In the research article recently published in ACS Analytical Chemistry journal the researchers led by Lucie Nováková from the Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, present a novel analytical approach for mixtures of natural products.This study addresses the complexity of plant extract analysis by developing a holistic two-injection method that integrates ultrahigh-performance <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=38">supercritical fluid chromatography</a> (UHPSFC) with multimodal ionization tandem mass spectrometry (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=32">MS/MS</a>). This method, which does not require manual intervention, significantly improves the efficiency and accuracy of analyzing various small molecules, such as terpenes, flavonoids, and phenolic acids, in plant extracts. The authors applied this method to Eucalyptus sp. extracts, demonstrating its potential as a robust tool for comprehensive plant metabolite analysis.<blockquote><h4>The original article</h4></blockquote><blockquote><h5><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c03599">Ultrahigh-Performance Supercritical Fluid Chromatography–Multimodal Ionization–Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts</a></h5>Kateřina Plachká, Veronika Pilařová, Štefan Kosturko, Jan Škop, Frantisek Svec, and Lucie NovákováAnalytical Chemistry 2024 96 (7), 2840-2848DOI: 10.1021/acs.analchem.3c03599<h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote><h5>Selected sections from the article follow.</h5><h3>Abstract</h3>Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=electrospray">electrospray</a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=atmospheric+pressure+chemical+ionization">atmospheric pressure chemical ionization</a> (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS two-injection approach were determined, and it was applied to the analysis of Eucalyptus sp. extracts prepared by <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=SFE">supercritical fluid extraction</a>. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Analytical_Chemistry_2024_96_7_2840_2848_cover_image_2a6c4c6d6b.jpg" alt="Analytical Chemistry 2024 96 (7), 2840-2848: graphical abstract."><h4>Subjects</h4><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=chromatography">Chromatography</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Flavonoids">Flavonoids</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Hydrocarbons">Hydrocarbons</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Ionization">Ionization</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=Solvents">Solvents</a></li></ul><h3>Experimental Section</h3><h4>Chromatographic Conditions</h4>All experiments were carried out using the supercritical fluid chromatography system <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=38&amp;mainauthorItems=5&amp;search=Acquity+UPC2">Acquity UPC2</a> (Waters, Milford, MA, USA) equipped with a binary pump, an autosampler, a column thermostat, a back pressure regulator (BPR), and a PDA detector. The system was coupled to a triple quadrupole mass spectrometer <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=38&amp;mainauthorItems=5&amp;search=Xevo+TQ-XS">Xevo TQ-XS</a> (Waters) via a commercially available SFC-MS dedicated pre-BPR splitter device with an additional isocratic or binary pump for makeup solvent delivery (Waters).Several stationary phases were tested during optimization including Viridis BEH (hybrid silica), Viridis BEH 2-ethylpyridine (2-EP), Torus 2-picolylamine (2-PIC), Viridis HSS C18 SB (C18), Torus 1-amino anthracene (1-AA), all in 3.0 × 100 mm, 1.7 μm, Torus Diol (diol) (3.0 × 50 mm and 100 mm, 1.7 μm), BEH HILIC (2.1 × 100 mm, 1.7 μm), CORTECS HILIC (silica) (3.0 × 100 mm, 1.6 μm), all from Waters, YMC Carotenoid C30 (4.6 × 100 mm, 5 μm) from YMC (Dinslaken, Germany, and porous graphitic carbon (PGC) columns Hypercarb (3.0 × 50 mm and 100 mm, 3.0 μm, Thermo Fisher Scientific Inc., Waltham, MA, USA) and Supel Carbon LC (3.0 × 150 mm, 2.7 μm, Merck KGaA, Darmstadt, Germany). Analyses were carried out using methanolic organic modifiers with or without an additive that included 10 mmol/L ammonia, 5% water, and 10 mmol/L formic acid. Final chromatographic conditions were as follows: Method 1: 150 mm Supel Carbon LC column at 60 °C, BPR pressure at 3300 psi (22.75 MPa), flow rate 1.5 mL/min, MeOH as organic modifier in gradient elution: 0% for 1.5 min, 0–40% in 1.5–4.0 min, 40–41% in 4.0–6.0 min, followed by 1.5 min equilibration at starting conditions. Method 2: 50 mm Torus Diol column at 20 °C and 5% water in MeOH as organic modifier with specific gradient conditions listed in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c03599#tbl1">Table 1</a>. The partial loop with needle overfill injection mode was used to inject 2 and 10 μL samples for methods 1 and 2, respectively. The autosampler was cooled to 5 °C to reduce the evaporation of volatile compounds.<h4>MS Conditions</h4>The four ionization sources, ESI, APCI, multimodal ESCi, and US, all from Waters were tested. MassLynx Software 4.1 was used for MS control, data acquisition, and processing. Optimization of the ionization sources conditions was carried out using a design of experiment (DoE) approach, followed by the optimization of the makeup solvent composition and flow rate. The makeup solvents tested included pure MeOH and MeOH with various additives, such as water, ammonia, formic acid, and their combinations in different concentrations. The makeup solvent flow rate was tested in the range of 0.0–0.6 mL/min. Selected reaction monitoring (SRM) transitions were determined for each analyte (Supporting Information S2), and their selectivity was verified. MODDE Software 13.0.1 was used to design the experiments for the ion source optimization and subsequent data evaluation. The parameters tested and their respective ranges for each ionization source are listed in Table 2. The DoE were selected as a compromise between the number of experiments required and the power of the study. Three replicates were always included to test the repeatability of the model. The data evaluation included its logarithmic transformation when necessary and the exclusion of outliers to achieve the highest possible linearity (R2), model validity, predictability (Q2), and reproducibility. The limits for the applicable model were set according to the MODDE software: R2 showing model fit &gt;0.5, Q2 estimating future prediction precision &gt;0.1 for significant model and &gt;0.5 for a good model, reproducibility &gt;0.5. Model validity tests diverse model problems and should be &gt;0.25. However, model validity can be lost for very good models with Q2 &gt; 0.9 due to high sensitivity in the test or extremely good replicates (&gt;0.9). Therefore, model validity was always carefully checked manually. The critical factors (marked with “!“ in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c03599#tbl2">Table 2</a>) were identified as having a significant effect on the model, i.e., contributing more than 50% to the final response. Therefore, these factors were optimized separately, and a second DoE was run.<h3>Results and Discussion</h3>The aim of this study was to develop a holistic method for the simultaneous analysis of compounds typically found in plant extracts, namely, flavonoids, terpenoic and phenolic acids, and terpenes. These compounds include molecules with a wide range of physicochemical properties, from small lipophilic terpenes to polar flavonoids, as listed in Table S1.First, a stationary phase had to be selected that would allow the retention of all of the target compounds. We expected that the retention and elution of hydrophilic flavonoids, and especially their glycosylated forms, i.e., rutin, hirsutrin, and hesperidin, would be the main challenge of the UHPSFC-MS method due to the low polarity of the CO₂-based mobile phase. On the other hand, less polar terpenes should be easily retained and eluted. A systematic column screening was carried out on a set of selected stationary phases, including hybrid silica, 2-EP, C18, 2-PIC, and diol with preferred H-bonding and π–π interactions. A generic gradient from 0 to 45% of organic modifier, i.e., MeOH, 10 mmol/L ammonia in MeOH, 10 mmol/L formic acid in MeOH, and 2% water in MeOH, was tested on all five columns. As expected, the addition of water to the organic modifier had a beneficial effect on the elution of polar flavonoids. However, it was necessary to increase the percentage of organic modifiers up to 58% to enable the elution of all target analytes. Unfortunately, these stationary phases were not able to retain most of the lipophilic terpenes, especially those without hydroxy groups. That was surprising as more lipophilic compounds are commonly analyzed by SFC. However, cymene, limonene, pinene, caryophyllene, and citronellal eluted in the dead volume even when using pure CO₂. Volatiles containing hydroxy groups were retained on C18, diol, silica, and 2-EP stationary phases but still coeluted in 1 peak near to the dead volume. Therefore, the retention and separation of terpenes became the main challenge. Accordingly, the 1-AA column with dominant π–π interactions was tested, where the higher retention of nonpolar compounds was expected. This hypothesis was confirmed. However, flavonoids were retained to such an extent that they could not be eluted within a reasonable analysis time under any conditions tested.Thus, a holistic approach to the analysis of a plant extract consisting of two UHPSFC methods carried out on two columns and the same system without any manual intervention was developed and tested as a proof of concept. Indeed, most commercially available SFC instruments use a column manager that allows two or more columns to be connected simultaneously. Thus, it is still possible to analyze a complex sample in just two injections, taking advantage of the two stationary phases with different selectivity.<h4>Application of UHPSFC-ESI/ESCi-MS/MS Method on Eucalyptus Extracts</h4><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Analytical_Chemistry_2024_96_7_2840_2848_Figure_4_Comparison_of_the_percentage_abundance_of_targeted_analytes_i_e_A_terpenes_B_terpenoic_acids_C_16211a715a.jpg" alt="Analytical Chemistry 2024 96 (7), 2840-2848: Figure 4. Comparison of the percentage abundance of targeted analytes, i.e., (A) terpenes, (B) terpenoic acids, (C).jpg"><h3>Conclusions</h3>UHPSFC-MS/MS is an interesting alternative to LC and GC methods, permitting the analysis of a wide range of compounds with a single instrument. Unfortunately, a single holistic method for the simultaneous analysis of terpenes and polar flavonoids remains unattainable due to two main challenges. No tested stationary phase enabled the retention and elution of both groups of analytes. However, new stationary phases are being introduced to the market every year, which is promising. Second, a universal ionization source combining GC and LC approaches is required for the simultaneous analysis of small lipophilic terpenes and polar glycosylated flavonoids.Despite these challenges, we were able to develop a two-injection approach for complex plant analysis as a proof-of-concept based on the ability of the UHPSFC to cover supercritical, subcritical, enhanced-liquid, and liquid conditions within one instrument. Here, volatile terpenes are measured using a PGC stationary phase within supercritical to subcritical conditions achieved by adding up to 40% of MeOH to CO₂. More polar phenolic and terpenoic acids and flavonoids are then analyzed on short diol column with gradient from 0 to 90% MeOH with addition of 5% water.The APCI source proved to be beneficial in the case of lipophilic volatiles when compared to ESI, but it was not possible to detect flavonoids using APCI. Conversely, US resulted in higher sensitivity for flavonoids, but similar to ESI, it was not possible to detect terpenes without hydroxy groups. The finally selected multimodal ESCi source was able to detect all targeted volatiles although the sensitivity was significantly lower than in the case of APCI. However, it was possible to switch from ESCi to ESI without any need for manual intervention, which was crucial for the applicability of the developed approach.Finally, the quantitative aspects of the two developed UHPSFC-ESI/ESCi-MS/MS methods were determined, and the approach was successfully applied for the analysis of SFE extract from Eucalyptus sp. The novel approach can be beneficial for analysis of complex samples containing analytes with a wide range of physicochemical properties.

Ultrahigh-Performance Supercritical Fluid Chromatography–Multimodal Ionization–Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts

This study presents novel LC-TQ-MS/MS and cost-effective UPLC methods for quantifying N-nitroso drug substance-related impurity (N-MFA) in mefenamic acid tablets and pediatric suspensions. Both methods were successfully validated according to ICH guidelines, demonstrating excellent sensitivity, linearity, and accuracy. LC-MS/MS achieved quantification at 0.01 ng/mL (1 mg/mL MFA), while UPLC quantified down to 0.036 μg/mL (10 mg/mL MFA). Linearity was confirmed across 0.01–125 ppm for LC-MS/MS and 3.6–125 ppm for UPLC with R² = 0.999.Accuracy recoveries ranged from 90.43% to 101.34% for LC-MS/MS and 92.12% to 101.21% for UPLC. Both methods were applied to commercial formulations and demonstrated suitability for routine use in R&amp;D and quality control labs. Additionally, sustainability and environmental impact were evaluated using tools like GAPI, AGREE, and RGB12, confirming their green analytical profile.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597">A novel LC-TQ-MS/MS method for quantifying mefenamic acid-NDSRI (N-nitroso drug substance-related impurity) in mefenamic acid tablet and pediatric suspension dosage forms: a comparative study with a cost-effective white, green, and blue UPLC method</a></h4>Srinivas Nakka, Naresh Kumar Katari, Siva Krishna Muchakayala, Sreekantha Babu Jonnalagadda, Surendra Babu Manabolu Surya&nbsp;RSC Advances, Volume 15, Issue 3, 2025, 1957-1969<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1039/d4ra08425j">https://doi.org/10.1039/d4ra08425j</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by-nc/3.0/">CC-BY 3.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Regulatory agencies have underscored the significance of predicting toxicity and precisely measuring the levels of nitrosamine contaminants in certain pharmaceuticals. These impurities, which include nitrosamines derived from active pharmaceutical ingredients, are referred to as Nitrosamine Drug Substance Related Impurities (NDSRIs). Nitrosamines, or N-nitrosamines, are molecules characterized by their N-nitroso functional group. They are classified as a group of concern due to their status as human carcinogens, with evidence showing that long-term intake above acceptable levels can increase cancer risks.These NDSRI substances are categorized into one of three “Cohort-of-Concern” (CoC) groups: aflatoxin-like, alkyl-azoxy, and N-nitroso. “CoC” denotes a collection of highly potent mutagenic compounds requiring special attention due to potential health risks. The focus of pharmaceutical regulatory authorities has shifted from common nitrosamines to NDSRIs because of their potential carcinogenicity and their presence in drug products at unacceptable levels.1–6 As a result, regulatory authorities recalled various drug products worldwide. Furthermore, the detection and quantification of NDSRIs are of significant concern to analytical researchers.7–11Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of medications that relieve pain and inflammation, including in renal transplants and chronic non-cancer pain in children.12,13 About 30 million people worldwide use NSAIDs every day, according to a report by Medical News Today. The worldwide market for NSAIDs was valued at USD 15.58 billion in 2019 and is envisioned to reach USD 24.35 billion by 2027.Mefenamic acid (MFA) is chemically known as 2-[(2,3-dimethylphenyl)amino]benzoic acid, with the chemical formula C15H15NO2 and a molecular weight of 241.28 g mol−1. MFA is classified as an anthranilic acid (phenolate) NSAID that exhibits both antipyretic and analgesic properties. This medication treats moderate pain and inflammation associated with osteoarthritis, post-operative pain, menstrual pain, rheumatoid arthritis, toothaches, and severe back and muscle pain. Additionally, it is also used to manage menorrhagia.14,15To our knowledge, this is the first UPLC-MS/MS and environmentally friendly UPLC method for quantifying the target compound N-MFA. As a result, there are no existing methods available to compare with the proposed UPLC-TQ-MS/MS and the cost-effective green UPLC method. Various regulatory agencies, including the US FDA and Health Canada, have proposed that the acceptable intake (AI) value for N-MFA is 78 000 ng per day.16,17 The interim control limit for N-MFA is 62.4 ppm, which can be derived from the AI value of the impurity and the maximum daily dose of the MFA. Hence, simple and highly sensitive UPLC-MS/MS and UPLC methods have been developed for the accurate quantification of N-MFA in MFA drug formulations, including tablets and pediatric suspensions.<div>The current study aimed to assess genotoxicity using (Q)-SAR (Quantitative Structure–Activity Relationship) models and to develop new LC-TQ-MS/MS and UPLC methods for quantifying N-MFA in both the MFA drug substance and its formulation dosage forms. Recent regulatory guidelines demonstrate that setting the quantitation limit (QL) at 30% is considered reasonably good and acceptable. If the values consistently fall below the QL across all process validation batches and corresponding stability samples (both accelerated and long-term), it is acceptable to proceed with skip lot testing. UPLC can detect the N-MFA impurity below the 30% control limit (∼19 ppm). Therefore, this cost-effective UPLC method is proposed alongside the LC-TQD-MS/MS for use in quality control laboratories. Both methods have been successfully validated regarding the QL, linearity, precision, accuracy, and robustness. The sustainability and eco-friendliness of the UPLC method were evaluated using greenness and whiteness assessment metrics, including GAPI, the analytical eco-scale, AGREE, AGREEprep, BAGI, and RGB12 tools.18–20 The results indicated that the proposed method exhibits a high level of greenness and whiteness. <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#fig1">Fig. 1</a> illustrates the chemical structures of MFA, N-DPA (N-nitroso diphenylamine), and N-MFA. The applicability of these methods was successfully verified through the analysis of commercial samples of APIs and dosage forms of MFA.<h4>Materials and method</h4><h5>Instrument and software</h5>The chromatographic investigation was accomplished on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/198">Waters H class UPLC system</a>, which includes a PDA-photo diode array detector model (L20UPD127A) modules, an Auto Sampler Manager with a Flow-Through Needle (FTN-L20FTP352G), Quaternary Solvent Manager (QSM-L20QSP358A) (Waters Corporation, USA). For the LC-TQ-MS/MS method (MRM analysis) a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/195">Waters Xevo TQ-XS MS</a> system equipped with an ESI ion source was utilized. A Mettler-Toledo (Model: XPE205, Columbus, Ohio, USA) analytical balance was employed to weigh the samples and the impurity standard. The analysis utilized high-purity water obtained from the Millipore Milli-Q purification system (Bedford, MA, USA). Centrifuge (Model: 5810R, Hemburg, Germany) from Eppendorf. ChemDraw Professional 15.0 sketched MFA, N-DPA, and N-MFA molecular structures. The two primary (Q)-SAR methodologies employed were the Derek Nexus (version 6.2.0, featuring the Derek KB 2022 1.0 knowledge base) and the Sarah Nexus (version 3.2.0, which includes the Nexus version 1.9 and the Sarah Model 2022.1). <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;search=TargetLynx">Target Lynx software</a> v 4.2 was used to compute all LC-TQ-MS/MS acquisition and process parameters.<h4>Results and discussion</h4><h5>Development and optimization of mass spectrometry conditions</h5>To develop an accurate quantification of N-MFA in MFA tablets and pediatric suspensions, optimizing mass parameters is a decisive step. We intended to attain a sensitive, precise, and accurate method. For N-MFA quantification, the MRM technique was utilized. For initial mass conditions optimization by injecting the 1000 ng mL−1 concentration of N-MFA into mass spectrometry using infusion mode to identify the prominent product ion in electron spray ionization in positive ion mode (m/z 271.13 → 241.14). Due to the pump flow rate being set at 0.7 mL min−1 and the recommended desolvation gas temperature and gas flow being 450 °C and 900 L per Hr, hence performed the analysis with the values mentioned earlier. Employing the Intellistart software, which is built into Waters mass lynx software, to fine-tune MS/MS parameters, including cone voltage, capillary voltage, and collision energy values with different mass transitions. Intellistart identified three major MRM transitions, including m/z 271.13 → 241.14, m/z 271.13 → 223.12, and m/z 271.13 → 208.10 (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#fig2">Fig. 2</a>). After careful optimization of cone voltage and collision energies finalized, the quantifier ion transition as m/z 271.13 → 241.14 and the qualifier ion transition as m/z 271.13 → 223.12 (Fig. S4<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#fn1">†</a>). A mass spectrometer was installed, a 50 μL loop, which improves the peak shape when injecting the samples containing a high percentage of organic solvents. The highest intensity of quantifier and qualifier ions are detected by using the below MS/MS optimized conditions: cone voltage is 18 V, capillary voltage is 2.48 kV, collision energy is 8 V and 18 V for qualifier and quantifier respectively, desolvation gas temperature is 450 °C, and gas flow 900 L h−1. In the MS/MS method, the events program flow state was directed from 0 to 5.4 min set to mass to detect the N-MFA and the flow state from 5.5 to 10 minutes to avoid the high concentration of MFA contamination triggered by formulation tablet and pediatric suspension samples 1 mg mL−1 concentration. The optimized MS/MS parameters are shown in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#tab2">Table 2</a>.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/RSC_Advances_Volume_15_Issue_3_2025_Pages_1957_1969_Fig_2_A_Mass_spectrum_of_NMFA_and_B_MSMS_fragmentation_pattern_of_N_MFA_09e3975748.jpg" alt="RSC Advances, Volume 15, Issue 3, 2025, Pages 1957-1969: Fig. 2. (A) Mass spectrum of NMFA, and (B) MS/MS fragmentation pattern of N-MFA." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_RSC_Advances_Volume_15_Issue_3_2025_Pages_1957_1969_Fig_2_A_Mass_spectrum_of_NMFA_and_B_MSMS_fragmentation_pattern_of_N_MFA_09e3975748.jpg 182w," sizes="100vw" width="1500" height="1286"><h5>Method validation</h5>The present proposed LC-TQ-MS/MS and UPLC methods have been fully validated by ICH Q2R2, USP 〈1225〉 guidelines,<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#cit23">23</a> and published journals,24–26 following the method validation parameters, such as specificity, limit of detection, limit of quantitation, linearity, precision, accuracy, robustness, and solution stability. <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#tab3">Table 3</a> presents the results of the validation study, demonstrating that the method is specific, precise, linear, and accurate.<h5>Specificity</h5>The current analytical method's specificity is demonstrated by the liquid chromatography system's capability to differentiate between the diluent, placebo, MFA, N-MFA, and N-MFA spiked at the specification level. Injected each 10 μL for LC-MS/MS and 50 μL for UPLC of diluent, placebo, N-MFA standard solution, and sample solution of MFA. The analysis showed that no co-eluting peaks were apparent at the retention time (tR) of N-MFA, allowing for the specific and accurate estimation of N-MFA in MFA tablet and pediatric suspension formulations. Overlay UV spectra of N-MFA and MFA are shown in Fig. S4,<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#fn1">†</a> and the typical LC-MS/MS and UPLC chromatograms indicating the specificity of the method can be found in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#fig3">Fig. 3</a> and <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/org/science/article/pii/S2046206925001597#fig4">4</a>, respectively.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/RSC_Advances_Volume_15_Issue_3_2025_Pages_1957_1969_Fig_3_Typical_MRM_chromatograms_of_A_placebo_B_N_MFA_standard_22464e21b1.jpg" alt="RSC Advances, Volume 15, Issue 3, 2025, Pages 1957-1969: Fig. 3. Typical MRM chromatograms of (A) placebo, (B) N-MFA standard at the specification level, and (C) UV spectra of N-MFA spiked to the MFA sample at the specification level, and (D) MRM of N-MFA spiked to the MFA sample at the specification level." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_RSC_Advances_Volume_15_Issue_3_2025_Pages_1957_1969_Fig_3_Typical_MRM_chromatograms_of_A_placebo_B_N_MFA_standard_22464e21b1.jpg 137w," sizes="100vw" width="1500" height="1710"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/RSC_Advances_Volume_15_Issue_3_2025_Pages_1957_1969_Fig_4_Typical_chromatograms_of_A_blank_B_placebo_C_N_MFA_standard_at_specification_level_1ba78e4737.jpg" alt="RSC Advances, Volume 15, Issue 3, 2025, Pages 1957-1969: Fig. 4. Typical chromatograms of (A) blank, (B) placebo, (C) N-MFA standard at specification level, and (D) N-MFA spiked to the MFA sample at specification level." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_RSC_Advances_Volume_15_Issue_3_2025_Pages_1957_1969_Fig_4_Typical_chromatograms_of_A_blank_B_placebo_C_N_MFA_standard_at_specification_level_1ba78e4737.jpg 125w," sizes="100vw" width="1500" height="1878"><h4>Conclusion</h4>In conclusion, two interdependent (Q)-SAR tools were utilized to assess and classify N-MFA as a Class-3 category, designated as a “CoC” which suggests potential genotoxicity. Diphenylamine is a precursor that can lead to the formation of N-MFA. The acceptable intake (AI) of N-MFA was derived from N-DPA, and the determined AI for N-MFA is 78 000 ng; hence the specification limit is 62.4 ppm. Proposed novel LC-TQ-MS/MS and cost-effective UPLC methods are proposed to accurately quantify N-nitroso mefenamic acid (N-MFA) in mefenamic acid (MFA) tablets and pediatric suspension dosage forms. The proposed method has been successfully validated in compliance with ICH guidelines. The developed methods were further applied to commercially available mefenamic acid formulations to determine accurate and precise quantification of N-MFA. For routine quality control laboratories, this cost-effective UPLC method can be used to detect 30% of the interim control limit (∼19 ppm). Furthermore, sustainability and eco-friendliness were evaluated using greenness and whiteness assessment metrics, including the analytical eco-scale, GAPI, AGREE, AGREEprep, BAGI, and RGB12 tools. The results indicated that the method proposed exhibits a high level of greenness and whiteness. Both methods can be used to determine N-MFA content in mefenamic acid formulation dosage based on the availability of instruments LC-TQ-MS/MS and UPLC in R&amp;D and quality control laboratories.</div>

A novel LC-TQ-MS/MS method for quantifying mefenamic acid-NDSRI (N-nitroso drug substance-related impurity) in mefenamic acid tablet and pediatric suspension dosage forms: a comparative study with a cost-effective white, green, and blue UPLC method

This study presents a novel and eco-friendly LC-QqQ-MS/MS method for the trace-level quantification of N-Nitroso Moxifloxacin (N-MOX) and di-Nitroso pyrrolopiperidine (DNPP) in moxifloxacin drug substances and products. As both NDSRIs fall into potency category 4 with an acceptable intake limit of 1500 ng/day, their control is vital for patient safety. The method achieves efficient chromatographic separation using a Cortecs T3 C18 column and gradient elution, and employs MRM transitions m/z 431.0 → 386.0 for N-MOX and 185.1 → 138.1 for DNPP.Validated per ICH guidelines, the method proved accurate, sensitive, and reproducible. Green assessment tools confirmed its low environmental impact in terms of time, solvent use, waste, and sample volume. Successfully applied to commercial moxifloxacin tablets and eye drops, this method offers a robust solution for routine NDSRI monitoring in pharmaceuticals.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971">A facile and eco-friendly simultaneous quantification LC-TQ-MS/MS approach for N-Nitroso Moxifloxacin and di-nitroso pyrrolopiperidine in Moxifloxacin tablets and eye drops</a></h4>Srinivas Nakka, Siva Krishna Muchakayala, Surendra Babu Manabolu Surya&nbsp;Green Analytical Chemistry, Volume 12, 2025, 100188<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1016/j.greeac.2024.100188">https://doi.org/10.1016/j.greeac.2024.100188</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.The emergence of nitrosamine impurities in active pharmaceutical ingredients (APIs) is a complex issue influenced by various factors related to chemical synthesis, raw material quality, and manufacturing processes. The concern of nitrosamine contamination in pharmaceuticals is quickly becoming more widespread, extending not only beyond smaller nitrosamines but also nitrosamine drug substance-related impurities (NDSRIs). These NDSRIs are particularly challenging because they are structurally related to the API, making their presence almost unavoidable [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0001">[1]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0002">[2]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0003">[3]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0004">[4]</a>]. Moreover, the lack of carcinogenicity or mutagenicity data for most NDSRIs complicates the establishment of acceptable intake levels (AIs). Based on the GSRS (Global Substance Registration System) database, 41.4 % of APIs and 30.2 % of API impurities are potential nitrosamine precursors. However, when tertiary amines are excluded from this classification, the percentages adjust to 14.7 %, and 12.8 % are for APIs and API impurities, respectively, that could potentially lead to nitrosamine formation [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0005">5</a>].In July 2023, the European Medicines Agency (EMA) introduced a novel assessment method known as the Carcinogenic Potency Categorization Approach (CPCA) for N-nitrosamines. This approach was designed to facilitate the determination of AI for nitrosamines, thereby simplifying the complexities associated with setting AI [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0006">6</a>,<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0007">7</a>]. Developing an analytical method with high sensitivity for regular analysis presents a considerable challenge, given the outstandingly low acceptable intake thresholds for these impurities and is fraught with sensitivity issues, while reactivity and stability concerns further complicate the analysis.Moxifloxacin (MOX) is a broad-spectrum antibiotic that acts against both Gram-positive and Gram-negative bacteria's from the fourth-generation fluoroquinolone class of medication and is prescribed for numerous bacterial ailments such as pneumonia, conjunctivitis, endocarditis, tuberculosis, and sinusitis. It can be administered orally, intravenously, or as eye drops. It obstructs the bacteria's DNA replication process, vital for their growth and proliferation [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0008">[8]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0009">[9]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0010">[10]</a>]. It is acknowledged worldwide for its efficacy in treating numerous bacterial infections and is listed among the World Health Organization (WHO)’s essential medicines.Green analytical chemistry (GAC) is dedicated to developing and applying methods that minimize environmental impact and improve sustainability. The core goal of this approach is to reduce the use of hazardous substances, minimize waste production, and encourage energy-efficient analytical processes. In 1998, Paul Anastas and his team introduced twelve principles to assess the environmental impact of chemical processes, which were later adapted for analytical methods [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0011">11</a>]. Gałuszka et al. developed twelve principles specifically for green analytical chemistry, incorporating the core concepts of green chemistry [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0012">12</a>]. It involves analyzing smaller sample quantities and reducing the usage or volume of toxic reagents, solvents, and energy consumption. Experts in green chemistry focus on the use of safe, environmentally friendly chemicals and low-waste assessment methods [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0013">[13]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0014">[14]</a>].<div>A comprehensive literature review reveals that, to date, there are no published research articles on the simultaneous trace-level quantification of N-MOX (1-cyclopropyl-6-fluoro-8-methoxy-7-((4aS,7aS)-1-nitrosooctahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), and DNPP {(4aS,7aS)-1,6-dinitrosooctahydro-1H-pyrrolo[3,4-b]pyridine}. The current study aimed to assess genotoxicity by utilizing Quantitative structure-activity relationship ((Q)-SAR) models that apply carcinogenic potency category classification, while also developing a straightforward, rapid, and novel UPLC-triple quadrupole (QqQ)-MS/MS method for the simultaneous trace-level quantification of N-MOX and DNPP impurities. The MOX, N-MOX, and DNPP chemical structures are illustrated in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#fig0001">Fig. 1</a>.<h4>2. Materials and methods</h4><h5>2.1. Instruments and software</h5>The chromatographic study was performed on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/198">Waters H class UPLC</a> (Ultra Performance Liquid Chromatography) system, which includes a PDA-photo diode array detector model (L20UPD127A) modules, an Auto Sampler Manager with a Flow-Through Needle (FTN- L20FTP352G), Quaternary Solvent Manager (QSM- L20QSP358A) (Waters Corporation, USA). A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/195">Waters Xevo TQ-XS LC-MS system</a> equipped with a triple quadrupole (QqQ) mass analyzer and Electrospray Ionization (ESI) ion source with multiple reaction monitoring (MRM) techniques was utilized (Waters Corporation, Milford, Massachusetts, USA). A Mettler-Toledo (Model: XPE205, Columbus, Ohio, USA) analytical balance was employed to weigh the samples and the impurity standard. The analysis utilized high-purity water consists of resistivity &lt;18.2 MΩ˙cm at 25 °C, TOC (Total Oxidizable Carbon) ≤5 ppb, Bacteria &lt;10 CFU (Colony-forming unit) / 100 mL obtained from the Millipore Milli-Q purification system (Bedford, MA, USA). Centrifuge (Model: 5810R, Hemburg, Germany) from Eppendorf. Millex-GV hydrophilic 0.22 µm PVDF filters (Burlington, Massachusetts, USA) have been obtained from Millipore. Using ChemDraw Professional 15.0, sketched the chemical structures of MOX, N-MOX, and DNPP. The two primary (Q)-SAR methodologies employed were the Derek Nexus (version 6.2.0, featuring the Derek KB 2022 1.0 knowledge base) and the Sarah Nexus (version 3.2.0, which includes the Nexus version 1.9 and the Sarah Model 2022.1). <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635">MassLynx</a> and TargetLynx software (v 4.2) were used for all LC-QqQ-MS/MS acquisition parameters and to process the data, respectively.<h4>3. Results and discussion</h4><h5>3.4. Development and optimization of MS/MS conditions</h5>Multiple Reaction Monitoring (MRM) is an analytical method of high sensitivity and specificity, gaining prominence in different fields, including drug development, clinical research, and metabolomics [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0017">17</a>]. It enables precise detection and accurate quantification of impurities at very low concentrations. During the current study, N-MOX and DNPP impurities were detected and quantified using the MRM approach in an electrospray ionization positive mode (ESI). The capillary voltage (kV), desolvation temperature ( °C), and desolvation gas flow (L/Hr) are the three major ESI source parameters. Concerning the low pump flow rate of 0.3 mL/min, the recommended desolvation gas temperature and gas flow are 450 °C and 900 L/Hr. Both N-MOX and DNPP showed maximum intensity at a capillary voltage of 3.0 kV. The MS parameters for MRM channels, such as collision energy (CE) and cone voltage (CV), were individually optimized to ensure a stable and robust signal for the compounds. Intellistart software, built into Waters mass lynx software, is employed to fine-tune MS/MS parameters, including cone voltage and collision energy values with different mass transitions. MS/MS fragmentation pattern for both impurities shown in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#fig0003">Fig. 3</a>. After careful optimization of cone voltage and collision energies finalized, the quantifier ion transition as m/z 185.1→ 138.1 and the qualifier ion transition as m/z 185.1 → 96.10 for DNPP; the quantifier ion transition as m/z 431→ 386 and the qualifier ion transition as m/z 431 → 319 for N-MOX (Fig. S2). A mass spectrometer, a 50 µL loop, was installed, which improves the peak shape when injecting samples containing a high percentage of organic solvents. Later, studies of the impact of collision energies varying from 5 to 30 V and optimized collision energy for N-MOX and DNPP were 20 and 15 V, respectively (Fig. S3). The MRM quantifier qualifier and ESI source parameters for both impurities were tabulated in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#tbl0002">Table 2</a>. In the MS/MS method; the events program was configured to direct the flow from 0 to 2.8 min. for mass detection of DNPP. To prevent contamination from high concentrations of MOX from tablets and eye drops (5 mg/mL concentration), the flow was diverted to waste from 2.9 to 4.5 min. From 4.6 to 8 min, the flow was directed again to detect N-MOX.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Green_Analytical_Chemistry_Volume_12_2025_100188_Fig_3_MS_MS_fragmentation_pattern_for_A_DNPP_and_B_N_MOX_fce810c628.jpg" alt="Green Analytical Chemistry, Volume 12, 2025, 100188: Fig. 3. MS/MS fragmentation pattern for (A) DNPP, and (B) N-MOX." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Green_Analytical_Chemistry_Volume_12_2025_100188_Fig_3_MS_MS_fragmentation_pattern_for_A_DNPP_and_B_N_MOX_fce810c628.jpg 232w," sizes="100vw" width="1338" height="900"><h5>3.9. Method greenness assessment of the proposed method</h5>The environmental impact or “greenness” of the recently developed LC-QqQ-MS/MS method was evaluated using insights from published journals [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0024">[24]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0025">[25]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0026">[26]</a>] and contemporary tools, including the Analytical Eco-scale, GAPI, AGREE, AGREEprep, and the Blue Applicability Grade Index (BAGI).<h5>3.9.2. Green analytical procedure index (GAPI) assessment tool</h5>In 2018, Płotka-Wasylka introduced the GAPI [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0028">28</a>], which uses a pictogram with red, yellow, and green colors to represent high, medium, and low environmental impacts, respectively. For the developed LC-QqQ-MS/MS method, pictograms 2, 3, 6, 8, 9, and 12 are green, indicating low environmental impact. Pictograms 4, 5, 10, 11, and 14 are yellow, reflecting medium impact, while pictograms 1, 7, 13, and 15 are red, indicating high impact. This visual representation highlights both qualitative and quantitative environmental impacts [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#bib0029">29</a>]. An illustration of the GAPI pictogram is provided in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#fig0007">Fig. 7</a>A. <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2772577424000971#tbl0006">Table 6</a> details the principles of GAPI, the selected method condition values, and their corresponding scores.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Green_Analytical_Chemistry_Volume_12_2025_100188_Fig_7_Pictograms_of_A_GAPI_B_AGREE_C_AGRE_Eprep_and_D_BAGI_11db2203ed.jpg" alt="Green Analytical Chemistry, Volume 12, 2025, 100188: Fig. 7. Pictograms of (A) GAPI (B) AGREE, (C) AGREEprep, and (D) BAGI." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Green_Analytical_Chemistry_Volume_12_2025_100188_Fig_7_Pictograms_of_A_GAPI_B_AGREE_C_AGRE_Eprep_and_D_BAGI_11db2203ed.jpg 106w," sizes="100vw" width="1090" height="1600"><h4>4. Conclusion</h4>A novel, highly sensitive, and eco-friendly UPLC-QqQ-MS/MS method has been developed for the simultaneous trace-level quantification of MOX NDSRI and DNPP in MOX drug substances, tablets, and eye drops. The method was rigorously validated according to ICH Q2(R2) guidelines, including parameters such as linearity, LOD, LOQ, accuracy, precision, and robustness, showing excellent performance with high recovery rates and lower % RSD values and detecting N-MOX from 0.1 ng/mL to 37.5 ng/mL and DNPP from 0.5 ng/mL to 37.5 ng/mL. The method's versatility and sensitivity were demonstrated through a successful application to the analysis of commercial MOX tablets and eye drops, thereby ensuring the safety of MOX formulations. Furthermore, the proposed LC-QqQ-MS/MS method was evaluated using advanced green tools such as Analytical Eco-scale, GAPI, AGREE, AGREEprep, and BAGI, which confirmed its sustainability. This method can be employed for the accurate trace-level quantification of N-MOX and DNPP in any of the drug substances and formulations adopting suitable sample preparation. This methodology offers several advantages, such as reduced sample and organic solvent consumption, as well as a shorter run time, allowing for the processing of more batch samples. Overall, this validated UPLC-QqQ-MS/MS method presents a precise, reliable, and environmentally friendly approach for the simultaneous quantification of N-MOX and DNPP, offering a valuable contribution to pharmaceutical research and development.</div>

A facile and eco-friendly simultaneous quantification LC-TQ-MS/MS approach for N-Nitroso Moxifloxacin and di-nitroso pyrrolopiperidine in Moxifloxacin tablets and eye drops

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