Development Of Sample Preparation Method Research Articles

Automated high throughput IgG N-glycosylation sample preparation method development on the Tecan Freedom EVO platform.

Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation. The study aimed to develop an automated method for sample preparation on the Tecan Freedom Evo 200 platform and compare its efficiency and precision with the manual counterpart. The initial method development included 32 pooled blood plasma technical replicates. An additional 24 pooled samples were used in the method comparison along with 78 random duplicates of plasma samples collected from 10,001 Dalmatians biobank to compare the manual and automated methods. The development resulted in a new automated method. For the automated method, glycan peaks comprising 91% of the total sample glycan showed a variation of less than 5% while 92% of the total sample showed a variation of less than 5% for the manual method. The results of the Passing-Bablok regression indicated no differences between the automated and manual methods for 12 glycan peaks (GPs). However, for 8 GPs systematic difference was present, while both systematic and proportional differences were present for four GPs. The developed automated sample preparation method for IgG glycan analysis reduced exposure to hazardous chemicals and offered a simplified workflow. Despite slight differences between the methods, the new automated method showed high precision and proved to be highly comparable to its manual counterpart.

Development of sample preparation methods for time-resolved cryogenic electron tomography and micro crystal electron diffraction

Development of sample preparation methods for time-resolved cryogenic electron tomography and micro crystal electron diffraction

Development of Sample Preparation Methods for Vitamin-Mineral Complexes and Determination of their Elemental Composition Using Total Reflection X-Ray Fluorescence Analysis

Development of Sample Preparation Methods for Vitamin-Mineral Complexes and Determination of their Elemental Composition Using Total Reflection X-Ray Fluorescence Analysis

Design of Experiments for Matrix-Assisted Laser Desorption/Ionization of Amphiphilic Poly(Ethylene Oxide)-b-Polystyrene Block Copolymers.

Matrix-assisted laser/desorption ionization (MALDI) has become a very popular ionization technique for mass spectrometry of synthetic polymers because it allows high throughput analysis of low amounts of sample while avoiding the complexity introduced by extensive multiple charging of electrospray ionization. Yet, fundamental mechanisms underlying this ionization process are not fully understood, so development of sample preparation methods remains empirical. Reliable prediction for the optimal matrix/analyte/salt system is indeed still not possible for homopolymers and it becomes even more challenging in the case of amphiphilic block copolymers where conditions dictated by one block are not compatible with MALDI requirements of the second block. In order to perform MALDI of copolymers composed of poly (ethylene oxide) (PEO) and polystyrene (PS) blocks, it was postulated here that experimental conditions suitable for both species would also be successful for PEO-b-PS. Accordingly, designs of experiments based on Quantitative Structure Activity Relationship (QSAR) analysis were first implemented, studying the influence of 19 matrices and 26 salts on the laser fluence requested for successful MALDI. This analysis first permitted to highlight correlations between the investigated 10 descriptors of matrices and salts and the analytical response, and then to construct models that permits reliable predictions of matrix/salt couples to be used for one or the other homopolymer. Selected couples were then used for MALDI of a PEO-b-PS copolymer but no general trend was observed: experimental conditions expected to work often failed whereas ionic adducts of the copolymer were clearly detected with some matrix/salt systems that were shown to badly perform for constituting homopolymers. Overall, this rules out the working assumption stating that the MALDI behavior of chains composed of PEO and PS segments should combine the behavior of the two polymeric species. Yet, although requiring a dedicated design of experiments, MALDI of the amphiphilic PEO-b-PS copolymer was achieved for the first time.

Recent sample pretreatment methods for determination of selective serotonin reuptake inhibitors (SSRIs) in biological samples

Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (FLU), sertraline (SER), paroxetine (PAR), fluvoxamine (FLV) and citalopram (CIT) have been the first treatment drugs for pregnant and breastfeeding women. Quantitative analysis of SSRIs in biological samples is extremely needed in public health and clinical practice. During the analysis, sample pretreatment is an important step that can obtain an accurate quantitative analysis of SSRIs in the complex samples. The present paper discussed the recent development of sample preparation methods for SSRI analysis. Traditional sample preparation techniques such as liquid liquid extraction (LLE) and solid phase extraction (SPE), which have been widely used in the separation of SSRIs in biological samples, were extensively presented. Moreover, the new sample preparation techniques including liquid phase microextraction (LPME), solid phase microextraction (SPME), electromembrane extraction (EME) and other miniaturized extraction techniques, which are becoming highly popular in SSRI analysis, were also critically reviewed. In this review, both the advantages and disadvantages of these sample pretreatment methods were addressed. As a summary, we prospected the challenges and promising directions for the future of sample pretreatment methods in SSRI analysis.

THE DEVELOPMENT AND THE VALIDATION OF THE METHOD FOR TRIMETOPRIM RESIDUES DETERMINATION IN TISSUES SAMPLES USING THE METHOD OF ENZYME-LINKED IMMUNOSORBENT ASSAY

The article presents the results of the development of sample preparation method of muscle and parenchymal tissues for their further testing on trimethoprim residues by enzyme-linked immunosorbent assay using Trimethoprim test kits from Kwinbon Biotechnology (China), designed to analyze another target matrix, viz. honey. The results of the comparison of sample preparation methods with the use of different techniques of analyte extraction from tissues, with the established percentage of extraction are presented. The most effective conditions are proposed: acetonitrile extraction using ultrasonic intensification, subsequent concentration of the analyte by evaporation, reconstitution of dried residue and degreasing. This approach makes it possible to determine trimethoprim residues in poultry tissues at the level of 5-10 μg/kg with an absolute recovery of about 75%. In accordance with the Decision of the European Commission 2002/657/EC, the validation of the proposed method was carried out taking into account the maximum permissible levels (MRL) of trimethoprim in the tissues of productive animals. The suitability of the method was confirmed on the basis of establishing of the main validation parameters for screening methods (technical threshold and cut-off factor) using control (pure) and spiked with a standard solution of trimethoprim at the level of ½ MRL samples of muscle and parenchymal tissues of broiler chickens. The main advantages of the developed technique are simplicity of performance, rapidity and cost effectiveness. The certainty of the results obtained using the developed method was verified using the confirmatory UPLC-MS/MS method. The article presents the results of a comparative study of pure samples of broiler chickens muscles and parenchymal tissues and spiked at the level of ½ MRL with a standard solution of trimethoprim. The developed screening technique of enzyme-linked immunosorbent assay is recommended for determination of residual amounts of trimethoprim in tissue samples and can be used for routine laboratory tests, as well as to study the withdrawal procedure of veterinary drugs based on this drug.

Актуальність проблеми визначення діоксинів і поліхлорованих біфенілів (ПХБ) у продуктах для дитячого харчування

The Aim of the Article. Analysis of the current state of the problem of the dioxins and polychlorinated biphenyls (PCBs) determination in baby food products and the development of sample preparation methods for determining these xenobiotics in food products for infants and young children. Dioxins form a group of structurally and chemically related chlorinated tricyclic oxygen-containing aromatic compounds (congeners), which includes 75 polychlorinated dibenzo-para-dioxins (PCDDs) and 135 polychlorinated dibenzofurans (PCDFs). The most toxic congeners of dioxins, in which chlorine atoms along with other positions are necessarily in 2,3,7,8 positions of benzene rings. Their total number is 17:7 PCDD congeners and 10 PCDF congeners. A group of polychlorinated biphenyls (PCBs) – chlorinated bicyclic aromatic compounds, which consists of 209 different congeners, 12 of which have a spatial and electronic structure and exhibit toxicological properties similar to dioxins, therefore, they are called dioxin-like PCBs (DL-PCBs). In addition, when monitoring food products as a marker, a group of 6 PCBs was selected that did not exhibit dioxin-like toxicity and therefore did not belong to dioxin-like PCBs (NDL-PCBs). Thus, of the total number of 419 PCDDs, PCDFs and PCBs, only 35 are toxicologically significant, therefore, these compounds are subject to control in baby food products. To control the content of the amount of dioxins, the amount of dioxins and DL-PCBs and the amount of NDL-PCBs in foods for infants and children from one to three years in Ukraine, the maximum levels of these compounds on the basis of the order of the Ministry of Health of Ukraine No. 368 of 05/13/2013 were harmonized with the maximum allowable levels in foods for infants and young children in accordance with Commission Regulation (EU) No. 1259/2011. Methods for the analysis of dioxins and PCBs. Two instrumental analysis methods are used to determine dioxins and dioxin-like PCBs in foods for baby food: 1) a combination of high-resolution (capillary) gas chromatography with high-resolution mass spectrometry (GC/MS); 2) a combination of GCHR with tandem mass spectroscopy (GC/MS/MS). High-resolution (capillary) gas chromatography is used to determine non-dioxin-like (marker) PCBs. The stage of sample preparation, including the stages of extraction and purification, is key in determining dioxins and PCBs. Conclusions. The stage of sample preparation using automatic devices (liquid extraction under pressure, an automated extract purification system) and chromatographic columns was developed to further determine the mass concentration of dioxins and PCBs in baby food products. The developed procedures using devices for the automatic extraction and purification of the obtained extracts from baby food samples will make it possible in the future to determine PCDDs/PCDFs, ortho-unsubstituted, mono-ortho-substituted and marker PCBs in one sample. Key Words: dioxins, polychlorinated biphenyls, baby food.

Development of sample preparation method for organochlorine pesticides analysis in soil samples

OCPs are the persistent organic pollutants which are highly toxic to the environment and can accumulate in humans by foods such as rice, vegetable, and fruits. The aim of this research was to develop a rapid and easy sample preparation method for determination 4 OCPs, aldrin, dieldrin, β-endosulfane, and p-p' DDT in soil samples by gas chromatography with electron capture detection. The analysis was performed by using HP-5 capillary column with the isothermal condition at 250 oC and carrier gas flow rate of 1 mL/min. The results show that the 4 OCPs and internal standard, pentachloronitrobenzene, were analyzed within 6 min. The optimized condition of the sample preparation method was using ultrasonic assisted solvent extraction with 20 mL of hexane and ethyl acetate (9:1 v/v) as an extractant for 15x2 min. The results obtained from the proposed sample preparation method have demonstrated that the limit of detection was 0.628–3.68 μg kg-1. Percent recoveries were in the range of 81.42 to 110.7% and RSD were 1.68–9.43%. Correlation coefficients of calibration graphs were more than 0.99. The developed sample preparation method exhibited simplicity rapid and low chemical reagent consumption with satisfactory results.

Biodistribution of polycyanoacrylate nanoparticles encapsulating doxorubicin by Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry Imaging (MSI)

Nanoparticles are widely used as carrier for the delivery of Active Principle Ingredients (API). From safety point of view, the fate of nanoparticles and API after administration becomes a key question. Today qualitative and quantitative biodistribution of such carriers or encapsulated API can be performed by chemical modification with a fluorescent probe or a radiolabeling and require a first acquisition step for visualization and a second step for quantification by tissues extraction. LC-MS/MS can be considered but the information about the localization of the API is lost.After a long period focused on the sample preparation method development, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) is nowadays recognized as the most promising technique for the study of biodistribution of small molecular weight API without additional labeling, combining a visual and quantitative approach.In this study, we extended the scope of use of MALDI-MSI to polycyanoacrylate nanoparticles loaded with doxorubicin. MALDI-MSI allowed the detection and quantification of doxorubicin and polycyanoacrylate, the polymer used as carrier, forming the nanoparticles in mouse liver section. Additionally, MALDI-MSI was able to detect doxorubicinol, the metabolite of doxorubicin.This Proof Of Concept was successful emphasizing the benefits of this technique (label free and quantitative approach) and offers the possibility to use a unique technique for the detection of encapsulated API and the carrier opening a wide field of application in drug delivery of formulated API.

Refinement of a Methodology for Untargeted Detection of Serum Albumin Adducts in Human Populations.

Covalently modified blood proteins (e.g., serum albumin adducts) are increasingly being viewed as potential biomarkers via which the environmental causes of human diseases may be understood. The notion that some (perhaps many) modifications have yet to be discovered has led to the development of untargeted adductomics methods, which attempt to capture entire populations of adducts. One such method is fixed-step selected reaction monitoring (FS-SRM), which analyses distributions of serum albumin adducts via shifts in the mass of a tryptic peptide [Li et al. (2011) Mol. Cell. Proteomics 10, M110.004606]. Working on the basis that FS-SRM might be able to detect biological variation due to environmental factors, we aimed to scale the methodology for use in an epidemiological setting. Development of sample preparation methods led to a batch workflow with increased throughput and provision for quality control. Challenges posed by technical and biological variation were addressed in the processing and interpretation of the data. A pilot study of 20 smokers and 20 never-smokers provided evidence of an effect of smoking on levels of putative serum albumin adducts. Differences between smokers and never-smokers were most apparent in putative adducts with net gains in mass between 105 and 114 Da (relative to unmodified albumin). The findings suggest that our implementation of FS-SRM could be useful for studying other environmental factors with relevance to human health.

Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography–mass spectrometry

The number and positions of disulfide linkages in a therapeutic monoclonal antibody (mAb) play a crucial role in forming and stabilizing a correct mAb structure that is critical to its function. Peptide mapping by liquid chromatography–mass spectrometry (LC–MS) analysis of enzymatically digested mAb under nonreducing condition is a powerful method for disulfide linkage characterization to ensure mAb drug function and quality. However, the development of a robust sample preparation method with improved digestion efficiency and minimized disulfide scrambling for disulfide linkage analysis is essential but challenging. In this study, a sample preparation method for analysis of correct disulfide linkages in therapeutic mAbs was developed. Instead of common trypsin digestion, Lys-C plus trypsin was used in this approach to improve digestion efficiency. In addition, lower digestion temperature (25 °C) and lower digestion pH (pH 6.8) were also examined to minimize disulfide scrambling. Our results showed that Lys-C plus trypsin digestion at pH 6.8 and 25 °C is a better sample preparation condition for all therapeutic mAbs tested in this study because of a better digestion efficiency (all expected disulfide linkages can be confidently observed) and minimal disulfide scrambling.

Small-molecule bioanalytical sample preparation method development starting from the BASICS.

To develop a robust small-molecule bioanalytical sample preparation method, systematic investigations of the bioanalytical handling conditions of analytes andIS of interest are preferred. So far, such investigations are done manually and are labor-intensive and error-prone. An automation-assisted system has been developed to facilitate such systematic investigations. The system takes experimental design and automates the majority of the wetlaboratory work. In addition, the system also automates the data extraction, recovery/loss computation, graphing and reporting. The automation-assisted system greatly reduces errors and labor involved in the systematic investigation of analytes andIS both for wet chemistry experiments and for data extraction and processing, enhances data processing efficiency and overall sample preparation method development productivity.

Bioanalytical laboratory automation development: why should we and how could we collaborate?

Bioanalytical laboratory automation development: why should we and how could we collaborate?

Plasma, blood and liver tissue sample preparation methods for the separate quantification of liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone

Besides the development of sample preparation methods for the determination of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine plasma and blood, this article also presents the first description of an accurate sample preparation method for the determination of such separate concentrations in the murine liver. The quantitative differentiation is based on the immediate hydrolysis of prednisolone phosphate (PP) into prednisolone (P) after its release from the liposomes in vivo: PP represents the encapsulated drug, while P represents the non-encapsulated drug. The use of 10 ml methanol/g tissue during homogenization of liver tissue ensures complete liposome rupture, prevention of the dephosphorylation of PP released during homogenization, sufficient clean supernatants, excellent extraction of P and sufficient extraction of PP and excellent accuracies and precision complying with the internal guidelines for pre-clinical studies (80–120% and maximal 20%, respectively). Similarly, the matching sample preparation methods for plasma and blood involve protein precipitation with four equivalents of methanol also ensuring accuracies and precision complying with the internal guidelines for pre-clinical studies. Application of these sample preparation methods is going to generate the first pharmacokinetic (PK) profile of a liposomal preparation, in which the encapsulated and non-encapsulated drug concentrations in a tissue are measured separately. Such separated concentration profiles can gain important insights into the PKs of liposomal PP and probably also with regard to liposomal formulations in general, like the quantification of the in vivo drug release from the liposomes.

Protein extraction from plant tissues for 2DE and its application in proteomic analysis

Plant tissues contain large amounts of secondary compounds that significantly interfere with protein extraction and 2DE analysis. Thus, sample preparation is a crucial step prior to 2DE in plant proteomics. This tutorial highlights the guidelines that need to be followed to perform an adequate total protein extraction before 2DE in plant proteomics. We briefly describe the history, development, and feature of major sample preparation methods for the 2DE analysis of plant tissues, that is, trichloroacetic acid/acetone precipitation and phenol extraction. We introduce the interfering compounds in plant tissues and the general guidelines for tissue disruption, protein precipitation and resolubilization. We describe in details the advantages, limitations, and application of the trichloroacetic acid/acetone precipitation and phenol extraction methods to enable the readers to select the appropriate method for a specific species, tissue, or cell type. The current applications of the sample preparation methods in plant proteomics in the literature are analyzed. A comparative proteomic analysis between male and female plants of Pistacia chinensis is used as an example to represent the sample preparation methodology in 2DE-based proteomics. Finally, the current limitations and future development of these sample preparation methods are discussed. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP17).

Development of sample preparation method for eleutheroside B and E analysis in Acanthopanax senticosus by ionic liquids-ultrasound based extraction and high-performance liquid chromatography detection

An ionic liquids-based ultrasonic-assisted extraction (ILUAE) method was successfully developed for extracting eleutheroside B and E from Radix Acanthopanax senticosus. Thirteen 1-alkyl-3-methylimidazolium ionic liquids with different cations and anions were investigated and 1-butyl-3-methylimidazolium bromide ([C4mim]Br) solution was selected as the solvent. The conditions for ILUAE, including the ionic liquid concentration, soaking time, ultrasonic power, ultrasonic time, solid–liquid ratio and number of extraction cycles, were optimized. With the proposed method, the energy consumption time was reduced to 30min, whereas conventional method requires about 4h. The proposed method had good recovery (97.96–103.39%) and reproducibility (RSD, n=5; 3.3% for eleutheroside B, 4.6% for eleutheroside E). ILUAE was an efficient, rapid and simple sample preparation technique that showed high reproducibility and was environmental friendly.

Development of sample preparation method for isoliquiritigenin, liquiritin, and glycyrrhizic acid analysis in licorice by ionic liquids-ultrasound based extraction and high-performance liquid chromatography detection

An ionic liquid-based ultrasonic-assisted extraction (ILUAE) method had been used for the effective extraction of isoliquiritigenin (IQ), liquiritin (LQ) and glycyrrhizic acid (GA) from licorice. The ionic liquids with different cations and anions were investigated in this work and 0.5M 1-butyl-3-methylimidazolium bromide solution was selected as solvent. In addition, the technical parameters including soaking time, solid–liquid ratio, ultrasonic power and time were optimized. Compared with the conventional solvent extraction, the proposed approach exhibited higher efficiency, which indicated the ILUAE was an efficient, rapid and simple sample preparation technique. There was no degradation of the target analytes had been observed at the optimum conditions which was evidenced by the stability studies performed with standard of IQ, LQ and GA. The proposed method also showed high reproducibility and was environmental friendly.

Flotation as a tool for indirect DNA extraction from soil

Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 microg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.

Development of Sample Preparation Method for Observation of Dopant Profile by Electron Holography

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Solid phase extraction conditions for the selective isolation of drugs from biological fluids predicted using liquid chromatography

A new method to predict the most suitable conditions for the solid phase extraction of 1,4-benzodiazepines and related compounds using C18 Sep-Pak carridges is proposed. The composition of the washing and elution solvents for the solid phase extraction of a test compound can be obtained from its capacity factor on a C18 HPLC column and an equation which relates capacity factors and solid phase extraction data of other similar compounds. The solid phase extraction data given in this paper can be used by others, there by saving considerable time and effort in the development of sample preparation methods. The suitability of the method was checked with two test compounds, showing good results.