Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

A versatile cryo-transfer system, connecting cryogenic focused ion beam sample preparation to atom probe microscopy.

Atom probe tomography (APT) is a powerful technique to obtain 3D chemical and structural information, however the 'standard' atom probe experimental workflow involves transfer of specimens at ambient conditions. The ability to transfer air- or thermally-sensitive samples between instruments while maintaining environmental control is critical to prevent chemical or morphological changes prior to analysis for a variety of interesting sample materials. In this article, we describe a versatile transfer system that enables cryogenic- or room-temperature transfer of specimens in vacuum or atmospheric conditions between sample preparation stations, a focused ion beam system (Zeiss Crossbeam 540) and a widely used commercial atom probe system (CAMECA LEAP 4000X HR). As an example for the use of this transfer system, we present atom probe data of gallium- (Ga)-free grain boundaries in an aluminum (Al) alloy specimen prepared with a Ga-based FIB.

Lab-on-a-Filter Techniques for Economical, Effective, and Flexible Proteome Analysis.

Effective and reliable protease digestion of biological samples is critical to the success in bottom-up proteomics analysis. Various filter-based approaches using different types of membranes have been developed in the past several years and largely implemented in sample preparations for modern proteomics. However, these approaches rely heavily on commercial filter products, which are not only costly but also limited in membrane options. Here, we present a plug-and-play device for filter assembly and protease digestion. The device can accommodate a variety of membrane types, can be packed in-house with minimal difficulty, and is extremely cost-effective and reliable. Our protocol offers a versatile platform for general proteome analyses and clinical mass spectrometry.

Glass Fiber-Supported Hybrid Monolithic Spin Tip for Enrichment of Phosphopeptides from Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are attracting increasing interest with their intriguing role in intercellular communications. Protein phosphorylation in EVs is of great importance for understanding intercellular signaling processes. However, the study of EV phosphoproteomics is impeded by their relatively low amount in limited clinical sample volumes, and it is necessary to have a sensitive and efficient enrichment method for EV phosphopeptides. Herein, a novel Ti(IV)-functionalized and glass fiber-supported hybrid monolithic spin tip, termed PhosTip, was prepared for enriching phosphopeptides from urinary EVs. Glass fiber as the stationary phase positions the hybrid monolith in a standard pipet tip and prevents the monolith from distortion during experiments. The preparation procedure for the new PhosTip is simple and time-saving. The hybrid monolithic PhosTip provides excellent enrichment efficiency of low-abundance phosphopeptides from cell digests and urinary EVs with minimum contamination and sample loss. Using the PhosTip, we demonstrate that 5373 and 336 unique phosphopeptides were identified from 100 and 1 µg of cell lysates, while 3919 and 217 unique phosphopeptides were successfully identified from 10 and 1 mL of urinary samples, respectively. The PhosTip was finally applied to enrich phosphopeptides in urine EVs from prostate cancer patients and healthy controls and quantify 118 up-regulated proteins with phosphosites in prostate cancer samples. These results demonstrated that the PhosTip could be a simple and convenient tool for enriching phosphopeptides from clinical samples and for broader applications in biomarker discovery.

Tandem Cation/Anion Exchange SPE Cartridge Method for Sample Desalting for HPLC Analysis of Soluble Dietary Fiber: Development and Inter-laboratory Validation.

In HPLC analyses of soluble dietary fiber, desalting processes using open, mixed-bed ion-exchange columns are time-consuming and labor-intensive. We developed and validated a simple desalting method using tandem cation/anion exchange SPE cartridges. We found that combining Bond Elut Jr SCX (upstream) and Bond Elut PSA (downstream) cartridges provided adequate desalting of test solutions. The developed method was then validated in an inter-laboratory study. Five test samples were prepared by mixing food matrixes with purified soluble dietary fiber and treated to generate solutions to test the desalting process. These solutions were then analyzed by eight different laboratories. The results demonstrated that the developed method is simple and reliable for desalting samples containing 140 to 945 mg/100 mL of soluble dietary fiber in preparation for HPLC analysis of soluble dietary fiber.

Fabrication of a polycarbonate microdevice and boronic acid-mediated surface modification for on-chip sample purification and amplification of foodborne pathogens.

In this study, we integrated sample purification and genetic amplification in a seamless polycarbonate microdevice to facilitate foodborne pathogen detection. The sample purification process was realized based on the increased affinity of the boronic acid-modified surface toward the cis-diol group present on the bacterial outer membrane. The modification procedure was conducted at room temperature using disposable syringe. The visible color and fluorescence signals of alizarin red sodium were used to confirm the success of the surface modification process. Escherichia coli O157:H7 containing green fluorescence protein (GFP) and Staphylococcus aureus were chosen as the microbial models to demonstrate the nonspecific immobilization using the microdevice. Bacterial solutions of various concentrations were injected into the microdevice at three flow rates to optimize the operation conditions. This microdevice successfully amplified the 384-bp fragment of the eaeA gene of the captured E. coli O157:H7 within 1 h. Its detection limit for E. coli O157:H7 was determined to be 1 × 103 colony-forming units per milliliter (CFU mL-1). The proposed microdevice serves as a monolithic platform for facile and on-site identification of major foodborne pathogens.

Comparison of extraction techniques for polycyclic aromatic hydrocarbons from lichen biomonitors.

Lichens are useful biomonitors for atmospheric polycyclic aromatic hydrocarbons (PAHs). Different sample preparation techniques were explored in this regard, including ultrasound-assisted solvent extraction, microwave-assisted extraction, Soxhlet, and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique. It was found that a QuEChERS technique using hexane:acetone (1:1, v/v), never reported before for application to lichens, provided the best recoveries of internal standards, the highest total peak area for all PAHs of interest, and %RSDs comparable with the other preparation techniques tested. The optimized sample preparation technique was found to be a comparatively fast method (45 min), with good recoveries (96%), using less solvents and minimal energy consumption. Strong matrix effects were found: both strong enhancement (for the lighter PAHs) and strong suppression (for the heavier PAHs). The use of matrix-matched standards is thus imperative for the accurate determination of PAH concentrations in the lichen samples. Graphical abstract "Note: This data is mandatory. Please provide."

Mass Spectrometry-Based Microbial Metabolomics: Techniques, Analysis, and Applications.

The demand for understanding the roles genes play in biological systems has steered the biosciences into the direction the metabolome, as it closely reflects the metabolic activities within a cell. The importance of the metabolome is further highlighted by its ability to influence the genome, transcriptome, and proteome. Consequently, metabolomic information is being used to understand microbial metabolic networks. At the forefront of this work is mass spectrometry, the most popular metabolomics measurement technique. Mass spectrometry-based metabolomic analyses have made significant contributions to microbiological research in the environment and human disease. In this chapter, we break down the technical aspects of mass spectrometry-based metabolomics and discuss its application to microbiological research.

Untargeted Soil Metabolomics Using Liquid Chromatography-Mass Spectrometry and Gas Chromatography-Mass Spectrometry.

The molecular composition of soil organic matter (SOM) sets the foundation for terrestrial microbial community structures and carbon cycling dynamics. However, the specific chemical constituents of SOM are underexplored. In this chapter we present a protocol for the extraction of small molecule metabolites from soil followed by compound detection and identification using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. There are options within the protocol to assess either the extracellular pool of metabolites or the total pool (including intracellular) and either polar or nonpolar metabolites, depending on the reader's research interests. These methods can be followed individually for a more targeted analysis or all methods can be combined to obtain a more comprehensive understanding of SOM metabolite composition (such as amino acids, nucleobases, organic acids, fatty acids, carbohydrates, secondary metabolites, and antibiotics).

Integration of sample preparation and analysis into an optofluidic chip for multi-target disease detection.

Detection of molecular biomarkers with high specificity and sensitivity from biological samples requires both sophisticated sample preparation and subsequent analysis. These tasks are often carried out on separate platforms which increases required sample volumes and the risk of errors, sample loss, and contamination. Here, we present an optofluidic platform which combines an optical detection section with single nucleic acid strand sensitivity, and a sample processing unit capable of on-chip, specific extraction and labeling of nucleic acid and protein targets in complex biological matrices. First, on-chip labeling and detection of individual lambda DNA molecules down to concentrations of 8 fM is demonstrated. Subsequently, we demonstrate the simultaneous capture, fluorescence tagging and detection of both Zika specific nucleic acid and NS-1 protein targets in both buffer and human serum. We show that the dual DNA and protein assay allows for successful differentiation and diagnosis of Zika against cross-reacting species like dengue.

Microfluidic deposition for resolving single-molecule protein architecture and heterogeneity.

Scanning probe microscopy provides a unique window into the morphology, mechanics, and structure of proteins and their complexes on the nanoscale. Such measurements require, however, deposition of samples onto substrates. This process can affect conformations and assembly states of the molecular species under investigation and can bias the molecular populations observed in heterogeneous samples through differential adsorption. Here, we show that these limitations can be overcome with a single-step microfluidic spray deposition platform. This method transfers biological solutions to substrates as microdroplets with subpicoliter volume, drying in milliseconds, a timescale that is shorter than typical diffusion times of proteins on liquid-solid interfaces, thus avoiding surface mass transport and change to the assembly state. Finally, the single-step deposition ensures the attachment of the full molecular content of the sample to the substrate, allowing quantitative measurements of different molecular populations within heterogeneous systems, including protein aggregates.

Evaluation of extraction and normalisation strategies for the analysis of lipids in placental vessels.

The analysis of lipids in tough or fibrous biological tissues can be challenging due to difficulties in obtaining a representative sample following homogenisation of the tissue. Furthermore, the choice of normalisation method can have a major effect on the quality of quantitative results. Therefore, a range of mechanical homogenisation techniques and normalisation strategies were evaluated for application to human placental vessels. The findings showed that rotor-stator homogenisation in a suitable solvent and wet weight normalisation were the best combination of procedures for quantitative analysis of lipids in placental blood vessels.

A radial calibration window for analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first-principles based method for studying macromolecules and particles in solution by monitoring the evolution of their radial concentration distribution as a function of time in the presence of a high centrifugal field. In sedimentation velocity experiments, hydrodynamic properties relating to size, shape, density, and solvation of particles can be measured, at a high hydrodynamic resolution, on polydisperse samples. In a recent multilaboratory benchmark study including data from commercial analytical ultracentrifuges in 67 laboratories, the calibration accuracy of the radial dimension was found to be one of the dominant factors limiting the accuracy of AUC. In the present work, we develop an artifact consisting of an accurately calibrated reflective pattern lithographically deposited onto an AUC window. It serves as a reticle when scanned in AUC control experiments for absolute calibration of radial magnification. After analysis of the pitch between landmarks in scans using different optical systems, we estimate that the residual uncertainty in radial magnification after external calibration with the radial scale artifact is ≈0.2 %, of similar magnitude to other important contributions after external calibration such as the uncertainty in temperature and time. The previous multilaboratory study had found many instruments with errors in radial measurements of 1 % to 2 %, and a few instruments with errors in excess of 15 %, meaning that the use of the artifact developed here could reduce errors by 5-to 10-fold or more. Adoption of external radial calibration is thus an important factor for assuring accuracy in studies related to molecular hydrodynamics and particle size measurements by AUC.

Freeze-thaw approach: A practical sample preparation strategy for residue analysis of multi-class veterinary drugs in chicken muscle.

Seven drugs from different classes, namely, fluoroquinolones (enrofloxacin, ciprofloxacin, sarafloxacin), sulfonamides (sulfadimidine, sulfamonomethoxine), and macrolides (tilmicosin, tylosin), were used as test compounds in chickens by oral administration, a simple extraction step after cryogenic freezing might allow the effective extraction of multi-class veterinary drug residues from minced chicken muscles by mix vortexing. On basis of the optimized freeze-thaw approach, a convenient, selective, and reproducible liquid chromatography with tandem mass spectrometry method was developed. At three spiking levels in blank chicken and medicated chicken muscles, average recoveries of the analytes were in the range of 71-106 and 63-119%, respectively. All the relative standard deviations were <20%. The limits of quantification of analytes were 0.2-5.0 ng/g. Regardless of the chicken levels, there were no significant differences (P > 0.05) in the average contents of almost any of the analytes in medicated chickens between this method and specific methods in the literature for the determination of specific analytes. Finally, the developed method was successfully extended to the monitoring of residues of 55 common veterinary drugs in food animal muscles.

A selective volatilization method for determination of chloride and sulfate in calcium carbonate pharmaceutical raw material and commercial tablets.

In this work a feasible method for chloride and sulfate determination in calcium carbonate pharmaceutical raw material and commercial tablets by ion chromatography after microwave-induced combustion was developed. The analytes were released from matrix by combustion in closed system pressurized with oxygen. Starch as volatilization aid, 100mmolL-1 HNO3 as absorbing solution and 5min of microwave irradiation time were used. Recovery tests using standard solutions were performed for the accuracy evaluation. A mixture of calcium carbonate pharmaceutical raw material or commercial tablets, starch and a certified reference material was also used as a type of recovery test. Recoveries ranging from 88% to 103% were obtained in both spike tests. Limits of detection (Cl-: 40µgg-1 and SO42-: 140µgg-1) were up to eighteen times lower than the maximum limits established for the analytes by Brazilian, British, European and Indian Pharmacopoeias. The limit tests recommended by the European Pharmacopoeia for Cl- and SO42- in CaCO3 were carried out to compare the results. Chloride and SO42- concentrations in the samples analyzed by proposed method were in agreement with those results obtained using the tests recommended by the European Pharmacopoeia. However, the proposed method presents several advantages for the routine analysis when compared to pharmacopoeial methods, such as the quantitative simultaneous determination, high sample preparation throughput (up to eight samples per run in less than 30min), reduced volume of reagents and waste generation. Thus, the proposed method is indicated as an excellent alternative for Cl- and SO42- determination in CaCO3 pharmaceutical raw material and commercial tablets.

Comparison of efficiencies of selected sample extraction techniques for the analysis of selected antiretroviral drugs in human plasma using LC-MS.

INTRODUCTION: Sample preparation in bio analytical chemistry poses a challenge because it can be compound dependent. We compared six sample extraction techniques i.e. QuEChERS (Q), liquid extraction (LE), protein precipitation (PPT), Q-PPT, Q-LE and LE-PPT for the extraction of antiretroviral drugs emtricitabine, tenofovir, efavirenz, lopinavir and rotinavir in human blood plasma. METHOD: A multiple reaction monitoring liquid chromatography- tandem mass spectrometry method for the determination of the same antiretroviral drugs developed and validated in this laboratory was used. Comparisons were based on the efficiencies of extraction, the precisions and accuracies. Using United States Food and Drug Administration guidelines, analytical performance characteristics i.e. limits of detection, lower limits of quantification and upper limits of quantification were also compared. RESULTS: The percent mean recoveries ranged between 68.8 and 81.2% for single modes and 52.4-70.5% for mixed mode techniques. The precisions of all the extraction techniques were within the Using United States Food and Drug Administration guidelines acceptable range of <15% at all concentration levels for all analytes. Accuracy ranged between 8.73 and 65.94% for single mode techniques and between 21.73 and 51.59% for mixed mode techniques. DISCUSSION: The mixed modes gave slightly lower recoveries but Q-LE compared well with the single modes at slightly higher spike levels. Limits of detection for all the six sample preparation techniques fell below the clinically relevant therapeutic range of approximately 3-8ppm. Therefore all techniques can be employed for routine therapeutic drug monitoring studies.

Critical Assessment of Analytical Techniques in the Search for Biomarkers on Mars: A Mummified Microbial Mat from Antarctica as a Best-Case Scenario.

The search for biomarkers of present or past life is one of the major challenges for in situ planetary exploration. Multiple constraints limit the performance and sensitivity of remote in situ instrumentation. In addition, the structure, chemical, and mineralogical composition of the sample may complicate the analysis and interpretation of the results. The aim of this work is to highlight the main constraints, performance, and complementarity of several techniques that have already been implemented or are planned to be implemented on Mars for detection of organic and molecular biomarkers on a best-case sample scenario. We analyzed a 1000-year-old desiccated and mummified microbial mat from Antarctica by Raman and IR (infrared) spectroscopies (near- and mid-IR), thermogravimetry (TG), differential thermal analysis, mass spectrometry (MS), and immunological detection with a life detector chip. In spite of the high organic content (ca. 20% wt/wt) of the sample, the Raman spectra only showed the characteristic spectral peaks of the remaining beta-carotene biomarker and faint peaks of phyllosilicates over a strong fluorescence background. IR spectra complemented the mineralogical information from Raman spectra and showed the main molecular vibrations of the humic acid functional groups. The TG-MS system showed the release of several volatile compounds attributed to biopolymers. An antibody microarray for detecting cyanobacteria (CYANOCHIP) detected biomarkers from Chroococcales, Nostocales, and Oscillatoriales orders. The results highlight limitations of each technique and suggest the necessity of complementary approaches in the search for biomarkers because some analytical techniques might be impaired by sample composition, presentation, or processing. Key Words: Planetary exploration-Life detection-Microbial mat-Life detector chip-Thermogravimetry-Raman spectroscopy-NIR-DRIFTS. Astrobiology 17, 984-996.

Natural Isotope Abundance in Metabolites: Techniques and Kinetic Isotope Effect Measurement in Plant, Animal, and Human Tissues.

The natural isotope abundance in bulk organic matter or tissues is not a sufficient base to investigate physiological properties, biosynthetic mechanisms, and nutrition sources of biological systems. In fact, isotope effects in metabolism lead to a heterogeneous distribution of 2H, 18O, 13C, and 15N isotopes in metabolites. Therefore, compound-specific isotopic analysis (CSIA) is crucial to biological and medical applications of stable isotopes. Here, we review methods to implement CSIA for 15N and 13C from plant, animal, and human samples and discuss technical solutions that have been used for the conversion to CO2 and N2 for IRMS analysis, derivatization and isotope effect measurements. It appears that despite the flexibility of instruments used for CSIA, there is no universal method simply because the chemical nature of metabolites of interest varies considerably. Also, CSIA methods are often limited by isotope effects in sample preparation or the addition of atoms from the derivatizing reagents, and this implies that corrections must be made to calculate a proper δ-value. Therefore, CSIA has an enormous potential for biomedical applications, but its utilization requires precautions for its successful application.

Applications of Isotope Ratio Mass Spectrometry in Sports Drug Testing Accounting for Isotope Fractionation in Analysis of Biological Samples.

The misuse of anabolic-androgenic steroids (AAS) in sports aiming at enhancing athletic performance has been a challenging matter for doping control laboratories for decades. While the presence of a xenobiotic AAS or its metabolite(s) in human urine immediately represents an antidoping rule violation, the detection of the misuse of endogenous steroids such as testosterone necessitates comparably complex procedures. Concentration thresholds and diagnostic analyte ratios computed from urinary steroid concentrations of, e.g., testosterone and epitestosterone have aided identifying suspicious doping control samples in the past. These ratios can however also be affected by confounding factors and are therefore not sufficient to prove illicit steroid administrations. Here, carbon and, in rare cases, hydrogen isotope ratio mass spectrometry (IRMS) has become an indispensable tool. Importantly, the isotopic signatures of pharmaceutical steroid preparations commonly differ slightly but significantly from those found with endogenously produced steroids. By comparing the isotope ratios of endogenous reference compounds like pregnanediol to that of testosterone and its metabolites, the unambiguous identification of the urinary steroids' origin is accomplished. Due to the complex urinary matrix, several steps in sample preparation are inevitable as pure analyte peaks are a prerequisite for valid IRMS determinations. The sample cleanup encompasses steps such as solid phase or liquid-liquid extraction that are presumably not accompanied by isotopic fractionation processes, as well as more critical steps like enzymatic hydrolysis, high-performance liquid chromatography fractionation, and derivatization of analytes. In order to exclude any bias of the analytical results, each step of the analytical procedure is optimized and validated to exclude, or at least result in constant, isotopic fractionation. These efforts are explained in detail.