Sample Preparation Approach Research Articles

On-site extraction of phenoxycarboxylic acid herbicides in environmental waters utilizing monolith-based in-tip microextraction technique

On-site extraction plays a significant role in the reliable quantification of strong polar phenoxycarboxylic acid herbicides (PCAs) in aqueous samples. In current study, a new technique for the field sample preparation of PCAs was developed by means of three channels in-tip microextraction device (TCIM). To capture PCAs effectively, an extraction phase based on monolith (EPM) using vinylimidazole and divinylbenzene/ethylene dimethacrylate as monomer and cross-linkers, respectively, was in-situ synthesized in pipette tips. The EPM fabricated at optimal conditions were characterized by a series of techniques and employed as the adsorbent of TCIM for the on-site extraction of PCAs. The adsorption isotherm was studied so as to inspect the extraction behaviors of EPM towards PCAs. Results revealed that the proposed EPM/TCIM presented satisfactory extraction performance towards PCAs through multiple interactions. The enrichment factors and adsorption capacity were 74-277 and 20 mg/g, respectively. Under the most beneficial extraction parameters, the developed EPM/TCIM was successfully employed to on-site extract PCAs, and then combining with HPLC equipped with diode array detector to monitor trace PCAs in actual waters. The limits of detection (LODs) towards investigated PCAs varied from 0.071 μg/L to 0.30 μg/L. In addition, the accuracy of established approach was inspected with documented method. Compared with existing lab-based sample preparation approaches, the introduced field sample preparation technique exhibits some merits such as avoidance of transporting large volume of water, prevention of analytes loss during sampling procedure, less usage of organic solvent and achievement of satisfactory efficient in sample preparation.

Oligonucleotide therapeutics in sports? An antidoping perspective.

Within the last two decades, the European Medicines Agency and the US Food and Drug Administration have approved several gene therapies. One category is oligonucleotide therapeutics, which allow for the regulation of the expression of target genes. Besides already approved therapeutics, there are several preclinical and clinical trials ongoing. The World Anti-Doping Agency prohibits the use of "nucleic acids or nucleic acid analogs that may alter genome sequences and/or alter gene expression by any mechanism" as a nonspecified method at all times. Hence, the administration of nucleic acids or analogs by athletes would cause an Anti-Doping Rule Violation. Herein, we discuss types of oligonucleotide therapeutics, their potential to be misused in sports, and considerations to sample preparation and mass spectrometric approaches with regard to antidoping analysis.

Influence of different sample preparation approaches on proteoform identification by top-down proteomics.

Top-down proteomics using mass spectrometry facilitates the identification of intact proteoforms, that is, all molecular forms of proteins. Multiple past advances have lead to the development of numerous sample preparation workflows. Here we systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification and fractionation. We found that all steps in sample preparation influence the subset of proteoforms identified (for example, their number, confidence, physicochemical properties and artificially generated modifications). The various sample preparation strategies resulted in complementary identifications, substantially increasing the proteome coverage. Overall, we identified 13,975 proteoforms from 2,720 proteins of human Caco-2 cells. The results presented can serve as suggestions for designing and adapting top-down proteomics sample preparation strategies to particular research questions. Moreover, we expect that the sampling bias and modifications identified at the intact protein level will also be useful in improving bottom-up proteomics approaches.

Development of an Untargeted Method for the Analysis of the Volatile Profile of Onions via Solid-Phase Microextraction Arrow-Headspace-Gas Chromatography-Mass Spectrometry Using Design of Experiments.

The distinctive aroma of onions, consisting primarily of sulfur-containing compounds, is one of the reasons for the popularity of the vegetable. The rapid enzymatic and chemical reactions that occur after the destruction of onion bulb tissue render the analysis of the volatile profile challenging. Therefore, sample preparation is a crucial step in the analysis of the onion volatilome, but it often does not receive the necessary attention in the literature. In this work, we focused on two aspects: Firstly, we compared different sample preparation approaches to maximize the volatile yield and to enable the description of the onion volatile profile as it would emerge after a solid-phase microextraction (SPME) Arrow sampling procedure. For headspace (HS)-gas chromatography-mass spectrometry analysis, onion juice with the addition of an ammonium sulfate solution proved to be the best option. Secondly, we optimized the HS sampling and desorption parameters (enrichment time, enrichment temperature, agitator speed, desorption time) for onion volatiles using the efficient design of experiments (DoE) approach. The optimal conditions for the analysis with HS-SPME Arrow were an enrichment time of 75min at 60°C with an agitator speed of 713rpm and a desorption time of 120s.

Determination of drugs of abuse and metabolites in plasma microsamples by LC-MS/MS after microQuEChERS extraction.

Aim: Identifying drugs of abuse and their metabolites in plasma is vital in both forensic and clinical toxicology. While the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method offers an efficient approach to sample preparation, its application is complex due to the wide-ranging properties of target analytes and the challenges posed by biological matrix interferences. This study aims to develop a microQuEChERS approach for the quantification of 14 drugs of abuse and metabolites utilizing minimal sample and solvent volumes.Methods: The microQuEChERS method involved using 10μl plasma samples, 25mg of a salt mixture and 150μl of acetonitrile. Extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with a 7.5min run. The assay was validated according to bioanalytical guidelines.Results: The accuracy was 96.8-112.4%. The within-assay precision was within 2.0-8.9% and the between-assay precision was within 3.2-8.2%. Matrix effects were found to range from -5.7 to 13.5%. The extraction yield was higher than 74.7%.Conclusion: This study described a microQuEChERS sample preparation approach for determining drugs of abuse and metabolites using plasma microsamples and LC-MS/MS. The approach is efficient, environmentally friendly and suitable for scenarios with limited amounts of biological samples.

Development and validation of a novel HPLC-MS/MS method for the quantitative determination of PFASs in non-hazardous solid waste samples

Perfluoroalkyl substances (PFASs) are a heterogeneous group of fluorinated synthetic compounds characterised by the presence of a hydrophobic carbonyl chain at different fluorination rates and by a terminal hydrophilic group. Because of their persistence and mobility, PFASs have been widely detected in ecosystems and living organisms, leading to their classification as persistent organic pollutants (POPs). Hence, monitoring the PFASs content in solid waste meant for disposal in non-hazardous landfills can be crucial, since leachate can pose a significant pollution threat to farmlands and aquifers.Therefore, a QuEChERS and SPE pretreatment with HPLC-MS/MS detection method for 10 different PFASs in solid waste samples was developed in this study. Different sample preparation approaches have been evaluated, taking into account the lack in the literature of this complex matrix.An innovative application using the Captiva EMR cartridge for the clean up (designed to be applied to lipidic matrix) was proposed and a multiresidual HPLC-MS/MS method for the analysis of PFASs in internal waters was extended to the investigation of non-lipidic samples as solid wastes. The method was then validated through 6 blanks and 6 spiked samples, according to the UNI EN ISO/IEC 17025. The optimized method showed satisfactory performance with recoveries ranging from 89.8 % to 106.5 % and with LOD and LOQ ranging between 0.0023 – 0.09 µg/L and 0.006 – 0.13 µg/L, respectively, for each of the PFASs under consideration. In addition, this approach demonstrated to be reliable and time consuming due to its capability for determining PFASs in waters and in solid wastes simultaneously in a single chromatographic run.

A high-throughput sample preparation approach for the determination of nonsteroidal anti-inflammatory drugs in urine samples through membrane-based microextraction employing a green supramolecular solvent

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs with antipyretic, analgesic, and anti-inflammatory effects widely used worldwide. This study aimed at developing an environmentally friendly and high-throughput sample preparation method for the determination of six NSAIDs in urine samples followed by high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD). The experimental workflow consisted of hollow-fiber microporous membrane liquid-liquid extraction (HF-MMLLE) using a supramolecular solvent immobilized into the porous of polypropylene hollow membranes (1 cm length). These membranes were attached to a 96-well plate system to permit multiple extractions simultaneously. Under the optimized conditions, limits of detection (LODs) and limits of quantification (LOQs) ranged from 6.06 to 30.3 µg/L and from 20 to 100 µg/L, respectively. The linear ranges varied from 20 to 1000 µg/L for indomethacin and diclofenac, from 50 to 1000 µg/L for ketoprofen and ibuprofen, and from 100 to 1000 µg/L for naproxen and fenoprofen. The coefficients of determination (R²) ranged from 0.9735 to 0.9997. Intraday precision ranged from 6.13 to 20.11 %, interday precision varied from 2.87 to 20.72 %, and accuracy ranged from 92.49 to 126.89 %. Moreover, the recently proposed Sample Preparation Metric of Sustainability (SPMS) was employed to evaluate the greenness of the sample preparation technique resulting in a score of 8.0, which is highly satisfactory according to the concepts of green analytical chemistry.

Artifact-free sample preparation of metal thin films using Xe plasma-focused ion beam milling for atomic resolution and in situ biasing analyses

The focused ion beam (FIB) technique using Ga ion beams is generally employed for transmission electron microscopy (TEM) sampling owing to its location-specific beam controllability on materials. However, Ga ion beam bombardment-induced damage hinders reliable structural analysis. Furthermore, Ga ions implanted in the damaged surface layer substantially lower the chemical stability of the samples, such as metal thin films. Here, aiming for atomic-level interface structural analysis, we propose a useful approach for the TEM sample preparation of heteroepitaxial Ag/Cu metal films without physical damage or chemical instabilities using the Xe plasma FIB (PFIB) system. Xe plasma-assisted sampling ensured that the interface structure of the Ag/Cu metal film remained intact; in contrast, the sample prepared using the focused Ga ion beam was damaged by elemental mixing. Furthermore, the sample prepared by Xe plasma milling exhibited robust structural stability over time after exposure to air in contrast to the sample prepared by Ga-ion milling, which induced structural decomposition owing to oxidation. In addition to the static structural analysis, the Cu thin film sample prepared by Xe PFIB exclusively exhibited pristine structural properties under in situ electrical biasing experiments, thus confirming the feasibility of conducting a fundamental study of Cu electromigration without artifacts.

Nanoparticle Assisted Fabric Phase Sorptive Extraction for Azo Dye Determination in the Industrial Sewage.

Currently, one of the significant environmental problems is the presence of azo dye materials in water sources. In this study, for the first time, a fast and sensitive sample preparation approach using nanoparticle-assisted fabric phase sorptive extraction (NFPSE) followed by high-performance liquid chromatography was examined to remove some azo dyes such as methyl red and sunset yellow from aqueous solutions. Primarily, the significance of several parameters affecting NFPSE, such as fabric type, the kind of sorbent, the number of contacts with sol-gel and the time of contact, was investigated. In addition, experiments were performed to determine the effect of different adsorption parameters, such as sample volume, adsorption time, adsorbent value, desorption time, ionic strength and pH. It was found that the calibration curve was linear within two ranges of concentrations (0.05-0.1 and 0.5-15ng/L for methyl red; 0.05-0.5 and 0.5-15ng/L for sunset yellow) with correlation coefficients better than 0.9683. The limit of detection was 0.014ng/L for methyl red and 0.015ng/L for sunset yellow. Repeatability Relative Standard Deviation (RSD) with three replicated experiments was 1.5-10% for methyl red and 2.5-5.8% for sunset yellow. Relative recovery percentages of 88-96% for methyl red and 62-92% for sunset yellow were obtained in the samples. Moreover, the results have shown that acceptable accuracy, precision and linearity make the "fabric phase sorptive extraction" a proper method for the determination of dyes from industrial sewage samples.

A facile lyophilisation-based sample preparation approach for the determination of selected wastewater-borne antiretroviral drugs and metabolites by SFC-MS/MS

The presence of pharmaceuticals in aquatic ecosystems is at the centre-stage of research after the realization that wastewater treatment processes are generally ineffective in the removal of these entities in wastewater given the high likelihood of effluent reuse after disposal. In addition, the continued efforts to scout for new and emerging aquatic contaminants has until recently elicited proliferation of numerous analytical methods for the determination of various (un)known contaminants, since emphasis is now placed in the development of environmentally benign approaches. Herein, we propose and discuss a novel and relatively eco-friendly analytical method based on lyophilization for sample preparation and SFC-MS/MS for the determination of eight (8) antiretroviral drugs (ARVDs) and three (3) metabolites in wastewater samples. This method proved useful in the improvement for the recoveries of lamivudine (3TC) and emtricitabine (FTC) by up to 99% compared to as low as 23% with solid phase extraction (SPE) method. Indeed, previous literature reports has reported poor recoveries for the polar ARVDs, especially on reversed phase (RP) SPE. In contrast, lyophilization promoted matrix effects as evidenced by ion suppression of up to 50% experienced on late eluting compounds. Despite this, lyophilization-SFC-MS/MS method was successfully validated for the quantification of all target analytes, partial exceptions were for ritonavir metabolite (RTVM) which could not be quantified using lyophilization possibly due to lyophilization-induced losses. Generally, the obtained data has proved that lyophilization is an alternative to SPE and SFC is a suitable alternative to LC.

Headspace gas chromatography with various sample preparation and chemometric approaches to improve discrimination of wild and feeding civet coffee

Abstract Civet coffee, or kopi luwak, has attracted significant attention within the coffee industry in certain regions due to its distinct flavor characteristics that arise from the digestive processes of the civet. The ability to discriminate between wild and feeding civet coffee is of major importance in upholding the industry’s established standards of quality and transparency. This study introduces an innovative method to differentiate between these two coffee types using Headspace Gas Chromatography-Mass Spectrometry (HS-GCMS) with advanced data analysis using machine-learning techniques. This study encompasses seven samples collected from various regions, all of which were subjected to analysis in both roasted and unroasted forms. The data analysis consisted of Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), which revealed clear trends that were mostly influenced by processing, indicating how roasting affects the chemical profiles of various coffee types. Further classification was conducted using Support Vector Machine (SVM) and Random Forest (RF) machine learning algorithms. SVM exhibited notable accuracy at 90%, effectively discriminating between wild and feeding civet coffee, whereas RF outperformed it with a remarkable 100% accuracy. This study contributes to the field of coffee characterization by presenting a robust approach to discriminate between roasted and unroasted wild and feeding civet coffee. This tool serves as a starting step for a valuable resource for both farmers and customers, as it promotes sustainable and ethical practices while retaining the distinct flavor characteristics of this exceptional specialty coffee.

Direct Observation of Trace Elements in Barium Titanate of Multilayer Ceramic Capacitors Using Atom Probe Tomography.

Accurately controlling trace additives in dielectric barium titanate (BaTiO3) layers is important for optimizing the performance of multilayer ceramic capacitors (MLCCs). However, characterizing the spatial distribution and local concentration of the additives, which strongly influence the MLCC performance, poses a significant challenge. Atom probe tomography (APT) is an ideal technique for obtaining this information, but the extremely low electrical conductivity and piezoelectricity of BaTiO3 render its analysis with existing sample preparation approaches difficult. In this study, we developed a new APT sample preparation method involving W coating and heat treatment to investigate the trace additives in the BaTiO3 layer of MLCCs. This method enables determination of the local concentration and distribution of all trace elements in the BaTiO3 layer, including additives and undesired impurities. The developed method is expected to pave the way for the further optimization and advancement of MLCC technology.

Impact of Sample Preparation Approach on Transmission Electron Microscopy Investigation of Sputtered AlNi Multilayers Used for Reactive Soldering

This work presents studies of sputtered Al/Ni reactive multilayers by transmission electron microscopy. They are prepared for these analyses by three methods (both Ga‐ and Xe‐based focused ion beam, FIB, and tripod polishing plus Ar+ ion milling in precision ion polishing system, PIPS) to check their impact on these materials. Every sample shows polycrystalline and mostly chemically pure Al/Ni layers. They also hint existence of intermetallic compounds, especially the tripod‐prepared sample. These intermetallics first originate from the sputtering process. The layers are increasingly rough along the growth direction. Other remarkable findings can be highlighted. First, the heating operations applied during the tripod polishing preparation lead to recrystallization and blurred Al/Ni interfaces due to increased metals reactions, although additional contributions by roughness and preparation thickness to the current compositional uncertainties must be distinguished by optimizing future sample fabrications and preparations. Second, Xe‐based FIB leads to lamellae with seemingly low contamination, although Ga‐FIB is a potentially good alternative. Finally, FIB is better to study cross‐section preparations of pristine Al/Ni multilayers, whereas the present tripod polishing procedures are unsuitable for this purpose but allow to observe the beginning of Al/Ni transformations upon heating, which is interesting for the technological optimization of these materials.

Approaches for Sampling and Sample Preparation for Microplastic Analysis in Laundry Effluents

The continuous growth in the production, unsustainable use, and disposal of plastics in recent decades has led to the emergence of a new type of pollutant, microplastics (MPs). In this article, the focus is on the form of MPs, which are produced by the fragmentation of textile fibres during washing processes. The problems associated with the characterisation and quantification of MPs in this type of sample are related to the wide range of concentrations, forms, and degree of degradation as well as physico-chemical and biological properties. Although the basic principles for the analysis of microplastics present in the environmental samples are known, there is the lack of standardised methods for the analysis of MPs in laundry effluents and domestic washing. Therefore, the continuous development of sophisticated analytical techniques and methodologies is required for the reliable collection and isolation, quantification, and characterisation of one of the most challenging analytes. The aim of this review is to outline the key steps of MPs analysis in laundry effluents and domestic washing, focusing on those steps that are underestimated in the current literature: sampling and sample preparation for analysis. Precisely these analytical steps, which can become the main source of analytical measurement system errors, ensure the quality of the analysis. This paper emphasises the importance of monitoring background contamination and presents guidelines to ensure quality control specifically for this type of analyte.

Diflubenzuron Mass Concentration Determination Method in Pesticides Using High-Performance Liquid Chromatography.

Pesticides are actively used in agriculture to protect crops from pests, and accurate determination of the concentration of their active ingredient is critical to ensuring their efficacy and safety. The research aims to develop a methodology that will enable the accurate determination of the mass concentration of diflubenzuron, a widely used active ingredient in pesticide formulations. This is achieved through the use of high-performance liquid chromatography (HPLC). The proposed method for determining the mass concentration of diflubenzuron using HPLC was developed through a series of experiments and optimisation steps. The sample preparation procedure was optimised to extract the maximum amount of diflubenzuron from the pesticide formulation. The chromatography conditions were optimised to separate diflubenzuron from other components in the sample matrix. A calibration approach was established for accurate quantification of diflubenzuron in the sample based on peak area measurements. The developed method can be widely used in agriculture, where accurate determination of diflubenzuron concentrations in pesticide formulations is critical for proper application and regulatory compliance. Both regulatory organizations and pesticide manufacturers can use it for batch-release testing and quality control to evaluate the efficacy and safety of pesticide products. In addition, the method can serve as a valuable tool for researchers and analysts working with pesticide residues in crops and environmental monitoring studies. The optimised sample preparation, chromatography conditions, and calibration approach provide reliable and reproducible results.

Critical evaluation of energy dispersive X-ray fluorescence spectrometry for multielemental analysis of coffee samples: Sample preparation, quantification and chemometric approaches

In the present contribution, a critical evaluation of the use of EDXRF (Energy dispersive X-ray fluorescence spectrometry) for elemental composition determination (K, Ca, Mn, Fe, Cu, Zn, Sr and Rb) of coffee samples is presented. Firstly, several sample treatment procedures (pressed pellets and loose powder) and quantification strategies (fundamental parameters and empirical calibration) were evaluated.In addition to quality and nutritional purposes, information of elemental content in coffee samples can be also useful for coffee traceability and provenance studies. In view of that, a comparison of different tools such as explorative principal component analysis (PCA) and classification by partial least square regression (PLS-DA) were also tested as discriminating tools of different types of coffee samples (i.e., pure, instant and coffee with additives). Chemometric analysis was performed using coffee elemental concentrations determined by the developed EDXRF method and, for the first time, using raw EDXRF spectra obtained, without the need of a fitting and quantification procedure.

A new approach for sample preparation to determine the multi-element composition of cobalt-rich crust samples by wavelength dispersive X-ray fluorescence spectrometry

A new approach for sample preparation to determine the multi-element composition of cobalt-rich crust samples by wavelength dispersive X-ray fluorescence spectrometry

Mass Spectrometry Imaging and Histology for the Analysis of Budding Intestinal Organoids.

Three-dimensional (3D) organoids have been at the forefront of regenerative medicine and cancer biology fields for the past decade. However, the fragile nature of organoids makes their spatial analysis challenging due to their budding structures and composition of single layer of cells. The standard sample preparation approaches can collapse the organoid morphology. Therefore, in this study, we evaluated several approaches to optimize a method compatible with both mass spectrometry imaging (MSI) and immunohistological techniques. Murine intestinal organoids were used to evaluate embedding in gelatin, carboxymethylcellulose (CMC)-gelatin-CMC-sucrose, or hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) solutions. Organoids were assessed with and without aldehyde fixation and analyzed for lipid distributions by MSI coupled with hematoxylin and eosin (H&E) staining and immunofluorescence (IF) in consecutive sections from the same sample. While chemical fixation preserves morphology for better histological outcomes, it can lead to suppression of the matrix-assisted laser desorption/ionization (MALDI) lipid signal. By contrast, leaving organoid samples unfixed enhanced MALDI lipid signal. The method that performed best for both MALDI and histological analysis was embedding unfixed samples in HPMC and PVP. This approach allowed assessment of cell proliferation by Ki67 while also identifying putative phosphatidylethanolamine (PE(18:0/18:1)), which was confirmed further by tandem MS approaches. Overall, these protocols will be amenable to multiplexing imaging mass spectrometry analysis with several histological assessments and help advance our understanding of the biological processes that take place in district subsets of cells in budding organoid structures.

Advancing (U-Th)/He Zircon Dating: Novel Approaches in Sample Preparation and Uncertainty Reduction

Abstract The laser microprobe (U-Th)/He dating method is a new and efficient technique that utilizes an interoperable and integrated suite of instruments, including the excimer laser system, quadrupole helium mass spectrometer, and quadrupole inductively coupled plasma mass spectrometer. To demonstrate the applicability of this new method, we applied both the conventional and laser microprobe techniques to the Sri Lanka zircon (LGC-1). We obtained twenty-two (U-Th)/He ages on nine shards using the laser microprobe method, showing an average (U-Th)/He age of 471.1 ± 16.6 Ma (1σ). This result is generally consistent with the mean conventional age (484.1 ± 9.6 Ma) for twenty-two zircon fragments. Both are nearly equal to the age value (~476 Ma) predicted by the He diffusion model and the thermal history model of Sri Lanka highland. The variations in the laser microprobe-derived ages are most likely caused by the uncertainties in volume measurements, which is also common in other studies. We used the Mahalanobis distance technique to reduce the volume measurement bias by identifying and eliminating abnormal data.

Enhanced molecular release from elderly bone samples using collagenase I: insights into fatty acid metabolism alterations

BackgroundBone is a metabolically active tissue containing different cell types acting as endocrine targets and effectors. Further, bone is a dynamic depot for calcium, phosphorous and other essential minerals. The tissue matrix is subjected to a constant turnover in response to mechanical/endocrine stimuli. Bone turnover demands high energy levels, making fatty acids a crucial source for the bone cells. However, the current understanding of bone cell metabolism is poor. This is partly due to bone matrix complexity and difficulty in small molecules extraction from bone samples. This study aimed to evaluate the effect of metabolite sequestering from a protein-dominated matrix to increase the quality and amount of metabolomics data in discovering small molecule patterns in pathological conditions.MethodsHuman bone samples were collected from 65 to 85 years old (the elderly age span) patients who underwent hip replacement surgery. Separated cortical and trabecular bone powders were treated with decalcifying, enzymatic (collagenase I and proteinase K) and solvent-based metabolite extraction protocols. The extracted mixtures were analyzed with the high-resolution mass spectrometry (HRMS). Data analysis was performed with XCMS and MetaboAnalystR packages.ResultsFast enzymatic treatment of bone samples before solvent addition led to a significantly higher yield of metabolite extraction. Collagenase I and proteinase K rapid digestion showed more effectiveness in cortical and trabecular bone samples, with a significantly higher rate (2.2 folds) for collagenase I. Further analysis showed significant enrichment in pathways like de novo fatty acid biosynthesis, glycosphingolipid metabolism and fatty acid oxidation-peroxisome.ConclusionThis work presents a novel approach for bone sample preparation for HRMS metabolomics. The disruption of bone matrix conformation at the molecular level helps the molecular release into the extracting solvent and, therefore, can lead to higher quality results and trustable biomarker discovery. Our results showed β-oxidation alteration in the aged bone sample. Future work covering more patients is worthy to identify the effective therapeutics to achieve healthy aging.