Sample preparation method development

P.D. McDonald and E.S.P. Bouvier, Solid Phase Extraction Applications Guide and Bibliography, A Resource for Sample Preparation Methods Development, Waters, Milford, MA... 

One of the most important trends to simplify these complications is the generation of simple, rapid, and reliable procedures for sample preparation. Method development and setup require the use of materials of known compositions, for example, certified reference materials. Therefore, spiking experiments have to be performed for method quality control. Under such experimental conditions, emphasis has to be placed on the spiking procedures as they exert an influence on the recovery values. Although present scientific knowledge is not perfect, the use of spiking experiments helps to minimize the errors. The integration and automation of all steps between sample preparation... 

The complex of the following destmctive and nondestmctive analytical methods was used for studying the composition of sponges inductively coupled plasma mass-spectrometry (ICP-MS), X-ray fluorescence (XRF), electron probe microanalysis (EPMA), and atomic absorption spectrometry (AAS). Techniques of sample preparation were developed for each method and their metrological characteristics were defined. Relative standard deviations for all the elements did not exceed 0.25 within detection limit. The accuracy of techniques elaborated was checked with the method of additions and control methods of analysis. 

The coupling of supercritical fluid extraction (SEE) with gas chromatography (SEE-GC) provides an excellent example of the application of multidimensional chromatography principles to a sample preparation method. In SEE, the analytical matrix is packed into an extraction vessel and a supercritical fluid, usually carbon dioxide, is passed through it. The analyte matrix may be viewed as the stationary phase, while the supercritical fluid can be viewed as the mobile phase. In order to obtain an effective extraction, the solubility of the analyte in the supercritical fluid mobile phase must be considered, along with its affinity to the matrix stationary phase. The effluent from the extraction is then collected and transferred to a gas chromatograph. In his comprehensive text, Taylor provides an excellent description of the principles and applications of SEE (44), while Pawliszyn presents a description of the supercritical fluid as the mobile phase in his development of a kinetic model for the extraction process (45). 

How critically interdependent matrix and analytical methods can be is illustrated in the example of the analysis of a soil sample. Table 7.1 shows the method dependent certified values for some common trace elements. The soil had been subjected to a multi-national, multi-laboratory comparison on a number of occasions (Houba et al. 1995) which provided extensive data. The data was subjected to a rigorous statistical program, developed for the USEPA by Kadafar (1982). This process allowed the calculation of certified values for a wide range of inorganic analytes. Uniquely, for the soil there are certified values for four very different sample preparation methods, as follows ... 

In order to increase the overall extraction efficiency during SFE sonication has been applied [352]. Ultrasound creates intense sinusoidal variations in density and pressure, which improve solute mass transfer. Development of an SFE method is a time-consuming process. For new methods, analysts should refer the results to a traditional sample preparation method such as Soxhlet or LLE. 

Indeed, a bDNA assay for diagnosis of African trypanosomiasis was developed and compared with buffy coat microscopy for detection of T brucei in human blood samples (Harris etal., 1996). Two repetitive DNA sequences found only in the T. brucei complex, a 177-bp satellite repeat and the ribosomal mobile element, were selected as targets in the bDNA assay. The assay used the standard bDNA components capture probes, target probes, amplifier molecules, and alkaline phosphatase-labeled probes. Various blood fractions and sample preparation methods were examined. Ultimately, buffy coat samples resulted in the highest sensitivity. Although typanosomes do not infect leukocytes, they cosediment with them. 

It needs to be pointed out, that the investigation of some technically important polymers like polyolefines has not been very successful so far. Owing to their inert nature they are difficult to dissolve and also difficult to ionize. Typically one needs for the ionization process some heterogeneities or double bonds in the polymer. For some insoluble substances a solvent-free sample preparation method has been developed that allows a characterization by MALDI-TOF mass spectrometry [93]. 

As shown in Figure 7.2, most assays involve a common series of steps that must be completed in order to report results. These steps include sample receipt, method development, sample preparation, analysis, data processing, and data reporting. While most researchers focus on speeding the analysis step, any of these steps can become bottlenecks. Thus it is important to optimize the whole process. 

When compared with conventional extraction and sample preparation methods, the developer claims that the MAP technology reduces production time, energy, solvent consumption, and waste production, while increasing extraction yields and product purity. 

As well as typical sample preparation methods such as filtration and liquid-liquid extraction, newer developments are now extensively used. The first of these is solid-phase extraction (SPE). This is a rapid, economical, and sensitive technique that uses several different types of cartridges and disks, with a variety of sorbents. Sample preparation and concentration can be achieved in a single step. Interfering sugars can be eluted with aqueous methanol on reversed-phase columns prior to elution of flavonoids with methanol. 

A systematical approach of sample preparation methods and optimisation of the quality aspects of sample preparation may enhance the efficiency of total analytical methods. This approach may also enhance the quality and knowledge of the methods developed, which actually enhances the quality of individual sample analyses. Unfortunately, in bioanalysis, systematical optimisation of sample preparation procedures is not common practice. Attention to systematical optimisation of assay methods has always been mainly on instrumental analyses problems, such as minimising detection limits and maximising resolution in HPLC. Optimisation of sample extraction has often been performed intuitively by trial and error. Only a few publications deal with systematical optimisation of liquid-liquid extraction of drugs from biological fluids [3,4,5]. 

Spiked sample a sample added with a known amount of related substances, prepared from authentic samples during method development or validation. 

Application of any analytical method is always easier when the matrix does not contain species that interfere with determination of the analyte. However, when an interference is expected, it is necessary to isolate the component to be measured from the matrix. Therefore, the quality of the result often depends on sample preparation. This preliminary step can have a more important influence on the end result than the measurement itself or the precision of the instrument used. Sample preparation, which follows the so-called sampling procedure, can often be tedious, delicate and time-consuming. Nonetheless, it has become an active area of study that benefits from the recent progress in chemistry and robotics. Currently used instruments that allow fast and selective measurements on very small amounts of sample have encouraged the development of new, rapid sample preparation methods. 

You need to decide the goal of an analysis before developing a chromatographic method. The key to successful chromatography is to have a clean sample. Solid-phase microextraction, purge and trap, and thermal desorption can isolate volatile components from complex matrices. After the sample preparation method has been chosen, the remaining decisions for method development are to select a detector, a column, and the injection method, in that order. 

Several other sample preparation methods were developed to simplify the solution-deposition procedures. For example, Cooks and coworkers studied adduct ion formation (cationization) of several organic compounds when the organic was burnished (rubbed) onto a metal foil, mixed with a metal salt and then burnished onto a metal foil, or just mixed with metal powders or salts and pressed into pellets. Not only did the SIMS spectra show dramatic differences is the efficiency of adduct ion formation for different metals, but the sample preparation methods had an equally dramatic effect on the SIMS spectra [18]. 

---