after the process has been determined to be safe without the risk of generating mutagenic and carcinogenic impurities.

Determination of trace methanesulfonates in drug matrix using derivatization and headspace single drop microextraction followed by high-performance liquid chromatography with ultraviolet detection

The selective and sensitive determination of potential genotoxic methanesulfonate impurities in drug substances is highly challenging. A new method is reported for testing of methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and isopropyl methanesulfonate (IPMS) in active pharmaceutical ingredients (APIs). Headspace single drop microextraction (HS-SDME) with room-temperature ionic liquid (RTIL) as extractant was employed to preconcentrate analytes and eliminate the drug matrix simultaneously. In order to increase volatilities for HS extraction and to improve their reactivity of the further derivatization at the same time, sodium iodide (NaI) was added to the sample to derivatize methanesulfonates to the corresponding iodoalkanes. The iodoalkanes in the extract were derivatized with N, N-diethyldithiocarbamate (DDTC) after HS-SDME, followed by separation and detection with high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Several important parameters, including reaction temperature, reaction time and concentration of NaI, sample volume, microdrop volume, stirring rate, salt addition, extraction time, concentration, reaction time and reaction temperature of DDTC were investigated. Under the optimal conditions, LODs and LOQs of all methanesulfonates were 15 ng mL−1 and 40 ng mL−1, respectively. Linearity (correlation coefficient values r > 0.999) and precision (the relative standard deviations were 1.0–4.6%) of six repeated injections were obtained. The recoveries at three spiked concentration levels were all in the range of 86.2–107.5% with the relative standard deviations <3.5%. The method reported here avoids interference of drug substances efficiently and detects methanesulfonate impurities in high sensitivity by HPLC-UV in Imatinib Mesylate and Levofloxacin Mesylate.

New Rapid Stability indicating RP-UPLC Method for the Determination of Olaparib, its Related Substances and Degradation Products in Bulk drug and Dosage Form

Abstract A rapid, specific, sensitive, and precise reverse-phase UPLC method was developed for the quantitative determination of a typical anticancer drug. Olaparib and its eight potential impurities in drug substances is described in this report. Statistical experimental designs (DOE) are used as advancement over conventional experimental approach .It provides more accurate results in fewer runs compared with conventional methods. Chromatographic separation was achieved on a Waters Acquity UPLC® BEH C18 (2.1 mm × 100 mm, 1.7 micron) column, using buffer (1.0ml Orthophosphoric acid in 1000ml water) and acetonitrile in gradient elution mode. The flow rate was kept at 0.4mL/min; thermostated at 35˚C with a short runtime of 15 min. The optimized method was found to produce symmetric and sharp peaks with good separation between processes related impurities and degradation impurities. Quantification is achieved with photodiode array detection at 220 nm over the concentration range of 10- 20 μg/mL. Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability-indicating capability of the developed UPLC method. Developed method is validated as per ICH guidelines and found to be linear, accurate, specific, selective, precise and robust. The method is useful during process development and quality of bulk manufacturing.

Genotoxic Impurities and Its Risk Assessment in Drug Compounds

The present work is based on a assess analysis of the literature in order to define the genotoxic impurities present in the drug substances and drug products. Starting from the definition of genotoxic impurities, sources of genotoxic impurities, their generation and classification based on their toxicity. The article also explains the various regulatory guidelines to control the genotoxic impurities in drug substances. Isolation, qualification of impurity and its identification along with quantification by the various analytical methods is also described in this review article.

Development and Validation of the Chiral Liquid Chromatography Method for Separation of Enantiomeric Impurity in Novel Oxazolidinone Antibacterial Agent WCK 4086.

A highly stereo-specific liquid chromatographic method was developed and validated for the quantification of enantiomeric impurity (R-enantiomer) in novel oxazolidinone antibacterial agent (WCK 4086), a drug substance. The separation was achieved on Chiralpak AD-H (amylose-based chiral stationary phase) using a mobile phase consisting of n-hexane:2-propanol:methanol:trifluoroacetic acid (80:10:10:0.4, v/v/v/v) at a flow rate of 1.0 mL min-1. Chromatographic resolution between two enantiomers was found to be more than 2.0. Method was extensively validated for the quantification of R-enantiomer in WCK 4086 and proved to be robust. Method was found to be highly specific as all other related impurities were separated from the enantiomers. The calibration curve for R-enantiomer showed an excellent linearity over the concentration range of 1-5 μg mL-1. Limit of quantitation (LOQ) and limit of detection (LOD) for R-enantiomer were 0.009 μg and 0.003 μg, respectively. Average recovery of the R-enantiomer was in the range of 94.55-109.67%. Analytical solutions were found to be stable up to 70 h at room temperature. Developed method was found to be specific, sensitive, precise and accurate for quantitative determination of R-enantiomer in WCK 4086 and useful for controlling the enantiomeric impurity in drug substance used for preclinical studies.

COMMON DEFICIENCIES RAISED BY VARIOUS REGULATORY AGENCIES

Timely Product registration is a challenge in today’s scenario. Regulatory agencies are keen in assessing CTD sections like redefining starting material of Drug substance manufacturing, Pharmaceutical development, impurities in Drug substance and Drug product, Container-closure. Common deficiencies are presented in this article.

Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 μg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

Simultaneous determination of eight short-chain aliphatic amines in drug substances by HPLC with diode array detection after derivatization with halonitrobenzenes.

Short-chain aliphatic amines are a class of hazardous impurities in drug substances. A simple method, involving derivatization followed by high-performance liquid chromatography with diode array detection, has been developed for residue determination of eight aliphatic amines simultaneously in drug substances. Different halonitrobenzenes derivatization reagents were systematically compared. As a result, 1-fluoro-2-nitro-4-(trifluoromethyl)benzene was selected since the derivatization effectively shifted the absorption wavelength to the visible region (400-450nm), where most drug substances, impurities and even the derivatization reagent absorb very weakly. Due to the redshift effect, interference was minimized and adequately low limits of quantitation were reached (0.24-0.80nmol/mL). Moreover, the derivatization reaction was readily carried out in dimethyl sulfoxide at room temperature for 1h using N,N-diisopropylethylamine as catalyst to achieve the highest yield. Without any pre-treatment, the derivatives were analyzed by high-performance liquid chromatography with diode array detection. The high stability of the derivatives within 24h at room temperature (RSD<1.04%) further facilitated the simultaneous preparation and consecutive analysis of quantities of samples. Finally, the proposed method was successfully applied for residue determination of eight aliphatic amines simultaneously in eight drug substance samples. This study could be helpful for the routine analysis and residue control of aliphatic amines in drug substances.