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Solid-Phase Extraction and Clean-Up Procedures in Pharmaceutical Analysis

Pharmaceuticals and Drugs

  1. P. Campíns-Falcó,
  2. A. Sevillano-Cabeza,
  3. R. Herráez-Hernández,
  4. C. Molins-Legua,
  5. Y. Moliner-Martínez,
  6. J. Verdú-Andrés

Published Online: 17 DEC 2012

DOI: 10.1002/9780470027318.a1920.pub2

Encyclopedia of Analytical Chemistry

Encyclopedia of Analytical Chemistry

How to Cite

Campíns-Falcó, P., Sevillano-Cabeza, A., Herráez-Hernández, R., Molins-Legua, C., Moliner-Martínez, Y. and Verdú-Andrés, J. 2012. Solid-Phase Extraction and Clean-Up Procedures in Pharmaceutical Analysis . Encyclopedia of Analytical Chemistry. .

Author Information

  1. Universitat de Valencia, Valencia, Spain

Publication History

  1. Published Online: 17 DEC 2012

1 Introduction

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

To understand the place of SPE or other sample clean-up procedures in the pharmaceutical field, the drug evolution cycle should be considered. This can be divided into different steps: discovery; chemical process development; metabolism, bioavailability, and pharmacokinetics; formulation studies; stability studies; toxicology and safety testing; and commercial production and marketing. This cycle gives different kinds of samples that may need a pretreatment before their measurement. These pharmaceutical samples are bulk-drug, preformulations, formulations, and biological samples. The last samples are also connected with topics such as therapeutic drug monitoring and toxicological analysis developed in many cases in the pharmaceutical field.

Nonvolatile species can be removed from either solid or liquid matrices by a number of techniques that range from distillation to dialysis, LLE, and SPE, including both ion exchange and sorption. The traditional LLE method, as a sample clean-up procedure for pharmaceutical samples, has been superseded by SPE. This approach with porous solid particles (Rohm and Haas Amberlite XAD resins) goes back to the early 1970s.1 Practical use of this method was undoubtedly hindered by the necessity of grinding, sizing, and purifying the early XAD resins. Now, with the ready availability of pure bonded-phase SI particles and, more recently, of pure polymeric resins of appropriate particle size, SPE has become the preferred method for isolation of organic components from predominantly aqueous samples.

Disposable cartridges filled with sorbents of a small particle size are generally employed, and the sample and the different mobile phases are flushed with a syringe as a low-pressure pump. The clean-up principles are similar to other open-column techniques. This method is quick and simple to operate, most sample handling losses are avoided and it is easily automated and coupled to LC.2, 3 LC has probably emerged as the method of choice in the pharmaceutical field. Gas chromatography (GC) has gained more prominence for drug screening and coupled with mass spectrometry (MS) for confirmation analysis too.

In recent years, an increasing number of LC methods incorporating on-line sample clean-up by SPE on disposable precolumns by use of column switching have been developed. Switching devices permit the off-line multistep methods to be transformed into single-step procedures by the on-line purification of the sample. The on-line coupling of SPE and capillary GC still poses a number of problems.4

A topic close to pharmaceutical analysis is residue analysis, as the determination of antibiotic residues in food products of animal origin. SPE on disposable cartridges or precolumns has also been proposed as a substitute for or combined with LLE5 in this field.

In systematic toxicological analysis, the substances present are not known at the start of the analysis. In such an undirected search, the extraction procedure must be a general procedure where a compromise must be reached in that the substances of interest are isolated at a yield as high as possible and the interfering substances from the biological material are removed.6 For a long time, the traditional sample work-up technique in analytical toxicology was LLE, often combined with sample pretreatment procedures such as conjugate hydrolysis, digestion, and protein removal. Although LLE proved to be suitable in a substantial number of cases, the disadvantages of this technique, for example, matrix interferences, emulsion formation, and use of large volumes of hazardous solvents, have troubled the analyst. The SPE approach can partially overcome the drawbacks of LLE.

The identification and quantification of pharmaceuticals in environmental waters is also a topic of increasing interest. Pharmaceuticals have become important emerging contaminants because of their continuous introduction into the environment (they are often incompletely removed in wastewater treatment plants), and effects both to wildlife and humans and development of bacterial resistance are major concerns. SPE is currently the most widely used technique for enrichment of multiclasses of pharmaceuticals in the analysis of environmental water.7, 8

Nowadays, SPE is a well-established technique with a broad range of applications. Research in the field of SPE is mainly focused on the synthesis of new sorbent materials for improving the selectivity and efficiency. The development of miniaturized formats of SPE, which reduce the consumption of solvents and facilitate on-line sample processing is also an area of increasing interest.9, 10 A timeline with the main contributions to the development of SPE is presented in Figure 1.

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Figure 1. Timeline with the main contributions to the development of SPE: sorbents (left) and formats (right).

2 Basic Theory

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

This section briefly summarizes the most used sorbents and methods in SPE. The same analyte/sorbent interactions that are exploited in high-performance liquid chromatography (HPLC) are used in SPE. Ion exchangers have also been included due to their use in sample pretreatment processes and because the process involved in the separation is a physicochemical process, as in sorption.

2.1 Sorbents and Interaction Mechanisms

The first sorbent used for SPE was XAD resin as mentioned earlier. This resin is a low-polarity styrene-divinylbenzene (SDB) copolymer that possesses the macroreticular characteristics essential for high sorptive capacity. In addition to polymeric resins, the solid-phase sorbents available include common inorganic sorbents (SI, alumina, Florisil), siloxane-bonded SI materials [octadecyl, octyl, ethyl, cyclohexyl (CH), phenyl (PH), 3-cyanopropyl (CN), diol (2OH), 3-aminopropyl (NH2), etc.], ion-exchange macroreticular resins, and specialty products such as resin-bonded phenylboronic acid (PBA) and activated carbon. Chemically bonded SI, usually with a C18 or C8 organic group, is the most commonly used material for SPE. Cross — linked polystyrene and other porous polymeric resins11 and mixed — mode SPE sorbents12 are also widely used.

Different sorbents are available for enhancing the selectivity of the extraction.13, 14 Among them, MIPs are the most popular.15, 16 This kind of sorbents is prepared by the polymerization of functional and cross — linking monomers in the presence of a template molecule (the analyte). Subsequent removal of the template creates recognition sites for the analyte or related compounds. The extraction is the result of multiple interactions (ion exchange and hydrogen bonding) between the polymer and the analyte. Today, specific MIP — based sorbents are commercially available for the selective extraction of different classes of compounds such as nonsteroidal anti — inflammatory drugs, β-blockers, or amphetamines. Another class of highly selective sorbents for SPE are immunoaffinity sorbents, which involve antigen–antibody interactions.17 The selected antibodies are usually immobilized onto an agarose gel or silica beds.

Another type of selective sorbent is restricted access material (RAM), which prevents the access of macromolecules to the region of the sorbent where the interaction with the analyte molecules occurs.18 In general, restricted access media are prepared by covering the internal surface with a bonded reversed — phase material, while the external surface is inert. Only small molecules (drugs and pharmaceuticals) access to the internal surface, while proteins are excluded by the outer.

As regards the improvement of the extraction capacity, most research effort is being devoted to the employment of nanoparticles as sorbents for SPE. Single — or multiwalled carbon nanotubes (CNTs) are by far the material most studied, because of their large surface — to — volume ratios, although other allotropic forms of carbon such as fullerene and graphene have also been used. In addition, CNTs can be functionalized to modify the selectivity.19 Until now, the described applications have been developed in laboratory — made cartridges, because no commercial cartridges or disks are available, and so, more developments in this area can be expected. Nanoparticles (NPs) seem especially well suited for miniaturized SPE.

Also, the employment of magnetic solids as sorbents for SPE is gaining popularity. In this type of sorbents, magnetic particles are covered with a material capable of interacting with the analyte (SI, polymer). In contact with the sample, the particles extract the analytes. For separation of the particles with the extracted analyte, an external magnetic field is applied. After matrix removal, the analytes are desorbed from the magnetic particles with an appropriate solvent. A schematic representation of this kind of extraction is shown in Figure 2. Examples of application of magnetic sorbents for extraction of pharmaceutical in biological20 and environmental waters21 can be found in the recent literature.

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Figure 2. Typical scheme of magnetic SPE.

SPE is typically carried out with cartridges filled with the sorbent. The packing material with particle sizes in the range of 30–60 µm is sandwiched between two porous frits with a pore diameter of about 20 µm in a column. The bottom end of most cartridges is terminated in a leur lock fitting. Cartridges are available in several sizes containing from 50 mg to 10 g of sorbent, with the 100–to 500–mg sorbent cartridges being the most used in the pharmaceutical field. Approximate values for the capacity and bed volume of the columns are 5% sorbent mass or 0.4–0.6 mequiv mL−1 for anion-exchange sorbents and 120 µL per 100 mg of sorbent, respectively.

A more recent approach to SPE is the use of a membrane (0.5-mm thick, 25 or 47 mm in diameter) loaded with an appropriate solid material immobilized within a stable, inert matrix of fibrillated poly(tetrafluoroethylene) (PTFE). These membranes are 90% by weight particle-loaded and 10% PTFE with very dense packing of the sorbent of a smaller average particle size (8 µm) than used in the conventional SPE cartridges. The capacity of the membranes is similar to that of the cartridges, but the small bed volume allows for reduced elution volumes, resulting in more concentrated eluates. Particle-loaded membranes are supplied in a disk format for large-volume use and are contained in disposable cartridges for the small volumes appropriate for pharmaceutical analysis.

Another format of SPE called bag-SPE has been recently proposed for the screening of pharmaceutical residues in environmental waters. The extraction device consists of a small amount of sorbent (20 mg of SDB) enclosed in a woven polyester fabric. This device can be used as a sampler, by immersing it into the sample. Equilibrium is reached in 4 h. After sampling, the bag is removed from the sample and immersed into methanol for desorption of the analytes.22

As mentioned earlier, during the past years, the development of miniaturized forms of SPE has gained much attention. In accordance with the principles of Green Analytical Chemistry, miniaturization reduces the consumption of solvents and sample, and the generation of wastes, resulting in more environmental-friendly and cost-effective methods.23, 24 In this sense, the most popular techniques is in-tip microextraction. In this miniaturized form of SPE, the sorbent is bonded at the working end of a pipette tip (10 µL) by means of an inert high-purity adhesive or immobilized with filters. The process involves the aspiration and discarding of the conditioning solvent, sample, washing solution, and desorbing solvent. Extraction can be carried out manually or automatically, and several samples can be handled in parallel. Most applications in the pharmaceutical field use reversed-phase or ion-exchange monolithic sorbents with large though pores because, as the amount of sorbent is very low, the back-pressure is also low. Different products (sorbents and sizes) for in-tip microextraction are now commercially available.

The development of other forms of miniaturized SPE such as in-needle solid-phase microextraction, which is based in the employment of a microsyringe as extraction device, is a growing research topic in many areas of applications.25

For the on-line mode, the majority of the applications use particle sizes in the 10- to 40-µm range, but monolithic sorbents are also increasingly used.16 The internal diameters of both the sample clean-up column and analytical column should be the same, to minimize extracolumn band broadening. The vast majority of studies employ an internal diameter in the 2–4.6-mm range. Short precolumns (between 5 and 10 mm) are preferable because the duration of the flushing needed to remove undesirable matrix compounds is minimized. Packed precolumns are commercially available from several manufacturers, but manual slurry or dry packing of a precolumn does not present any real problem and, in addition to rapid exchange, allows the easy screening of new packing materials. In the past years, capillary columns packed with the sorbent as well as open capillary columns coated with an extractive phase have gained popularity for effecting on-line sample conditioning. The methodology is called capillary microextraction or IT-SPME.26 IT-SPME with packed columns typically uses organic and inorganic monolithic sorbents, including MIPs, and immunoaffinity sorbents, whereas in IT-SPME with open capillary columns, polymeric coatings are generally used. When using IT-SPME with open capillary column, the extraction yields are generally lower, but the analytes can be extracted reproducibly.

There are three general extraction mechanisms used in SPE: polar, nonpolar, and ion exchange. In the last decade, increasing attention has been paid to the development of mixed — mode polymeric sorbents with combine a polymeric skeleton with ion — exchange groups, so they can combine two types of interaction mechanisms, reversed phase and ion exchange. These sorbents are more suitable for systematic toxicological analysis and for the screening of multiclasses of pharmaceuticals in environmental water.27 A wide variety of mixed — mode polymeric sorbents can now be found in the market (e.g. Oasis MCX, Oasis WAX, Strata — X, and many others). The main retention mechanisms for the most used sorbents are shown in Table 1.

Table 1. Sorbents and Interaction Mechanisms in SPE
InteractionSorbentChromatographic modeAnalyte functional groupsMatrix
  1. C18, octadecyl; C8, octyl; C2, ethyl; SXC, benzenesulfonic acid; PRS, propylsulfonic acid; CBA, carboxylic acid; SAX, quaternary amine; PSA, primary/secondary amine; DEA, diethylaminopropyl.

 SI-based bonded phase   
HydrophobicC18Reversed phaseAromatic ringsAqueous
Nonpolar–nonpolarC8 Alkyl chains 
Van der Waals forcesC2   
Hydrophilic polar–polar hydrogenCNNormal phaseHydroxylsNonpolar
Bridges p–p bond2OH Amines 
Dipole–dipoleNH2 Heteroatoms (S, O, N) 
Induced dipole–dipole    
Ion-exchangeSXCIon exchangeAminesAqueous
 PRS Pyrimidines 
 PSA Sulfonic acids 
 NH2 Phosphates 
Multiple hydrophobic/ion exchangeMixed bonded SIReversed phase and Ion exchangeAcidic, neutral, and basic compoundsAqueous
 No bonded phase   
Hydrophilic/ hydrophobicSINormal phaseGenerally used to provide class separation by dividing the sample into fractions containing a similar number and type of functional groupsNonpolar
 Diatomaceous earth   
 Activated carbon   
Hydrophobic/ hydrophilicCopolymer resinsReversed phaseAromatic ringsAqueous
   Alkyl chains 
CovalentPBAReversed phaseNucleotides, nucleosidesAqueous

Although the same type of SPE material can be obtained from different manufacturers, the results using SPE material from different manufacturers, and even results obtained from different batches from the same manufacturer, may show significant differences in behavior, i.e. in particle size distribution and flow velocities.

2.2 Method Development

It is recommended to find the sorbent that will provide the best results. For that, the potential interactive properties of the isolate(s) and matrix, taking note of functional groups, should be considered (Table 1). Also, other known influences that may affect the extraction negatively are important, such as protein binding and properties of the isolate(s) such as stability, solubility, and pKa.

If the analyte(s) has nonpolar (alkyl chains and aromatic rings), polar (hydroxyls, amines, and keto groups), or ionic [amines and carboxylic acids (CBAs)] functional groups, then these properties can be selectively employed to facilitate retention. The undesired constituents of the matrix may interfere with retention of the analytes, requiring consideration of a different mechanism.

The same operational modes as in HPLC are used in SPE. Table 1 shows the option generally chosen for each sorbent.

The sample processing involves four distinct steps. Pretreatment of the sorbent with an activating solvent (such as methanol for chemically bonded SI and porous polystyrene resins) must be carried out to obtain better surface contact and to remove impurities from the sorbent. It is returned to the ready condition by washing with three or four bed volumes of a solvent as similar to the sample solvent in polarity, ionic strength, and pH as possible. Nevertheless, different sorbents such as the sulfonated polymers and vinylpyrrolidone copolymer have been developed that do not need previous conditioning. The sample is then sorbed onto the cartridge. The maximum volume that can be sampled depends on the breakthrough volume for the analyte(s). The cartridge is then rinsed with a weak solvent to remove undesirable matrix components. In the final step, the analytes are eluted by a solvent of sufficient strength to desorb them in a small volume without displacing more strongly sorbed matrix components.

The typical solvents used for nonpolar extraction are methanol, acetonitrile, ethyl acetate, chloroform, acidic methanol, and hexane. Methanol, isopropanol, and acetone are used for polar extraction. Alkaline and acidic buffers and high ionic strength solutions are employed for ion-exchange extraction. Acidic buffer and acidic methanol are the solvents selected for covalent extraction.

The sample and solvents are flushed into the cartridge manually by use of a syringe, by centrifugation, or in a vacuum manifold that permits the processing of between 10 and 24 columns at one time generally. SPE membranes require a filtration manifold or special apparatus for multiple extraction of several disks. For the on-line option, switching valves and pumping systems are necessary.

Different strategies exist to improve the sample clean-up by SPE, taking into consideration the sorbent used. Table 2 summarizes these strategies.

Table 2. Strategies for Improving Sample Clean-Up by SPE
Inadequate analyte retentionPolarChange to more nonpolar solvent
 NonpolarChange to more polar solvent
  Ionic strength ≤0.1
  Bad activation
  Ionic analyte
 Ion exchangerDilution
  Change the pH
  Decrease the ionic strength
InadequatePolarStudy the secondary mechanism
ElutionNonpolarModify the ionic strength
  Increase the nonpolar solvent
 Ion exchangerIncrease the elution volume
Poor sample clean-upPolar, nonpolar, ion exchangerIncrease the strength of the matrix washing solvent
Poor reproducibilityPolar, nonpolar, ion exchangerProtein binding
  Analyte absorption on the sorbent pH

3 Off-Line Versus On-Line Procedures

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

Off-line SPE may be converted into an on-line procedure using column-switching techniques. Its use in HPLC was first reported in 1973.28 The term ‘column-switching’ or ‘coupled column chromatography’ includes, in the widest sense, all techniques by which the direction of the flow of the mobile phase is changed by valves, so the effluent from a primary column is passed to a secondary column for a defined period of time. Switching can be effected manually or by automated controllers.

Coupling LC/LC is a well-established technique for sample clean-up in the analysis of pharmaceutical samples. Its success depends on the type of sample, the precolumn, the eluent, and the column-switching configuration.2 The only significant difference with the off-line SPE is the switching network.

In the simplest configuration, only a switching valve is required in addition to a basic chromatograph. It should be indicated that during the sampling and clean-up steps, the eluent in the analytical column is stagnant. Therefore, this configuration may lead to considerable baseline fluctuations depending on the elution conditions, which limits its applicability. More powerful systems can be achieved at a reasonable cost by using an additional pumping system, as depicted in Figure 3. In such a way, the analytical column is equilibrated during the clean-up stage (valve in position 1), and only the fraction of eluent containing the compounds of interest is transferred to the analytical column when the valve is changed to position 2.

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Figure 3. Typical scheme of a column-switching system used for on-line SPE.

In spite of the level of instrumentation, different transfer modes can be used. In forward flush (also called straight flush) configurations, the flow direction in the precolumn is not changed during the transfer stage, which prevents solid particles retained at the head of the precolumn from being sent to the second column. However, for samples containing compounds that are strongly retained in the precolumn, backflush configurations (in which the flow direction of the mobile phase through the precolumn is reversed for the transfer onto the secondary column) are preferable, because the desorption of the most retained components after every injection is easier.

To improve sample clean-up, several columns can be coupled easily in the on-line mode by using several switching valves. In linear coupled-column systems, the peak capacities of the individual columns are summed. A review of commonly used switching networks is given by Ramsteiner.29

One of the most obvious trends in analytical laboratories is the growing need for automation. SPE has been traditionally performed in non- (or semi)automated mode. However, an important reason for growth is that SPE is amenable to automation, and today, there are several examples of commercially available systems for automated off-line and on-line SPE.

Compared with manual SPE, automated SPE also improves the reproducibility because variations in experimental parameters are reduced or even eliminated. Another important advantage of automated SPE in pharmaceutical analysis is safety, because workers are protected from toxic substances and from samples obtained from patients with a range of diseases (HIV or hepatitis B, for instance).

There are different approaches to automation of SPE, which involve different levels of apparatus cost. These approaches can be categorized into off-line and on-line systems.30

3.1 Off-Line Automated Solid-Phase Extraction

In off-line methods, the SPE cartridges are not directly connected to instrument, normally a liquid or a gas chromatograph. Unattended sample work-up and analysis can be performed as a result.

The simplest approach to automation is based on the employment of liquid-handling devices that include SPE in their functions. These workstations usually incorporate precision pumps (which deliver measured volumes of liquid to test tubes) with multisolvent capabilities, replaceable pipettes, and random access to vessels. The system also includes a SPE rack, with the cartridges or disks, and a liquid-handling probe. The probe runs in the trays (usually xyz), so it can reach any of the positions: samples, tubes, or SPE cartridges, manipulating the sample and providing air or nitrogen (to move liquids through the cartridges) and the extraction or washing solvents. Specially designed probes or column caps create a tight seal in the cartridges. The syringe pump delivers conditioning solvents, which are then pushed through the cartridges by means of the pressurization line. Next, the sample is added and the pressurization step is repeated. If required, one or more washing solvents are also added, and pressure is again applied. Finally, the cartridge (which is over the waste container) is moved, so the sample is eluted into a clear container for further processing. Optional equipment such as a barcode reader is also available in many commercial systems. All operations are controlled by a keypad controller or by a computer. In semiautomated systems, the extracts collected are manually injected onto the chromatograph. However, total automation is possible by connecting the workstation to the analytical column, for instance, by means of a loop injector. Examples of this approach to automation are the Gilson ASPEC or the Hamilton Microlab SPE systems.

A more sophisticated form of SPE automation is that using flexible or semiflexible robotic manipulation. Semiflexible systems incorporate a robot-like device that performs only a few of the steps involved in sample preparation. Some modern chromatography autosamplers fit into this category. These devices extend the possibilities of unattended sample work-up because operations typically involved in sample conditioning (internal standard addition, dilution, digestion, or derivatization) can also be performed in a fully automated way. The Prep-Station (Hewlett Packard) system is a typical example of this kind of instrument.

In flexible systems, the robot can be programmed to perform all operations involved in the sample conditioning procedure: weighting, dilution, and of course, those required in SPE. Nevertheless, robots are too expensive to be used in SPE operations. For this reason, in normal applications, the robot is interfaced to a SPE workstation. The robot may move cartridges from a dispenser to racks holders, remove spent cartridges, or transfer collection tubes, but rarely does the robot wait for liquids to be dispensed or for air to pass through the cartridges; instead, the SPE workstation performs these operations. In membrane-based SPE and in-tip microextraction, automation can be easily achieved by commercially available systems using 96-well plates and robots.31 This allows the users to process up to 96 samples in parallel.

Robots compatible with SPE have been commercialized by different companies such as Zymark Corporation or Hewlett Packard, among others.

3.2 On-Line Automated Solid-Phase Extraction

In on-line systems, the SPE device is inserted into the liquid or gas stream of the chromatograph, which means that it becomes part of the chromatograph. Automated sample preparation with on-line SPE is accomplished by using column switching. When the precolumn is loaded with the sample (normally a biofluid), the analytes are selectively retained. The precolumn is then washed to remove endogenous interferents, so that they are sent to waste. The analytes are finally desorbed and transferred by means of an electronically controlled switching valve, which passes the mobile phase through the precolumn, and the analytes are eluted out to the analytical LC or GC column. In such a way, off-line sample manipulation is reduced to a minimum and the entire analysis can be easily automated. Hence, SPE/LC or SPE/GC can be considered particular forms of multidimensional LC/LC and LC/GC chromatography, respectively.

The SPE/LC approach is particularly well suited for pharmaceutical analysis, as a vast majority of the samples of interest are compatible with liquid chromatographic systems (especially those operating under reversed-phase conditions). Besides the inherent advantages of automation, this methodology offers some advantages over off-line automated systems, for example, protection of light-sensitive or oxidation-sensitive analytes, because during sample processing, they are kept away from light and air, respectively. Moreover, implementation is relatively inexpensive compared with off-line SPE devices. In many applications, the cost is little more than a switching valve and a precolumn unit over the chromatograph needed for a manual method. However, an additional pump may sometimes be necessary for washing the precolumn. Although good stability is normally achieved for the repetitive injection of 10–100 µL of untreated samples, a clear disadvantage is that periodic regeneration or replacement of the precolumn may be needed to prevent clogging, especially when processing biofluids with a large amount of proteins (for example, blood or plasma). Some commercial systems incorporate cartridge holders that can be loaded with several cartridges. The individual cartridges (which act as a precolumn) are connected to the analytical column by means of a switching arrangement. Each cartridge is periodically replaced by a fresh one, so clogging or memory effects are minimized. Alternatively, SPE in the individual cartridges is performed off-line, and then the cartridges are automatically inserted into the flow stream. Examples of this type of device are the AASP (Varian) and Prospect (Spark Holland) systems.

While on-line SPE/GC is quite popular in environmental analysis, its use is not widespread in the pharmaceutical field. This can be explained by the successful performance of SPE/LC methods, and also by the inherent difficulties of coupling SPE to GC, with either solvent or thermal desorption. Moreover, in pharmaceutical analysis, care must be taken to prevent water-containing eluents from being transferred to the GC part of the system. This problem may be overcome by drying the cartridge with an inner gas stream before the transfer step. Another possibility is to place a drying cartridge between the SPE precolumn and the GC system. Nevertheless, with modern equipment, the introduction of up to 100 µL of solvent can be achieved.

A precolumn can also be used to couple a SPE workstation to a GC chromatograph, as illustrated in Figure 3. Larger sample volumes (up to several milliliters) can be processed in the SPE cartridges. The analytes are subsequently reconcentrated in the precolumn, which facilitates the complete transfer of the relevant fraction of solvent to the GC system. A proper selection of the precolumn packing would also improve the selectivity, for example, by using a packing material with retention capabilities different from those of the SPE cartridges. If required, the precolumn can be dried before connection to the GC unit. As a result, excellent sensitivity and selectivity can be achieved. For example, the system described in Figure 4 enables the detection of some benzodiazepines at low parts per billion levels from only 1 mL of plasma.32 Similarly, a precolumn can be used to couple a dialyzer module to a GC chromatograph (also to a LC chromatograph). The dialysis step adds selectivity as sample macromolecules do not reach the SPE unit.

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Figure 4. Set-up for on-line SPC/GC (a), and the chromatograms obtained from 1 mL of plasma spiked with 100 ng mL−1 of trazodone (T) (b) and blank plasma (c) V1, V2, and V3 are switching valves; SVE, solvent vapor exit; FID, flame ionization detector (reproduced by permission of Vieweg Publishing from Ref. 32.)

A very interesting alternative for on-line automated systems with GC and HPLC is SPME, because difficulties derived from the injection of large volumes of solvents can be overcome. Automated SPME has been achieved by using polymer-coated fused-SI fibers. The fiber previously fixed to a microliter syringe is automatically inserted into the sample vial. The analytes diffuse and partition in the polymeric phase. Next, the fiber is removed from the vial, and the analytes are thermally desorbed, on-column, in the injector of the gas chromatograph. For HPLC, an interface consisting of a six-port switching valve and a desorption camera are needed. This interface replaces the injection circuit of the liquid chromatograph. SPME with fibers is nowadays a very popular technique for sample preparation, and the literature shows hundred of applications in the pharmaceutical field.25 IT-SPME is another format of SPME, which uses capillary columns instead of fibers for extracting the analytes. Among the advantages of IT-SPME over fiber SPME it should be mentioned that the extraction can be easily effected in an on-line mode.26 As stated earlier, packed and open tubular capillary columns can be used in IT-SPME. Configurations with packed capillary columns are similar to those used to implement extractive precolumns (Figure 5a), whereas open capillary columns are generally placed between the needle and the loop of a programmable sample injector (Figure 5b). In the later instance, the analyte is extracted from the sample to the capillary coating by repeated draw/eject cycles of the sample solution until equilibrium or until sufficient analyte is extracted. Next, the analytes are desorbed from the coating transferred to the analytical column by aspirating a desorption solvent or by flushing the capillary with a stream of the mobile phase. Alternatively, the capillary column can be used as the loop of an injector valve (in-valve IT-SPME) as depicted in Figure 5c. The analytes are extracted into the capillary coating during the sample loading (the sample volume can be higher than the internal volume of the capillary). Next, the sample remaining in the capillary is removed by flushing the proper solvent. Finally, the valve is rotated, so the analytes are desorbed from the capillary coating with the mobile phase and transferred to the analytical column.

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Figure 5. Typical configurations of IT-SPME: extraction with packed capillary columns (a), extraction by draw/eject cycles (b), and in-valve IT-SPME (c).

4 Pharmaceutical Analysis

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

4.1 Quality Control

The control of pharmaceutical quality is one of the primary objectives of drug manufacturers. Quality control (QC) has been defined as actions such as testing, monitoring, and inspecting taken to detect and control defects. The control of drug substances and drug product quality involves developing sets of tests and specifications for raw materials, intermediates of interest, and final products, performing the tests on the subject material, compiling the results and submitting them to regulatory authorities on a regular basis. From the synthesis of a drug onward, its identity, strength, purity, and quality are continuously assessed and monitored.

The British and US pharmacopoeias recommend microbial assay, ultraviolet (UV) spectrophotometric and HPLC methods for the determination of pharmaceutical preparations. HPLC is currently the most popular technique for impurity and dissolution testing and formulation determination, and an increasing number of publications using this technique in combination with SPE have been described for QC or quality assurance studies.

In the development of new drugs, the determination of the purity is important to establish the acceptability of batches for safety assessment and clinical trials. In general, process impurities include trace substances resulting from the drug substance synthesis and trace side-reaction products or contaminants not removed by the isolation and purification scheme. Regulatory authorities set various levels for individual and total impurities in both drug substance and drug product that must be reported and investigated. Ideally, the total amount of impurity, as well as the quantity of each individual impurity, should be monitored in the bulk drug. Prabhu et al.33 described the isolation and concentration of impurities in 3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-6-methyl-2(1H)-pyridone (L-696229) using sequential preparative HPLC, SPE, and LLE. Three SPE cartridges connected in series were used.

An example of the use of SFE in studies related to dissolution testing can be found in the study by Kenney et al.34 The antiarrhythmic drug L-768673 in pharmaceutical preparations was retained on a C18 SPE column and was analyzed by HPLC and UV detection at 245 nm.

We have proposed spectrophotometric procedures that integrate sample clean-up and derivatization in the same process by using C18 solid supports. The potential of this technique was evaluated determining the content of dl-amphetamine sulfate in centramine tablets using 1,2-naphthoquinone-4-sulfonic acid (NQS).35 This methodology has also been used for the determination of cephalexin in capsules and oral suspension (both singly and in combination with bromhexine).36

Another important area of application is enantiomeric analysis. While the manufacturing processes may lead to racemic drugs, it is often observed that both enantiomers have different biological activities or only one enantiomer is active and the other one is completely inactive. Enantiomeric purity is controlled in drug manufacture, storage, and shipment for both bulk drug and product for molecular entities with chiral centers. The specific chiral LC methods have involved three general techniques, namely precolumn derivatization to yield diastereomers, mobile phases with constituents to afford separation of enantiomer pairs, and chiral stationary phases. Lots of references can be found on these techniques and several reviews have been written.3, 37, 38 We have used the first option for chiral analysis of amphetamine in pharmaceutical samples using a mixture of o-phthaldialdehyde and N-acetyl-l-cysteine as derivatizing reagent.39 The assay uses a 20 mm ×2.1 mm inner diameter precolumn packed with a Hypersil C18 (30 µm) material for purification and derivatization. The resulting derivatives were subsequently separated in a C18 achiral column using a mobile phase of acetonitrile–methanol–acetate buffer and detected by fluorescence.

4.2 Drug Monitoring

The analysis of drugs in a biofluid matrix by HPLC or any other separation–detection scheme is usually difficult owing to the large number of substances present in the sample and because the compounds of interest are often present at very low concentration. The most problematic fluids are those that contain a large fraction of proteins: blood, plasma, and serum. Cerebrospinal and interstitial fluids, as well as urine, are generally more compatible with LC, owing to their low protein content. Solid samples can be processed after solubilization and homogenization.

Many methods have been published for the determination of drugs in biological matrices using SPE cartridges for sample clean-up. Table 3 shows representative examples using different sorbents for screening analysis and determination of several drugs. For each analysis, Table 3 also gives the separation and detection techniques employed. Several kinds of drugs have been included, such as anticonvulsants, antiasthmatics, antiarrhythmics, antibiotics, diuretics, and illicits. Although the most used detector in HPLC is the UV/visible detector, examples of other detectors are shown in Table 3. Some reports with GC are also recommended. As an example of an analysis that needs a derivatization step, amphetamine and methamphetamine determination has been included in Table 3. SPE has been proposed for both reversed-phase and normal-phase HPLC. An overview of screening procedures using diatomaceous earth, polystyrene–divinylbenzene copolymers, and mixed-mode bonded SI sorbents can be found in Franke and de Zeeuw.6

Table 3. Representative Examples of the Use of SPE Cartridges for Sample Clean-Up in Drug Monitoring
AnalyteStudied sorbentMatrixSeparation and detection techniquesReference
Screening of amiloride, acetazolamide, hydrochlorothiazide, triamterene, chlorthalidone, furosemide, cyclothiazide, bendroflumethiazide, bumetanide, ethacrynic acid, probenecid, and spironolactoneC18, C8, C2, CH, PH and cyanoUrineHPLC with photodiode arrayCampíns-Falcó et al.40, 41
Screening of stimulants, narcotics, sedative hypnotics, antidepressants, antipsychotics, antihistamines, cardiovascular drugs, analgesics, and some derivativesBond Elut Certify (mixed-mode)Urine or serumHPLC with photodiode arrayLai et al.42
Screening of cephalexin, cefotaxime, cefazolin, cefuroxime, and cefoxitinC18, C8, C2, CH, PH, cyano cartridges, and 3 M Empore disk C18UrineHPLC with diode arrayGallo-Martinez et al.43
Screening of benzodiazepines and imidazo-pyridines, antidepressants, antipsychotics, central analgesics, anticonvulsants, antimalarials, b-blocking and b-against agents, drugs to treat cardiac diseases, alkaloids, anticoagulants, analgesics and anti-inflammatory drugs, barbiturates, and carbamatesC18Powdered hairHPLC with photodiode array and GC/MSGaillard and Pepin44
Determination of teophiline, paraxanthine, caffeine, and cefoxitinC18UrineHPLC with diode arrayCampíns-Falcó et al.45, 46
Determination of ethacrynic acidC18UrineHPLC with diode arrayCampíns-Falcó et al.47
Determination of probenecidC18UrineHPLC with diode arrayCampíns-Falcó et al.48
Determination of cefuroxime3 M Empore disk C18UrineHPLC with diode arrayGallo-Martinez et al.43
Determination of cocaine, benzoylecgonine, and cocaethyleneCopolymeric phase combining C8 and strong cation exchangeUrineHPLC with diode arrayClauwaert et al.49
Amphetamine and methamphetamine with NQS as derivatizing reagentC18, C18, C8, C2, CH, PH, and cyano cartridgesUrineHPLC with diode arrayCampíns-Falcó et al.50
    Molins-Legua et al.51
MethadoneHydrophobic and cation-exchange disk (mixed-mode)SerumHPLC with UVRudaz and Veuthey52
Determination of antimalarial drug proguanil and its metabolites, cycloguanil, and 4-chlorophenyl biguanideCN endcappedPlasma, whole blood, and urineHPLC with UVBergqvist et al.53
PhenytoinC18Human breast milk, maternal plasmaHPLC with UVShimoyama et al.54
OxycodoneC18PlasmaHPLC and electrochemicalWright et al.55, de la Torre et al.56
Tricyclic antidepressantsBond Elut TCAPlasmaCapillary GC with nitrogen–phosphorus 
Determination of ecstasy (methylenedioxymethamphetamine), methamphetamine, and methylenedioxy-ethamphetamineC8UrineGC/MSSzignan et al.57

As mentioned earlier, the use of column-switching techniques has become an area of major interest. Its application to drugs in biological samples by HPLC methods incorporating on-line sample clean-up by SPE using column switching has been reviewed.2 The time required for the sample clean-up process using this technique is drastically reduced, as minimum off-line sample manipulation is involved. Therefore, this technique can be very useful where a large series of samples must be processed. As a result, the precision is generally improved. Some examples of that improvement in the analytical results are given in papers about on-line sample clean-up with an octadecylsilica-C18 SPE precolumn and using column switching for screening of diuretics and probenecid58, 59 and the determinations of acetazolamide60 and triamterene61 in urine samples using an octadecylsilica-C18 SPE precolumn.

Antibiotics are used in food-producing animals not only for treatment of disease but also to maintain health and promote growth. The use of unauthorized antibiotics or the failure to follow label directions for approved antibiotics could result in unsafe antibiotic residues in food products. Therefore, monitoring antibiotic residues in food forms part of a general policy to prevent unapproved uses of antibiotics. Different review articles can be found that show current trends in sample preparation and clean-up procedures for the analysis of drugs residues in foods, including SPE-based procedures.5, 62

5 Comparison with Liquid–Liquid Extraction

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

LLE has been the most used technique for sample clean-up, but SPE is now becoming the preferred method for this step of the analysis. It should be remarked that, as stated earlier, SPE is clearly advantageous over LLE as regards the consumption of organic solvents and generation of wastes. This section focuses mainly on a comparison of the two methods in terms of the instrumentation required, as well as in the recoveries and precision attainable.

5.1 Instrumentation

The equipment involved in using a sorption column for off-line processing can be very simple for manual use as described in earlier sections. For automated off-line SPE, more sophisticated apparatus is required. On-line assemblies require only a switching valve for the simplest configuration and additional pumps and valves for specific applications. The exception is in-valve IT-SPME, as this technique only requires the replacement of the inner loop of the injection valve by the extractive capillary column. In summary, SPE can work with or without expensive apparatus, and the partial or total automation of the on — line mode is relatively easy.

In the simplest form of LLE, a separatory funnel is needed. When the distribution constant of the solutes are very small, continuous extractors or countercurrent distribution apparatus is required. A continuous liquid–liquid extractor can be coupled to a gas or liquid chromatograph in three different ways: indirectly, by collecting the flow of the phase of interest in vials after the sample has been aspirated or injected into a flow injection manifold and subsequently injecting their contents manually into the chromatograph; on-line, via a simple turntable acting as an interface between the two continuous separation systems; and on-line, but directly, with no interface or discontinuity between the extractor and the chromatograph. Simplicity and the degree of automation of these three alternatives increase in the same order as they have been mentioned. In all cases, SFE is generally easier and cheaper than LLE.

5.2 Recovery and Precision

Disadvantages of LLE are matrix interferences, emulsion formation, and the use of large volumes of hazardous solvents. SPE can overcome these drawbacks. The methods used for breaking an emulsion in LLE are generally time-consuming. The most common method is the addition of salt (e.g. NaCl) either as a solid or a saturated aqueous solution. Settling of the lower phase can be accelerated in a separatory funnel by the application of heat, as with an infrared lamp. More difficult emulsions may require high-speed centrifugation to separate two phases. Another technique is the use of low-melting-point organic solvents, such as ether, for the extraction of aqueous samples. Emulsions in this case are first separated by centrifugation, then the mixture is frozen in dry ice or liquid nitrogen. Finally, the organic phase is simply poured off, leaving the frozen aqueous phase behind. Another option is to use filter papers.

Where extraction gives low recovery of the desired simple components despite optimum adjustment of solvent polarity and pH, recovery can be increased by using large volumes of solvent for the same sample quantity and by using successive or continuous extraction. Following LLE of the sample, the solvent phase is normally filtered and may be further dried over anhydrous sodium sulfate, concentrated by evaporation, and subjected to further clean-up procedures. The rate and extent of extraction may be different for a solute in a test system and in the presence of a sample matrix. Partial association of drug substances with proteins in plasma samples has been recognized.

Diuretics can provide a good example for testing the recoveries obtained for acidic, basic, and neutral drugs in urine samples processed by LLE and off-line and on-line SI-based bonded-phase SPE.40, 41, 58, 59 Acidic diuretics present better recoveries by LLE in acidic conditions, while basic diuretics increase their recoveries in basic conditions. Neutral diuretics present similar values for both acidic and basic extractions. The urine blanks obtained by SPE in different SI-based bonded-phase cartridges and water as a washing solvent are similar to those obtained by LLE in acidic conditions (Figure 6). The standard deviations obtained are similar to those obtained by LLE, but the recoveries achieved are better, although only a unique cartridge has been used for sample clean-up. C18 sorbent provides higher recoveries for all the analytes. The efficiency of the on-line method is similar to that found with the C18 cartridges (Figure 6). The recoveries obtained for standards and spiked urine are the same, indicating that the sample matrix does not affect the recoveries of diuretics and probenecid. The precision is excellent with coefficients of variation below 6% in spiked urine samples. Also, it is greatly improved in comparison with LLE or SPE on disposable cartridges, as no on-line operation is involved.

thumbnail image

Figure 6. Typical chromatograms obtained at 230 nm from a urine sample by (a) acidic LLE, (b) basic LLE, (c) off-line SPE into a C18 column cartridge, and (d) on-line SPE with column switching using a precolumn packed with a C18 packing.

SDB resins are especially interesting for analyzing urine samples, because sulfate and glucoronide conjugates can be isolated. Extraction yields are in the order of 80%, with a precision of 7–27%, which are on average rather high.6 These types of compounds are not amenable for classic solvent extraction. The SDB resin is also used for isolating substances from other biological matrices such as blood, serum, bile, gastric content, and tissues.

The extraction yields of substances isolated from different biological samples may vary considerably, however. The resin has to be cleaned very carefully. SDB-based SPE columns (e.g. Bond Elut ENV, Varian) have become available, which overcome the above drawbacks. Moreover, SDB material in extraction disks can minimize volumes of elution solvents, while still allowing relatively large sample volumes; this is a main characteristic of the SDB sorbents. Mixed-mode polymeric resins are well suited for the fractionated extraction of acidic, basic, and neutral drugs from urine samples.27

6 Other Applications of Solid-Phase Extraction

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

6.1 Concentration

In pharmaceutical testing (stability or dissolution tests, for example), high concentrations of analytes are usually involved. Consequently, dilution of the samples is often necessary before the actual measurement. In contrast, many other applications usually require some form of analyte enrichment: identification and/or quantification of drug impurities in pharmaceutical preparations (byproducts from the synthesis or degradates); pharmacokinetic studies; therapeutic drug monitoring; and in those situations where the sample volume is restricted, particularly if the sample has to be divided between several screening tests. It should be noted that, in a majority of such cases, the analytes also require purification owing to the large number of substances present in the samples.

Analyte enrichment with SPE column or disk cartridges is based on collecting the analytes in a volume of solvent lower than that of the sample. Unfortunately, in this process, undesirable matrix components may also be concentrated, especially if low selective packings (such as common reversed-phase materials) are used for retention of the compounds of interest. For this reason, washing of the cartridges with an appropriate solvent or mixture of solvents is often needed to improve the selectivity.

In principle, the enrichment factor is only limited by the losses of the analytes from the cartridges due to breakthrough during both the sampling and the washing steps. Therefore, the success of the enrichment procedure depends on a variety of factors: sample volume and type, characteristics of the cartridge, nature and volume of the washing eluent, and volume of solvent used to collect the analytes.

To achieve maximum enrichment, a careful selection of the SPE cartridges should be made according to the basic principles indicated in previous sections. The main parameters to be considered are type of packing material, particle size, and loading capacity. Cartridges packed with 100 mg of packing material are generally suitable for most applications involving the analysis of up to 1 mL of the most common biofluids. In many instances, apolar packings (C18 or C8) provide nearly quantitative recoveries of the analytes and the required selectivity, as salts and other very polar matrix constituents are wasted out with a relatively small volume of washing solvent (normally water or a buffer). However, more selective packings (MIP-based sorbents, restricted access media, immuno-sorbents, and protein-coated phases) may be required for some applications.

For a given SPE cartridge, the volume of washing solvent should be as low as possible to prevent breakthrough. Therefore, the use of large volumes of samples may not compensate the losses of interesting compounds during the washing stage. With respect to the sample type, the most problematic fluids are those containing a high content of particulate material. In those cases, the employment of large sample volumes may be limited by the low flow rates attainable not only during the sample loading but also during the washing and desorption steps. This effect can be minimized if the samples are previously filtered or centrifuged to prevent blocking of the cartridge frits.

However, to achieve the maximum enrichment factor, the volume of solvent used to collect the analytes should be as low as possible. This volume is mainly determined by the amount of packing material in the cartridge, provided that a strongly eluotropic solvent is used for desorption. Cartridges containing 100–200 mg of packing permit the collection of the analyte in relatively small volumes, typically a few hundred microliters. Lower volumes lead to unsatisfactory reproducibility because, after the desorption step, small (and variable) fractions of the eluent may remain in the cartridges. As in LLE, the enrichment factor can be improved by evaporating the collected extracts to dryness, followed by redissolution. Evaporation, however, is time-consuming and may be inadequate if volatile compounds are going to be analyzed. It should be noted that, if chromatography is the final step of the analysis, the sensitivity attainable with the SPE column or with disk cartridges is limited by the fact that only a fraction of the collected extracts is finally injected into the chromatograph.

On-line SPE is an attractive alternative for enrichment of the analytes in chromatography (especially in LC).2 In this approach, enrichment is based on retention of the analytes in a precolumn when a large volume of sample is flushed through it; the precolumn is then flushed with an appropriate solvent, and the enriched analytes are finally transferred to the analytical column.

The main problems associated with the direct injection of biofluids in chromatographic systems are precolumn clogging and irreversible adsorption of matrix constituents to the stationary phases. For these reasons, the volume of samples that can be processed with on-line SPE are significantly lower than those that can be handled with SPE cartridges. Otherwise, rapid pressure development occurs, and the performance of both the precolumn and the analytical column may seriously deteriorate after a few injections. Although special packings (such as RAMs) have been designed for the on-line SPE in the biomedical field,63 sample volumes of 50–100 µL can be adequately processed with conventional packings. Clogging is minimized in IT-SPME configurations with open capillary columns. Nevertheless, the sensitivity attainable with on-line SPE is similar to that of the off-line methods, as in the latter approach only a fraction of analyte is introduced in the chromatographic system. For this reason, on-line SPE is the best alternative for trace enrichment when the sample volume is restricted.

Moreover, careful selection of working conditions allows the injection of large sample volumes with suitable stability for most applications. For example, a straight-flush configuration (in which the flow direction of the mobile phase through the precolumn is not changed for the transfer onto the analytical column during the transfer step) protects the analytical column from solid particles or matrix components retained at the head of the precolumn, thus making possible the injection of relatively large volumes of biofluids. In addition, the precolumn lifetime can be extended by including a regeneration step in the final procedure. For regeneration, the precolumn is disconnected from the analytical column, and then it is flushed with a strongly eluotropic solvent. As regeneration can be carried out during the chromatographic separation, the total analysis time is not increased. In many instances, occasional cleaning (e.g. when daily work is finished) allows the analysis of several samples with suitable stability and performance of the system for routine work. This is illustrated in Figure 7, which shows the effect of the successive injection of 250 µL of untreated plasma on back-pressure. Although back-pressure in the precolumn (packed with a 40-µm C18 stationary phase) is approximately duplicated every 25 injections, the pressure at the top of the analytical column remains constant. Cleaning of the precolumn with an organic solvent (e.g. acetonitrile) every 25 injections reduces pressure and permits the system to be used for the analysis of many other samples. Under these conditions, quantification at sub-parts per million levels is possible for a large number of compounds in plasma or urine.

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Figure 7. Effect of the number of injections on the back-pressure of the precolumn (+), the analytical column (×), and on the two columns linked (•). The dashed line indicates cleaning both columns with acetonitrile (approximately 15 mL).

Enrichment via a precolumn can also be used to overcome dilution, if dialysis is used to remove macromolecular constituents in biofluids (Section 3.2).

6.2 Derivatization

In pharmaceutical analysis, chemical derivatization is often needed to enhance the sensitivity and/or the selectivity of the overall analytical process, particularly when using LC. In this respect, most of the described procedures are based on precolumn solution derivatization because these reactions do not suffer from kinetic limitations and can be conducted under very flexible conditions (pH and temperature). In the analysis of biological samples, a preliminary step is analyte purification because the reagent can be reactive toward matrix constituents. In addition, derivatization generally involves extra sample handling aimed at removing unreacted reagent or to preconcentrate the derivatives. As a result, the final procedure may be very tedious and time-consuming.

Precolumn derivatization may be simplified in some instances with SPE materials. Krull et al.64 proposed a very interesting approach based on using solid-phase reagents for simultaneous clean-up and derivatization.

The solid-phase reagent contains ionic or covalent labile tags that possess specific detector enhancement properties. The choice of the pore size of the solid support (or the percentage of cross-linking when using a polymer network) permits size exclusion, thus enabling retention of small molecules but hindering the large ones. After purification, the adsorbed analytes are made to react with the reactive part of the solid-phase material. Finally, the derivatives formed are desorbed for further analysis. This methodology has been successfully used for the derivatization of several amines with a variety of reagents in both off-line and on-line modes.

Another approach to derivatization with SPE supports is based on the successive addition of the SPE material (normally a resin) and the derivatization reagent to the samples. The derivatives are retained on the SPE material and isolated from the matrix and from the excess of reagent by filtration. Next, the derivatives are desorbed from the resin, which is then separated by filtration. This methodology has been applied, for example, to simplify multiple derivatization required for the analysis of prostaglandins in plasma by GC.65

Alternatively, derivatization can be carried out in conventional SPE cartridges or precolumns (in on-line SPE) as has been developed in our group. In the former approach, the SPE cartridge is used to selectively retain and/or preconcentrate the analytes and then to retain the derivatives formed when an aliquot of the reagent is flushed though it. The analyte and the reagent are made to react for a given period of time. Then, the excess of reagent can be removed (if required) by flushing the cartridge with an appropriate solvent. Finally, the derivatives are collected for further processing. The on-line methodology is based on similar principles, but the time of reaction is controlled by stopping the flow rate in the precolumn after injecting the reagent. It is important to note that, under this approach, sample clean-up, enrichment, and derivatization can be performed without manual intervention. In addition, minimum instrumentation is needed over conventional equipments: an isocratic pump, a precolumn, and a switching valve. This methodology has been applied to the LC determination of different types of compounds such as amino acids, amphetamines, and polyamines in plasma and urine, in combination with reagents typically used to improve UV or fluorescence detection, as can be seen in Table 4. Chemical derivatization onto extractive sorbents can also be effected with SPME fibers, as illustrated for amphetamine and related drugs.66 For this purpose, the amphetamines were extracted from urine or plasma by immersing a carbowax-templated resin-coated fiber into the sample. Next, the fiber was removed from the sample and immersed into a solution of the derivatization reagent for the required time. Finally, the derivatives were desorbed into a SPME/HPLC interface and chromatographed. Derivatization is also compatible with IT-SPME. The reagent is loaded into the capillary coating, and then, the sample is passed through the capillary for the extraction and derivatization of the analytes. After the required reaction time, the remaining solution is removed from the capillary by passing water through it. Next, by rotating the injection valve (Figure 5c), the derivatives are desorbed with the mobile-phase and transferred to the separative column. This extraction/derivatization scheme has been applied for the determination of short-chain aliphatic amines in water,67 but applications in the pharmaceutical filed have not yet been described. However, the methodology could be of interest in the analysis of pharmaceuticals with derivatizable groups in environmental waters, as IT-SPME enables the introduction of large sample volumes into the extractive capillary, and therefore, excellent sensitivity can be achieved.

Table 4. Methods for Determination of Drugs in Urine, which Integrate Clean-Up and Derivatization in the Same Process
AnalyteStudied sorbentDerivatizing reagentSeparation and detection techniquesReference
Amphetamine and methamphetamineC18 cartridge and precolumnNQSHPLC with diode arrayCampíns-Falcó et al.68
    Herráez-Hernández et al.69
Norephedrine, ephedrine, pseudo-ephedrine, amphetamine, 3-phenylpropylamine, and methamphetamineC18, C8, C2, CH, PH, CN cartridges, and 3 M Empore C18 disk3,5-Dinitrobenzoyl chlorideHPLC with diode arrayHerráez-Hernández et al.70
Norephedrine, ephedrine, pseudo-ephedrine amphetamine, methamphetamine, and 3-phenylpropylamineC18 cartridge and precolumn9-Fluorenylmethyl chloroformateHPLC with fluorescenceHerráez-Hernández et al.71
Amphetamine and methamphetamineC18 cartridge and precolumnDansyl chlorideHPLC with fluorescence and chemiluminescenceMolins-Legua et al.72
Putrescine, cadaverine, spermidine, and spermineC18 cartridgeDansyl chlorideHPLC with fluorescenceMolins-Legua et al.73

7 Future Trends

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

Because during the past decade interest in implementing green chemistry principles has grown dramatically, research efforts are focused on designing and using environment-friendly sample treatment methods that reduce the time of analysis, the consumption of chemicals, and the generation of wastes. In this context, current research in SPE is clearly aimed toward development of miniaturized and automated systems, particularly for the analysis of pharmaceuticals in biological samples.74 Future advances can be expected in development of miniaturized forms of SPE that can be coupled on-line to chromatographic systems15 or integrated in microfluidic devices.75 Additional benefits in this area will be reduction of the volume of sample required and higher sample throughput.

As regards the sorbents, most attention is being focused in the development of new sorbents (i.e. mixed-mode polymeric sorbents) that combine high capacity and selectivity in a single material.13 Efforts are also put into exploiting the unique properties of nanoparticles in SPE.76 Studies on the development of miniaturized formats of SPE using nanoparticles and sol-gel coatings are of particular interest,77 and substantial progress in this area can be expected in near future.

8 Acknowledgments

  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References

The authors are grateful to the Spanish Ministerio de economia y competitividad (project CTQ 2011–26760) Y.M.M. expresses her gratitude for a JdC research contract (Ministerio de Ciencia e Innovación).

Abbreviations and Acronyms

Carboxylic Acid








Gas Chromatography


High-performance Liquid Chromatography


In-tube Solid-phase Microextraction


Liquid Chromatography


Liquid–Liquid Extraction


Mass Spectrometry




1,2-Naphthoquinone-4-sulfonic Acid


Phenylboronic Acid




Propylsulfonic Acid


Primary/Secondary Amine




Quality Control


Restricted Access Media


Quaternary Amine






Solid-phase Extraction


Solid-phase Microextraction


Benzenesulfonic Acid






  1. Top of page
  2. Introduction
  3. Basic Theory
  4. Off-Line Versus On-Line Procedures
  5. Pharmaceutical Analysis
  6. Comparison with Liquid–Liquid Extraction
  7. Other Applications of Solid-Phase Extraction
  8. Future Trends
  9. Acknowledgments
  10. Related Articles
  11. References
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