Quantify Trace Levels Research Articles

Development and Validation of Trace Level Quantification of Nine Azido Genotoxic Impurities in Losartan Potassium Active Pharmaceutical Ingredients by Liquid Chromatography‐Tandem Mass Spectrometry

ABSTRACTThe objective of the study is to develop a sensitive and simple liquid chromatography‐tandem mass spectrometry for the determination of nine azido impurities in Losartan potassium active pharmaceutical ingredients. An advanced analytical method should be developed to determine these nine Azido impurities at 0.5 ppm level. Currently, no such method is reported to control these impurities in losartan potassium drug substance. The nine impurities separation was achieved using Hypersil ODS stationary phase with gradient elution and mobile phase of 0.1 M ammonium acetate as mobile phase A and methanol as mobile phase B. The developed method was found to be sensitive to the limit of detection and limit of quantitation levels as per the International Council for Harmonization M7 guidelines. The method was linear from 0.5 ppm to 15 ppm for all nine azido impurities. This is a single method for the simultaneous determination of all azido impurities in the presence of losartan potassium drug substance at 0.5 ppm and the same method used in the quality control laboratory for sample analysis.

Development and validation of an ICP-MS method and its application in assessing heavy metals in whole blood samples among occupationally exposed lead smelting plant workers.

Occupational exposure to heavy metals affects various organ systems and poses a significant health risk to workers. Consequently, its precise estimation is of clinical concern and warrants the need for an analytical method with reliable precision and accuracy. The current study aimed to develop an analytical method using inductively coupled plasma‒mass spectrometry (ICP-MS) to detect trace to elevated levels of potentially toxic elements in human blood. The sample preparation was optimized using a two-step ramp temperature microwave acid digestion program. The toxic elements were quantified using ICP-MS operating in kinetic energy discrimination (KED) mode, adjusting the data acquisition parameters and instrumental settings. The analytical method was validated using standard performance parameters. Each validation parameter was aligned with the acceptable criteria outlined in standard guidelines. The method achieved optimal linearity (r2 > 0.99), recovery (85.60-112.00%), and precision (1.35-7.03%), was capable of detecting the lowest concentrations of 0.32, 0.28, 0.28, and 0.19µg/L, and was capable of quantifying trace levels of 1.01, 0.88, 0.90, and 0.62µg/L for arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb), respectively. Post-validation, the method was applied to estimate heavy metals in blood samples from 250 Pb-smelting plant workers, revealing potential health implications of occupational exposure. The cohort analysis revealed that demographic and employment factors were associated with elevated blood Pb levels, leading to symptoms and health risks. Clinical analysis revealed that 33.6% of the participants experienced hypertension. These findings highlight the significant health risks associated with elevated blood Pb levels. The weak but significant correlation with systolic blood pressure underscores the need for improved monitoring and workplace safety. This emphasizes the importance of continuous monitoring, targeted interventions, and enhanced occupational hygiene to protect workers' well-being.

Large scale screening and quantification of micropollutants in fish from the coastal waters of Cochin, India: Analytical method development and health risk assessment

Urban estuarine and coastal water receive several micropollutants through industrial and agricultural influxes. The bioaccumulation of these micropollutants in fish and their entry into the coastal population's food chain raises significant food safety concerns. Hence, a comprehensive analytical method was developed for ultra-trace level quantification of 345 micropollutants in fish. The optimized sample preparation method could extract compounds suitable for both GC–MS/MS and LC-MS/MS analysis simultaneously. The target list of contaminants included 278 agricultural pesticides and also 102 endocrine disruptors covering polyaromatic hydrocarbons, polychlorinated biphenyls, organochlorines, and endocrine-disrupting pesticides. The GC–MS/MS with large volume injection (LVI) technique, and LC-MS/MS operating in MRM mode, achieved an LOQ of <2.00 ng/g for most of the analytes. The extraction strategy involved tri-phase partitioning between water, acidified acetonitrile, and hexane, followed by salting out. Dispersive solid phase cleanup (dSPE) with C18, Z-Sep+, CaCl2, and MgSO4 was able to reduce the matrix influence, and the method achieved satisfactory recovery in the range of 70.0–120.0 % for all the target analytes. The repeatability and reproducibility relative standard deviation values of the measured analytes were <20.0 %, and the Horwitz ratio values were well below 2. The method was used to accurately measure the target micropollutants in fish from the Cochin estuary, the highly urbanized portion of the Vembanad Lake, and an important Ramsar site. At least one or more of the 41 different micropollutants were identified and quantified in about 90.7 % of the 108 samples analyzed. The importance of large-scale screening and trace-level quantification methods in environmental monitoring and risk assessment is underscored by the results. The risk assessment showed a moderate risk of exposure to the nearby coastal population through the food chain.

Simultaneous multianalyte trace-level quantification of eight genotoxic nitrosamine impurities in valsartan Active Pharmaceutical Ingredient and tablet formulation using UFLC-MS/MS and greenness assessment

Valsartan, a widely prescribed antihypertensive drug, was identified for the presence of carcinogenic nitrosamine impurities in 2018, leading to product recalls worldwide and necessitating sensitive liquid and gas chromatographic methods for trace level detection of nitrosamine impurities. This study developed and validated an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the quantification of eight genotoxic nitrosamines (NDMA, NDEA, NDPA, NEIPA, NDIPA, NMBA, NDBA, and NMPA) in valsartan drug substance and tablet formulations. The method employed a triple quadrupole analyzer, atmospheric pressure chemical ionization (APCI) ionization source and multiple reaction monitoring (MRM) scan mode for the analysis. Chromatographic separation was achieved on a Phenomenex biphenyl column (150 × 4.6 mm, 3 µm) maintained at 55 °C. The mobile phase consisted of a binary gradient of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in methanol), with a total run time of 20 min for the sensitive and specific detection of the analytes. Method validation demonstrated excellent linearity, recovery, precision, and sensitivity. The greenness of the developed analytical method was critically assessed through comprehensive evaluations using the GAPI, AGREE, and AES metrics and the results obtained are compared with the reported methods in the literature. This novel UFLC-MS/MS method offers a reliable and sensitive approach for routine quality control analysis of nitrosamine impurities in Valsartan drug substance and tablet formulations.

Spectrophotometric quantification of trace levels of cyanide in water-insoluble drug substances and confirmation of specificity through an orthogonal comparison approach

Some pharmaceutical ingredients use cyanide in their synthesis, and this impurity must be controlled as per ICH, FDA and EDQM regulatory compliance. Spectrophotometric approaches for measuring cyanide levels are confined to water or water-soluble compounds with poor solution stability and low sensitivity. No method is available for the cyanide in water-insoluble drug substances through spectrophotometry. To address this gap, our study proposed a spectrophotometric approach for measuring trace cyanide in water-insoluble drugs such as Primidone and Clobazam. This method utilizes chloramine-T, which reacts with cyanide to form cyanogen chloride (CNCl), consequently reacting with pyridine-barbituric acid to produce a coloured derivative (C13H10N4O6, MW: 318.06 g/mol). This protocol also involves the selection of a suitable solvent and optimizing the pH for sensitive/accurate quantification of cyanide impurity. Mass spectrometry confirms cyanide derivatization as part of the orthogonal procedure comparison of ICH Q2 (R2). Method validation follows ICH Q2 (R2) guidelines in determining specificity, LOD (0.396 ppm), LOQ (1.200 ppm), linearity (1.2-45 ppm), and recovery (91-99% with %RSD<10%) of the method. The stability of the pyridine-barbituric acid reagent and derivatized compound was also evaluated. This approach effectively measures cyanide in water-insoluble drugs, meeting essential regulatory standards.

Quantitative analysis of microplastics in beach sand via low-temperature solvent extraction and thermal degradation: Effects of particle size and sample depth

Quantifying trace levels of microplastics in complex environmental media remains a challenge. In this study, an approach combining field collection of samples from different depths, sample size fractionation, and plastic quantification via pyrolysis-gas chromatography–mass spectrometry (Py-GC–MS) was employed to identify and quantify microplastics at two public beaches along the northeast coast of the U.S. (Salisbury beach, MA and Hampton beach, NH). A simple sampling tool was used to collect beach sand from depth intervals of 0–5 cm and 5–10 cm, respectively. The samples were sieved to give three size fractions: coarse (>1.2 mm), intermediate (100 μm–1.2 mm), and fine (1.2 μm–100 μm) particles. Following density separation and wet peroxide oxidation, a low-temperature solvent extraction protocol involving 2-chlorophenol was used to extract polyester (PET), polystyrene (PS), polyamide (PA), and polyvinyl chloride (PVC). The extract was analyzed using Py-GC–MS for the respective polymers, while the solid residue was pyrolyzed separately for polyethylene (PE) and polypropylene (PP). The one-step solvent extraction method significantly simplified the sample matrix and improved the sensitivity of analysis. Among the samples, PET was detected in greater quantities in the fine fraction than in the intermediate size fraction, and PET fine particles were located predominantly in the surface sand. Similar to PET, PS was detected at higher mass concentrations in the fine particles in most samples. These results underscore the importance of beach environment for plastic fragmentation, where a combination of factors including UV irradiation, mechanical abrasion, and water exposure promote plastic breakdown. Surface accumulation of fine plastic particles may also be attributed to transport of microplastics through wind and tides. The proposed sample treatment and analysis methods may allow sensitive and quantitative measurements of size or depth-related distribution patterns of microplastics in complex environmental media.

Novel multiple extraction thermal desorption approach prior to gas chromatography for the determination of residual solvents applied to modified cellulose

In this work, multiple extraction thermal desorption (METD), as a sample introduction method for GC, was developed. This technique was used for the determination of residual solvents (RS) in modified cellulose, because it is practically impossible to dissolve or distribute it uniformly in water and common organic solvents. Moreover, METD facilitates the optimization of the desorption time and it is more sensitive to quantify trace level volatiles in insoluble material, compared to direct dynamic desorption (DDD). In addition, METD provides diagnostic information about the sample-sorbent interaction. Three solvents (methanol, ethanol and tert-butanol) were determined in two types of modified cellulose (dialdehyde cellulose (DAC) and DAC-ethylenediamine (DAC-EDA)). It was shown that good linearity over a wide concentration range was achieved. The limits of detection (LOD) and limits of quantification (LOQ) for the different solvents ranged from 0.1 to 0.3 μg and from 0.3 to 0.9 μg per tube, respectively. Accuracy of the METD method was verified by using an alternative method based on the decomposition of the modified celluloses by Trichoderma reesei cellulase, followed by headspace-trap-GC (HS-trap-GC). The results obtained from the two validated methods were found to be similar (relative deviation < 17.0 %). However, the developed METD-GC method is preferable for the analysis of RS in modified cellulose since it does not require sample pretreatment and possesses higher sensitivity.

A Novel Reversed Phase HPLC Assay Method for Simultaneous Estimation of Glucose, Sodium Citrate and Chlorides in Pharmaceutical Formulations and Drug Solution for Oral Rehydration

A rapid, straightforward, sensitive, efficient, and cost-effective reverse-phase high-performance liquid chromatographic method was employed to simultaneously determine Glucose, Sodium Citrate, and chloride in a drug solution for oral solution. Glucose, Sodium Citrate, and Chlorides are all listed in the USP monograph. While various assay methods are available, titrimetric methods are ineffective for trace-level quantification. Although IC, AAS, and ICP-MS offer high accuracy, they are expensive and often inaccessible to many testing facilities. When selecting methods, it’s crucial to consider quality control requirements and user-friendly techniques. A simple proprietary HPLC method has been developed to simultaneously quantify Chlorides, Glucose, and Sodium Citrate, with a shorter run time. The separation was carried out using a Shim-pack SCR-102(H) ion exclusion analytical column (7.9 mm x 300 mm, 7 µm) with a flow rate of 0.6 mL/min. The column compartment was kept at 40°C, and the injection volume was 10 µL, with detection at 200 nm. All measurements were performed in a 0.1% phosphoric acid solution. The analytical curves showed excellent linearity (r &gt; 0.9999) within the concentration ranges of 0.74 to 1.74 mg/mL for Sodium Citrate (SC), 1.0 to 2.5 mg/mL for Sodium Chloride (SC), and 3.0 to 7.3 mg/mL for Glucose. The method was validated according to the guidelines of the International Conference on Harmonization (ICH Q2B) and USP&lt;1225&gt;. The method demonstrated precision, robustness, accuracy, and selectivity. During accelerated stability testing over six months, no significant variations were observed in organoleptic analysis and pH. Consequently, the developed method is considered suitable for routine quality control analyses, enabling the simultaneous determination of Sodium Citrate, Chloride, and Glucose in pharmaceutical formulations and solutions for oral rehydration.

Development and validation of LC-MS/MS method for trace analysis of acrylamide, acrylic acid and N, N-methylene bis acrylamide in sandy loam soil

Acrylamide and N, N-methylene bis acrylamide are most commonly used monomer and crosslinker compounds employed in synthesis of super absorbent hydrogels. When applied as soil conditioners, there are apprehensions that these hydrogels degrade over time and thus may release the toxic monomers in the soil. A method was thus developed using Liquid Chromatography tandem mass spectrometry (LC-MS/MS) for the trace level quantification of acrylamide (AD), acrylic acid (AA) and N,N-methylene-bis-acrylamide (MBA) in sandy loam soil amended by two test hydrogels the Pusa Hydrogel and SPG 1118 hydrogel prepared using AD and MBA. The MRM (multiple reaction monitoring) transitions were optimized for both the compounds. Soil samples were extracted using dispersive solid-phase extraction (dSPE) with a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) technique, employing acetonitrile. All analytes were quantified at trace levels within a five-minute run using UHPLC equipped with a C-18 column.Single laboratory validation of the developed method in soil matrix was conducted based on specificity, linearity, sensitivity, accuracy, precision, matrix effect and measurement of uncertainty. LC-MS/MS exhibited a linear response in the concentration range of 0.001 to 1 µg mL−1, with correlation coefficient >+0.99. Acceptable recovery (within 70–120 %) with repeatability (%RSD ≤20 %) was obtained at 0.01 to 1 µg g–1 fortification levels. LOQ (Limit of quantification) of the method for AD, AA and MBA in soil matrix were 0.05, 1 and 0.01 µg g–1, respectively. Both intra-laboratory repeatability and intermediate precision at LOQ suggested well acceptable precise (HorRat≈ 0.3) method for quantification. Matrix enhancement effect was observed in the order: AA>AD>MBA. The Expanded Uncertainty (EU) in soil matrix at LOQ was 21.64 %, 28 % and 19 % for AD, AA and MBA respectively. Groundnut and wheat grown with application of the hydrogels showed no detectable residues of monomers in soil samples (total n = 60) near the root zone at the time of crop harvesting.

A novel LC-QTOF-MS/MS method for trace level identification and quantification of potential genotoxic impurity, 5-Nitro-2-(propylthio)pyrimidine-4,6-diol in ticagrelor drug substance

An important objective of the present work is to develop and validate a novel LC-QTOF-MS/MS method to identify and quantify 5-Nitro-2-(propylthio) pyrimidine-4,6-diol genotoxic impurity at trace level (∼8.33 ppm) in ticagrelor drug substance, is an antiplatelet medicine used in the treatment of heart attacks and strokes. The potential genotoxic impurities must be controlled below acceptance limit during process of ticagrelor synthesis and release. In reversed-phase mode, the ACQUITY UPLC CSH C18 (2.1 mm x 100 mm, 1.7 μm) with ambient column temperature was used. 0.1% formic acid in Milli-Q water and methanol in the ratio of 30:70 v/v used as mobile phase A and mobile phase B with isocratic elution mode and the run time was 8.0 min. The flow rate was maintained at 0.25 mL/min, autosampler temperature was 5°C, Injection volume was 20 μL. The Retention time of 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was found at 1.345 minutes. Limit of detection (LOD) and limit of quantification (LOQ) were found at 0.56 ppm and 1.67 ppm, respectively. The 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was linear (R2=0.9986) over the concentration range 1.666 to 12.495 ppm. The method was accurate and precise, it was demonstrated by calculating the %mean recovery values ranging from 98.0-106.9%.

Trace Level Quantification of Genotoxic Impurities through Atmospheric Pressure Chemical Ionization (APCI) Coupled with Triple Quardrapole Analyzer in Combination Diabetic Drug Product

N-Nitrosamine impurities have been found in several drug products, showing a concern for regulatory aspects. The study presents the development and validation of a sensitive LC-MS/MS method for detecting eight nitrosamines in vildagliptin and metformin drug products. The developed method was specific and linearity was ranged 3.53-55.92 ppb for NDMA, 3.18-50.37 ppb for NMBA, 0.97-15.41 ppb for NDEA, 0.98-15.52 ppb for NEIPA, 1.00-15.86 ppb for NDIPA, 0.98-15.46 ppb for NDBA, 0.98-15.58 ppb for NMPA and 0.7-15.32 ppb for NDPA with correlation coefficient (r) was more than 0.98 and square of correlation coefficient (r2) was found to be greater than 0.96. The LOQ were obtained in the range of 0.97-3.53 ppb with %RSD in the range of 2.5-10.9% for eight nitrosamines showed good sensitivity. Accuracy was found in the range of 98.63 ± 5.77-125.17 ± 3.68% at LOQ level, 74.77 ± 0.83-88.17 ± 0.15% at 50% level, 79.93 ± 8.47-89.00 ± 0.61% at 100% level and 74.67 ± 2.31-84.03 ± 0.93% at 150% level for the eight nitrosamine impurities that were well within acceptance criteria of not less than 70% and not more than 130%. The method validation results demonstrated that the method is precise, accurate and linear, can be applied to quantify the nitrosamines in vildagliptin and metformin drug products.

Analyzing Alkyl Bromide Genotoxic Impurities in Febuxostat Based on Static Headspace Sampling and GC-ECD.

Herein, a sensitive and selective gas chromatography-electron capture detector (GC-ECD) method was developed and validated for the quantification of trace levels of five bromo-containing genotoxic impurities in Febuxostat active pharmaceutical ingredient (API) after headspace sampling (HS). Multivariate experimental designs for the optimization of static headspace parameters were conducted in two stages using fractional factorial design (FFD) and central composite design (CCD). The optimum headspace conditions were 5 min of extraction time and a 120 °C extraction temperature. Baseline separation on the analytes against halogenated solvents was carried out using an Agilent DB-624 (30 m × 0.32 mm I.D., 1.8 μm film thickness) stationary phase under isothermal conditions. The method was validated according to ICH guidelines in terms of specificity, linearity, the limits of detection and quantification, precision and accuracy. The linearity was assessed in the range of 5-150% with respect to the specification limit. The achieved LOD and LOQ values ranged between 0.003 and 0.009 and 0.01 and 0.03 μg mL-1, respectively. The accuracy of the method (expressed as relative recovery) was in the range of 81.5-118.2%, while the precision (repeatability, inter-day) was less than 9.9% in all cases. The validated analytical protocol has been successfully applied to the determination of the impurities in various Febuxostat API batch samples.

Trace level quantification of N-nitrosorasagiline in rasagiline tablets by LC-TQ-MS/MS

Parkinson's disease is a chronic, progressive neurological disease that currently affects about more than 10 million population worldwide. Rasagiline is a selective, irreversible monoamine oxidase type B inhibitor used as monotherapy in early Parkinson's disease. Rasagiline tablets have been recalled from market due to the presence of unacceptable levels of nitrosamine impurity. European Medical Agency has set up very stringent limit 100ng/day of N-nitrosorasagiline (NSRG) in drug product based on its mutagenicity. The analytical methods need to be sufficiently sensitive in order to adequately detect and quantify trace levels of NSRG. A highly sensitive LC-MS/MS method for determination of NSRG in rasagiline tablet formulation was developed by effectively separating on zorbax eclipse XDB C18 column using 0.1% formic acid in mixture of water and acetonitrile (35:65 v/v) in an isocratic mode at 0.5mL/min flow rate. The measurement of NSRG was performed using triple quadrupole mass detection accompanied by electrospray ionization in the multiple reaction monitoring mode. The validation of the method was comprehensive, demonstrating strong linearity across the concentration spectrum of 2 to 200ng/mL for NSRG. The obtained correlation coefficient exceeded 0.998, signifying a robust relationship. Recoveries spanning from 80.0% to 120.0% for NSRG were deemed satisfactory. The developed method was able to detect and quantitate NSRG at a concentration level of 1 to 2ng/mL respectively (1 to 2ppm with respect to 1mg/mL of rasagiline tablet sample concentration). The developed and validated method can be employed for routine quality control testing of rasagiline tablets.

Identification, trace level quantification, and in silico assessment of potential genotoxic impurity in Famotidine

Potential genotoxic impurities in medications are an increasing concern in the pharmaceutical industry and regulatory bodies because of the risk of human carcinogenesis. To prevent the emergence of these impurities, it is crucial to carefully examine not only the final product but also the intermediates and key starting material (KSM) used in drug synthesis. During the related substances analysis of KSM of Famotidine, an unknown impurity in the range of 0.5–1.0% was found prompting the need for isolation and characterization due to the possibility of its to infiltrate into the final product. In this study, the impurity was isolated and characterized as 5-(2-chloroethyl)-3,3-dimethyl-3,4-dihydro-2H-1,2,4,6-thiatriazine 1,1-dioxide using multiple instrumental analysis, uncovering a structural alert that raises concern. Considering the potential impact of impurity on human health, an in silico genotoxicity assessment was established using Derek and Sarah tool in accordance with ICH M7 guideline. Furthermore, molecular docking and molecular dynamics simulation were performed to evaluate the specific interaction of the impurity with DNA. The findings reveal consistent interaction of the impurity with the dG-rich region of the DNA duplex and binding at the minor groove. Both in silico prediction and molecular dynamic study confirmed the genotoxic character of the impurity. The newly discovered impurity in famotidine has not been reported previously, and there is currently no analytical method available for its identification and control. A highly sensitive HPLC-UV method was developed and validated in accordance with ICH requirements, enabling quantification of the impurity at trace level in famotidine ensuring its safe release.

Mitigating matrix effects for LC-MS/MS quantification of nitrosamine impurities in rifampin and rifapentine

Since the discovery of potentially mutagenic nitrosamine impurities in pharmaceuticals in 2018, liquid chromatography-mass spectrometry (LC-MS) methods have been widely applied for the trace level quantification of nitrosamines. As more nitrosamines and drug samples are analyzed, method accuracy is increasingly recognized as a major analytical challenge. Matrix effects can have a significant adverse impact on method accuracy but have not attracted much attention in nitrosamine quantification literature. In this study, we observed severe matrix effects when adapting LC-MS method conditions from a published and validated method for the analysis of nitrosamines in tuberculosis drugs. Several approaches were explored to mitigate these matrix effects, including using stable isotope-labeled internal standards, modifying LC conditions and sample concentration, and quantifying with standard addition instead of an external calibration method. The results underscore the importance of monitoring matrix effects and validating nitrosamine quantification procedures for specific sample matrices. Finally, two LC-MS/MS methods are presented with validation data for the quantification of 1-methyl-4-nitrosopiperazine (MNP) in rifampin and 1-cyclopentyl-4-nitrosopiperazine (CPNP) in rifapentine and revealed high nitrosamine levels in the tested samples that far exceeded recommended acceptable intake limits.

Synthesis and Trace-Level Quantification of Mutagenic and Cohort-of-Concern Ciprofloxacin Nitroso Drug Substance-Related Impurities (NDSRIs) and Other Nitroso Impurities Using UPLC-ESI-MS/MS-Method Optimization Using I-Optimal Mixture Design.

Globally, the pharmaceutical industry has been facing challenges from nitroso drug substance-related impurities (NDSRIs). In the current study, we synthesized and developed a rapid new UPLC-MS/MS method for the trace-level quantification of ciprofloxacin NDSRIs and a couple of N-nitroso impurities simultaneously. (Q)-SAR methodology was employed to assess and categorize the genotoxicity of all ciprofloxacin N-nitroso impurities. The projected results were positive, and the cohort of concern (CoC) for all three N-nitroso impurities indicates potential genotoxicity. AQbD-driven I-optimal mixture design was used to optimize the mixture of solvents in the method. The chromatographic resolution was accomplished using an Agilent Poroshell 120 Aq-C18 column (150 mm × 4.6 mm, 2.7 μm) in isocratic elution mode with 0.1% formic acid in a mixture of water, acetonitrile, and methanol in the ratio of 475:500:25 v/v/v at a flow rate of 0.5 mL/min. Quantification was carried out using triple quadrupole mass detection with electrospray ionization (ESI) in a multiple reaction monitoring technique. The finalized method was validated successfully, affording ICH guidelines. All N-nitroso impurities revealed excellent linearity over the concentration range of 0.00125-0.0250 ppm. The Pearson correlation coefficient of each N-nitroso impurity was >0.999. The method accuracy recoveries ranged from 93.98 to 108.08% for the aforementioned N-nitrosamine impurities. Furthermore, the method was effectively applied to quantify N-nitrosamine impurities simultaneously in commercially available formulated samples, with its efficiency recurring at trace levels. Thus, the current method is capable of determining the trace levels of three N-nitroso ciprofloxacin impurities simultaneously from the marketed tablet dosage forms for commercial release and stability testing.

Systematic optimization of reverse phase ultra‐high‐performance liquid chromatography method for quantification of nilotinib and its related substances in bulk drug and pharmaceutical formulation using quality by design approach

AbstractA sensitive ultra‐high‐performance liquid chromatography (UHPLC) method was developed for the quantification of trace levels of nilotinib and its related substances (RSs) in the nilotinib capsule dosage form. Critical method parameters were optimized using the design of experiments, employing the ACQUITY UHPLC BEH Phenyl column (2.1 × 100 mm, 1.7 μm) at a constant flow rate of 0.6 mL/min. The isocratic mobile phase, consisting of aqueous and organic phases, achieved efficient chromatographic separation within 8 min at 261 nm. The mean retention time for nilotinib was 6.1339, and for RSs, it ranged from 2.10 to 6.91 min, with a resolution exceeding 2 for all peaks. The method demonstrated robustness, separating known impurities and degradation products. The linearity was assessed over a concentration range of 0.02–80 ppm for nilotinib and 0.015–0.12 ppm for impurities. The limit of detection and limit of quantitation (LOQ) for nilotinib were 0.01 and 0.02 μg/mL, respectively, and 0.01 and 0.015 μg/mL for individual impurities. Recovery studies at LOQ, 100%, and 150% of the specification limit yielded percent recoveries ranging from 92.27% to 102.45%. Precision results showed low relative standard deviations, below 2% for nilotinib and below 8% for impurities. This method is deemed suitable for pharmaceutical formulation quantification.

A systematic review of analytical methodologies capable of analysing phytocannabinoids in cosmetics.

As cannabidiol (CBD) is not considered to be a drug and because of its potential health claims, it is an interesting compound that is often found in cosmetics. However, the safety of CBD, as well as the presence of trace amounts of other phytocannabinoids, including the psychoactive substance ∆9-tetrahydrocannabinol (THC), is still being debated. A robust analytical technique capable of analysing cosmetic products and determining their phytocannabinoid content will be crucial in assessing the safety of these products. This systematic review aims to highlight the current analytical tools that could be used to analyse phytocannabinoids in cosmetics. The ideal method would be able to analyse high levels of CBD in combination with trace levels of THC and their acids. The method should provide good recoveries and accuracies in a variety of matrices while providing information on up-coming phytocannabinoids such as cannabichromene (CBC), cannabigerol (CBG) and cannabinol (CBN). The systematic review approach was based on the Preferred Reporting Items for Systematic review and Meta-Analyses method. The research focused on studies published from January 2010 to December 2022 in PubMed and Scopus. A total of 15 datasets met the inclusion and exclusion criteria and were tabulated to allow easy comparison. Although some of the reviewed methods can handle multiple matrices and provide satisfactory recoveries, this review process did not identify an ideal method. The most suitable methods either could not quantify phytocannabinoid acids or were not sensitive enough to quantify trace levels of psychoactive phytocannabinoids.

Optimization of LC-MS/MS Analytical Method for Trace Level Quantification of Potential Genotoxic Impurities in Siponimod Pure Drug and Formulations

Optimization of LC-MS/MS Analytical Method for Trace Level Quantification of Potential Genotoxic Impurities in Siponimod Pure Drug and Formulations

Simultaneous and trace-level quantification of four benzene sulfonate potential genotoxic impurities in doxofylline active pharmaceutical ingredients and tablets using high-performance liquid chromatography with ultraviolet detection.

In the production of doxofylline, the common occurrence of toxic p-toluene sulfonate generation prompted the development and validation of a method using HPLC with ultraviolet detection (HPLC-UV). This method is designed for detecting four potential genotoxic impurities (PGIs) present in both doxofylline drug substance and tablets, with a focus on the UV-absorbing group p-toluene sulfonate. The four impurities were methyl 4-methylbenzenesulfonate (PGI-1), ethyl 4-methylbenzenesulfonate (PGI-2), 2-hydroxyethyl 4-methylbenzenesulfonate (PGI-3), and 2-(4-methylphenyl)sulfonyloxyethyl 4-methylbenzenesulfonate (PGI-4). In this method, chromatographic separation was achieved using a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm). The mobile phases consisted of 20% acetonitrile as mobile phase A and pure acetonitrile as mobile phase B, operating in gradient elution mode at a flow rate of 1.0 mL/min. According to the guidelines of the International Conference on Harmonization, it was determined that this method could quantify four PGIs at 0.0225 μg/mL in samples containing 60 mg/mL. The validated approach demonstrated excellent linearity (R2 > 0.999) across the concentration range of 30%-200% (relative to 0.075 μg/mL doxofylline) for the four PGIs. The accuracy of this method for the four PGIs ranged from 94.8% to 100.4%. The reverse-phase-HPLC-UV analytical method developed in this study is characterized by its speed and precision, making it suitable for the sensitive analysis of benzene sulfonate PGIs in doxofylline drug substances and tablets.