Search Results (15)

Nitrosamine impurities have provoked numerous global medicine recalls due to their possible presence during drug manufacturing or storage. Regarding formulation of nitrosamine impurities, a key risk involves reactions between nitrosating agents (nitrite) in excipients and vulnerable amines as impurities or degradants. Rapid detection across various sample types is essential to support pharmaceutical manufacturing. In this study, two methods were developed to detect nitrite in excipients and crucial secondary amines in active ingredient metformin hydrochloride at trace levels, respectively. The former method was developed based on the reaction of nitrite ions with 2,3-diaminonaphthalene to form 1-[H]-naphthotriazole (NAT), whereas the latter was based on amine tosylation. Mass spectrometric conditions were optimized using electrospray ionization in the positive mode. Multiple reaction monitoring transitions were determined at m/z 170 → 115 for NAT, and m/z 200.1 → 91 for dimethylamine (DMA) and 228.1 → 91 for diethylamine (DEA). These methods were validated using selected eight excipients or metformin hydrochloride in terms of specificity, linearity, accuracy, precision, robustness, limit of quantification (LOQ), and limit of determination according to the ICH guidelines. The results of the validation were within the acceptable criteria. Applicability of the methods was evaluated using 170 pharmaceutical samples donated by industries. The nitrite content in the excipients ranged from <LOQ to 4.74 ppm, with observed levels 1.3 to 6 times higher than the average (0.8 ppm) in the samples. The DMA levels in the metformin hydrochloride were within the limit (500 ppm) but varied significantly (0.2–209.2 ppm) among manufacturers. DEA was detected at lower levels (0.7–0.9 ppm). To mitigate the nitrosamine content in the metformin products, the excipient compositions were investigated by selecting those with low nitrite levels. As the types of impurities detected have become increasingly diverse and detection cycles have become more frequent, the requirement for preemptive safety management to relieve public anxiety is essential for regulatory aspects. Nitrite and secondary amines are crucial precursors to N-nitrosamine, and the suggesting approach could be a means to mitigate N-nitrosamine contamination. Full article

Many studies are being conducted on the detection of nitrosamine impurities in solid formulations. However, research on semi-solid formulations such as gels, ointments and creams is not common. In particular, excipients used to increase viscosity and add fragrance can significantly impact the sample preparation. Volatile compounds derived from natural fragrances are composed of a wide variety of complex components, making them very difficult to handle and completely separate from the analytes. Due to the complex composition of these formulations, an analytical method was developed to accurately separate and analyze nine nitrosamine impurities (NDMA, NDEA, NMEA, NDPA, NDBA, NPIP, NMOR, DIPNA and EIPNA) simultaneously. To overcome challenges in the sample preparation of excipients with physical and chemical properties, the sample was prepared using solvents such as methanol, hexane, water and dichloromethane. The target analytes were extracted with dichloromethane for the final preparation for GC–MS/MS and the optimal conditions were established. While multiple GC columns were tested, peak overlapping interferences were observed, leading to the use of a 60m-long column to overcome peak overlap. The GC–MS/MS condition was set for optimal performance and ionization energy, with parameters adjusted for each analyte. The developed method was validated in accordance with guidelines to ensure its reliability and suitability. As a result, all nine nitrosamine impurities were simultaneously analyzed, confirming excellent performance. The sample preparation method and procedure, column specification and GC–MS/MS conditions have the potential to be adapted not only for semi-solid formulations of pharmaceuticals and cosmetics but also for other formulations such as solid and liquid samples, rendering them suitable for the analysis of nitrosamine impurities. Full article

The presence of mutagenic and carcinogenic N-nitrosamine impurities in medicinal products poses a safety risk. While incorporating antioxidants in formulations is a potential mitigation strategy, concerns arise regarding their interference with drug absorption by inhibiting intestinal drug transporters. Our study screened thirty antioxidants for inhibitory effects on key intestinal transporters—OATP2B1, P-gp, and BCRP in HEK-293 cells (OATP2B1) or membrane vesicles (P-gp, BCRP) using ³H-estrone sulfate, ³H-N-methyl quinidine, and ³H-CCK8 as substrates, respectively. The screen identified that butylated hydroxyanisole (BHA) and carnosic acid inhibited all three transporters (OATP2B1, P-gp, and BCRP), while ascorbyl palmitate (AP) inhibited OATP2B1 by more than 50%. BHA had IC₅₀ values of 71 ± 20 µM, 206 ± 14 µM, and 182 ± 49 µM for OATP2B1, BCRP, and P-gp, respectively. AP exhibited IC₅₀ values of 23 ± 10 µM for OATP2B1. The potency of AP and BHA was tested with valsartan, an OATP2B1 substrate, and revealed IC₅₀ values of 26 ± 17 µM and 19 ± 11 µM, respectively, in HEK-293-OATP2B1 cells. Comparing IC₅₀ values of AP and BHA with estimated intestinal concentrations suggests an unlikely inhibition of intestinal transporters at clinical concentrations of drugs formulated with antioxidants. Full article

Rifampicin is an essential medicine for treating and preventing tuberculosis (TB). TB is a life-threatening infectious disease and its prevention and treatment are public health imperatives. In the time of a global crisis of nitrosamine contamination of medicinal products, patient safety and a reduction in the number of drug recalls at the same time are crucial. In this work, the LC-MS/MS method was developed for the determination of the 1-methyl-4-nitrosospiperazine (MNP), a genotoxic nitrosamine impurity in various products containing rifampicin at a 5.0 ppm limit level according to Food and Drug Administration (FDA). Extraction with neutralization was necessary due to the matrix and solvent effect associated with the complexity of the rifampicin product. The developed method was validated in accordance with regulatory guidelines. Specificity, accuracy, precision, limit of detection, and limit of quantification parameters were evaluated. The recovery of the MNP was 100.38 ± 3.24% and the intermediate precision was 2.52%. The contamination of MNP in Rifampicin originates in the manufacturing process of the drug. Furthermore, the results of the forced degradation experiments show that the formation of MNP is possible by two mechanisms: through degradation of rifampicin and the oxidation of 1-amino-4-methyl-piperazine. This article points out that it is necessary to monitor and describe degradation products and the mechanism of degradation of potentially affected active pharmaceutical ingredient (API) with respect to the formation of nitrosamines during stress testing, as it was done in the following work for rifampicin in multicomponent products. Full article

Risk control for nitrosamine impurities in drug products is currently a major challenge in the industry. Nitrosamines can form during drug product manufacturing and storage through the reaction of nitrites with amine-containing APIs or impurities. The level of nitrites in excipients and the rate of reaction often control the build-up of nitrosamine. Although the variability in nitrite levels across excipient types and suppliers is well recognized, the impact of excipient selection on the level of nitrosamine formed has not been systematically studied. This gap of knowledge is addressed in the current work. We present theoretical case studies of formulations where microcrystalline cellulose (MCC), or lactose supplier, or superdisintegrant type are changed in pursuit of lower levels of nitrite. The impact of the average, maximum, and minimum levels of nitrites in each excipient on nitrosamine formation in the dosage form is calculated. The input data for this calculation are the formulation composition, nitrosamine molecular weight (MW), percentage of conversion, and nitrite levels per excipient. The percentage of conversion (based on the formulation and manufacturing variables) and nitrite levels were taken from the recent literature. We show that changing the supplier of a single excipient, or of the three most critical excipients, can reduce nitrosamine formation by up to −59% and −89%, respectively. We also show that high-risk formulations, e.g., high MW nitrosamines, high dosage weights, and high percentages of conversion (e.g., wet granulation), can often be de-risked below regulatory acceptable daily intake via careful excipient selection. Finally, we provide an open-access tool that enables users to calculate the theoretical formation of nitrosamines in their specific formulations. This calculation template can be used for (i) the preliminary screening of the risk of nitrosamine formation in drug products and (ii) the preliminary assessment of the impact of excipient selection for risk mitigation. Full article

N-nitrosamines are a class of carcinogenic chemical compound. Considering the large-scale application of agrochemicals globally, the elimination of N-nitrosamines from pesticides should be a priority for manufacturers and regulators. A set of methods was developed and validated for the determination of the toxicologically relevant N-nitrosamine impurity of ethalfluralin (ethyl-N-(2-methylallyl) N-nitroso amine—EMANA) in 33% w v⁻¹ emulsifiable concentrate (EC) formulations. Solid Phase Extraction (SPE) was compared with the “dilute and shoot” approach. Gas chromatography (GC) was combined with Flame Ionization Detection (FID) and mass spectrometry (MS). For MS, two mass filtering modes (Selective Ion Monitoring—SIM, tandem mass spectrometry—MS/MS) and two ionization modes (Electron Ionization—EI, Positive chemical ionization—PCI) were applied. It was concluded that, in the case of samples with high nitrosamine concentration (>90 μg g⁻¹), the “dilute and shoot” approach can be applied without compromising the quality of the results. SPE, however, is required to attain the LOQ (0.33 μg g⁻¹) with good recovery (97.4–110.67%), linearity (R > 0.99) and precision (%RSD 0.68–1.74). The LOQ supersedes the limit set by EFSA (1 μg g⁻¹) in the Technical Active Substance—TAS. The concentration range of the methods is 0.05–110 μg g⁻¹. The methods were applied for the official surveillance program of the Greek agrochemicals market. Full article

Nitrosamines occur widespread in food, drinking water, cosmetics, as well as tobacco smoke and can arise endogenously. More recently, nitrosamines have been detected as impurities in various drugs. This is of particular concern as nitrosamines are alkylating agents that are genotoxic and carcinogenic. We first summarize the current knowledge on the different sources and chemical nature of alkylating agents with a focus on relevant nitrosamines. Subsequently, we present the major DNA alkylation adducts induced by nitrosamines upon their metabolic activation by CYP450 monooxygenases. We then describe the DNA repair pathways engaged by the various DNA alkylation adducts, which include base excision repair, direct damage reversal by MGMT and ALKBH, as well as nucleotide excision repair. Their roles in the protection against the genotoxic and carcinogenic effects of nitrosamines are highlighted. Finally, we address DNA translesion synthesis as a DNA damage tolerance mechanism relevant to DNA alkylation adducts. Full article

(1) Background: The aim of this study was to identify the degradation product of ramipril (RAM) formed under dry air and to verify its potential modes of carcinogenicity. We intended to check whether its formation and presence in final dosage forms could pose a cancer risk to humans who are treated with RAM due to cardiological indications. The carcinogenicity of this compound was evaluated with respect to two mechanisms: a potential direct DNA-damage and indirect toxicity, secondary to forming mutagenic N-nitroso metabolites. (2) Methods: Firstly, a forced ageing test under dry air was conducted for pure RAM in order to induce its degradation. The validated HPLC system was used to describe the kinetic order of this reaction. The emerging degradation impurity was identified by HPLC-MS. In the second stage, the cancer risk of the identified RAM degradant was predicted using a structure-based assessment by in silico QSAR model, employing three endpoints: carcinogenicity, genotoxicity and mutagenicity. In the third stage, the obtained QSAR results were experimentally verified. To verify genotoxicity prediction, in vitro micronucleus assay was employed. It enabled us to assess the potential direct DNA-damaging properties of RAM degradant at high concentrations (as screening series) and at concentrations usually observed in human blood (to mimic the clinical scenario). To verify the QSAR mutagenicity prediction, an in vitro Ames test was carried out. It was designed so as to detect two mechanisms of mutagenicity: a direct one (for pure degradant) and an indirect one (via N-nitroso-metabolites formation). N-nitroso-metabolites for mutagenicity assessment were obtained using NAP test. (3) Results: The kinetic mechanism of RAM degradation was first-order, the degradation rate constant was k = 1.396 ± 0.133 × 10⁻⁵ s⁻¹ (T = 373 K), thus the formation of impurity was rapid. Energy of activation was 174.12 ± 46.2 kJ/mol, entropy was positive, thus reaction was bimolecular and favored; enthalpy was 171.65 ± 48.7 kJ/mol, thus reaction was endothermic. Only one degradation impurity was formed, and it was identified as RAM diketopiperazine derivative (DKP). QSAR simulation predicted that DKP could be carcinogenic and genotoxic, but this result had only moderate reliability. DKP was also predicted to be non-mutagenic and this prediction was strong (endpoint score 0.2). The confirmatory micronucleus experiment for genotoxicity prediction suggested that DKP was cytotoxic and it could be also aneugenic at a high concentration (0.22 mg/mL), evidenced by a three-fold increase in micronuclei relative to the control (11.86:33.33%, p = 0.0184). At physiologic concentrations, its cytotoxicity and genotoxicity did not occur. This means that the genotoxicity of DKP was limited by a threshold mechanism. In the mutagenicity in vitro assessment, pure DKP was not mutagenic, but its nitrosation product induced base substitutions mutations in test bacteria TA100 following metabolic activation at a concentration of 4.5 mg/mL, confirming its mutagenicity. (4) Conclusions: RAM rapidly cyclizes to diketopiperazine derivative under dry air. This impurity resides in drugs administered to patients. DKP is potentially aneugenic and cytotoxic at high concentrations, yet at concentrations typically occurring in human blood, this effect is unlikely. The exposure of patients to high concentrations of DKP, exceeding the typical blood level and standard RAM dosing, could lead to cancer development, thus the safe threshold for human exposure to DKP must be verified in follow-up in vivo experiments. Based on our results, it is impossible to establish the maximum safe dose of pure DKP to humans. Furthermore, DKP itself is not mutagenic, but it is liable to the formation of mutagenic nitroso-metabolites in vivo. Nitroso-derivatives of DKP are in vitro mutagens and their real-life impact on humans must be further evaluated in in vivo studies. Until this is carried out, RAM should not be formulated by manufacturers using dry procedures to minimize DKP formation and reduce risk of human carcinogenesis, since DKP could cause cancer via two independent mechanisms: direct genotoxicity when the exposure over standard RAM dosing occurs, and indirect mutagenicity via in vivo N-nitrosamine formation. Full article

The ability to effectively detect N-nitrosamine compounds by liquid chromatography–tandem mass spectrometry presents a challenge due to the problems of high detection limits and difficulty in simultaneous N-nitrosamine compound detection. In order to overcome these limitations, this study reduced the detection limit of N-nitrosamine compounds by applying n-hexane pre-treatment to remove non-polar impurities before the conventional process of column extraction. In addition, ammonium acetate was used as the mobile phase to enhance the retention of nitrosamine target substances on the chromatographic column, with formic acid added to the mobile phase to improve the ionization level of N-nitrosodiphenylamine, to achieve the simultaneous detection of multiple N-nitrosamine compounds. Applying these modifications to the established detection method allowed the rapid and accurate detection of N-nitrosamine in water within 12 min. The linear relationship, detection limit, quantification limit and sample spiked recovery rate of nine types of nitrosamine compound were investigated, showing that the correlation coefficient ranged from 0.9985–0.9999, while the detection limits of the instrument and the method were 0.280–0.928 µg·L⁻¹ and 1.12–3.71 ng·L⁻¹, respectively. The spiked sample recovery rate ranged from 64.2–83.0%, with a standard deviation of 2.07–8.52%, meeting the requirements for trace analysis. The method was applied to the detection of N-nitrosamine compounds in nine groundwater samples in Wuhan, China, and showed that the concentrations of N-nitrosodimethylamine and NDEA were relatively high, highlighting the need to monitor water bodies with very low levels of pollutants and identify those requiring treatment. Full article

The purpose of this research study was to develop an analytical method for the quantification of 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4] triazolo [4,3-a] pyrazine (7-nitroso impurity), which is a potential genotoxic impurity. Since sitagliptin is an anti-diabetic medication used to treat type 2 diabetes and the duration of the treatment is long-term, the content of nitroso impurity must be controlled by using suitable techniques. To quantify this impurity, a highly sensitive and reproducible ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed. The analysis was performed on a Kromasil-100, with a C18 column (100 mm × 4.6 mm with a particle size of 3.5 µm) at an oven temperature of approximately 40 °C. The mobile phase was composed of 0.12% formic acid in water, with methanol as mobile phases A and B, and the flow rate was set to 0.6 mL/min. The method was validated according to the current International Council for Harmonisation (ICH) guidelines with respect to acceptable limits, specificity, reproducibility, accuracy, linearity, precision, ruggedness and robustness. This method is useful for the detection of the impurity at the lowest limit of detection (LOD), which was 0.002 ppm, and the lowest limit of quantification (LOQ), which was 0.005 ppm. This method was linear in the range of 0.005 to 0.06 ppm and the square of the correlation coefficient (R²) was determined to be > 0.99. This method could help to determine the impurity in the regular analysis of sitagliptin drug substances and drug products. Full article

The ubiquitous presence of mutagenic and potentially carcinogenic N-nitrosamine impurities in medicines has become a major issue in the pharmaceutical industry in recent years. Rigorous mitigation strategies to limit their amount in drug products are, therefore, needed. The removal of nitrite, which is a prerequisite reagent for the N-nitrosation of amines, has been acknowledged as one of the most promising strategies. We have conducted an extensive literature search to identify nineteen structurally diverse nitrite scavengers and screened their activity experimentally under pharmaceutically relevant conditions. In the screening phase, we have identified six compounds that proved to have the best nitrite scavenging properties: ascorbic acid (vitamin C), sodium ascorbate, maltol, propyl gallate, para-aminobenzoic acid (PABA), and l-cysteine. These were selected for investigation as inhibitors of the formation of N-methyl-N-nitrosoaniline (NMA) from N-methylaniline and N-nitroso-N’-phenylpiperazine (NPP) from N-phenylpiperazine in both solution and model tablets. Much faster kinetics of NMA formation compared to NPP was observed, but the former was less stable at high temperatures. Vitamin C, PABA, and l-cysteine were recognized as the most effective inhibitors under most studied conditions. The nitrite scavenging activity does not directly translate into N-nitrosation inhibitory effectiveness, indicating other reaction pathways may take place. The study presents an important contribution to identifying physiologically acceptable chemicals that could be added to drugs to prevent N-nitrosation during manufacture and storage. Full article

In order to meet the analytical requirements of the European Medicines Agency (EMA), a new HPLC-FLD method was successfully developed using dansyl chloride for the derivatization and determination of the genotoxic impurities N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) in Lisinopril API and its final product. Samples’ pretreatment includes liquid–liquid microextraction, denitrosation, and derivatization steps. To optimize the process, the parameters contributing to high sensitivity and yielding reliable results were thoroughly studied and optimized using one-factor-at-a-time and experimental design approaches. The analytes were pre-column derivatized with Dansyl-Cl and analyzed by HPLC-fluorescence (λ_em/λ_em = 340/530) using a C₁₈ column and a mixture of phosphate buffer (pH = 2.8; 20 mM)/acetonitrile 55:45 v/v as the mobile phase. The six-level concentration calibration was shown to be linear, with R equal to 0.9995 for both analytes. The limit of detection (LOD) was satisfactory and equal to 4.7 and 0.04 ng/mL for NDMA and NDEA, respectively. Precision was less than 13.4% in all cases, and the average recoveries were equal to 109.2 and 98.1% for NDMA and NDEA, respectively. The proposed procedure is relatively easy, rapid, and suitable for the determination of the two nitrosamines in routine analysis tests. Full article

N-nitroso-N-methyl-4-aminobutyric acid (NMBA) is the third N-nitrosamine impurity found in sartans. Herein, a sensitive and stable LC-MS/MS method with multiple reactions monitoring mode has been developed for the quantitative determination of NMBA in four sartan substances. The effective separation of NMBA and sartan substances was achieved on a C18 column under gradient elution conditions. The mass spectrometry method of the atmospheric pressure chemical ionization source and internal standard method was selected as the quantitative analysis method of NMBA. Then, this proposed LC-MS/MS analysis method was validated in terms of specificity, sensitivity, linearity, accuracy, precision and stability. Good linearity with correlation coefficient over 0.99 was obtained at the NMBA concentration of 3–45 ng/mL, and the limit of quantification was 3 ng/mL. Additionally, the recoveries of NMBA in four sartan substances ranged from 89.9% to 115.7%. The intra-day and inter-day relative standard deviation values were less than 5.0%. In conclusion, this developed determination method for NMBA through liquid chromatography–tandem mass spectrometry showed the characteristics of good sensitivity, high accuracy and precision, which will be of great help for the quantitative analysis of NMBA in sartan products. Full article

In 2018, the discovery of carcinogenic nitrosamine process related impurities (PRIs) in a group of widely used drugs led to the recall and complete withdrawal of several medications that were consumed for a long time, unaware of the presence of these genotoxic PRIs. Since then, PRIs that arise during the manufacturing process of the active pharmaceutical ingredients (APIs), together with their degradation impurities, have gained the attention of analytical chemistry researchers. In 2020, favipiravir (FVR) was found to have an effective antiviral activity against the SARS-COVID-19 virus. Therefore, it was included in the COVID-19 treatment protocols and was consequently globally manufactured at large-scales during the pandemic. There is information indigence about FVR impurity profiling, and until now, no method has been reported for the simultaneous determination of FVR together with its PRIs. In this study, five advanced multi-level design models were developed and validated for the simultaneous determination of FVR and two PRIs, namely; (6-chloro-3-hydroxypyrazine-2-carboxamide) and (3,6-dichloro-pyrazine-2-carbonitrile). The five developed models were classical least square (CLS), principal component regression (PCR), partial least squares (PLS), genetic algorithm-partial least squares (GA-PLS), and artificial neural networks (ANN). Five concentration levels of each compound, chosen according to the linearity range of the target analytes, were used to construct a five-level, three-factor chemometric design, giving rise to twenty-five mixtures. The models resolved the strong spectral overlap in the UV-spectra of the FVR and its PRIs. The PCR and PLS models exhibited the best performances, while PLS proved the highest sensitivity relative to the other models. Full article

The wide range and complexity of cosmetic formulations currently available on the market poses a challenge from an analytical point of view. In addition, during cosmetics manufacture, impurities coming from raw materials or formed by reaction of different organic compounds present in the formulation may be present. Their identification is mandatory to assure product quality and consumer health. In this work, micro-matrix solid-phase dispersion (μMSPD) is proposed as a multi-target sample preparation strategy to analyze a wide number of unexpected families of compounds including polycyclic aromatic hydrocarbons (PAHs), pesticides, plasticizers, nitrosamines, alkylphenols (APs), and alkylphenol ethoxylates (APEOs). Analytical determination was performed by gas chromatography-mass spectrometry (GC-MS) for the determination of 51 target compounds in a single run, whereas liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed for the analysis of six APs and APEOs. Both methodologies were successfully validated in terms of linearity, accuracy, and precision in leave-on and rinse-off cosmetics. Limits of detection (LODs) were calculated in the low ng g⁻¹, showing their suitability to determine trace levels of impurities and banned compounds with different chemical natures, providing useful tools to cosmetic control laboratories and companies. Full article