An Overview of Sample Preparation Techniques for Food Analysis
S. Kathirvel*, R. Raju, K.K. Aneesha Fasla, V.P. Shahana
Department of Pharmaceutical Analysis, National College of Pharmacy, Manassery, Mukkam post, Kozhikode, Kerala
*Corresponding Author E-mail: firstname.lastname@example.org
This write-up provides a review on the various sample preparation techniques involved in the analysis of the food substances and food products. The awareness about food safety is highlighted. The sampling procedure involved in food analysis is discussed. A different sample preparation method including its advantages and disadvantages are addressed. The applicability of the all the methods involved in sample preparation techniques of food analysis is also touched upon.
Foods are diverse group of products that are often supplemented with additional items such as preservative, anti-oxidants, vitamins and also some time contaminated with harmful substances such as pesticides residues. The awareness about food safety and food analysis has grown to a greater extent as a result of stringent regulation. It is absolutely necessary to carry out a detailed analysis of the food product for its composition, nutritional value and toxicological aspect. It must be emphasized that a successful food analysis cannot be achieved without an appropriate, convenient and reliable sample preparation methodology. The food analysis procedure should start with sampling. An ideal sample should be true representative of the bulk material. Grinding, chopping, digestion and centrifugation are some of the procedures utilized when performing sampling. When a homogeneous sampling has been carried out, then the sample is ready for the subsequent analysis. A computer- assisted literature survey revealed the availability of various methods involved in the food analysis [1-3]. However the present review article encompasses the recent techniques involved in the sample preparation methods of food analysis.
SAMPLE PREPARATION METHODS:
Essentially, sample preparation can be carried out in two steps: 1.Extraction of the target analytes and 2.Removal of interfering substances. Traditional techniques used for sample preparation are often time consuming and require large amount of solvent. Of late, lots of developments have taken place which has resulted in the miniaturization of the techniques used. Today we have many micro extraction techniques along with automated, fast, cheap and solvent less sample preparation methods. Different sample preparation methods are available today. The user can select an appropriate method suitable for the sample and the as per the requirement of analysis. Each method will be having its own advantages as also few disadvantages .Some of the important techniques are described below.
1. Pressurized Liquid Extraction (PLE):
This technique utilizes pressurized solvents that are brought to a temperature higher than their boiling point, without being vaporized because of the pressure applied. This procedure brings the solvent to a physical state that is closed to that of a supercritical fluid, with a positive increase in its extraction efficiency. Usually in PLE process, the temperature ranges between 80 deg and 200 deg with a corresponding pressure range of about 1000-2500 psi.PLE has been applied to a variety of food sample. The advantages of this technique include reduced solvent conception and improved extraction efficiency. But it also requires expensive equipments and pressure proof vessels. Food sample with high moisture content should be pre-treated with desiccants for water removal.
2. Microwave assisted Extraction (MAE):
Chemicals absorb microwave in a manner that is directly proportional to their value of dielectric constant. The higher the dielectric constant, stronger the absorption. Based on this principle, microwave can be used as an extraction aid in a procedure that can be theoretically placed between SFE and PLE. When performing a MAE, the analyst can choose to work with closed or open PTFE vessels, depending on the solvent used. This technique can be used for the extraction of variety of food constituent such as vitamins, pesticides, and lipids etc .One major drawback of the technique is the possible co-extraction of interfering compounds causing a lack of selectivity.
3. Solvent- Assisted Flavour Evaporation (SAFE):
Solvent-assisted flavor evaporation (SAFE) was introduced in 1999.SAFE has already been applied to food matrices such as milk, fruit pulps, juices etc. volatiles from the food sample are stripped and collected in to flask which are liquid nitrogen cooled. One of the disadvantages of SAFE is the equipment maintenance, which is time consuming especially when dealing with fatty matrices.
4. Liquid Phase Micro Extraction (LPME):
The most recent sample preparation techniques have been modified in to miniaturized versions which allows for both less conception of solvents and analyte concentration. Hence, liquid-liquid extraction has given birth to liquid phase micro exraction (LPME) introduced jeannot and cantwell in1996. This technique is also known as Single Drop Micro Extraction (SDME). This technique exploits a drop, at the tip of a microsyringe, of a water- immiscible solvent immersed in a aqueous sample containing analytes. Usually a few microlitres of solvent are enough to allow the extraction. As soon as the extraction is considered complete, the drop is withdrawn in to the needle and can be injected into the system (for example, a GC system). The simplicity of technique and its low cost have made SDME an attractive to classical liquid-liquid extraction. An acceptable choice of solvent (1-octanol, ethylene glycol, xylene, hexane etc) contributes selectivity to SDME. The technique can be easily automated which improves the repeatability of data. A disadvantage of the technique is low sensitivity depending upon sample complexity. Several applications on food analysis using SDME have been reported.
5. Solid Phase Extraction (SPE):
This extraction technique, better known as “sample cleanup” technique is one of the most widely used in food analysis and the technique dates back to 1980s. It is based on the use of disposable cartridges or discs packed with sorbent material, the most common being silica derivatized with alkyl octadecyl chains. The sample, dispersed in a liqued, is passed through the sorbent bed, where analytes remain adsorbed, while undesired elements are not retained or selectively flushed away in second step. The procedure is fast, simple easily automated and environment friendly. Recent advances in SPE can be attributed to the increasing availability of different sorbent materials which improves the selectivity of the technique.
6. Dispersive Solid Phase Extraction (DSPE):
A novel SPE approach was introduced in 2003 by anastassiades. This technique involves a preliminary food extraction by means of acetonitrile. In a second step, a salt (magnesium sulphate, sodium chloride and/or sodium sulphate) is added to drive partitioning between the aqueous and acetonitrile phases. The acetonitrile layer is then removed further SPE, the sorbent (generally PSA, primary-secondary amine), salt and solvent extract are mixed together and centrifuged. The supernatant layer is then recovered and this can be injected into the chromatographic system. DSPE is linked to a standardized AOAC extraction method for pesticides in food: QuEChERS, acronym of quick, Easy, Cheap, Effective, Rugged, and Safe. This method has gained popularity worldwide because it is simple, fast, robust and covers a wide range of pesticides.
7. Matrix Solid Phase Dispersion (MSPD):
This technique was introduced to improve the effectiveness of SPE on biological matrices. MSPD basically involves three steps: 1. Sample blending with a sorbent material, 2. packing of a pre-fritted SPE cartridge with the blend, 3. Elution of analytes with suitable solvents. Usually the sample is crushed in a mortar where a sorbent is placed; thereby causing it’s dispersion over the surface of the sorbent. With subsequent solvent elution, it is possible to selectively get rid of undesired compounds. Common solvents used in MSPD include methanol, acetone and dichlormethane , sometime added with triethylamine or acetic acid.
8. Solid-Phase Dynamic Extraction:
This technique exploits a principle similar to that used in solid-phase micro extraction (SPME). It is based on the use of thin film sorbent, coated on the inside wall of a hollow needle. First introduced as INCAT (Inside Needle Capillary Adsorption Trap), this technique can extract and concentrate analytes directly onto the GC syringe that is then injected onto the GC system causing analytes to be thermally desorbed.
9- Head Space Sorptive Extraction (HSE):
Solid-Phase Micro Extraction (SPME) has become a popular sample preparation technique. Inspite of its popularity, SPME suffers from some drawbacks, one being the low concentration capability. To overcome this deficiency, new techniques like Stir Bar Sorptive Extraction (SBSE) and Head Space Sorptive Extraction (HSSE) were introduced. SBSE utilizes a glass encapsulated stir bar coated with layer of PDMS (25- 250L). This device can be easily used to extract analytes from solution. In HSSE, the same device is suspended in the sample head space. Analytes accumulate on the coating and are then transferred into the thermal desorber connected to the GC system. Alternatively, analytes can be removed from the stir bar by means of liquid-liquid extraction. A disadvantage of this technique is the use of PDMS as sorbent material which strongly limits it’s selectivity. HSSE has been applied to a wide variety of food matrices.
10- Solid-Phase Aroma Concentrate Extraction (SPACE):
This sample extraction technique is a modified version of headspace SPME, basically exploiting a stainless steel rod coated with a mixture of sorbent material (mainly graphite carbon). The extraction consists of fixing the rod at the head of a closed flask, where it adsorbs the aroma. As in SPME, the adsorbed analytes are released in to the GC by thermal desorption. The advantage of this technique is mainly the increased extraction surface which allows enhanced sensitivity.
Once a sample preparation technique is chosen, the laboratory must validate the procedure to prove that the chosen method is reliable and fit-for-purpose, which can be done by participating in proficiency testing/inter-laboratory comparison programmes. Participation in at least one proficiency test/inter-laboratory comparison is required in order to achieve and/or maintain accreditation according to ISO/IEC 17025:2005
Traditionally in food safety testing laboratories, liquid-liquid extraction (LLE) has been the most popular sample preparation choice. LLE relies upon the partitioning of analytes and interfering compounds into immiscible organic and aqueous layers. During the separation of organic and aqueous solvents, it is sometimes impossible to avoid the formation of emulsions that make it difficult to collect the layer that contains the target analytes, and the repeatability and accuracy of the method can be compromised. Two of the more rapidly growing sample preparation techniques are QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and solid phase extraction (SPE). QuEChERS was developed by Steven Lehotay and Michelangelo Anastassiades 4as a means to extract and analyse multi-residue pesticides from food samples. QuEChERS is performed in two steps — extraction, which utilizes various salts to partition organic compounds into one layer, followed by a dispersive SPE step that relies on SPE sorbents to remove unwanted interferences such as lipids and organic acids. This is a non-selective technique that can be adapted to analyse just about any food matrix (e.g., fatty, waxy or pigmented), with a minimal amount of method development. Each chemical class of pesticide has its own unique properties. A targeted sample preparation technique would not be able to extract the different classes because there is no single shared chemical or physical property that can be targeted. For this reason, a nonspecific technique such as QuEChERS will allow analysts to perform a cleanup step by selectively targeting unwanted compounds, leaving the various pesticides untouched and ready to be analysed. Although the QuEChERS sample preparation approach is an excellent solution for laboratories that wish to analyse many compounds of different chemical classes, the technique does not allow for easy concentration of analytes. When the laboratory needs to analyse a particular compound or a specific class of compounds, SPE is an excellent clean-up choice. SPE targets compounds based on their chemical or physical properties, allowing interferences to be washed away, resulting in cleaner extracts compared to non-selective techniques such as QuEChERS. SPE also allows concentration of the sample, which improves the signal-to-noise ratio. SPE does, however, require that method development be performed to ensure the most suitable sorbent is chosen. In another application analysing aflatoxins in peanut butter, SPE was chosen as the most appropriate sample preparation technique to increase sensitivity as a result of high signal-to-noise ratio. For laboratories that wish to achieve better extraction efficiency while analysing target compounds that have similar physical and chemical properties, SPE will be the most appropriate sample preparation method.
Food analysis is a complex issue that requires continuous modification of current approaches. Much emphasis is to be given for improving selectivity and sensitivity, automation to improve repeatability and micro systems to reduce chemical consumptions. The rapid development of sample pre-treatment indicates the need for methods which are compatible with modern analytical techniques. The continuously increasing number of samples to be tested, related to the growing concern over food safety, require methods that must be simple, reliable, cheap and, not least, take in account chemical laboratory waste problems. Because of the increased regulation for food safety, laboratories must be diligent when selecting their means of analysing for contaminants or other harmful substances. Sample preparation is perhaps the most important step in the analysis because it can affect the analyte concentration and the cleanliness of the sample prior to further analysis. In order to achieve the most reliable analysis of chemical hazards, the analyst must first consider how the sample preparation step will affect the following one — the instrumental analysis. Food matrices are notoriously complicated because they contain components such as carbohydrates, lipids and proteins. It has been estimated that about 30 percent of analytical errors originate from the sample preparation step, making it even more important that the technique chosen is reliable and repeatable. Whenever possible, official analytical methods provided by international organizations should be followed. However, it is sometimes necessary for a laboratory to create its own documented in-house method. In this case, the analyst will need to determine the most appropriate preparation technique for the samples at hand.
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3. Angelika Beyer, Marek Biziuk, Applications of sample preparation techniques in the analysis of pesticides and PCBs in food. Food Chemistry.2008; 108: 669-680.
4. M. Anastassiades, S.J. Lehotay, D. Tajnbaher and F.J. Schenk. J.AOAC International.2003; 86: 2.
Received on 17.10.2016 Accepted on 27.12.2016
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