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Sample Preparation for Mass Spectrometry Applications
... 2 Ideally sample preparation strategies will repeatedly provide extracts containing high amounts of the target analyte(s) for measurement without interference, while using low amounts of resource and time to do so. 1 However, a compromise is often encountered during development with only some of these desirable characteristics achieved, with these depending on whether a targeted preparation or a "screen" of the sample for unknown materials (i.e. biomarker studies) is required. ...
... biomarker studies) is required. 1 Example complex mixtures include environmental and clinical samples and these have traditionally used multistep preparations with a range of procedures and equipment, resulting in time and resource consuming methods. 1 With greater focus on links between environmental and public health, and increased regulation for pollution and waste management, the selection of environmental matrices, target chemistries and their (trace) amounts for analysis 3,4,5 make the analytical challenge more difficult to address without appropriate sample preparation solutions. For example, the introduction of the Water Framework Directive 3 and Circular Economy legislation (waste and landfill 6 ) has increased the types of matrices requiring molecular (pollutant) characterization to include organisms, wastewater, sludges, and receiving sediments. ...
... 1 Example complex mixtures include environmental and clinical samples and these have traditionally used multistep preparations with a range of procedures and equipment, resulting in time and resource consuming methods. 1 With greater focus on links between environmental and public health, and increased regulation for pollution and waste management, the selection of environmental matrices, target chemistries and their (trace) amounts for analysis 3,4,5 make the analytical challenge more difficult to address without appropriate sample preparation solutions. For example, the introduction of the Water Framework Directive 3 and Circular Economy legislation (waste and landfill 6 ) has increased the types of matrices requiring molecular (pollutant) characterization to include organisms, wastewater, sludges, and receiving sediments. ...
Accurate measurement of the composition of complex samples is key for the safety and efficacy of a range of products used in daily life, with sample preparation a critical step in this workflow. QuEChERS is one such method, however published protocols do not explicitly address acidic, basic, neutral, and amphiphilic species in a single protocol and often use extra steps or an alternative preparation to recover the breadth of chemical types. Our work addresses this need by investigating the use of QuEChERS for monitoring this wide range of chemistries within environmental solids and blood plasma, using a protocol that can accommodate both milliliter and microliter sample volumes. While published methods can require significant resource and time, our approach offers a reduction in preparation time (for environmental samples), with the “micro‐QuEChERS” protocol offering a further reduction in cost. The analytical performance of these methods were assessed using reversed‐phase LC‐MS and showed good accuracy, precision, and sensitivity for the expected concentrations in the tested applications. Target analytes of variable lipophilicity/acidity were extracted and isolated from soil, with largely repeatable matrix effects < 15%RSD and recoveries of 39‐100%. An initial “proof‐of‐concept” investigation using the “micro‐QuEChERS” protocol showed reduced matrix enhancement (median value of 90%ME) for soil, and improved matrix effects and recovery (>65%) for blood plasma. This novel sample preparation method can therefore offer an improved approach with wider applicability providing “cleaner” extracts than other methods used for high‐throughput clinical analysis.
... Sample pretreatment steps are critical for hyphenated-MS-based analysis (Wells, 2023). This is especially true for urine specimens because of the complexity of the urine matrix. ...
... Obviously, diluted specimens contain more matrix components than post-LLE/SPE extracts; in other words, diluted specimens are dirtier than post-LLE/SPE extracts. Thus, the diluteand-shoot method will contaminate inside the LC-MS instrument (e.g., protein buildup in the LC part and ionization source) more than post-LLE/SPE extracts, causing service disruption because of erratic test results and/or more frequent downtime, unless adequate maintenance work is given to the instrument (Wells, 2023). ...
Toxicology testing is performed in clinical settings, forensic settings, and for controlling doping. Drug screening is a toxicology test to determine if drugs are present in biological samples. The most common specimen type for drug testing is urine, as drugs and/or their metabolites are often more concentrated in the urine, extending the detection window of drugs. The dilute-and-shoot method is a simple procedure used in toxicology testing, where a sample is diluted before being directly injected into the liquid chromatography-mass spectrometry (LC-MS) system. This method is easy, quick, and cost-saving, and can be used for protein-poor liquid specimens such as urine. Thus, it is reasonable and attractive for busy toxicology laboratories to combine the dilute-and-shoot method with high-resolution hyphenated-MS for urine drug screening. This method has several disadvantages, including a suboptimal detection capability for certain analytes, as well as interference from co-eluting matrix components called matrix effects, in which co-eluting matrix molecules alter the ionization efficiency of the analyte molecules at the ionization source in LC-MS, altering (mostly reducing) the analyte detection capability. The matrix effect testing is essential for the validation of LC-MS-based assays. A reasonable approach to addressing these undesirable effects would be to minimize these components. The most straightforward approach is to reduce the amounts of matrix components by using a higher dilution of the specimen and a lower volume for specimen injection. Optimization of the chromatographic separation is another reasonable approach for reducing co-eluting matrix components with the analyte.
... For measuring the concentration of FLX in biological matrices, few HPLC, gas chromatography mass spectrometry (GCMS) and liquid chromatography mass spectrometry (LCMS) methods are available. [10,14,15] These methods, however, have some drawbacks, therefore, LC-MS/MS approach for measuring FLX was considered by utilizing the RapidTrace ® automated SPE system, [16,17] and detailed processes are given in Table 1. To create a reliable, consistent, and reproducible LC-MS/MS method chromatographic, separation conditions were optimized after MS/MS optimization to offer adequate separation and peak symmetry with an appropriate response. ...
A wide-range, specific, and precise liquid chromatography tandem mass spectrometric (LC-MS/MS)technique for quantifying fluoxetine (FLX) in human plasma was developed using the RapidTrace® automated solid-phase extraction (SPE) method; the analyte and internal standard (IS) were extricated on Oasis MCX SPE cartridges. Acetonitrile and 5 mM ammonium formate buffer (90:10 v/v) were used as mobile phase to achieve chromatographic separation on the reverse phase (C18 column). The analyte and IS were ionized using +ve electrospray ionization approach which was further traced by multiple-reaction monitoring on a tandem mass spectrometer. To quantify the FLX and FLX-d5, the parent-to-daughter ion transition of m/z of 310.0/44.1 and 315.0/44.0 was used, respectively. The method demonstrated a linear active limit of 0.20-30 ng/ml with recoveries ranging from 63.04% to 79.39% for quality control samples and 61.25% for IS samples. The concentrations over the calibration range demonstrated acceptable precision and accuracy. Due to the high inconsistency of the FLX concentration data, the minimum threshold of the assay was kept at 0.20 ng/ml. The flow rate was maintained at 500 μL/min, and the time for sample analysis for each injection was 3.5 min. The method was found to be specific, sensitive, and faster with minimum utilization of organic solvents and was utilized further for metabolic and pharmacokinetic studies.
... 35,45 However, thorough cleanup of the sample digests by offline solid phase extraction (SPE), may be preferred. 74 Specific anion-or cation-exchange cartridges have been shown useful for peptide purification. 75 SPE with hydrophilic lipophilic balanced (HLB) cartridges has been applied in various studies. ...
Protein mass spectrometry (MS) is an enabling technology that is ideally suited for precision diagnostics. In contrast to immunoassays with indirect readouts, MS quantifications are multiplexed and include identification of proteoforms in a direct manner. Although widely used for routine measurements of drugs and metabolites, the number of clinical MS-based protein applications is limited. In this paper, we share our experience and aim to take away the concerns that have kept laboratory medicine from implementing quantitative protein MS. To ensure added value of new medical tests and guarantee accurate test results, five key elements of test evaluation have been established by a working group within the European Federation for Clinical Chemistry and Laboratory Medicine. Moreover, it is emphasized to identify clinical gaps in the contemporary clinical pathways before test development is started. We demonstrate that quantitative protein MS tests that provide an additional layer of clinical information have robust performance and meet long-term desirable analytical performance specifications as exemplified by our own experience. Yet, the adoption of quantitative protein MS tests into medical laboratories is seriously hampered due to its complexity, lack of robotization and high initial investment costs. Successful and widespread implementation in medical laboratories requires uptake and automation of this next generation protein technology by the In-Vitro Diagnostics industry. Also, training curricula of lab workers and lab specialists should include education on enabling technologies for transitioning to precision medicine by quantitative protein MS tests.
... From the different types of PP methods, the use of a miscible organic solvent is the most convenient approach to prepare complex matrices (like plasma), because of its reduced cost and minimal requirements for method development, even when compared with SPE and LLE procedures [45]. However, one limitation of this method is the fact that it does not allow to concentrate the analyte [46], except if an evaporation step is added [47]. In spite of this, PP can also be used as a complementary technique, being followed by another sample preparation procedure, such as SPE. ...
Since dopamine (DA) was discovered as an essential neurotransmitter, with a profound impact on motor control, memory, and behavioral impulses, the pathogenesis of several neurodegenerative and neuropsychiatric disorders have been associated with the dysfunction of the dopaminergic system. Regarding this, the most common drugs used to treat these pathologies act on the dopaminergic neurons. Therefore, the measurement of DA and its precursors and metabolites levels can be a useful tool to help the diagnosis and development of targeted therapeutic approaches to neurological disorders. Furthermore, monitoring and detecting DA metabolism (DA, precursors, and metabolites) in biological samples, like plasma, urine, and cerebrospinal fluid, constitute an interesting subject from a clinical perspective. However, the development of suitable and efficient methods to determine these compounds in biological samples remains a challenge. Thus, this review provides an overview of the recent advances and available methodologies to quantify DA and its precursors and metabolites in plasma samples focusing on previous reports which used less than two milliliters. Also, it deals with the actual extraction and separation techniques, as well as detection modes; and it gives a perspective, on the present-day, about the use of analytical methods as a helpful tool to improve diagnosis.
... -isolate the analyte of interest from the biological sample and place it in an LC-MS/MS compatible matrix, -concentrate or dilute the analyte of interest, depending on the detection limit of the LC-MS/MS system being used, and -separate the analyte from matrix components and other compounds that could interfere with the measurement [4,17,[53][54][55]. ...
Tandem mass spectrometry (tandem MS) is a powerful technique that directly measures compounds based on their molecular weight. Liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS), a type of tandem MS, has been increasingly adopted by clinical laboratories in recent years. While it is complicated and challenging, it has selectivity and accuracy advantages over more traditional laboratory techniques such as immunoassay. Able to measure a wide range of compounds, when correctly utilized and implemented, LC-MS/MS can benefit the clinical laboratory and improve patient care. This mini review is an introduction to clinical LC-MS/MS for the novice user.
Functionalized nanoparticles, such as drug-loaded liposomes and extracellular
vesicles (EVs) are widely used as nanocarriers for disease biomarkers due to their
excellent biocompatibility. These nanoparticles hold significant potential for both
therapeutic and diagnostic applications, particularly in biomedical research. However,
the physical and chemical heterogeneity within a population of nanocarriers poses
challenges, as it can impact bio-distribution and targeting. Since the chemical
composition and size of nanoparticles critically influence their function, precise
characterization techniques are essential. Standard nanoparticle tracking analysis
(NTA), a more recent technique, is widely used to address these challenges, but it has
limitations, including being time-intensive, requiring skilled operators, and involving
complex theoretical considerations. This review begins by introducing liposomes and
EVs as nanoparticle systems. It then discusses the theory of optical trapping-based
measurements, principles of standard NTA technique, compares it to other
characterization methods, examines existing algorithms, and highlights current
challenges. Additionally, it explores the potential of deep learning techniques for
enhancing optical trapping-based measurements and standard NTA technique, and
concludes with a discussion on the challenges faced by deep learning approaches in
this context.
Clinical and diagnostic tests in laboratories are useful in screening, diagnosis, and prognosis. These tests require grinding specimens. Commonly, this is done by hand using mortars and pestles. With this method, only one sample can be done at a time, and the results are not uniform. A novel device for simultaneously grinding multiple tissue samples without cross-contamination is described below. The device consists of a first unit having several pestles and Eppendorf housings separated from each other, a second unit containing a planetary gear system connected to operate these Eppendorf tubes and pestles, and a third unit containing a motor and controller. These units are geometrically configured and dimensioned so that when they are stacked on top of one another they are in lock position and consequently the pestles are lined up with the Eppendorf tubes. The device was built so that 12 samples can be grinded at a time. A programable controller allowed users to select the grinding time at will. A mechanism permitted users to eject conveniently the pestles into the Eppendorf tubes after grinding to help avoid cross contamination. Many tests with shrimp leg tissues infected by white spot syndrome virus (WSSV) against controlled samples consisted of pure water were conducted. The grinded samples were inspected by eyes and checked for contamination using Polymerase Chain Reaction (PCR) machine. The results showed that device allows grinding tissues homogeneously in an effective way and reducing handiwork without cross-contamination. Small industrial production were conducted.
Sedentary lifestyle, malnutrition and intense work hours resulting in high stress are challenging the human mind and psychology. Many people are genetically predisposed, but many, unfortunately, are unable to withstand this life and need professional support. Although technology is produced to make life easier, in practice it causes many people to live outside the border. Antidepressants, which are used as a result of professional support, benefit in the short term, but they cause serious damage to environmental and aquatic life on the invisible side of the iceberg due to personal waste, irregular use and overdose. If there is no follow-up and environmental removal, all non-target creatures other than humans will be damaged in a very short time, and hence this damage will return to the human being, like a boomerang. Although many methods have been developed for the treatment of these types of pollutants today, it is clear that new agents and methods should be developed that will enable mobile study in natural environment where the pollution emerges. Accordingly, the aim of this review is to make a critical analysis of studies on the determination, removal, and elimination of antidepressants in recent years and to inspire future studies.
Sample preparation is an important step in forensic toxicological analysis. With the technological advancements and the availability of mass spectrometers with increasing sensitivities, the need to remove potential interferences, such as matrix components or non-relevant analytes to the analysis, is essential to optimizing an analytical method. Forensic toxicological analysis involves diverse analytes with a variety of chemical properties that need to be considered when selecting an appropriate sample preparation technique. The push for broad standards and best practice documents for forensic toxicology demonstrates the need for laboratories to ensure they meet the required capabilities to not only detect specific classes of drugs commonly encountered in forensic case work, but also ensure method validity. The objective of this article is to provide the forensic toxicology community a high-level overview of biological matrices and their relevant components that should be considered during sample pre-treatment as well as traditional and non-traditional sample preparation techniques and present general applications.
This article is categorized under:
• Toxicology > Drug Analysis
Abstract
This chapter presents the mechanical processing in sample preparation which includes grinding, sieving, common filtration, microfiltration, and ultrafiltration with a discussion about membrane materials and filtering devices. It also discusses centrifugation and its use in connection with filtration and ultrafiltration.
A high throughput sample preparation method was developed utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. Plasma was mixed with 3% aqueous ammonia and loaded into each well of 96-well plate. After washing with water and methanol sequentially, 3% of formic acid in acetonitrile was used to elute arachidonic acid. The collected fraction was injected onto a reversed phase column at 30°C with mobile phase of acetonitrile/water (70 : 30, v/v) and detected by LC-MS/MS coupled with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r
2 = 0.9999). The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%. The limit of detection is 3 ng/mL.
Urine is one of the most important biofluids in clinical proteomics, and in the past decades many potential disease biomarkers have been identified using mass spectrometry-based proteomics. Current studies mainly perform analyses of the urine supernatant devoid of cells and cell debris, and the pellet (or sediment) fraction is discarded. However, the pellet fraction is biologically of interest. It may contain whole human cells shed into the urine from anatomically proximal tissues and organs (e.g., kidney, prostate, bladder, urothelium, and genitals), disintegrated cells and cell aggregates derived from such tissues, viruses and microbial organisms which colonize or infect the urogenital tract. Knowledge of the function, abundance, and tissue of origin of such proteins can explain a pathological process, identify a microbe as the cause of urinary tract infection, and measure the human immune response to the infection-associated pathogen(s). Successful detection of microbial species in the urinary pellet via proteomics can serve as a clinical diagnostic alternative to traditional cell culture-based laboratory tests. Filter-aided sample preparation (FASP) has been widely used in shotgun proteomics. The methodology presented here implements an effective lysis of cells present in urinary pellets, solubilizes the majority of the proteins derived from microbial and human cells, and generates enzymatic digestion-compatible protein mixtures using FASP followed by optimized desalting procedures to provide a peptide fraction for sensitive and comprehensive LC-MS/MS analysis. A highly parallel sample preparation method in 96-well plates to allow scaling up such experiments is discussed as well. Separating peptides by nano-LC in one dimension followed by online MS/MS analysis on a Q-Exactive mass spectrometer, we have shown that more than 1,000 distinct microbial proteins and 1,000 distinct human proteins can be identified from a single experiment.
Lipids play multiple key roles in biological systems, including acting as secondary energy reserves, being components of cellular membranes and participating in cell-signaling pathways. As a result, lipid defects have been implicated in the development of diseases. Consequently, the detailed analysis of lipids found in clinical samples, known as clinical lipidomics, is crucial for improving our understanding of disease mechanisms and progression, and the development of potential treatment strategies. This review documents the lipidomics workflow, beginning with a description of approaches to preparation of samples of lipids. We also describe of progress in mass spectrometry technologies that led to the development of several widely-used techniques of lipid analysis. The review concludes with a discussion on the major challenges that exist in clinical lipidomics studies and looks at potential solutions that can address them.
Dried blood spot (DBS) sampling, the collection of whole blood samples on paper, is an emerging technique used for bioanalytical methods. Several analytical challenges, such as possible effects of spotting volume, hematocrit and spot inhomogeneity are identified for these methods, however, no regulatory-based guidelines for the specific validation of DBS-based assays are available hitherto. To date, 68 validation reports concerning methods for the quantitative determination of drugs in human DBS could be traced in the literature, with large differences in the extensiveness of the reported validations. This review aims to present an overview of these published validations. Additionally, the different challenges of DBS-based assays are discussed and recommendations on how to perform validation tests addressing these challenges are provided.
A simple and selective ultra performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) assay was developed for the determination of the human plasma protein binding of four bioactive flavonoids (such as orientin and vitexin) in Polygonum orientale. Protein precipitation was used for sample preparation. Equilibrium dialysis technique was applied to determine the plasma protein binding under physiological conditions. The separation was achieved through a Waters C18 column with a mobile phase composed of 0.1% formic acid in acetonitrile and 0.1% aqueous formic acid using step gradient elution at a flow rate of 0.35 mL/min. A Waters ACQUITY™ TQD system was operated under the multiple reaction monitoring (MRM) mode of positive electrospray ionization. All of the recovery, precision, accuracy and stability of the method met the requirements. Good correlations (r>0.99) of the four compounds were found, which suggested that these compounds can be simultaneously determined with acceptable accuracy. Results showed that the plasma protein bindings of the four bioactive flavonoids were in the range of 74–89% over the six concentrations studied. The binding parameters containing protein binding affinity, protein binding dissociation constant, and protein binding site were studied. The maximum ability to bind with protein was also determined in the assay in order to understand the drug-protein binding of each compound better.
Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), mRNA, noncoding RNA (ncRNA; enriched in miRNA) and protein from the same tissues. After tissue selection, about 12-16 extractions of DNA, RNA or protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins.
Ketoconazole, an imidazole‐piperazine compound, is an orally active antimycotic agent. In addition, ketoconazole is a specific inhibitor of cytochrome P450 3A4. As about 60% of oxidized drugs are biotransformed by this isoform, the potential effect of a concomitant administration of ketoconazole on drug disposition may be of interest during drug development. The present paper describes three different approaches (methods A, B, and C) to attain high‐throughput sample preparation and analysis in the quantification of ketoconazole in human plasma. Method A consisted of acetonitrile precipitation in a 96‐well plate, transfer of the supernatant via a Tomtec Quadra 96 Model 320, and subsequent injection onto a 50 × 4.6 mm (i.d.) Develosil Combi‐RP‐5 column (packed with C30 bonded silica particles). Method B consisted of an identical sample preparation to method A with the exception that a Michrom Magic Bullet™ column, 2.0 → 0.50 mm (i.d., tapered bore) ×25 mm length, was used. Lastly, in method C, a turbulent‐flow chromatography (TurboFlow LC/APCI‐MS/MS) module was used for the direct analysis of ketoconazole in human plasma. A Sciex API 3000 was used in methods A and B, while a Micromass Quattro LC was employed in method C. Based on the values obtained for the calibrator (standard) and quality control samples, all three protocols yielded satisfactory accuracy, precision, and reduced manual sample preparation time. Copyright © 2000 John Wiley & Sons, Ltd.
The book is the first authoritative reference on high throughput bioanalytical sample preparation methods, strategies, products, and automation strategies for small molecule analysis. The book opens with the role of bioanalysis in the drug development process, describes the many available sample preparation techniques, details 96-well microplate product information, and explains how to perform high throughput methods using protein precipitation, liquid-liquid extraction, solid-phase extraction, and online sample preparation techniques. Each technique includes information on method development and automation strategies.
Educators and practitioners of analytical chemistry as well as researchers and graduate students of analytical chemistry, medicine, environmental science, biochemistry, pharmacology, geology, and food science.
Chemistry, Molecular Sciences and Engineering deals with a centrally located field; this field is located in between Earth and Environmental Sciences (EES), BIOMED and Materials. In this field the focus is largely based on Molecules, often with atomic resolution, and deals with Synthesis, Structures, Properties and their Applications. This topical introduction on the concept of Chemistry, Molecular Sciences and Engineering deals with an overview of the field and also marks the borders with other relevant science areas.
The application of a new silica-based, monolithic-type HPLC-column for fast separations is presented. The column is prepared according to a new sol-gel process, which is based on the hydrolysis and polycondensation of alkosysilanes in the presence of water soluble polymers. The method lends to "rods" made of a single piece of porous silica with a defined pore structure, i.e. macro- and mesopores. The main feature of silica rod columns is a higher total porosity, about 15% higher than of conventional particulate HPLC columns. The resulting column pressure drop is therefore much lower, allowing operation at higher flow rates including flow gradients. Consequently, HPLC analysis can be performed much faster, as it is demonstrated by various applications.
Shotgun proteomics has become a routine method for identifying, characterizing, and quantifying proteins on a large scale. Essential to these experiments is the digestion of the proteins within a sample to peptides using proteases, most commonly trypsin. Proteases generate peptide fragments suitable in length for separation using liquid chromatography and gas-phase fragmentation, and identification using tandem mass spectrometry. The use of proteases other than trypsin allows for further characterization of protein posttranslational modifications and topology. This chapter provides a summary of the proteases used for digestion of proteins for shotgun proteomics and the types of experiments for which they are best exploited. Since a protease usually requires access to specific protein domains, also described are common protein solubilization methods that maintain protease activity and are compatible with liquid chromatography-mass spectrometry. Potential analysis interferants that can be introduced during the digestion process and the steps that can be taken to minimize or eliminate them are discussed. In general, this chapter guides the reader through a discussion of theory relevant to protease digestions, and protocols for the analysis of proteins, both on a large scale and in a more targeted fashion, on purified protein complexes and membrane-embedded proteins.
In environmental and food chemistry, sampling is a multistage process, which includes collection of the sample itself and a series of steps, so-called sample preparation, aimed to provide a representative measurement of the sample lot/area. Homogenization is part of the sample preparation procedure and includes mixing and blending of the sample, particle size reduction, and mass reduction. Yet because homogenization techniques are tedious and laborious, some laboratories do not incorporate it as part of their analytical protocol. Consequently, biased results can be obtained as a result of poor sample homogeneity. Diverse equipment and several procedures are available on the market, such as grinding with the common procedure based on mortar and pestle, as well as other techniques such as shearing, beating, and use of other instrumentation. Depending on the type of sample and objectives to be accomplished, the homogenization procedure should be such that it provides uniform and repeatable results, using instrumentation that is easy to clean and maintain, is adapted to hold sample sizes of variable magnitude, and, preferably, that can be automated. In this chapter, several aspects related to sample homogenization are discussed in terms of needs, available techniques, and applications for environmental and food analysis. The final objective is to provide the necessary tools to achieve homogenate samples, and especially to implicate environmental and analytical chemists in this subject so as to contribute toward obtaining more exact, precise, and comparable analytical results.
Microdialysis is a powerful tool for the continuous monitoring neurotransmitters and other substances in the brain. This chapter reviews approaches to sampling and calibration as well as the methods that are employed for the analysis of the microdialysis samples. Both off-line and on-line systems are discussed as well as microfluidic applications.
The proteome of various species covers tens of thousands of proteins with their sequences annotated from their genomes. Proteome analysis of these species involves detailed qualitative and quantitative characterization of these proteins, including the associated modifications and interactions. Therefore, the speed and throughput of the proteome analysis technologies are the key components in governing the outcome of a comprehensive proteome analysis. This is particularly true for the shotgun approach where the proteome is digested followed by peptide sequencing using tandem mass spectrometry (MS/MS). Traditional protocols for enzyme digestion of protein mixtures may take 6-48 hours to complete and may not be able to handle all types of proteins. The speed and applicability of digestion methods may become the bottleneck of throughput improvement and proteome coverage, compared to fast, accurate, and sensitive MS analysis and powerful bioinformatics tools. Microwave energy has been employed to accelerate the protein sample workup for proteome analysis in the past decade or so. Enzyme- or chemical-induced proteolysis can be accomplished in minutes or even seconds with the assistance of microwave irradiation. Proteins difficult to handle by traditional methods can be processed using microwave-assisted sample preparation protocols. In this chapter, development and applications of microwave technologies to facilitate both enzymatic digestion and chemical digestion are reviewed. Approaches of microwave-assisted enzymatic digestion, particularly trypsin digestion, adapted to both in-solution- and in-gel-based protein analysis are discussed. The practical aspects of microwave-assisted chemical hydrolysis using different acids and base as a complementary or alternative tool for protein degradation are also addressed.
Ultrafiltration (UF) uses a semipermeable membrane to fractionate molecules in liquids of about 1000 daltons in molecular weight to 500 000 daltons. The membrane is made of either polymeric or inorganic materials and contains pores of a defined size distribution. UF is a simple process involving only the pumping of fluids.
The sample preparation method plays a critical role in untargeted profiling of biological fluids and tissues for biomarker discovery. In this chapter, commonly used sample preparation methods in global metabolomics by nuclear magnetic resonance and mass spectrometry, such as solvent precipitation, ultrafiltration, and solid-phase extraction, are critically discussed with an emphasis on how well they meet the requirements of an ideal metabolomic method, including (1) depth of metabolite coverage, (2) simplicity and minimal handling to prevent metabolite loss and/or degradation and reduce introduction of extraneous contaminants, (3) reproducibility, and (4) incorporation of a metabolism quenching step in order to reflect the true metabolome composition at the time of sampling. Recent developments in this field are also examined including in vivo methods such as microdialysis and solid-phase microextraction, turbulent flow chromatography, and dried blood spot sampling. Finally, recent comprehensive studies comparing the performance of various sample preparation methods are highlighted to aid in the selection of the most appropriate method for a given application.
Methods of sample preparation for the use of liquid chromatography (LC) coupled to mass spectrometry (MS), in order to produce the metabolic profiles required for metabonomic/metabolomic research, are described. These methods cover the collection, storage, and preparation of a range of sample types, including biofluids (e.g., plasma, serum, and bile) and tissues. The methods covered range from the relatively simple procedures required for samples such as urine, which generally involve no more than centrifugation and dilution, through more complex methods such as solvent precipitation, solid-phase extraction, or turbulent flow chromatography that can be applied to blood plasma up to tissue preparation.
Using restricted-access media, chromatographers can analyze low molecular weight compounds in biological fluids by direct injection. In part I, guest columnists Boos and Rudolphi classified and reviewed restricted-access media and its various chemistries. In part II, they compare several restricted-access media packings used in both single- and coupled-column modes and discuss the advantages and disadvantages of the two approaches.
In the first of a two-part series, "Sample Prep Perspectives" looks at restricted-access media and the chemistries of its many varieties. Using restricted-access media, chromatographers can analyze low molecular weight compounds in biological fluids by direct injection. Sample preparation and analysis times are reduced. In Part II, guest columnists Boos and Rudolphi will demonstrate the usefulness of restricted-access media in HPLC columns and column-switching experiments.
Monoliths are chromatography sorbents cast as a homogenous phase into chromatography columns as a single, continuous piece. In contrast, regular chromatographic sorbents are packed as individual particles. In this month's "Column Watch," the guest columnists compare three of these novel sorbents with a conventional packed-particle column in terms of porosity, static, and dynamic capacity. Monoliths show flow rate-independent separation efficiency and dynamic capacity as well as higher porosity than conventional columns.
Quantitative bioanalysis of dried plasma spots (DPS) is not subject to the impact of hematocrit and sample non-homogeneity that are often encountered in dried blood spot (DBS) assay. In the present report, an evaluation of plasma microsampling for DPS has been conducted for the first time using ritonavir as a model compound orally administered to dogs. For this evaluation, an LC-MS/MS method was developed and validated according to the current health authorities' guidance and industry practice for the analysis of ritonavir in DPS samples. The measured ritonavir concentrations in the DPS samples prepared using SAFE-TEC devices and directly from the conventional wet plasma using standard pipette were compared with each other and against those of conventional wet plasma. Both DPS results correlated well with each other and were comparable to those of the wet plasma. Good incurred sample reanalysis results were obtained for the two sets of DPS samples and wet plasma as well. The current plasma microsampling for DPS can serve as an alternative to DPS sampling via standard pipetting and wet plasma in in vivo studies.
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Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation.
Background:
Lung cancer claims highest rate of cancer related mortality worldwide, mainly due to late diagnosis and distant metastasis. Sputum cytology is the simplest, non-invasive and cost effective technique but it has low sensitivity due to lack of robust processing methods to retrieve all the diagnostic materials clogged in mucus, inflammatory exudates and blood.
Methods:
This study have compared conventional pick and smear method of sputum processing with samples prepared by homogenization methods using N-acetyl-l-cysteine, Dithiothreitol (DTT), CytoRich red solution and cell blocks (CBs) with respect to screening time, quality of staining, cellularity, smear background, nuclear and cytoplasmic morphology preservation, and diagnostic efficacy. The significance of CB prepared from homogenised samples for immunocytochemistry, protein extraction, Genomic DNA and RNA extraction were also evaluated on a cohort 3,185 samples. The significance of the morphological features in each of the techniques was statistically analysed using SPSS 11 software.
Results:
The smear background clarity, staining quality and diagnostic efficacy of samples processed in red solution was found to be superior to the conventional method (P < 0.0001), where as samples homogenized in DTT showed a better cellularity (P < 0.0001). CBs prepared from samples homogenized in red solution were found to be very significant (P < 0.0001) in increasing the diagnostic efficacy compared to other two methods. Immunocytochemistry and DNA extraction were found possible in CBs as well as from the cell suspension. A combined analysis of smears and CBs found to improve the sensitivity of sputum cytology.
Conclusion:
The study suggests homogenization of sputum in CytoRich ® red solution and cellblock preparations routinely for all samples to improve the sensitivity of sputum cytology. IHC and DNA extraction can be performed in sputum samples suggesting the role of sputum samples for ancillary techniques.
The global metabolic profiling of feces represents a challenge for both analytical chemistry and biochemistry standpoints. As a specimen, feces is complex, not homogenous and rich in macromolecules and particulate, non-digested, matter that can present problems for analytical systems. Further to this, the composition of feces is highly dependent on short-term dietary factors whilst also representing the primary specimen where co-metabolism of the host organism and the gut-microbiota is expressed. Thus the presence and the content of metabolites can be a result of host metabolism, gut microbiota metabolism or co-metabolism. Successful sample preparation and metabolite analysis require that the methodology applied for sample preparation is adequate to compensate for the highly variable nature of the sample in order to generate useful data and provide insight to ongoing biochemical processes, thereby generating hypotheses. The current practices for processing fecal samples for global metabolic profiling are described with emphasis on critical aspects in sample preparation: e.g., homogenization, filtration, centrifugation, solvent extraction and so forth and also conditions/parameter selection are discussed. The different methods applied for feces processing prior to metabolite analysis are summarized and illustrated using selected examples to highlight the effect of sample preparation on the metabolic profile obtained.
Copyright © 2015 Elsevier B.V. All rights reserved.
The direct dilution of blood with alkali has been introduced as an alternative to acid digestion for improvement of the analytical productivity when measuring trace elements using inductively coupled plasma mass spectrometry (ICP-MS). This study compared these two sample preparation methods for the ICP-MS determination of multiple elements in human blood and serum.
Aliquots (0.2 or 0.5mL) of human whole blood and serum samples, including reference samples (whole blood and serum), were subjected to alkali dilution (ammonia solution) or acid digestion (nitric acid). The samples were then analysed for their concentrations of Li, B, Mg, P, S, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Cd, Sb, Cs, Ba, Pb and U with a quadrupole ICP-MS instrument equipped with a collision/reaction cell.
Analysis of the reference materials showed that the alkali dilution and acid digestion methods provided equally good agreement with the reference values for Mg, Mn, Co, Ni, Cu, Zn, As, Se, Rb, Mo, Cd, Sb and Pb. The alkali dilution method generally gave better agreement with the reference values for Li, B, P, S, K, Cr and U, whereas acid digestion gave better agreement for Ca, Fe, Sr and Cs. Strong associations (R(2)>0.90) between the two methods were obtained for the concentrations of Li, B, Mn, Co, Cu, As, Se, Rb, Sr, Mo, Cd, Cs and Pb in the collected human whole blood and for Li, B, Mg, P, S, K, Fe, Co, Cu, Zn, As, Se, Rb, Sr, Sb and Cs in the collected serum.
The results suggest that the alkali dilution method is suitable for the determination of Li, B, Mn, Co, Cu, Zn, As, Se, Rb and Sr in whole blood and serum; Mo, Cd and Pb in whole blood; and Sb in serum by ICP-MS. Acid digestion is preferred for Fe and for low concentrations of Cs.
Copyright © 2014. Published by Elsevier Inc.
Expressions have been derived for plate height and minimum analysis time in open tubular columns in which laminar and turbulent flow of the mobile phase may be employed. Using these equations the role of various column parameters in fast analysis has been studied in the laminar and turbulent flow regions of gas and liquid chromatography. In the gas chromatography turbulent flow can lead to analysis times about one tenth of those obtained under comparable circumstances by using laminar flow. In liquid chromatography turbulence can shorten analysis times by as much as a factor of 104. In general turbulent flow chromatography entails the use of slightly longer column lengths and much larger pressure drops than would be needed under similar conditions in laminar chromatography.
This study presents an on-tissue proteolytic digestion and peptide extraction method using microwave irradiation for in situ analysis of proteins from spatially defined regions of a tissue section. The methodology utilizes hydrogel discs (1 mm diameter) embedded with trypsin solution. The hydrogel discs are applied to a tissue section, directing enzymatic digestion to a spatially confined area of the tissue. By applying microwave radiation, protein digestion is performed in 2 minutes on-tissue, and the extracted peptides are then analyzed by MALDI MS and LC-MS/MS. The reliability and reproducibility of the microwave-assisted hydrogel mediated on-tissue digestion is demonstrated by the comparison with other on-tissue digestion strategies, including comparisons with conventional heating and in-solution digestion. LC-MS/MS data were evaluated considering the number of identified proteins as well as the number of protein groups and distinct peptides. The results of this study demonstrate that a rapid and reliable protein digestion can be performed on a single thin tissue section while preserving the tissue architecture, and the resulting peptides can be extracted in sufficient abundance to permit analysis using LC-MS/MS. This approach will be most useful for samples that have limited availability but are needed for multiple analyses, especially for the correlation of proteomics data with histology and immunohistochemistry.
Sample preparation is a critical step in large-scale multiclass analysis such as sport drug testing. Due to the wide heterogeneity of the analytes and the complexity of the matrix, the selection of a correct sample preparation method is essential, looking for a compromise between good recoveries for most of the analytes and cleanliness of the extract. In the present work, seven sample preparation procedures based on solid-phase extraction (SPE) (with 5 different cartridges), liquid-liquid extraction (LLE) and sorbent-supported liquid extraction (SLE) were evaluated for multiclass sport drug testing in urine. The selected SPE sorbents were polymeric cartridges Agilent PLEXA™ and Oasis HLB™, mixed mode cation and anion exchange cartridges Oasis MAX™ and MCX™, and C18 cartridges. LLE was performed using tert-butyl methyl ether and SLE was carried out using Agilent Chem Elut™ cartridges. To evaluate the proposed extraction procedures, a list of 189 compounds were selected as representative from different groups of doping agents, including 34 steroids, 14 glucocorticosteroids, 24 diuretics and masking agents, 11 stimulants, 9 beta-agonist, 16 beta-blockers, 6 Selective Estrogen Receptors Modulators (SERMs), 24 narcotics and 22 other drugs of abuse/sport drugs. Blank urine samples were spiked at two levels of concentration, 2.5 and 25μgL(-1) and extracted with the different extraction protocols (n=6). The analysis of the extracts was carried out by liquid chromatography electrospray time-of-flight mass spectrometry. The use of solid-phase extraction with polymer cartridges provided high recoveries for most of the analytes tested and was found the more suitable method for this type of application given the additional advantages such as low sample and solvent consumption along with increased automation and throughput.
Volumetric Absorbtive Micro Sampling is a novel approach to obtaining a dried blood sample for quantitative bioanalysis that overcomes the area bias and sample homogeneity issues associated with conventional DBS when a sub-punch is taken from the sample. The VAMS sampler absorbs a fixed volume of blood (~10 µL) in 2 to 4 seconds with less than 5% volume variation across the hematocrit range 20 to 70% with low tip to tip variability. There is no evidence of selective absorption by the tip of the plasma component over whole blood. Recommendations for best practice when collecting samples were developed based upon the results of tests examining a number of potential abuse scenarios.
Liquid chromatography-mass spectrometry (LC-MS) is one of the most prominent analytical techniques, due to its inherent selectivity and sensitivity. In LC-MS, chemical derivatizations are frequently used to enhance the MS ionization efficiency and selectivity, to facilitate structure elucidation, and to improve the chromatographic separation. In this review, we present an overview of derivatization-based LC-MS analysis. We summarize the reaction mechanisms of representative derivatization reagents and the selection strategy to guide and to stimulate future studies. Furthermore, we emphasize applications of derivatization in peptide and protein analysis, metabolite analysis, environmental analysis, pharmaceutical analysis, food-safety evaluation and MS imaging.
Irbesartan (IRB) and hydrochlorothiazide (HCT) are angiotensin-II receptor antagonist and thiazide-class diuretic compounds, respectively, which are in use in the treatment of hypertension. A novel dilute-and-shoot HPLC assay method for simultaneous quantification of IRB and HCT in fixed-dose combination tablets and urine samples was described. The separation of IRB, HCT and agomelatine (internal standard) was carried out using a second generation C18-bonded monolithic silica column (Chromolith(®) High Resolution RP-18e, 100×4.6mm, Merck KGaA), utilizing both mobile phase and flow rate gradient elution programs. The analytes were detected at 230 nm wavelength using photodiode array detector within 24 minutes with high resolution, observing about 50 percent more peak capacity when using second generation C18-bonded monolithic silica column. Urine samples were introduced into the system effortlessly, with only filtration and subsequent dilution. Validation studies were performed according to the official recommendations of USP and ICH, and the developed method was successfully applied to pharmaceutical tablets and urine samples.
Urine is an important, non-invasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ~ 10 µg total protein were processed by 96FASP and LC-MS resulting in 700 to 900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1,700 proteins (± 398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics.
Although chemical derivatization for signal enhancement in drug testing is most often associated with gas chromatography, it also has the potential to improve the detection of analytes poorly ionized by atmospheric pressure ionization techniques, such as electrospray ionization used in liquid chromatography-mass spectrometry. A number of acidic compounds, namely drug glucuronides (e.g. conjugates of temazepam, oxazepam, lorazepam, morphine, testosterone, epitestosterone, 5-α-dihydrotestosterone, androsterone, p-nitrophenol, and paracetamol) were successfully derivatized with tris(trimethoxyphenyl) phosphoniumpropylamine to introduce a quaternary cation functionality to the analytes. Benzodiazepine glucuronides were more specifically investigated, and following positive mode electrospray ionization mass spectrometry, average improvements to peak areas as a result of derivatization were 67-, 6-, and 7- fold for temazepam, oxazepam, and lorazepam glucuronides. Average improvements to the signal-to-noise ratios for temazepam, oxazepam, and lorazepam glucuronides were 1336-, 371- and 217-fold, respectively. The values obtained for the derivatized conjugate were also typically higher than those for the underivatized parent drug. Urine containing benzodiazepine glucuronides was also successfully derivatized. The data indicates potential for the use of charge derivatization to improve the detection of molecules with acidic functionalities by liquid chromatography-mass spectrometry (LC-MS) techniques in certain scenarios. Copyright © 2014 John Wiley & Sons, Ltd.
In the past 20 years, liquid chromatography-mass spectrometry (LC-MS) has become a standard analytical technique in doping control and toxicology laboratories. Research groups have successfully applied it to detect substances by direct injection, or “dilute-and-shoot”-LC-MS (DS-LC-MS).
However, some urinary components can precipitate into the vial, hampering the injection. Dissolved urinary matrix is responsible for shifted retention times and ion suppression or ion enhancement. To compensate for the effect of the matrix, an isotope-labeled internal standard (IL-ISTD) is the best choice.
Dilution can also minimize the matrix effect, but can result in reduced analyte detectability, so DS-LC-MS methods are predominantly available for substances for which the required urinary detection levels are high and that show good ionization efficiency.
Taking into account the progressive increase in instrument sensitivity, we expect that the application of DS-LC-MS will also come available for substances with low required detection levels or limited ionization efficiency.
Continuous glucose monitoring (CGM) is an important aid for diabetic patients to optimize glycemic control and to prevent long-term complications. However, current CGM devices need further miniaturization and improved functional performance. We have coupled a previously described microfluidic chip with enzymatic microreactor (EMR) to a microdialysis probe and evaluated the performance of this system for monitoring subcutaneous glucose concentration in rats. Nanoliter volumes of microdialysis sample are efficiently reacted with continuously supplied glucose oxidase (GOx) solution in the EMR. The hydrogen peroxide produced is amperometrically detected at a (polypyrrole (PPy)-protected) thin-film Pt electrode. Subcutaneous glucose concentration was continuously monitored in anesthetized rats in response to intravenous injections of 20% glucose (w/v), 5 U/kg insulin, or saline as a control. In vitro evaluation showed a linear range of 2.1-20.6 mM and a sensitivity of 7.8 ± 1.0 nA/mM (n = 6). The physical lag time between microdialysis and the analytical signal was approximately 18 min. The baseline concentration of blood glucose was 10.2 ± 2.3 mM. After administering glucose to the rats, glucose levels increased by about 2 mM to 12.1 ± 2.3 mM in blood and 11.9 ± 1.5 mM in subcutaneous interstitial fluid (ISF). After insulin administration, glucose levels decreased by about 8 mM relative to baseline to 2.1 ± 0.6 mM in blood and 2.1 ± 0.9 mM in ISF. A microfluidic device with integrated chaotic mixer and EMR has been successfully combined with subcutaneous microdialysis to continuously monitor glucose in rats. This proof-of-principle demonstrates the feasibility of improved miniaturization in CGM based on microfluidics.
Achieving sufficient selectivity in bioanalysis is critical to ensure accurate quantitation of drugs and metabolites in biological matrices. Matrix effects most classically refer to modification of ionization efficiency of an analyte in the presence of matrix components. However, nonanalyte or matrix components present in samples can adversely impact the performance of a bioanalytical method and are broadly considered as matrix effects. For the current manuscript, we expand the scope to include matrix elements that contribute to isobaric interference and measurement bias. These three categories of matrix effects are illustrated with real examples encountered. The causes, symptoms, and suggested strategies and resolutions for each form of matrix effects are discussed. Each case is presented in the format of situation/action/result to facilitate reading.
Monoliths were first used as materials for chromatographic separation 20 years ago and as extraction sorbents for solid-phase extraction over 10 years ago.Recently, monolithic silica and polymers were modified to suit devices for extraction and enrichment of analytes in matrices for environmental, food and biological analyses. This approach contributed to miniaturization and automation, which can reduce the time and the cost of sample preparation. Numerous applications were demonstrated for on-line and in-line preconcentration with monoliths, and many kinds of off-line device were designed and developed.This review discusses trends in these device and applications.
This article reviews recent developments in in-vivo sampling and sample preparation for analysis of biological systems. The trend towards microanalytical techniques is justified by the small amount of sample that is available from some biological systems and the need to minimize interference with the system that is being studied or analyzed.Different approaches to direct in-vivo sampling and sample preparation are described, including microsampling, ultrafiltration, microextraction, microdialysis, solid-phase microextraction, biosensors, ambient mass spectrometry, spectroscopy, and microfluidic devices.
Two important aspects of peptide and protein quantification by LC-MS/MS, the enzymatic digestion step and the internal standardization approach, were systematically investigated with a small protein, salmon calcitonin, which could be analyzed both without and with digestion. Quantification of undigested salmon calcitonin, after solid-phase extraction from plasma, resulted in a lower limit of quantification of 10 pg/mL, while introduction of a tryptic digestion step, followed by quantification of a signature peptide, increased this to 50 pg/mL. The sensitivity was reduced by interferences in the SRM-transition of the signature peptide due to the increase in sample complexity caused by the digestion and a less selective SRM transition of the signature peptide as compared to undigested salmon calcitonin. Eight internal standardization approaches were compared with respect to accuracy and precision in work-flows with and without digestion. Analogue and stable-isotope labeled (SIL) internal standards were evaluated including an in-house created 18O-labeled peptide, a cleavable SIL peptide and an internal standard created by differential derivatization of the signature peptide. We conclude that the best internal standard for the work-flows both with and without digestion was the SIL-form of the analyte, although the use of several SIL-signature peptides and a differentially derivatized signature peptide also resulted in methods with performances which meet the FDA guidelines.
Background:
The influence of matrix effects in LC-MS/MS analysis of biological samples can be enormous and has to be evaluated during method development. Phospholipids, which are present in considerable quantities in biological fluids, are supposed to cause matrix effects when co-eluting with analytes. Therefore, the reduction of phospholipids should lead to the minimization of matrix effects. METHODOLOGY & RESULTS: Here, a polymeric reversed-phase (PRP) SPE cartridge was compared with a combination of mixed-mode-anion-exchange (MAX) and mixed-mode-cation-exchange (MCX) SPE cartridges regarding elimination of matrix effects during sample clean-up. For evaluation of matrix effects post-column infusion experiments were performed. Phospholipid amount in the sample extract and matrix effects are enhanced using PRP in contrast to the combination of MAX/MCX.
Conclusion:
For an efficient elimination of phospholipids during sample preparation and to improve method accuracy and precision it is advisable to use a combination of MAX/MCX SPE cartridges.