Sample Preparation Techniques Research Articles

Elastin-like polypeptide-functionalized nanobody for column-free immunoaffinity purification of aflatoxin B1.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B1 (AFB1) was developed using an AFB1-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (Tt) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB1 as well as phase transition when the temperature rose above Tt. To establish the optimal conditions for cleaning AFB1, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods.

Melt-blown polypropylene nonwoven as an efficient and eco-economic sorbent for pipette tip micro-solid phase extraction for the determination of tyrosine kinase inhibitors

BackgroundThe detection of tyrosine kinase inhibitors (TKIs) in biological fluids is essential due to their critical role in cancer therapy and the variability in individual drug metabolism, which necessitates precise dosing. Traditional methods for analyzing TKIs in biological fluids, such as blood plasma, typically involve complex sample preparation techniques that can be resource-intensive, environmentally burdensome, and not sufficiently sensitive for low-concentration analytes. There is a pressing need for more efficient, economical, and environmentally friendly methods that can enhance sensitivity and throughput without compromising accuracy. ResultsThis study explores the use of melt-blown polypropylene nonwoven (MBPP), commonly found in face masks, as a novel sorbent for pipette-tip micro-solid phase extraction (PT-μSPE). MBPP demonstrated excellent hydrophobicity and significant mesoporous adsorption capacity. An extraction device was fashioned by inserting a segment of MBPP (15 mg) into a 200 μL disposable plastic pipette tip, which was then attached to a 2.5 mL disposable plastic syringe. The MBPP's fabric form removes the need for a frit, allowing the extraction process to be completed in just 3 min through simple plunger manipulation. The method achieved extraction recoveries ranging from 60.5 % to nearly 100 %. Subsequent method validation using liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed satisfactory linearity (coefficient of determination R2 > 0.993), accuracy (relative recoveries: 86.3%–114.8 %), and precision (relative standard deviation: 3.4%–11.3 %), with detection limits between 0.022 and 0.135 ng mL−1. SignificanceThe introduction of MBPP for PT-μSPE represents a significant advancement in the bioanalytical detection of TKIs, offering a highly efficient, cost-effective, and environmentally sustainable method. It compares favorably with existing techniques, offering advantages in terms of cost, environmental impact, and ease of use. This approach has the potential to be widely adopted for routine monitoring of TKIs in clinical settings.

On the determination of charge and nitrogen content in cellulose fibres modified to contain quaternary amine functionality

Research interest in quaternization of cellulose fibres has increased considerably over the past decades. However, there is little or no consensus regarding how to characterize the material in terms of degree of substitution (DS), and the literature suggests a range of different methods focusing on charge determination as well as nitrogen content quantification. This work aims to fill the knowledge gap regarding how the different methods perform in relation to each other, and for what cellulosic systems each method has advantages, disadvantages and even potential pitfalls. FT-IR and NMR measurements are used to establish successful modification and determine the relative number of substituent groups. Another six methods are compared for the determination of the DS of cellulosic fibres and nanofibrils. The methods include Kjeldahl measurements, nitrogen determination by chemiluminescence, determination of molecular nitrogen by the Dumas method, colloidal titration, conductometric titration and polyelectrolyte adsorption. It can be concluded that most techniques investigated are reliable within certain ranges of DS and/or when using appropriate post-treatment of the quaternized material and suitable sample preparation techniques. The results from the present work hence provide recommendations to make an educated choice of method, and experimental protocol, based on the technique at hand.

Applications of Lightsheet Fluorescence Microscopy by High Numerical Aperture Detection Lens.

This Review explores the evolution, improvements, and recent applications of Light Sheet Fluorescence Microscopy (LSFM) in biological research using a high numerical aperture detection objective (lens) for imaging subcellular structures. The Review begins with an overview of the development of LSFM, tracing its evolution from its inception to its current state and emphasizing key milestones and technological advancements over the years. Subsequently, we will discuss various improvements of LSFM techniques, covering advancements in hardware such as illumination strategies, optical designs, and sample preparation methods that have enhanced imaging capabilities and resolution. The advancements in data acquisition and processing are also included, which provides a brief overview of the recent development of artificial intelligence. Fluorescence probes that were commonly used in LSFM will be highlighted, together with some insights regarding the selection of potential probe candidates for future LSFM development. Furthermore, we also discuss recent advances in the application of LSFM with a focus on high numerical aperture detection objectives for various biological studies. For sample preparation techniques, there are discussions regarding fluorescence probe selection, tissue clearing protocols, and some insights into expansion microscopy. Integrated setups such as adaptive optics, single objective modification, and microfluidics will also be some of the key discussion points in this Review. We hope that this comprehensive Review will provide a holistic perspective on the historical development, technical enhancements, and cutting-edge applications of LSFM, showcasing its pivotal role and future potential in advancing biological research.

Measurement Techniques for Low-Concentration Tritium Radiation in Water: Review and Prospects.

Tritium (3H) is one of the most critical nuclides for environmental monitoring, yet it is challenging to measure. Its high natural mobility and its potential to enter the human body through the food chain underscore the importance of not overlooking the radiation safety risks associated with tritium. The need for the online measurement of tritium at low concentrations is becoming increasingly apparent. This review examines the two principal stages of current measurement methodologies: sample preparation and radiation signal detection. It provides a summary of the tritium sample preparation and detection techniques, highlighting advances in the research with potential applications in online monitoring. The review concludes with an analysis of the issues inherent in the current techniques and offers perspectives on possible technological enhancements and future trajectories for the development of online monitoring systems for trace tritium levels.

Discrete Element Study on Mechanical Properties of MICP-Treated Sand under Triaxial Compression

Microbial-induced calcium carbonate precipitation (MICP) has attracted much attention as a promising technology for soil improvement in the infrastructures of marine engineering. This paper introduces a novel numerical sample preparation technique for MICP-treated sand, with particular attention paid to the distribution patterns of calcium carbonate, including contact cementing, bridging, and grain coating. The effect of calcium carbonate content (CCC) on the deformation and failure mechanism is studied at macroscopic and granular scales. The findings show that a small amount of calcium carbonate can quickly increase the strength of sand. The strength improvement and deformation control of MICP technology are better than those of traditional compaction treatment. As the calcium carbonate content increases, the mechanical coordination number of the sand also increases, indicating a more stable microstructure of the sand phase. In the contact bonding mode, initial failure occurs as shear failure between sand and calcium carbonate. In the bridge mode, initial failure manifests as shear failure between calcium carbonate particles. In the coating mode, initial failure occurs as tensile failure between sand and calcium carbonate. Calcium carbonate contributes to a reduction in both sliding and rolling movements among sand particles.

A tutorial on optical photothermal infrared (O-PTIR) microscopy.

This tutorial reviews the rapidly growing field of optical photothermal infrared (O-PTIR) spectroscopy and chemical imaging. O-PTIR is an infrared super-resolution measurement technique where a shorter wavelength visible probe is used to measure and map infrared (IR) absorption with spatial resolution up to 30× better than conventional techniques such as Fourier transform infrared and direct IR laser imaging systems. This article reviews key limitations of conventional IR instruments, the O-PTIR technology breakthroughs, and their origins that have overcome the prior limitations. This article also discusses recent developments in expanding multi-modal O-PTIR approaches that enable complementary Raman spectroscopy and fluorescence microscopy imaging, including wide-field O-PTIR imaging with fluorescence-based detection of IR absorption. Various practical subjects are covered, including sample preparation techniques, optimal measurement configurations, use of IR tags/labels and techniques for data analysis, and visualization. Key O-PTIR applications are reviewed in many areas, including biological and biomedical sciences, environmental and microplastics research, (bio)pharmaceuticals, materials science, cultural heritage, forensics, photonics, and failure analysis.

Applications of Sample Preparation Techniques in the Analysis of New Psychoactive Substances

The global rise of new psychoactive substances (NPSs) poses challenges for their analysis in biological matrices due to their complex chemistries and short market lifespan. A comparative study for the simultaneous extraction, separation, and detection of 19 NPSs was conducted. Six solid-phase extraction (SPE) methods and one supported liquid extraction method (SLE) were compared for the extraction of analytes from blood, serum, plasma, and urine. Comparisons of four derivatization agents were conducted, at four temperatures and two incubation times. Extraction methods were assessed by precision, sensitivity, and extraction efficiency. Derivatizing agents were assessed on their selectivity and sensitivity, and a three-way ANOVA was conducted to determine statistical significance. CSDAU SPE cartridges were shown to be the most efficient when extracting analytes from blood, serum, and plasma, whereas Xcel I cartridges performed the strongest when extracting analytes from urine. SPE extraction efficiencies, when utilizing the best-performing cartridges, ranged from 49 to 119%. SLE successfully extracted all analytes from all matrices (ranging from 22 to 120%). Pentafluoropropionic anhydride: ethyl acetate was the most successful derivatizing agent, allowing all analytes to be detected, with the highest peak area responses and more unique spectra. The optimum temperature for incubation was 37 °C, with no statistical difference found between the two incubation times.

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.

Recent advances in solid phase microextraction with various geometries in environmental analysis.

Solid phase microextraction (SPME) has emerged as a versatile sample preparation technique for the preconcentration of a broad range of compounds with various polarities, especially in environmental studies. SPME has demonstrated its eco-friendly credentials, significantly reducing the reliance on solvents. The use of biocompatible materials as a coating recipe facilitates the acceptance of SPME devices in analytical chemistry, primarily in the monitoring of environmental pollutants such as persistent organic pollutants (POPs), volatile organic compounds (VOCs), and pesticides from the various environmental matrices. During the last few years, investigators have reported an improvement in the SPME enrichment technique after changing the coating recipe, geometries, and sampling procedure from the complex matrices. Furthermore, the development of various geometries of SPME with large surface areas has enhanced the extraction efficiency of environmental pollutants. As a miniaturized sample preparation technique, SPME significantly reduces the solvent usage, suggesting a potential platform for green chemistry-based research for water, air, and soil analysis. This review article summarizes the evolution of SPME, its various modes, the application of SPME, recent innovations, and prospects for the determination of water, air, and soil pollution. The advantages and disadvantages of SPME in comparison to other extraction techniques have been discussed here. This review serves as a valuable resource for investigators working in sustainable environmental research.

Investigation of Automated Sample Preparation Techniques for Gas Chromatography-Mass Spectrometry Analysis of Volatile Organic Compounds in Thai Green Curry Paste

Investigation of Automated Sample Preparation Techniques for Gas Chromatography-Mass Spectrometry Analysis of Volatile Organic Compounds in Thai Green Curry Paste

Analytical methods for the determination of xylazine in pharmaceutical, clinical and forensic matrices - A review.

Xylazine, a non-opioid veterinary anaesthetic tranquillizer that is not licensed for human use, has been linked to an increase in overdose fatalities worldwide. The study delves into the forensic aspects of xylazine usage, emphasizing on chemical, clinical and toxicological analyses of drug seizures, bodily fluids and tissues. It advocates for validated analytical methods for determining xylazine. This study provides supporting material to pave the path for the usage and development of relevant and verified alternative screening and confirmation methods for laboratories. Google Scholar, Scopus, Science Direct and PubMed were searched for relevant articles and case reports in relation to xylazine misuse and established analytical methods for forensic investigation until April 2023. A total of 79 articles were evaluated, and 40 publications met the inclusion criteria. The most prevalent xylazine exposures recorded were incidental and intentional misuse/abuse. Common symptoms upon presentation were hypotension, bradycardia, drowsiness and lethargy, although mortality was less prevalent. Solid-phase extraction and liquid-liquid extraction are two extensively used sample preparation techniques. These techniques are used to extract desired analytes from complex matrices. Several analytical techniques have been stated, including GC-MS, LC-MS/MS, HPLC-DAD and others. The analytical procedures used are determined by the matrices involved, the amount of xylazine present, interfering compounds, the degree of precision required and the laboratory infrastructure. In the present context, the LC-MS/MS methods are preferred as the gold standard. In the near future, many analytical techniques such as capillary electrophoresis, PSI-MS, immuno-analytical techniques and SERRS may show significant potential.

Advanced Characterization and Sample Preparation Strategies for Nanoformulations

The escalating impact and remarkable progress of nanotechnology have shifted the paradigms of medicine and the healthcare system. Nanosystems have emerged, extensively holding the potential to advance disease diagnosis and treatment specificity. The extraordinary attributes imparted by nano-systems have helped in overcoming the limitations of conventional interventions to an extent and led to targeted therapy, to name one. The role of nanotechnology in diagnosis is another breakthrough in its appellation. This article aims to address the current characterization and sample preparation techniques for the analysis of nanosystems and provide insights into novel methodologies and in situ instrumentation that have eased sampling procedures.

Optimization of the QuEChERS-UPLC-APCI-MS/MS method for the analysis of vitamins D and K nanoencapsulated in yogurt

A novel approach was developed to simultaneously determine the contents of vitamins D2, D3, K1, and K2 in yogurt fortified with nanoencapsulated vitamins D and K. This method combines QuEChERS extraction with UPLC-APCI-MS/MS analysis. Optimization of the QuEChERS process included fine-tuning the addition of salts using response surface methodology based on the Box–Behnken design. Under the optimized conditions, the developed method exhibited an excellent linearity (R2 > 0.999) across concentrations ranging from 0.5 to 500 µg/L. The limits of detection and quantification (LOD and LOQ) were found to be 0.01–0.04 µg/L and 0.04–0.11 µg/L, respectively, with precision, accuracy, and recovery rates exceeding 94.88 %, and accompanied by acceptable relative standard deviations. Comparative analysis with traditional methodologies revealed the significant advantages of the proposed approach. Previous techniques such as liquid–liquid extraction combined with saponification are time-consuming and require high sample quantities. In addition, dispersive liquid–liquid microextraction requires a long analysis time and exhibits a poor sensitivity, particularly in terms of its LOD and LOQ values. In contrast, our method offers a straightforward, efficient, and reliable sample preparation technique suitable for detecting vitamins D2, D3, K1, and K2 in a yogurt matrix. This study not only demonstrates the feasibility of applying the QuEChERS method for stable vitamin quantification in yogurt, but it also represents an innovative contribution to enhancing the detection sensitivity and efficiency in food analysis. By emphasizing these methodological advancements and comparative benefits, this research underscores the significance of adopting advanced analytical approaches in food science.

Effects of Sample Handling on Visible and Near-Infrared Soil Reflectance

Soil reflectance is a cumulative attribute determined by interactions between light (photons) and the physical, chemical, and biological properties of soil. Soil properties such as organic matter, moisture, mineral oxide contents, soil texture, and surface roughness all influence soil reflectance at unique wavelengths. Many standard sample preparation techniques are designed to alter soil properties, so as to homogenize samples to improve the consistency of reflectance data collected. This study aims to quantify the effects of one standard sample preparation activity, drying, and repeated wetting–drying cycles on visible–near-infrared soil reflectance, by collecting reflectance data from nine samples which were dried then wetted for a total of three times. This study demonstrates the major, permanent effects of a drying and wetting cycle on soil reflectance, and then presents a model which can be used to correct for these effects. These results have direct implications for remote sensing activities, soil libraries, and soil spectral libraries. These results show that without correction, data from soil spectral libraries, spectral data collected from stored samples, and spectral data transferred between studies, have limited utility for characterizing soils as they exist in the field. Soil spectral data collected in the lab requires correction before it may be used to predict soil properties in the field, as standard sample handling procedures change intrinsic soil properties and introduce systematic error into these data.

Challenges of Extracting and Determining Cannabinoids in Different Matrices.

Introduction: Accurate and precise analysis of cannabinoids is important for elucidating their therapeutic potential and developing therapies, which are targeted toward different medical conditions. A wide range of cannabis products are present on the market and are available in different dosage forms, including dried flowers, extracts, and consumables. The aim of this article is to provide an updated narrative review of literature on challenges of analyzing cannabinoids in plant material, oils, and edibles. Method: Literature search was conducted to identify sample preparation and analytical techniques for determination of cannabinoids in plant material, oils, and edibles and associated challenges. Results: Challenges related to determination of cannabinoids in plant material include matrix complexity, co-extraction of unwanted compounds during sample preparation, and differences in matrix composition between calibration standards and sample extracts. During analysis of cannabinoids in oil, the unique properties of carrier oils need to be taken into consideration. Analysis of cannabinoids in edibles can be considered to be challenging due to the wide range of matrix types that are available on the market, rendering analysis resource-intensive, time-consuming, and impractical. Discussion: Analysis of cannabinoids in plant material, oils, and edibles requires a multifaceted approach that includes regulatory guidance, method development, and technological innovation. In the face of an evolving analytical landscape where novel cannabinoids are being identified and require determination, there is a need for the development and validation of standardized accurate and precise analytical methods, which are specifically tailored for each matrix.

Advances and Applications of Hybrid Graphene-Based Materials as Sorbents for Solid Phase Microextraction Techniques.

The advancement of traditional sample preparation techniques has brought about miniaturization systems designed to scale down conventional methods and advocate for environmentally friendly analytical approaches. Although often referred to as green analytical strategies, the effectiveness of these methods is intricately linked to the properties of the sorbent utilized. Moreover, to fully embrace implementing these methods, it is crucial to innovate and develop new sorbent or solid phases that enhance the adaptability of miniaturized techniques across various matrices and analytes. Graphene-based materials exhibit remarkable versatility and modification potential, making them ideal sorbents for miniaturized strategies due to their high surface area and functional groups. Their notable adsorption capability and alignment with green synthesis approaches, such as bio-based graphene materials, enable the use of less sorbent and the creation of biodegradable materials, enhancing their eco-friendly aspects towards green analytical practices. Therefore, this study provides an overview of different types of hybrid graphene-based materials as well as their applications in crucial miniaturized techniques, focusing on offline methodologies such as stir bar sorptive extraction (SBSE), microextraction by packed sorbent (MEPS), pipette-tip solid-phase extraction (PT-SPE), disposable pipette extraction (DPX), dispersive micro-solid-phase extraction (d-µ-SPE), and magnetic solid-phase extraction (MSPE).

Versatile injector for inline renewable solid-phase extraction: Application to cyclodextrin-based bioaccessibility assessment in environmental solids

BackgroundSolid phase extraction (SPE) is a standard sample preparation technique in HPLC workflows. Inline cartridges are high-performance alternatives to manual or robotic systems but at long term, they suffer from irreversible sorption of matrix components and sorbent compaction. Bead injection (BI) is a niche fluidic technique that allows renewing a sorbent bed through the manipulation of its suspension. However, there is a need for a versatile and reliable tool in HPLC that can exchange the inline sorbent automatically, resorting to inexpensive and assorted bulk sorbents. ResultsWe present a new flow path for a liquid chromatographic injector to perform inline micro-solid phase extraction. The sample is processed at real time, trapping the analytes and discarding the matrix. Cleaning the matrix and injecting 10 μL of sample takes 70 s, comparable with the injection in commercial HPLC systems. If the aim is to preconcentrate the analytes, average enrichment factors of 250 have been obtained after processing sample volumes of 3200 μL in 16 min (interleavable with the chromatographic step), keeping the peak position and width independent of the injected volume (compared to large volume direct injection).The desired bed mass is automatically and pressure-driven manipulated in the valve, retained by an inline frit, and optionally, after the analysis, removed by forward flow.The chromatographic performance of the new design is compared to the standard 6-port, 2-position HPLC injector. As a case study, we have monitored the extraction kinetics of a cyclodextrin-based bioaccessibility extraction test of persistent organic contaminants in soil, by extracting several fractions in valve, process them with inline SPE with a balanced hydrophilic-hydrophobic reversed-phase sorbent, and inject the bioaccessible compounds into HPLC. Aiming at avoiding carryover, the sorbent bed (ca. 3 mg) is exchanged before every run. It should be noted that this contribution focuses on HPLC, but other non-separative techniques, such as Flow Injection Analysis, can equally benefit from this injection platform. SignificanceThis contribution reports the first use of inline BI-solid phase extraction in HPLC workflows, without heart-cut eluate injection, in which the sorbent can be exchanged automatically by forward flow. This performance is enabled by prototyping a valve that can autonomously swap sorbents in real-time for diverse samples, as a cartridge exchanger, but using cost-effective and environmentally friendlier bulk sorbents (bed masses from sub-mg to 5 mg) without requiring additional hardware.

Electromembrane extraction of human chorionic gonadotropin – A case study on mass transfer of intact protein versus signature peptide

For the first time, we present targeted protein detection by tryptic digestion of human chorionic gonadotropin (hCG) followed by electromembrane extraction (EME). Operational parameters were optimized, and urine and serum samples spiked with hCG underwent tryptic digestion followed by EME of the βT5 signature peptide. The liquid membrane comprised nitrophenyl octyl ether (NPOE), carvacrol, and di(2-ethyl hexyl) phosphate (DEHP) at ratios of 49:49:2 (w/w/w). Extractions were performed in a conductive vial format for 45 min at 5 V. Even from highly complex digested samples of serum and urine, the signature peptide βT5 was extracted by EME and detected by LC-MS/MS. While attempts to extract intact hCG protein were unsuccessful, the extraction of the signature peptide was efficient. The extraction recovery from undigested and digested urine was 71 % (RSD = 17 %) and 116 % (RSD = 17 %), respectively. For serum, the extraction recoveries were 11 % (RSD = 23 %) for undigested samples and 110 % (RSD = 14 %) for digested samples. This study demonstrates both the potential and challenges of EME for protein analysis. Experiments regarding EME of intact proteins provided new insights into protein phase distribution. This fundamental case study underscores the potential of EME as a sample preparation technique for the targeted determination of protein biomarkers and drugs.

Ionic liquid-assisted sample preparation mediates sensitive proteomic analysis of Bacillus subtilis spores

Endospore-forming bacteria are ubiquitous. Bacterial endospores are multilayered proteinaceous structures that protects the bacterial genome during stress conditions. They are also responsible for a wide range of critical clinical infections in humans. Precise analysis of spore-forming pathogens remains a major challenge in the field of proteomics because spore structures are highly resistant to conventional solubilizers and denaturing agents, such as sodium dodecyl sulfate and urea. We present an ionic liquid-assisted (i-soln) technique of sample preparation, called pTRUST, which enables shotgun analysis of Bacillus subtilis spores even when the starting materials are in the sub-microgram range. In proteomic analysis, this technique shows 50–2000-fold higher sensitivity than other conventional gel-based or gel-free methods (including one-pot sample processing). Using this technique, we identified 445 proteins with high confidence from trace amounts of highly pure spore preparations, including 52 of the 79 proteins (approximately 70%) previously demonstrated to be localized in spores in the SubtiWiki database and detected through direct protein analysis. Consequently, 393 additional proteins were identified as candidates for spore constitutive proteins. Twenty of these newly identified candidates were produced as green fluorescent protein fusion proteins, and each was evaluated for authenticity as a spore constituent using fluorescence microscopy analysis. The pTRUST method's sensitivity and reliability using the i-soln system, together with hitherto unreported proteins in spores, will enable an array of spore research for biological and clinical applications.