Sample Preparation Perspectives
The sample preparation step in an analytical process typically consists of an extraction procedure that results in the isolation and enrichment of components of interest from a sample matrix. Extraction can vary in degree of selectivity, speed, and convenience and depends not only on the approach and conditions used but on the geometric configurations of the extraction phase. Increased interest in sample preparation research has been generated by the introduction of nontraditional extraction technologies. These technologies address the need for reduction of solvent use, automation, and miniaturization and ultimately lead to on-site in situ and in vivo implementation.
These extraction approaches are frequently easier to operate but provide optimization challenges. More fundamental knowledge is required by an analytical chemist not only about equilibrium conditions but, more importantly, about the kinetics of mass transfer in the extraction systems. Optimization of this extraction process enhances overall analysis. Proper design of the extraction devices and procedures facilitates convenient on-site implementation, integration with sampling, and separation/quantification, automation, or both. The key to rational choice, optimization, and design is an understanding of the fundamental principles governing mass transfer of analytes in multiphase systems. The objective of this perspective is to summarize the fundamental aspects of sample preparation and anticipate future developments and research needs.
1. Why Sample Preparation is needed?
Sample preparation has, for a long time, been a relatively neglected part of the systems for chemical analysis. Often, sample preparation has been performed by highly manipulative multi-step procedures using methods such as conventional liquid-liquid extraction. As other parts of the analytical procedures, e.g. separation and detection, have been further developed, sample preparation has, in many cases, become somewhat of a bottleneck in the total procedures. Because of this, sample preparation has attracted increasing attention during recent years, and the need for new improved methods has been largely recognized. Indeed, there has been a major change in the general attitude towards sample preparation. Frequently, it was earlier considered as a separate procedure prior to the real analysis while it nowadays has become a, more or less, integrated part of the analytical procedure. To be able to develop sample preparation methods to new much more advanced levels that are, or will be, required, it is necessary to base their development on a systematic and scientific approach. Thus fundamental understanding of the different processes involved in a sample preparation method should serve as a basis for its optimization. This is in contradiction with the earlier oftentimes applied practical approach. In the opinion of the author, a key to the recent development is, in the first place, miniaturization, thus adapting to the general trend in analytical chemistry. Indeed, this is a presupposition for the integration of sample preparation in the total analysis systems. Such matching of the different parts of the analysis system has led, and will lead, to improved over-all performance. This concerns qualities such as accuracy and precision, sensitivity, selectivity, throughput and cost-effectiveness. The current interest in new sample preparation methods has resulted in the recent publication of a number of books and reviews on this topic.
In many application areas, there is an urgent need for faster analyses—high-throughput analyses. In the pharmaceutical industry, approaches such as combinatorial chemistry and parallel synthesis provide a means of production of large numbers of compounds to be used for drug-discovery experiments. For determination of the physiological performance of a drug it has to be determined in various biological fluids. This inevitably leads to an increase in the number of biological samples for pharmacokinetic analysis. At the same time, desired concentration levels for quantitative analysis have been reduced. All this puts bioanalytical chemistry under pressure to provide methods for high-throughput analysis at low analyte concentration levels. This situation has been described in a number of reviews. It seems that the need for high throughput can be met by application of automated integrated analytical systems which include the sample clean-up. As increased throughput is achieved for sample preparation, the bottleneck will move to another part of the analysis chain!
The major goal of sample preparation is to prepare the sample for the separation/ detection part of the analysis. For drug analysis in plasma or whole-blood samples, the sample preparation should thus remove the drug from the matrix for quantification, separate the drug from endogenous interfering components, and, if needed, concentrate the drug. To anyone practicing analytical chemistry, a sample preparation method should fulfil the following requirements: it should be easy to handle, be fast and robust, automated, and operated at a low cost/sample. The recovery, accuracy, and precision should be within internationally accepted limits. Further, high throughput should be provided. These goals cannot be met by traditional methods and that is why these are currently being further developed.
- Source: Bloomberg, Anal. Bioanal. Chem. 2009, 393, 797-807
2. Sample preparation geared to LC-MS
The sample preparation method must be adapted to the demands of the separation/ detection part that follows. Current methods for quantitative bioanalysis, in general, include LC coupled to tandem mass spectrometry, LC-MS-MS. Often, multiple reaction monitoring (MRM) is used. Due to the high selectivity of the MRM, it might be considered unnecessary to separate the analytes in the LC column. This would facilitate the use of generic LC methods, which are often called for. Thus it would usually not be necessary to develop LC methods tailored to different types of sample. However, the analysis is complicated by the possible presence of ion suppression, sometimes called matrix ionization, at the LC-MS interface. Interferences co-eluting with the analyte from the LC-column may thus compete in the analyte ionization process leading to reduced or enhanced analyte signal. For plasma samples, the ion suppression may, depending on the matrix, differ from sample to sample and quantitative analysis thereby becomes compromised. The degree of ion suppression depends on type of ionization technique used, where ESI is usually more sensitive to this effect than APCI. Further, the type of interface matters, where Z-spray < orthogonal spray linear spray geometries. The risk of ion suppression puts high demands on the sample preparation. In fact, the risk of ion suppression is a factor that sets the currently necessary level of sample preparation for LC-MS-MS. Interferences remaining after the sample preparation may be removed from the analytes in the LC separation step. Interferences are likely to be eluted in the LC front where unretained compounds are eluted or at the end of the elution gradient, where strongly retained compounds are eluted; therefore it is desirable to have the analytes well away from the front and the end of the gradient. To achieve this, the gradient may have to be adjusted in some cases. A certain degree of separation in the LC is thus desirable, which may come into conflict with the wish for very short LC-run times.
About matrix components and endogenous materials in human plasma:
Biological matrices include plasma, serum, cerebrospinal fluid, bile, urine, tissue homogenates, saliva, seminal fluid, and frequently whole blood. Quantitative analysis of drugs and metabolites containing abundant amounts of proteins and large numbers of endogenous compounds within these matrices is very complicated. Direct injection of a drug sample in a biological matrix into a chromatographic column would result in the precipitation or absorption of proteins on the column packing material, resulting in an immediate loss of column performance (changes in retention times, loss of efficiency and capacity). Similar damage can occur to different components of the ESIMS/MS system commonly utilized for analyzing drugs. Matrix components identified by different analytical techniques are shown in the Table below. Major classes encountered in plasma consist of inorganic ions/salts, proteins and/or macromolecules, small organic molecules, and endogenous materials.
3. Sample preparation for herbal medicines/supplements
Plants are naturally gifted at the synthesis of medicinal compounds. The extraction and characterization of active compounds from medicinal plants have resulted in the discovery of new drugs with high therapeutic value. A classic example is aspirin, which was initially discovered as salicylic acid in willow bark and leaves; another noted example is taxol, recently proven to be effective against breast and ovarian cancers, which was initially discovered in bark of yew trees.
The use of medicinal plants (herbs) has a long history throughout the world and herbal preparations, including herbal extracts, can be found in the pharmacopoeias of numerous countries. In recent years there have been a renaissance of interest in natural or herbal remedies worldwide, partly because of the realization that modern medicine is not capable of providing a “cure-all” solution against human diseases and that the presence of unwanted side-effects is almost unavoidable. Unlike modern drugs that invariably comprise a single active species, herb extracts and/or prescriptions contain multiple active constituents. Interestingly, natural compounds contained in these “herbal cocktails” can act in a synergistic manner within the human body, and can provide unique therapeutic properties with minimal or no undesirable side-effects.
A key factor in the widespread acceptance of natural or alternative therapies by the international community involves the “modernization” of herbal medicine. In other words, the standardization and quality control of herbal materials by use of modern science and technology is critical. At present, however, quality-related problems (lack of consistency, safety, and efficacy) seem to be overshadowing the potential genuine health benefits of various herbal products, and a major cause of these problems seems to be related to the lack of simple and reliable analytical techniques and methodologies for the chemical analysis of herbal materials.
Sample preparation is the crucial first step in the analysis of herbs, because it is necessary to extract the desired chemical components from the herbal materials for further separation and characterization. Thus, the development of “modern” sample preparation techniques with significant advantages over conventional methods (e.g. reduction in organic solvent consumption and in sample degradation, elimination of additional sample clean-up and concentration steps before chromatographic analysis, improvement in extraction efficiency, selectivity, and/or kinetics, ease of automation, etc.) for the extraction and analysis of medicinal plants is likely to play an important role in the overall effort of ensuring and providing high quality herbal products to consumers worldwide.
- Source: Carmen W. Huie, Anal. Bioanal. Chem. 2002, 373, 23-30
4. Sample preparation for food samples and complexity of food matrices
The term “food” refers to the broad range of edible materials that comprise the essential body nutrients required for life and growth, such as proteins, carbohydrates, fats, vitamins, or minerals. Foodstuffs are described variously as “liquid” or “solid”, and “wet” or “dry”, depending on the amounts of water and fat they contain. Samples of plant origin are classified for analytical purposes as having a high or medium water content and a lower content of saccharides (from 5% to 15%), very low water content (dry), or a high content of oils. Similarly, food samples can be divided into four main groups based on water and fat content. Food samples of biological origin (liquid or solid) have been divided generally into the five categories: milk, eggs, other samples of animal origin (e.g. muscle, liver, and fat), plant material, food (meat, fish, cereals, wine, juice, oils, sugar, etc). This coarse division is important when considering the choice of isolation technique, extraction solvent, and sample clean-up method during an analytical procedure.
5. Sample preparation in Cosmetics
Recent concerns about UVA and UVB radiation from the sun contributing to skin cancers have highlighted the importance of applying an effective sunscreen before exposure to these harmful rays. As sunscreens are categorized as drug products by the U.S. Food and Drug Administration (FDA), they must undergo rigorous testing before being released to market. Proposed changes to the FDA regulations governing the labeling of sunscreen products are also expected to come into force by the end of 2009, which will require manufacturers to supply information to regulators on the UVA screening provided by its products. As a result of regulatory concerns, an efficient means of analysis is needed that can provide fast and dependable measurements of UV-absorbing compounds.
There are many available techniques for analyzing these compounds, including thin layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC). However, the oily matrix of sunscreen lotions coupled with the high UV absorbance of the analytes makes ultra high performance liquid chromatography (UHPLC) the method of choice for analyzing sunscreens. By additionally using photodiode array detection, the complete absorption spectrum of each compound is obtained as it elutes. The sensitivity of HPLC/PDA is also sufficient to measure these compounds in environmental water samples, which facilitates research on exposure and environmental fate.
SAMPLE PREPARATION PROTOCOLS IN USE
A number of advances have been made during the past decade to convert sample preparation techniques used for about 30 years for the clean-up of drugs in biological matrices into formats that are amenable for high volume processing with or without automation. Detailed accounts about the fundamentals of these techniques can be found in several books, reviews and articles published in the literature. Therefore, only brief descriptions of the principles of these methods will be summarized. For isolating drugs and metabolites from biological and other complex matrices, several approaches have been reported, which consist of:
- Solid phase extraction (SPE)
- Liquid-liquid extraction (LLE)
- Protein precipitation (PPT)
- Affinity separations (MIP)
- Membrane separations
- Preparative high performance liquid chromatography (HPLC)
- Solid phase micro extraction (SPME)
- Ultrafiltration and micro dialysis
SPE, LLE, and PPT are the most commonly used sample preparation techniques and hence most of the discussion will be devoted to them. All of these methods have certain ultimate goals summarized below.
- Concentrate analyte(s) to improve limits of detection and/or quantitation
- Exchange analyte from a non-compatible environment into one that is compatible with chromatography and mass spectrometric and/or other instrumental analytical techniques
- Remove unwanted matrix components that may interfere with the analysis of the desired compound
- Perform selective separation of individual components from complex mixtures, if desired
- Detect toxins in human system or in environment (air, drinking water, soil)
- Identify stereochemical effects in drug activity and/or potency
- Follow drug binding to proteins
- Determine stability and/or absorption of drugs and follow their metabolism in human body
How to select the most appropriate Sample Preparation Method
Should depend on three specific criteria:
- Requirements of the assay
- Time allowed to run sample prep method
- Possible investment towards method development time
1. Late Discovery/Early Development (Pharmaceutical Arena)
- Requires rapid sample turn around
- Higher limits of quantitation
- Very little method development time (1-2 days)
- Protein Precipitation may be ideal choice
2. Development (pre-clinical and clinical) (Pharmaceutical Arena)
- Drugs more potent and dosed at lower levels
- Requires ultra-sensitivity, great selectivity and rugged method development
- Greater method development time (3-5 days)
- SPE is the more ideal choice