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An RP-HPLC Method for the Simultaneous Analysis of Selected Antiviral Drugs

CHROMATOGRAPHIA 2025年第3期88卷 179-191页

作者： Ozdemirhan, Mehmet Emre Ozen, Guerkan nemutlu, emirhan Turkish Med & Med Devices Agcy Dept Anal & Control Labs Ankara Turkiye Baskent Univ Fac Pharm Dept Analyt Chem Etimesgut Ankara Turkiye Hacettepe Univ Fac Pharm Dept Analyt Chem Ankara Turkiye

A rapid and efficient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of six antiviral drugs: emtricitabine (EMT), entecavir (ENT), favipiravir (FAV), ganciclovir (GAN), valaciclovir (VAL), and valganciclovir (VLG). Chromatographic separation was achieved on an ExsilTM Mono 100 C18 column using a mobile phase of 50 mM phosphate buffer (pH 4): acetonitrile (90:10, v/v) at a flow rate of 0.5 mL/min. UV detection was performed at 215 nm. The method was validated according to ICH Q2(R2) guidelines, demonstrating excellent linearity (R-2 > 0.9938) across the concentration ranges of 1.0-150 mu g/mL for EMT, ENT and VLG, and 5.0-200 mu g/mL for FAV, GAN and VAL. The LOD and LOQ values ranged from 0.02 to 0.48 mu g/mL and 0.05 to 1.45 mu g/mL, respectively. Intra-day and inter-day precision, expressed as relative standard deviation (RSD), were less than 1.82%, while accuracy, expressed as relative error (RE), was within +/- 1.34%. Recovery studies yielded values ranging from 98.12% to 101.60%, further confirming method accuracy. The method proved to be selective, robust, and suitable for the quantitative analysis of these antiviral drugs in pharmaceutical formulations, with results obtained for commercially available tablets demonstrating excellent agreement with labeled contents. Notably, stability studies revealed the susceptibility of EMT, ENT, and FAV to degradation under refrigerated storage, highlighting the need for timely analysis of these compounds. In addition, the greenness, blueness, and whiteness of the method were evaluated, and the developed method performs strongly across environmental (AGREE score: 0.79), practical (BAGI index: 85), and sustainability (whiteness value: 74.7) criteria, reinforcing its viability as a sustainable approach for antiviral analysis. This validated RP-HPLC method provides a reliable and efficient tool for the quality control of these essential ant

关键词： Antivirals HPLC Validation Pharmaceuticals

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Tracing of Amino Acids Dynamics in Cell Lines Based on ¹⁸O Stable Isotope Labeling

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ANALYTICAL CHEMISTRY 2025年第4期97卷 2143-2152页

作者： Eylem, Cemil Can Baysal, Ipek Ciftci, Samiye Yabanoglu nemutlu, emirhan Hacettepe Univ Fac Pharm Dept Analyt Chem TR-06230 Ankara Turkiye Hacettepe Univ Vocat Sch Hlth Serv TR-06230 Ankara Turkiye Hacettepe Univ Fac Pharm Dept Biochem TR-06230 Ankara Turkiye

Metabolite levels and turnover rates are necessary to understand metabolomic dynamics in a living organism fully. Amino acids can play distinct roles in various cellular processes, and their abnormal levels are associated with pathological conditions, including cancer. Therefore, their levels, especially turnover rates, may provide enormous information about a phenotype. 13C- or 13C,15N-labeled amino acids have also been commonly used to trace amino acid metabolism. This study presented a new methodology based on 18O labeling for amino acids that relied on monitoring mass isotopologues to calculate the turnover rates of amino acids. The method optimization studies were carried over for selective amino acid monitoring. This methodology provides a rapid, robust, and simple GC-MS method for analyzing the fluxes of amino acid metabolism. The developed method was applied to fetal human colon (FHC) and human colon carcinoma (Caco-2) cell lines to determine cancer-induced shifts in the turnover rates of amino acids. These results defined metabolic reprogramming in Caco-2 cells through increased glutamate and serine turnovers and sharply decreased turnovers of aspartate, threonine, and methionine, therefore pointing to some metabolic vulnerabilities in the metabolism of cancerous cells. The simple mechanism of the developed methodology, the availability of affordable 18O-enriched water, and the ease of application can open a new arena in fluxomics analysis.

关键词： Metabolism

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Patient-derived cornea organoid model to study metabolomic characterization of rare disease: aniridia-associated keratopathy

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BMC OPHTHALMOLOGY 2025年第1期25卷 1-17页

作者： Koc, Ali Can Sari, Vedat Kocak, Gamze Recber, Tuba nemutlu, emirhan Aberdam, Daniel Guven, Sinan Izmir Biomed & Genome Ctr TR-35340 Izmir Turkiye Dokuz Eylul Univ Izmir Int Biomed & Genome Inst TR-35340 Izmir Turkiye Hacettepe Univ Fac Pharm Dept Analyt Chem TR-06100 Ankara Turkiye Sorbonne Paris Cite Univ Ctr Rech Cordeliers INSERM U1138 Paris France Dokuz Eylul Univ Fac Med Dept Med Biol & Genet TR-35340 Izmir Turkiye

BackgroundAniridia is a rare panocular disease caused by gene mutation in the PAX6, which is essential for eye development. Aniridia is inherited in an autosomal dominant manner, but its phenotype can vary significantly among individuals with the same mutation. Animal models, such as drosophila, zebrafish, and rodents, have been used to study aniridia through Pax6 deletions. Recently, patient-derived limbal epithelial stem cells (LESCs) and human-induced pluripotent stem cells (hiPSCs) have been used to model the disease in vitro, providing new insights into therapeutic *** this study, corneal organoids were generated from hiPSCs derived from aniridia patients with three different PAX6 nonsense mutations, allowing for a detailed comparison between diseased and healthy control models. These organoids structurally mimicked the human cornea and were used to investigate histologic and metabolomic differences between healthy and aniridia-derived *** metabolomic analysis revealed significant metabolic differences between wild-type (WT) and aniridia-associated keratopathy (AAK) hiPSCs. Further metabolomic profiling at different time points demonstrated distinct metabolic shifts, with amino acid metabolism pathways being consistently enriched in AAK *** study emphasizes the profound impact of AAK mutations on metabolism, particularly in amino acid biosynthesis and energy metabolism pathways.

关键词： Aniridia Pax6 HiPSCs (Human induced pluripotent stem cells) Cornea organoids Metabolomics Aniridia-associated keratopathy (AAK)

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Endocannabinoid system upregulates the enrichment and differentiation of human iPSC- derived spermatogonial stem cells via CB2R agonism

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BIOLOGICAL RESEARCH 2025年第1期58卷 1-22页

作者： Gizer, Merve Onen, Selin Erol, Ozgur Dogus Aerts-Kaya, Fatima Recber, Tuba nemutlu, emirhan Korkusuz, Petek Hacettepe Univ Grad Sch Hlth Sci Dept Stem Cell Sci TR-06100 Ankara Turkiye METU MEMS Ctr TR-06530 Ankara Turkiye Hacettepe Univ Ctr Stem Cell Res & Dev PEDI STEM TR-06100 Ankara Turkiye Hacettepe Univ Adv Technol Applicat & Res Ctr HUNITEK Ankara Turkiye Hacettepe Univ Lab Anim Res & Applicat Ctr HUDHAM Ankara Turkiye Hacettepe Univ Fac Pharm Dept Analyt Chem TR-06100 Ankara Turkiye Hacettepe Univ Fac Med Dept Histol & Embryol TR-06100 Ankara Turkiye

BackgroundMale factor infertility (MFI) is responsible for 50% of infertility cases and in 15% of the cases sperm is absent due to germ cell aplasia. Human induced pluripotent stem cell (hiPSC)-derived spermatogonial stem cells (hSSCs) could serve as an autologous germ cell source for MFI in patients with an insufficient sperm yield for assisted reproductive technology (ART). The endocannabinoid system (ECS) has been implicated to play a role in mouse embryonic stem cells (mESCs) and the human testicular environment. However, the contribution of the ECS in hiPSCs and hiPSC-derived hSSCs is currently unknown. Here, we aimed to assess whether hiPSCs and hiPSC-derived hSSCs are regulated by components of the ECS and whether manipulation of the ECS could increase the yield of hiPSC-derived SSCs and serve as an autologous cell-based source for treatment of *** reprogrammed human dermal fibroblasts (hDFs) to hiPSCs, induced differentiation of hSSC from hiPSCs and evaluated the presence of ECS ligands (AEA, 2-AG) by LC/MS, receptors (CB1R, CB2R, TRPV1, GPR55) by qPCR, flow cytometry and immunofluorescent labeling. We then examined the efficacy of endogenous and synthetic selective ligands (ACPA, CB65, CSP, ML184) on proliferation of hiPSCs using real-time cell analysis (RTCA) and assessed the effects of on CB2R agonism on hiPSC pluripotency and differentiation to *** from hDFs expressed the pluripotency markers OCT4, SOX2, NANOG, SSEA4 and TRA-1-60;and could be differentiated into ID4+, PLZF + hSSCs. hiPSCs and hiPSC-derived hSSCs secreted AEA and 2-AG at 10- 10 - 10- 9 M levels. Broad expression of all ECS receptors was observed in both hiPSCs and hiPSC-derived hSSCs, with a higher CB2R expression in hSSCs in comparison to hiPSCs. CB2R agonist CB65 promoted proliferation and differentiation of hiPSCs to hiPSC-hSSCs in comparison to AEA, 2-AG, ACPA, CSP and ML184. The EC50 of CB65 was determined to be 2.092 x 10- 8 M for support of pluripotency an

关键词： Human induced pluripotent stem cells Human spermatogonial stem cells Endocannabinoid systems CB2R CB65 Male infertility

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Integrative Multi-Omics Approach in Human Colon Adenocarcinoma Cell Line: Identification of Phenotypic Changes Observed in Cisplatin and 5-Fluorouracil Resistance

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MICROCHEMICAL JOURNAL 2025年 212卷

作者： Ozen, Gurkan Eylem, Cemil Can Baysal, Ipek Kocak, Engin Yabanoglu-Ciftci, Samiye Kir, Sedef nemutlu, emirhan Baskent Univ Fac Pharm Dept Analyt Chem Ankara Turkiye Hacettepe Univ Fac Pharm Dept Analyt Chem Ankara Turkiye Hacettepe Univ Vocat Sch Hlth Serv Ankara Turkiye Hlth Sci Univ Fac Gulhane Pharm Dept Analyt Chem Ankara Turkiye Hacettepe Univ Fac Pharm Dept Biochem Ankara Turkiye Hacettepe Univ Vaccine Inst Dept Vaccine Technol TR-06230 Ankara Turkiye Hacettepe Univ Turkish Doping Control Ctr TR-06230 Ankara Turkiye

Aims: Today's omics technologies enable complex analyses to better understand phenotypes. Characterization of metabolic phenotypes requires the identification of large numbers of proteins, metabolites and simultaneous as well as comprehensive analysis of their turnover rates. In this study, alterations in the phenotype of human colon adenocarcinoma cell lines (Caco-2) resulting from resistance to chemotherapeutics were investigated by multiomics analysis. Main methods: Drug resistance to cisplatin (CIS) and 5-fluorouracil (5-FU) was induced by long-term exposure of Caco-2 cells to minimum lethal dose. Integrated omics analysis was performed using proteomics, metabolomics, lipidomics and O-18-based fluxomics analyses. Key findings: As a result of aforementioned analyses, 650 proteins, more than 300 metabolites/lipids were determined and the turnover rates of a total of 38 metabolites, including intermediates in the Krebs Cycle, amino acids, fatty acids and phosphonucleotides, were calculated. Multivariate analyses revealed significant differences in phenotype between the cell group induced with either chemotherapeutic agent and the control cells. The joint pathway analysis performed by profiling metabolites, lipids and proteins with significant (p < 0.05) differences between groups showed that malate-aspartate shuttle, amino acid, ammonia metabolism, glucose-alanine and urea cycle pathways were altered due to resistance to the chemotherapeutic agent. Significance: Data from comprehensive omics (proteomics, metabolomics, lipidomics and fluxomics) studies of Caco-2 cells induced either with CIS or 5-FU, showed that resistance to chemotherapeutics is due to differences in cell dynamics. Furthermore, the phenotype behind drug resistance depends on the chemotherapeutic agent.

关键词： Colon adenocarcinoma 5-fluorouracil Cisplatin Proteomics Metabolomics Lipidomics and Fluxomics

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A novel injectable nanotherapeutic platform increasing the bioavailability and anti-tumor efficacy of Arachidonylcyclopropylamide on an ectopic non-small cell lung cancer xenograft model: A randomized controlled trial

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INTERNATIONAL JOURNAL OF PHARMACEUTICS 2025年 670卷 125153页

作者： Boyacioglu, Ozge Varan, Cem Bilensoy, Erem Aykut, Zaliha Gamze Recber, Tuba nemutlu, emirhan Kilic, Nedret Korkusuz, Petek Hacettepe Univ Grad Sch Sci & Engn Dept Bioengn TR-06800 Ankara Turkiye Atilim Univ Fac Med Dept Med Biochem TR-06830 Ankara Turkiye Hacettepe Univ Grad Sch Sci & Engn Dept Nanotechnol & Nanomed TR-06800 Ankara Turkiye Hacettepe Univ Fac Pharm Dept Pharmaceut Technol TR-06100 Ankara Turkiye Bilkent Univ Fac Sci Dept Mol Biol & Genet TR-06800 Ankara Turkiye Hacettepe Univ Fac Pharm Dept Analyt Chem TR-06100 Ankara Turkiye Hacettepe Univ Fac Med Dept Histol & Embryol TR-06100 Ankara Turkiye METU MEMS Ctr TR-06530 Ankara Turkiye

Rapid progressing non-small cell lung adenocarcinoma (NSCLC) decreases treatment success. Cannabinoids emerge as drug candidates for NSCLC due to their anti-tumoral capabilities. We previously reported the controlled release of Arachidonylcyclopropylamide (ACPA) selectively targeting cannabinoid 1 (CB1) receptor in NSCLC cells in vitro. Hydrophobic polymers like polycaprolactone (PCL) offer prolonged circulation time and slower drug clearance which is suitable for hydrophobic molecules like ACPA. Thus, the extended circulation time with enhanced bioavailability and half-life of nanoparticular ACPA is crucial for its therapeutic performance in the tumor area. We assumed that a novel high technology-controlled release system increasing the bioavailability of ACPA compared to free ACPA could be transferred to the clinic when validated in vivo. Plasma profile of ACPA and ACPA-loaded PCL-based nanomedicine by LC-MS/MS and complete blood count (CBC) was assessed in wild-type Balb/c mice. Tumor growth in nanomedicine-applied NSCLC-induced athymic nude mice was assessed using bioluminescence imaging (BLI) and caliper measurements, histomorphometry,immunohistochemistry, TUNEL assay, and Western blot on days 7-21. Injectable NanoACPA increased its systemic exposure to tissues 5.5 times and maximum plasma concentration 6 times higher than free ACPA by substantially improving bioavailability. The potent effect of NanoACPA lasted for at least two days on ectopic NSCLC model through Akt/PI3K, Ras/MEK/Erk, and JNK pathways that diminished Ki-67 proliferative and promoted TUNEL apoptotic cell scores on days 7-21. The output reveals that NanoACPA platform could be a chemotherapeutic for NSCLC in the clinic following scale-up GLP/GMP-based phase trials, owing to therapeutic efficacy at a safe low dose window.

关键词： Non-small cell lung cancer Arachidonylcyclopropylamide (ACPA) Polycaprolactone (PCL) Cannabinoids Nanomedicine Nanoparticle-based drug delivery system

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Detailed characterization of bone marrow adipose tissue mesenchymal stem cells in healthy donor, Fanconi anemia, and acute myeloid leukemia

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BONE 2025年 193卷 117413-117413页

作者： Muratoglu, Bihter Ozdemir, Cansu Eylem, Cemil Can Recber, Tuba nemutlu, emirhan Alpdundar-Bulut, Esin Vargel, Ibrahim Uckan-cetinkaya, Duygu Hacettepe Univ Inst Hlth Sci Dept Stem Cell Sci Ankara Turkiye Hacettepe Univ Ctr Stem Cell Res & Dev PEDI STEM Ankara Turkiye Hacettepe Univ Dept Analyt Chem Ankara Turkiye Hacettepe Univ Dept Pediat Div Hematol Ankara Turkiye Hacettepe Univ Fac Med Dept Plast Reconstruct & Aesthet Surg Ankara Turkiye

Bone marrow is a complex tissue featuring distinct cellular organization and diverse cell types. Bone marrow adipose tissue (BMAT) is a dynamic component crucial for tissue function and disease processes. This study explores differences between bone marrow-derived mesenchymal stem cells (BM-MSCs) and BMAT-derived mesenchymal stem cells (BMAT-MSCs), isolated from the same cavity, examining their differentiation potential and secretory profiles. BM-MSCs and BMAT-MSCs both exhibit classical mesenchymal characteristics, with over 90 % positivity for markers such as CD105 and CD29. Notably, BMAT-MSCs display significantly higher differentiation potential than BM-MSCs, with enhanced osteogenic and adipogenic capabilities, as indicated by increased calcium accumulation and lipid storage. In Fanconi anemia (FA) and acute myeloid leukemia (AML), osteogenic potential is limited, indicating impaired differentiation under these pathological conditions. Gene expression analysis of adipogenic molecules and metabolic regulators revealed significant differences in expression profile between BM- and BMAT-MSCs, particularly during adipogenic differentiation, indicating distinct characteristics that were more notable in FAs and AMLs. Furthermore, metabolomic profiling of BM plasma, using GC-MS for in-vivo niche reflection, and lipid analysis via LC-qTOF-MS show significant lipidomic alterations in patient samples, highlighting metabolic dysregulation and lipid remodeling. Lipid-mediated signaling and membrane composition changes appear integral to disease mechanisms. In conclusion, this study highlights the distinctive molecular and metabolomic profiles and adaptive mechanisms of BM- and BMATMSCs in bone marrow pathologies.

关键词： Bone marrow adipose tissue Acute myeloid leukemia Fanconi anemia Mesenchymal stem cells Metabolomics Lipidomics

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Optimized high-throughput protocols for comprehensive metabolomic and lipidomic profiling of brain sample

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TALANTA 2025年 282卷 126953页

作者： Eylem, Cemil Can nemutlu, emirhan Dogan, Aysegul Acik, Vedat Matyar, Selcuk Gezercan, Yurdal Altintas, Suleyman Okten, Ali Ihsan Akduman, Nursabah Elif Basci Hacettepe Univ Fac Pharm Dept Analyt Chem TR-06100 Ankara Turkiye Adana City Training & Res Hosp Dept Neurosurg Adana Turkiye Adana City Training & Res Hosp Dept Pathol Adana Turkiye Univ Med Sci Adana City Training & Res Hosp Dept Biochem Adana Turkiye

Establishing direct causal and functional links between genotype and phenotype requires thoroughly analyzing metabolites and lipids in systems biology. Tissue samples, which provide localized and direct information and contain unique compounds, play a significant role in objectively classifying diseases, predicting prognosis, and deciding personalized therapeutic strategies. Comprehensive metabolomic and lipidomic analyses in tissue samples need efficient sample preparation steps, optimized analysis conditions, and the integration of orthogonal analytical platforms because of the physicochemical diversities of biomolecules. Here, we propose simple, rapid, and robust high-throughput analytical protocols based on the design of experiment (DoE) strategies, with the various parameters systematically tested for comprehensively analyzing the heterogeneous brain samples. The suggested protocols present a systematically DoE-based strategy for performing the most comprehensive analysis for integrated GC-MS and LC-qTOF-MS from brain samples. The five different DoE models, including D-optimal, full factorial, fractional, and Box-Behnken, were applied to increase extraction efficiency for metabolites and lipids and optimize instrumental parameters, including sample preparation and chromatographic parameters. The superior simultaneous extraction of metabolites and lipids from brain samples was achieved by the methanol-water-dichloromethane (2:1:3, v/v/v) mixture. For GC-MS based metabolomics analysis, incubation time, temperature, and methoxyamine concentration (10 mg/mL) affected metabolite coverage significantly. For LC-qTOF-MS based metabolomics analysis, the extraction solvent (methanol-water;2:1, v/v) and the reconstitution solvent (%0.1 FA in acetonitrile) were superior on the metabolite coverage. On the other hand, the ionic strength and column temperature were critical and significant parameters for high throughput metabolomics and lipidomics studies using LC-qTOF-MS. In

关键词： Brain Experimental design Metabolomics Lipidomics Multi-omics Sample preparation

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Recent advancements in the bioactive alkaloids analysis in plant and biological specimen: From the perspective of activity, sample preparation, and analytical method selection☆

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JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 2025年 1258卷 124592页

作者： Erdogan-Kablan, Sevilay Yayla, Seyda Hurkul, M. Mesud Cetinkaya, Ahmet nemutlu, emirhan Ozkan, Sibel A. Hacettepe Univ Fac Pharm Dept Analyt Chem Ankara Turkiye Ankara Univ Fac Pharm Dept Pharmaceut Bot Ankara Turkiye Ankara Univ Fac Pharm Dept Analyt Chem Ankara Turkiye

Alkaloids are a diverse group of naturally occurring organic compounds. They are known for their significant pharmacological properties. This review provides an up-to-date analysis of bioactive alkaloids in plant and biological samples, emphasizing their biological activities, extraction techniques, and analytical methods. The study focuses on significant alkaloids such as morphine, codeine, vinblastine, vincristine, berberine, quinine, quinidine, caffeine, nicotine, ephedrine, and atropine, highlighting their chemical structures, therapeutic applications, and mechanisms of action. Recent advances in extraction methods, including conventional and modern green techniques such as supercritical fluid extraction, microwave-assisted extraction, and solid-phase microextraction, are discussed in detail. In addition, the review provides an overview of state-of-the-art analytical techniques used for alkaloid quantification, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, and novel spectroscopic methods. Emphasis is placed on the challenges associated with alkaloid analysis, including matrix effects, stability, and structural diversity. The results contribute to the growing body of knowledge in alkaloid research, providing insights into their potential therapeutic applications and analytical improvements for more accurate and efficient detection in various biological and plant matrices.

关键词： Plant alkaloids Biological activities Sample preparation Analytical methods

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Greenness assessment of 174 CEN, ISO, and pharmacopoeia standard methods and their sub-methods used for environmental, food, trace element and pharmaceutical analyses

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ADVANCES IN SAMPLE PREPARATION 2025年 14卷

作者： Benede, Juan L. Cagliero, Cecilia nemutlu, emirhan Pena-Pereira, Francisco Bicchi, Carlo Carrasco-Correa, Enrique Javier Celeiro, Maria Chisvert, Alberto Gentili, Alessandra Godfrey, A. Ruth Gumustas, Mehmet Krokos, Fragoulis Kumkrong, Paramee Llompart, Maria Locatelli, Marcello Mester, Zoltan Ozkan, Sibel A. Pedersen-Bjergaard, Stig Segundo, Marcela A. Tobiszewski, Marek Psillakis, Elefteria Univ Valencia Dept Analyt Chem GICAPC Res Grp Burjassot 46100 Valencia Spain Univ Turin Dept Drug Sci & Technol I-10125 Turin Italy Hacettepe Univ Fac Pharm Dept Analyt Chem TR-06320 Ankara Turkiye Univ Vigo Ctr Invest Marina Dept Quim Analit & Alimentaria Grp QA2 Edificio CC Expt Vigo 36310 Spain Univ Valencia Dept Analyt Chem CLECEM Grp Burjassot 46100 Valencia Spain Univ Santiago de Compostela Dept Analyt Chem Nutr & Food Sci CRETUS Santiago De Compostela 15872 Spain Sapienza Univ Dept Chem Ple Aldo Moro 5 I-00185 Rome Italy Swansea Univ Med Sch Swansea SA2 8PP Wales Ankara Univ Inst Forens Sci Dept Forens Toxicol TR-06620 Ankara Turkiye Hellen Org Standardizat Natl Qual Infrastruct Syst GR-12133 Peristeri Greece Natl Res Council Canada Ottawa ON Canada Univ Chieti Pescara G dAnnunzio Dept Sci I-66100 Chieti Italy Ankara Univ Fac Pharm Dept Analyt Chem TR-06560 Ankara Turkiye Univ Oslo Dept Pharm Box 1068 Blindern N-0316 Oslo Norway Fac Hlth & Med Sci Dept Pharm Univ Pk 2 DK-2100 Copenhagen O Denmark Univ Porto Fac Pharm Dept Chem Sci LAQVREQUIMTE Rua Jorge Viterbo Ferreira 228 P-4050313 Porto Portugal Gdansk Univ Technol GUT Fac Chem Dept Analyt Chem Ul G Narutowicza 11-12 PL-80233 Gdansk Poland Gdansk Univ Technol GUT EcoTech Ctr Ul G Narutowicza 11-12 PL-80233 Gdansk Poland Tech Univ Crete Sch Chem & Environm Engn Lab Aquat Chem GR-73100 Khania Crete Greece

This work evaluates the greenness of 174 standard methods with a sample preparation step and their 332 sub-method variations from CEN, ISO, and Pharmacopoeias, used in laboratories for environmental/organic, food, trace element, or pharmaceutical analyses. The widely adopted AGREEprep metric was applied to assess the greenness of the standard methods. The discussion begins with the overall scores of each method, followed by a detailed analysis of individual criteria, that highlights the strengths and weaknesses of the evaluated sample preparation methods in terms of greenness. The results revealed a generally poor greenness performance, as 67 % of the methods scored below 0.2 on the AGREEprep scale, where 1 represents the highest possible score. Specifically, the percentage of methods scoring below 0.2 was 86 % for methods related to the environmental analysis of organic compounds, 62 % for methods used in food analysis, 62 % for those applied to inorganic and trace metals analysis, and 45 % for methods used in pharmaceutical analysis. The findings obtained in this work reveal that many official methods still rely on resource-intensive, outdated techniques, scoring poorly on key greenness criteria. This discrepancy highlights the urgent need to update standard methods by including contemporary and mature sample preparation methods, as the traditional methodologies currently used often conflict with global sustainability efforts and increase regulatory and societal pressures. As such, this contribution serves not only as a critique of the current state of official standard methods but also as a call to action for their reform. This work was conducted within the framework of the IUPAC project "Greenness of official standard sample preparation methods" (2021-015-2-500).

关键词： AGREEprep Official standard methods Sample preparation

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