Sample preparation for mass spectrometry

Background

Biological samples such as blood, plasma, serum, and urine contain contaminants that are not suitable for direct analysis and must be removed before chromatographic separation and detection by mass spectrometry. This chapter discusses the classic sample preparation techniques performed in drug analysis laboratories in clinical and research settings and introduces high-throughput applications for improved efficiency.

Content

The general techniques discussed are dilution, centrifugation, sonication, and homogenization. Separation techniques discussed are filtration and ultrafiltration, dialysis and microdialysis, desalting, buffer exchange, enzymatic hydrolysis, and acid-base digestion. Protein precipitation is the precipitation technique discussed. Enrichment techniques described consist of evaporation, solvent exchange, and derivatization. Extraction techniques reviewed are liquid-liquid extraction, solid-supported liquid-liquid extraction, salt-assisted liquid-liquid extraction, and solid-phase extraction (offline and online sample processing). The chromatographic techniques discussed include column-switching (single and dual column modes) for turbulent flow chromatography, restricted access media, monolithic columns, and immunoaffinity extraction. The evolving techniques described are dried blood spots, capillary microsampling, and tissue imaging.

The clinical chemistry laboratory encounters a wide variety of samples in its day-to-day operations of chemical analysis. Although an automated clinical chemistry analyzer can detect analytes directly from only a small volume of serum, it should be kept in mind that the plasma came from a biological fluid (whole blood) that required a sample preparation step before it was added to the sample queue. Therefore the act of centrifuging a whole blood sample in the collection tube to prepare either serum or plasma constitutes a sample preparation step. When red blood cells break within a whole blood sample and hemolysis occurs, that sample will require further sample cleanup because the cell contents are no longer separated from the cells during centrifugation, and the resulting red coloration from hemoglobin and cell contents may interfere with detection. Urine may appear to the novice as a clean sample matrix because it is often translucent; however, this matrix contains proteins and salts that must be removed or diluted before analysis. The objective of this chapter is to discuss the goals for sample preparation and describe the many different methodologies that the clinical chemistry laboratory encounters, with a focus on those procedures performed before quantitation by mass spectrometry (MS) instrumentation.

Dilution

Sample dilution is an important pretreatment step in separation methods for the following reasons: (1) reduces the concentration of salts and endogenous materials in a sample matrix, (2) reduces the viscosity or ionic strength of the sample, (3) enhances the compatibility of the sample with the mobile phase, (4) reduces the concentration of the sample to fall within the range of the standard curve, and (5) protects the analytical column from overloading. Sample dilution is most commonly performed with urine because drug concentrations are fairly high and dilution can be accomplished without a detrimental effect on sensitivity. A simple dilution approach is preferred when the primary goal is to spend minimal time preparing samples for analysis. However, dilution can also be one component within a multistep sample preparation protocol for additional sample cleanup. Although the amount of protein in a sample is usually a concern, the protein concentrations in urine are almost negligible under normal physiologic conditions. However, repeated injections over time can result in protein buildup within the chromatographic separation column and the ion source. When diluted urine is injected repeatedly, system downtime must be anticipated to clean the ion source.

A very simple protocol involves the dilution of urine with an aqueous or organic solvent and subsequent injection of an aliquot onto a chromatographic column with MS detection; this technique is sometimes referred to as “dilute-and-shoot.” However, this approach is not without challenges because large amounts of endogenous components within urine may potentially coelute with a target analyte. Although they are not seen in the selected ion monitoring mode, the presence of interferences may significantly affect the efficiency and reproducibility of the ionization process within the mass spectrometer interface. In a report by Fu and associates, the ionization behavior of indinavir in human urine is presented as an example in which endogenous matrix components were found to interfere with the ionization of the target analyte, leading to increased variation in tandem MS (MS/MS) responses. The sample preparation for this method used a volume of 1 mL urine diluted with 650 μL acetonitrile so that the resulting concentration of organic in the sample was equal to or less than that of the mobile phase. An aliquot of 6 μL was injected into a liquid chromatography (LC)-MS/MS system. The characterization of this effect and methods to overcome these interferences are described in the report. Deventer and associates discuss urine analysis in doping control, in which 24-hour turnaround times are mandatory in major sports events and minimal time for sample preparation is a major objective. Whether dilution is acceptable within the analytical method depends on two factors: (1) ionization behavior of the target analyte(s) and (2) detection level requirement. Analytes that are efficiently ionized are more likely to be diluted before injection and examples include opiates and stimulants (the basic nitrogen atom is easily ionized) and diuretics (sulfonamide groups, carboxylic acids, or basic amines can be protonated or deprotonated). The minimum limits of detection (cutoff points) depend on the drug class, and this factor is a major concern because no preconcentration step is employed. When the cutoff point cannot be met using diluted urine, an extraction step must be included. Dilution is not the only sample preparation step used in doping control because crystallization of salts (calcium phosphate, calcium oxalate) and proteinuria (which can be 10 times higher in performance athletes) can clog the chromatographic system if not removed; therefore centrifugation is first employed to sediment these materials to the bottom of the sample tube and yield a supernatant free of particulates and sediment. Filtration is an alternative to centrifugation but involves additional cost for the disposable filter unit. The analytical considerations of ionization behavior and minimum detectable limits are also important in analytical toxicology including workplace drug testing.

Advances in LC column technology using monolithic silica rods (single piece of porous silica) have recently made dilute and shoot sample preparation more attractive. Columns made with this monolithic stationary phase having a defined pore structure (macropores and mesopores) can accommodate the injection of diluted urine samples because they have high permeability, porosity, and capacity, as well as faster equilibration times. The total porosity of monoliths is approximately 15% higher than that of conventional particulate high-performance liquid chromatography (HPLC) columns, and so the resulting column pressure drop is therefore much lower, allowing operation at higher flow rates, including flow gradients. for additional discussion on chromatography, refer to Chapter 19 . The chemistry of organic, inorganic, and hybrid (organic-inorganic) monoliths and the application of these columns for sample preparation are reviewed by Nema and associates. In addition to outlining the chemical synthesis and variety of monolithic LC columns, Nema and associates describe the fabrication of silica monoliths into polypropylene syringe barrels, spin columns, and pipette tips to replace traditional solid-phase extraction media. Koyuturk and colleagues illustrate the use of carbon-18 ( ¹⁸ C)-bonded monolithic columns in a method for the quantification of irbesartan and hydrochlorothiazide in urine, in which diluted urine samples were injected directly into the chromatographic system. The sample preparation method involves centrifugation of 10 mL of urine at 4000 rpm (3220 RCF) for 10 minutes to pellet particulates and removal of 2 mL of supernatant that is then filtered through a 0.22-μm polyvinylidene difluoride (PVDF) membrane. A portion (1 mL) of the filtrate is diluted to 10 mL with water, and an aliquot (5 μL) is injected into the chromatographic system. The ease of sample preparation in this approach provided an advantage, and the column was reported to have a long lifetime. After hundreds of dilute-and-shoot injections, the authors reported there was nearly no change in the retention, resolution, and peak morphology of the analytes.

Dilution followed by injection, in an offline sample preparation approach, is rarely used for plasma because analyte concentrations are not as high as in urine, so any dilution negatively affects sensitivity. Moreover, this sample matrix contains high amounts of protein that must be greatly reduced. However, knowing that dilution can be very attractive for the minimal effort and total time involved, it is possible to dilute and inject an aliquot from plasma when the sensitivity needs are met, small injection volumes are used, and the proper choice of an LC column is made. A report by McCauley-Myers describes a method in which plasma samples are first centrifuged, pipetted into wells of a microplate, and then placed on a liquid-handling workstation where a volume of 15 μL plasma supernatant is removed and diluted with 485 μL of a solution of water/methanol/formic acid (70 : 30 : 0.1, v/v/v) containing internal standard. The samples were sealed and mixed; the dilution resulted in a slightly viscous solution with no observed precipitation. An aliquot of 5 μL was injected into an LC-MS/MS system. The lower limit of quantitation for the dilution assay (2 ng/mL) was 400 times higher than that of a more selective procedure that also concentrated the analyte (liquid-liquid extraction [LLE]; 5 pg/mL lower limit of quantitation). However, the advantage offered by the dilution procedure was a 50 times greater throughput. In this case, throughput was a more important consideration than analyte sensitivity. The preferred method for direct injection of neat plasma samples is by online procedures for LC analysis that employ specialty columns constructed from restricted-access materials or system approaches such as turbulent flow chromatography (TFC) or online solid-phase extraction (SPE); these procedures are discussed in the “Chromatographic Techniques” section.

In the analytical laboratory, the process of preparing samples in one or more 96-well plates using manual pipetting with a dilution protocol can be labor-intensive and time-consuming. Liquid-handling workstations that automate this entire process are ideally suited for these tasks. Jiang and coworkers describe a fully automated and validated sample dilution and preparation process using a Tecan Freedom EVO150 liquid-handling workstation (Tecan, Research Triangle Park, NC) equipped with an eight-channel liquid-handler arm and a 96-multichannel arm. A robotic sample preparation program was validated that contains a dilution calculation spreadsheet and a Visual Basic macro to automatically transform sample information from a laboratory information management system work list into executable comma-separated values (CSV) work lists that contain each sample’s dilution scheme, source well positions, destination well positions, and liquid classes. A preprogrammed robotic script within the Freedom EVO software having several executable commands transforms CSV files to executable work lists and then executes the specified sample pipetting and dilution scheme. The dynamic dilution range is reported to be 1- to 1000-fold and is divided into three dilution steps: 1- to 10-, 11- to 100-, and 101- to 1000-fold. The entire process is accomplished within 1 hour for two racks of samples (96 samples/rack) and includes pipetting samples, diluting samples, and adding internal standard(s).

Centrifugation

The process of centrifugation allows for the sedimentation and separation of particulates in solution or as part of heterogeneous mixtures when centrifugal force is applied in the radial direction for a given time. Particles in solution, when subjected to a given centrifugal force, will migrate away from the axis of rotation at a rate that depends on size and density. Factors such as the rotational speed and the distance from the axis of rotation influence this process. The centrifugal force applied to the sample can be much greater than the force of gravity, which allows for even very fine particulates to settle out of the solution. Centrifugation can separate or fractionate fragile components such as platelets and does not damage their function or induce their activation. In its simplest application, centrifugation will sediment large particles or molecules to the bottom of a tube as a visible pellet. Centrifugation at a combination of different forces and times will allow the differential fractionation of materials, such as separation of nucleus cytoskeleton from mitochondria, plasma membranes, and ribosomes because endogenous materials have varying intrinsic densities. Adding trichloroacetic acid or acetonitrile to a sample of plasma precipitates proteins out of solution and a subsequent centrifugation procedure pellets the proteins to the bottom while an aliquot of the solution above the pellet is nearly (>95%) protein-free and is analyzed. Sample preparation methods for urine that are useful to produce the metabolic profiles required for metabolomic research usually involve only centrifugation and dilution. Typical dilution factors with water range between 1 : 1 and 1 : 10 before LC-MS analysis. Mild centrifugation (1000 to 3000 relative centrifugal force × 5 minutes) immediately after sample collection is recommended to remove cellular components. LC-MS–based methods for blood, plasma, serum, CSF, other biofluids, and tissues require more complex procedures than those for urine. ^,

Density gradient separations involve a gradient of high concentrations of a small molecule, such as sucrose, which are distributed along the axis of the centrifugally generated force. Low concentrations are at the top of the tube, and high concentrations are at the bottom. When concentrations of the molecule are high enough, the density and viscosity of the solution vary along with the gradient. A dense molecule such as cesium chloride (CsCl) actually forms a density gradient during the process of centrifugation. When a sample undergoes centrifugation in a density gradient, the different components within the sample will sediment toward the bottom if their buoyant density is greater than that of the solution or they will float to the top if their density is lower than that of the solution. In another approach, centrifugation in the gradient is performed to the point at which the solution density of the molecule equals its buoyant density; then the molecule does not sediment or float, and separation occurs by differences in their buoyant densities. Sucrose density gradient ultracentrifugation is a valuable technique for fractionating DNA, RNA, and proteins. Jasinski and colleagues described a methodology for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by CsCl equilibrium density gradient ultracentrifugation and large-scale purification of RNA nanoparticles by sucrose gradient ultracentrifugation.

Sonication

The action of directing sound waves (mechanical vibrations) toward a liquid or solid causes molecular disruption and is useful as a sample preparation step to remove an analyte from or disrupt its binding to a sample matrix. Sonication is also referred to as ultrasound-assisted extraction. Typically, sample preparation methods use sonication more for solids than liquids, although sonication applied to a liquid sample does aid in analyte dissolution. Sound waves are delivered by an ultrasonic probe placed beside or in proximity with the sample, within an ultrasonic bath, or using a cup horn, and the overall effect achieved is the removal of the analyte from its association with the sample matrix. A frequent application of sonication is leaching minerals, chemicals, and natural compounds from plant tissues, soils, fruits, and vegetables. Applications involving biological matrices are also encountered, such as the extraction of mercury from human urine and cobalamins from animal tissues and biological fluids. Overviews on ultrasound-assisted extraction for the pretreatment of solid samples are summarized by Bendicho and associates. ^, Applications are presented, and the variables that influence ultrasound-assisted extraction are discussed, such as sonication time, ultrasound amplitude, extraction solvent, particle size, and solid concentration in liquid. Ultrasound is also useful as an adjunct to assist with tissue homogenization ^, and digestion procedures in the clinical or bioanalytical setting.

Homogenization

The process of homogenization involves aggressive mixing and blending of two immiscible materials (e.g., liquid and tissues) to create a homogenous composition. Note that homogenization is a disruptive procedure for the sample matrix and may involve crushing, grinding, pulverizing, and/or cutting processes to breakdown the solid matrix. The goals of the homogenization process are to provide uniform and repeatable results, use instrumentation that is easy to clean, be able to accommodate a range of sample sizes, and be automatable if at all possible. Solid and semisoft sample matrices such as tissues and fecal samples are commonly homogenized with a buffer solution of known pH to disrupt or release analyte bound to the solid matrix material and create a suspension of extremely small particles distributed uniformly throughout a liquid. After a filtration or centrifugation step to remove particulates, an aliquot of the liquid is analyzed or undergoes additional sample preparation, depending on the cleanliness and sensitivity needs of the assay.

The classic method of homogenization is the use of a mortar and pestle, which can homogenize small amounts of sample; however, the procedure is manual and limited in scale. When a larger quantity of tissue requires homogenization, an instrument such as a blender is appropriate, but this approach is also a time-consuming and a manual procedure. Instrumentation has been introduced to provide for homogenization of multiple samples to remove the manual component, whether adapted within a laboratory or a commercial product such as the Autogizer (Tomtec, Hamden, CT). ^, An application using a liquid-handling workstation is described that transfers homogenized brain tissue samples from individual test tubes into a 96-well microplate for further sample purification and analysis.

Typical applications requiring homogenization techniques are the isolation of RNA, DNA, and proteins from tissues for proteomic and genomic analyses, oligopeptide analysis from tissues, metabolomics (fecal samples provide insight to gut microbiota) and screening of drugs from various tissues. ^, A sample preparation method for analysis of cell morphology in sputum samples, which are viscous and not miscible in buffer solution, employs homogenization using mucolytic agents such as N -acetylcysteine or dithiothreitol. Semisoft matrices such as feces can sometimes use only water for the homogenization procedure, as in the isolation and purification of metabolites of the cytotoxic drug paclitaxel. Solid biological tissues are commonly homogenized in saline or various buffers at a temperature of 4 °C to retain structural integrity of proteins and/or analyte stability. Liquid organic acids such as trifluoroacetic acid or trichloroacetic acid are sometimes added in a 10% (v/v) concentration as a protein precipitating agent. The end result of the homogenization process is a uniform mixture of tiny fragments of tissues, ready for subsequent sample preparation before analysis.

Filtration

Filtration as a sample preparation method is a pressure-driven process that effectively removes particulates from biological fluids that can potentially foul LC lines, column frits, and/or the MS interface. Liquid-handling workstations and pipetting systems also benefit from filtration so that all liquids are free of materials that may introduce pipetting challenges, especially plasma clots. Filtration for bioanalysis applications is more appropriately named microfiltration and employs membrane pore sizes that are typically in the range of 0.1 to 10 μm. Within this text and the published bioanalytical literature, filtration is the common terminology, however. Filtration is attractive as a first or second step in sample preparation because the sample concentration is not diluted and no organic solvents are used; filtration is often followed by additional techniques that concentrate the sample. Applications that commonly benefit from a filtration step include the separation of tissues from liquid after homogenization, removal of a mass of precipitated proteins or cellular debris from neat plasma before use with any of the traditional sample preparation techniques, and direct injection techniques (TFC, restricted access media [RAM], and online SPE). Filtration at the end of a sample preparation method is also effective, such as to clarify eluates and/or reconstituted extracts before chromatographic analysis. Methods for precipitating proteins from plasma use microfiltration, and these procedures are discussed in the “Precipitation Techniques” section.

Membranes used for filtration are generally composed of a porous polymer such as polyethersulfone, PVDF, polypropylene, or a cellulose derivative (e.g., nitrocellulose) and are available in a range of porosities for exclusion by pore size. Filtration membranes are configured as single-use disposable units that accommodate from one sample at a time (syringe filters or spin filters) to 96 and 384 samples in the microplate format. The term ultrafiltration typically refers to a pore size less than 0.1 μm, whereas nanofiltration specifies a pore size less than 0.01 μm and a molecular weight cutoff of approximately 1000 Da ( Table 21.1 ).

Passive filtration through a membrane using gravity can be performed, but vacuum is a more practical approach, except for protein-containing and viscous samples. When using vacuum to pull proteinaceous samples through the membrane, the force achieved is not always sufficient to completely pull liquids through a fine-porosity membrane and residual liquid can be left above the membrane or adsorbed onto the plastic inside wall, below the membrane but above the collected liquid. Centrifugation is superior to vacuum because it attains higher forces and effectively passes the full volume of liquid through a fine-porosity exclusion filter. Passive ultrafiltration was compared with centrifugal ultrafiltration in a study by Blanco and coworkers. Detection of Legionella pneumophila antigen in urine by enzyme immunoassay or immunochromatographic testing is specific for diagnosing legionellosis, and the use of concentrated urine obtained by selective ultrafiltration has been shown to significantly improve the sensitivity of antigen detection. When antigen from 4 mL urine is concentrated by centrifugal ultrafiltration (Amicon Ultra-4, Millipore, Bedford, MA) using a force of 3000 × g for 15 minutes, the sensitivity of antigen detection was shown to be equal to that obtained using traditional passive ultrafiltration (Urifil-10, Millipore), which requires 1 to 3 hours. Therefore great time saving results from the use of centrifugation as part of the sample preparation method.

Ultrafiltration uses a semipermeable membrane to separate molecules in liquids having a molecular weight range of 1000 to 500,000 Da. The membrane is made of either polymeric or inorganic materials and contains pores of a defined size distribution. Note that the molecular weight cutoff (MWCO) rating for a membrane is a nominal number that is not absolute in terms of capability to exclude proteins by size. Ultrafiltration is commonly used to clarify protein solutions, and in proteomics the technique filter-aided sample preparation (FASP) describes the on-filter cleanup and digestion of protein samples for MS analyses. This method allows for gelfree processing of biological samples solubilized with detergents for proteomic analysis by MS. Using FASP, detergents are removed by ultrafiltration, and after protein digestion, peptides are separated from undigested material and recovered in the eluate. Manza and colleagues described the use of commercially available microcentrifugation devices (cellulose spin filters with a 5000 MWCO) for this purpose. The protein sample is added to the upper chamber of a spin filter with a MWCO membrane, and contaminating species are washed away. Proteins are resuspended in a buffer compatible with digestion. Proteins are then reduced, alkylated, and digested on the filter, and the resulting peptides are isolated in the eluate by centrifugation. The method significantly reduces the complexity and the time required for sample preparation and minimizes the loss of sample. The results obtained are reported as equivalent to those for in-solution digestions.

In clinical proteomics, the urine supernatant obtained after centrifugation is devoid of particulates and endogenous materials, including the pellet fraction. However, it can also be important to analyze the pellet fraction when looking for whole human cells that may have been shed into the urine from proximal tissues and organs or viruses and any microbes that may have infected the urogenital tract. The detection of microbes in the isolated pellet after centrifugation provides diagnostic information that is complementary to traditional cell culture–based laboratory tests. Yu and Pieper describe a FASP method used in shotgun proteomics that effectively lyses cells present in urinary pellets isolated after centrifugation, solubilizes the majority of proteins resulting from microbial and human cells, and yields protein mixtures that are compatible with enzymatic digestion. The subsequent use of desalting procedures yields a peptide fraction suitable for analysis by LC-MS/MS. The methodology is scaled to higher throughput using parallel sample preparation in 96-well plates, peptide separation using nano-LC in one dimension, and analysis via a Q-Exactive benchtop quadrupole Orbitrap MS (Thermo Scientific, Waltham, MA). Using this technique, it was shown that more than 1000 distinct microbial proteins and 1000 distinct human proteins can be identified from a single experiment. Michalski and associates discuss the technical and performance advantages of coupling of a quadrupole mass filter to an orbitrap analyzer and its application to MS-based proteomics.

A porous hollow fiber membrane is an attractive alternative to conventional flat membranes because it increases the membrane area for diffusion (i.e., high surface-to-volume ratio) and is easy to incorporate into flow streams. An application of centrifugal ultrafiltration based on molecular weight separation uses a hollow fiber membrane for the measurement of the drug amoxicillin in human plasma; in this case the membrane acts as a dialysis membrane to isolate free (non–protein bound) drug for analysis. The separation device is constructed using a slim glass tube (6 cm × 5 mm) and a U-shaped hollow fiber membrane. After addition of a plasma sample followed by centrifugation (e.g., 1.25 × 10 ³ g for 10 minutes), small molecules pass through the ultrafiltration membrane without the use of additional buffer or high centrifugal force. The filtrate is withdrawn from the hollow fiber, and an aliquot is free of proteins and suitable for direct injection into a MS.

Dialysis and microdialysis

Dialysis, also known as equilibrium dialysis, is a concentration-driven separation technique that allows the selective diffusion of low molecular weight solutes from larger macromolecules through a semipermeable membrane made from a synthetic polymer or cellulose. The MWCO of the membrane is determined by the size of its pores, and the analyst chooses the appropriate specification for the separation. Dialysis is commonly used to separate and measure the concentrations of protein-bound drug and free drug within a liquid sample. There is no enrichment or concentration factor involved in the dialysis process. The analytes become diluted because the driving force of the mass transfer process is simply the difference in concentration across a semipermeable membrane. The sample is placed on one side of the membrane, and a buffer solution or dialysate is placed on the opposite side. Molecules such as protein-bound drugs that are larger than the pores of the membrane are retained, and smaller molecules pass freely through the membrane. Over time, small molecules approach equilibrium with the entire dialysate volume. The classic dialysis technique is clearly a useful separation technology to capture non–protein-bound (free) drugs from protein-bound drugs, but it is not commonly used as the only sample preparation step before MS analysis because the analyte concentration is greatly diluted. An aliquot of the liquid volume representing free drug concentration is then subjected to an enrichment technique such as SPE and then is amenable to chromatography and MS detection. Dialysis is also useful for desalting and exchanging buffer, techniques that are useful with gel filtration chromatography because they also use MWCO limits for separation. Gel filtration is much faster than dialysis (a few minutes compared with longer than 1 hour) and can remove contaminants from small sample volumes, as long as the column size and format are matched appropriately to the sample. Dialysis techniques are generally focused on determining plasma protein binding for drug analytes under physiologic conditions. ^, Note that the choice of buffer used in the equilibrium dialysis method plays an important role in the quality of the data obtained using this technique. A major disadvantage of dialysis is the dilution of the sample, thus reducing analyte concentration; also, in some cases, analyte binding to the chosen membrane may be observed.

Microdialysis is a particular application of dialysis that measures free (unbound) analyte and is used for the real-time monitoring of physiologic events occurring within the interstitial fluid in living tissues. A great advantage of microdialysis is that the technique can be connected directly to online MS analysis. The dialysis membrane is contained within a probe that is placed in proximity to tissues in vitro or in vivo. Microdialysis does not add or remove any fluids, because only molecules equilibrate across the semipermeable membrane in response to a concentration gradient, enabling the diffusion of substances from the interstitial space into the dialysis probe. This technique is valuable for serial sampling or continuous monitoring in animals in which the probe is mechanically positioned so that they can move freely without sedation or impairment in any way. The inlet of the probe is connected to a microsyringe pump that can provide ultralow flow rates; the other end of the probe is connected to either a collection vial or an LC micro valve that enables online analysis. There are different shapes, sizes, and varieties of the probes depending on the tissue and the region being investigated. The use of hollow fiber membranes increases the surface area available for diffusion. Microdialysis methodology has been shown useful for serial sampling of the extracellular fluid for drug analysis applications in pharmacokinetics, as well as for the continuous monitoring of brain chemicals such as neurotransmitters and peptides and other small molecule substances such glucose, lactate, pyruvate, and glycerol. The dialysate requires no further sample cleanup and is analyzed directly by LC-MS/MS.

Tang and colleagues describe the dynamic, continuous, and simultaneous analysis of multiple neurotransmitters to research the complex interactions between neuronal and intercellular communications. Online microdialysis is coupled with hydrophilic interaction chromatography–MS/MS for the simultaneous measurement of the transmitters acetylcholine, serotonin, dopamine, norepinephrine, glutamate, γ-aminobutyric acid (GABA), and glycine, toward the goal of understanding transmitter release from embryonal carcinoma stem cells in vitro. The limit of detection was determined as the concentration of analytes with a signal-to-noise ratio of at least 3 and are described in pg of microdialysates; limits are reported as 2 pg for acetylcholine, serotonin, and glutamate and 10 pg for dopamine, norepinephrine, GABA, and glycine.

Microdialysis sampling in the brain is summarized by Ducey and colleagues, who discuss approaches to sampling and calibration, as well as the methods employed for the analysis of the microdialysis samples. Many applications of offline and online microdialysis systems, and some microfluidic applications, are published for the monitoring of pharmacokinetics or biological events using LC separation (many with MS detection). Korf and associates review the current status of microdialysis and microfiltration, which can be combined easily with other analytical techniques, and focus on the use of small volumes with ultraslow sampling to provide advantages. Applications discussed are quantitative pharmacokinetics, glucose metabolism in the brain, cytokines, and proteomics (tumor secretomes), both in vivo and in vitro. Although the sampling of interstitial fluid using microdialysis has become common, the small sample volumes, low concentration of analytes, and many different low molecular weight molecules assayed all present great analytical challenges. A review by Guihen and O’Connor highlights these challenges and discusses the importance of proper evaluation of conditions and needs to select the most suitable analytical methodology for chromatography and detection. Microdialysis tools have been adapted for protein, oligopeptide, and peptide analyses in proteomics sample preparation as well, using sample volumes from 10 to 100 μL. ^,

Desalting and buffer exchange

The process of desalting separates salts and small molecules from larger proteins in a sample so they are removed from subsequent analysis steps. Buffer exchange replaces the original equilibration buffer pair of the sample with a different buffer pair, one more suitable for subsequent processing or analysis by ion exchange or affinity chromatography. Desalting and buffer exchange represent a particular type of size exclusion chromatography using porous particles that are characterized by a maximum effective pore size. Size exclusion chromatography is also referred to as gel permeation chromatography, gel filtration, or molecular sieve chromatography. The larger proteins in a sample are excluded by pore size of the resin beads and quickly flow around the beads and exit the column first, along with the matrix components. Salts and small molecules enter the resin beads and flow through, but their migration is slowed and they elute from the column at a later time with the column equilibration liquid or buffer. The choice of effective pore size in resin beads can be varied to target certain analytes; for example, peptides can be separated from proteins using resin beads having larger pore sizes. The technique can be useful for sequential fractionation based on molecular size, in addition to simply removing larger molecules from smaller molecules. A wide selection of resins or gels is available to accomplish a range of sample preparation needs. For additional discussion on this subject, refer to Chapter 19 .

The common formats for performing gel filtration on the laboratory bench are gravity-flow columns and centrifuge columns, also known as spin columns. The sample is loaded onto the top of a gravity-flow column, and it slowly passes through the resin bed; the column is positioned upright. The sequential addition of one or more buffer solutions (ionic strength and pH are varied) to the column, during wash and elution steps, applies gentle pressure to move existing liquids slowly through the gel matrix and out. As fractions elute from the bottom of the column, they can be collected to isolate proteins or macromolecules of interest. Gravity-flow columns can be prepared individually with chromatographic resin or they can be purchased already configured. A variation of the gravity-flow column is one that is a closed system and the sample and buffer are sequentially forced through the cartridge using pressure from a syringe. Centrifuge or spin columns use a much greater centrifugal force to push a sample through the gel filtration matrix, and this procedure takes far less time than using the gravity-flow format. In the case of centrifugation, the sample is not diluted because no additional liquids are needed to force it through the gel matrix. A 96-well plate configuration of gel filtration columns is commercially available for higher throughput applications.

Chromatographic techniques are also used in an online mode with LC and MS for desalting and buffer exchange of proteins using size exclusion chromatography. Four different primary approaches are used: (1) TFC, (2) RAM, (3) monolithic columns, and (4) immunoaffinity extraction (IAE). These techniques are discussed further in the “Chromatographic Techniques” section.

Enzymatic hydrolysis