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Chapter 3 Accelerated Solvent Extraction Devanand Luthria a, *, Dutt Vinjamoori b , Kirk Noel c , and John Ezzell d a USDA/ARS/FCL, Beltsville, MD 20705; b Monsanto, St. Louis, MO 63167; c Monsanto, Ankeny, IA 50021; d Dionex Corporation, Salt Lake City Technical Center, Salt Lake City, UT 84119 Abstract Extraction of solid and semisolid samples using liquid solvents is a common practice in nearly every analytical laboratory. Years of empirical testing have resulted in rugged and reproducible methodologies for a wide range of analyte classes. However, recent concerns regarding the volumes of organic solvents used (with the associated human exposure), along with increased purchase and disposal costs, have emphasized the need for more efficient sample extraction methods. In response to these concerns, accelerated solvent extraction (ASE ® , Dionex Corporation, Salt Lake City, UT) was introduced. Since its introduction in 1995, ASE has grown rapidly as an accepted alternative to traditional extraction methods. Accelerated solvent extraction takes advantage of the enhanced solubilities that occur as the temperature of a liquid solvent is increased. Increasing the temperature of solvent results in a decrease in viscosity, allowing better penetration of the sample matrix. In addition, analyte diffusion from the sample matrix into the solvent and overall solvent capacity are increased. In tradi- tional Soxhlet extraction, the solvent that comes into contact with the sample has passed through a cooling condenser, and is therefore close to room temperature at the point of contact. The time required to complete Soxhlet extractions ranges from 6 to 48 h. Semi-automated Soxhlet systems that immerse the sample into boiling solvent are available. This increase in the temperature of the contacting solvent shortens the required extraction time to ~2 h. Using these systems, a further increase in tempera- ture beyond the boiling point of the solvent is not possible due to solvent loss because these systems operate at atmospheric pressure. However, a continued increase in the temperature should continue to enhance the extraction process. This can be accom- plished by applying pressure, which maintains the solvent in its liquid state beyond its atmospheric boiling point. This is the theoretical basis for ASE technology and repre- sents the next step in liquid solvent extraction of environmental samples. There are, of course, limits to which raising the temperature is feasible, due to thermal degradation concerns. However, as evidenced by data published to date, there is room to continue raising the temperature, thereby improving the extraction efficiency, without risking analyte degradation in environmental samples. As the extraction efficiency is *The research work was done at Monsanto. Copyright © 2004 AOCS Press increased, the time required to perform extractions and the amount of solvent needed is reduced. When performing ASE, a 10-g sample can be extracted in ~12 min using 12–15 mL of solvent. As the sample size is increased (33 mL volume maximum), the amount of solvent used increases proportionally (45–50 mL maximum), but the total extraction time remains unchanged. The short extraction times and small solvent usage make this technique amenable to automation. Samples loaded into stainless steel extraction vessels (11, 22, or 33 mL internal volume) are extracted sequentially into standard 40- or 60-mL glass collection vials. After extraction, the spent sample remains in the cell, whereas the extract is immediately ready for processing. The sys- tem is designed to extract up to 24 samples unattended. Because existing solvent-based extraction methods can be readily transferred to ASE technology, methods develop- ment is greatly simplified. Existing sample preparation and postextraction processing steps remain in place because the extracts generated by ASE will be of very nearly the same composition as those generated with the existing solvent-based extraction tech- nique. The large range of polarities and solvent strengths available when using liquid solvents, including solvent mixtures, allows a high degree of flexibility and selectivity when developing methods for new sample matrices. Introduction Accelerated solvent extraction (ASE), also referred to as pressurized fluid extrac- tion (PFE) and pressurized liquid extraction (PLE), is a liquid solvent extraction technique that uses aqueous and organic extraction solvents at elevated tempera- tures and pressures. Although the initial applications focus of this technique was the environmental area, the versatility and ease of use of the approach have proven useful for laboratories performing extractions in the food and polymer industries, as well as in the pharmaceutical and consumer products areas. Traditional reflux-based extraction techniques such as Soxhlet extraction can take from 4 to 48 hour to perform, and 24-h extractions are common. Other liquid solvent-based extraction techniques such as wrist shaker, hot plate boiling, and sonication require copious amounts of solvent and often involve extensive labor steps such as filtering or concentration before extract analysis. One thing that they all have in common is operation at ambient pressure. An increase in temperature beyond the boiling point of the solvent is not possible due to solvent evaporation. Accelerated solvent extraction is performed by using the same solvents as in the traditional approaches, but at higher temperatures than is possible in these techniques. This increase in temperature improves the kinetics of the process, resulting in more efficient extractions (faster and using less solvent) compared with traditional approach- es. The solvents are used under pressure so that their liquid state is maintained under heated conditions. For example, solvents such as water, methanol, acetone, or hexane are routinely used in ASE at temperatures ranging from 75 to 150°C. The solvents are maintained as liquids under pressure, normally at 1500 psi (10.4 MPa). ASE is per- formed, therefore, using very hot liquids to expedite the extraction process. Copyright © 2004 AOCS Press The flow-through design of the technique results in extracts that do not require the extended labor of filtration as a means of separating the sample matrix from the extracted analytes. In further contrast to traditional extraction approaches, all of the basic steps are amenable to automation, freeing the analyst from the labor-inten- sive nature of most sample preparation protocols. Automated ASE systems can extract up to 24 sample cells, and have built in the necessary safety considerations for unattended operation. Instrumentation A schematic diagram of an ASE system is shown in Figure 3.1. The extraction proce- dure consists of a combination of dynamic and static flow of the solvent through a heated extraction cell containing the sample. These cells must be capable of safely withstanding the pressure requirements of the system, and are normally constructed of stainless steel, with frits in the end caps to allow the passage of solvent while maintain- ing the solid sample within. Cell sizes range from 1 to 100 mL. The pore size of the frit should not allow passage of the matrix particles (5–10 µm is typical). The sample cell is interfaced to the solvent flow path, where it is filled with solvent. It is important to ensure that all of the void volume has been filled with solvent to have good contact between the sample matrix and the solvent, and to avoid possible analyte oxidation, which may occur in the presence of air at elevated temperatures. The sample cell is then heated by direct contact with a heat source (heating the cell before solvent intro- duction can result in the loss of volatile compounds). To maintain the extraction sol- vents in their liquid state, a pressure source must be applied. The system pressure must be above the threshold required to maintain the liquid state of the solvent at the set temperature and be able to move the solvent through the sample cell in a reasonable time period. This is normally accomplished with an HPLC-type pump, which can Fig. 3.1. Schematic diagram of an ASE system. Copyright © 2004 AOCS Press maintain a constant fluid pressure of 1000–3000 psi (6.9–20.7 MPa). Once thermal equilibrium has been reached, the sample cell is maintained at the set temperature for an additional time period of 5–10 min. During this static phase, analyte diffusion from the matrix into the solvent is believed to occur. After this static hold step, the outlet valve is opened and a measured volume of fresh solvent, usually 40–60% of the cell volume, is allowed to flush over the sample, discharging the previous volume into the collection vial or bottle. Last, compressed nitrogen gas is used to force all of the sol- vent from the lines and cell into the collection vessel. It is important that all of the liq- uid solvent used in the extraction process be collected for analysis. The collection ves- sels normally used are standard 40- or 60-mL vials, or 250-mL bottles, sealed with Teflon-coated septa. This allows the extracts to be collected and maintained in a sealed, inert environment (under a nitrogen blanket) to prevent sample loss while wait- ing for quantification. Sample Preparation Proper sample preparation is essential to obtain efficient and reproducible extrac- tions. The ideal sample for extraction is a dry, finely divided solid, through which the extraction solvent can easily flow and thoroughly penetrate the matrix particles. Whatever can be done, within reason, to make samples approach this definition will be beneficial to the extraction process. Generally, samples should be prepared for ASE extraction in the same manner as traditional extraction techniques. Samples with large particle sizes (>1 mm) should be ground so as to increase the surface interaction of the solvent and matrix. Wet or sticky samples should be mixed with drying agents such as sodium sulfate or pelleted diatomaceous earth, or with dispersing agents such as Ottawa sand before extraction. Typical sample sizes used in ASE are 1–50 g of solid or semisolid material. Sample Extraction Parameters Extraction Solvent. As extraction parameters, solvent choice and temperature have the greatest effect on extraction efficiency with ASE. An extraction solvent that would solubilize the target analyte(s) but leave the majority of the sample matrix intact should be chosen. This is normally done by matching the polarity of the solvent and target analyte. ASE extraction can be performed with the entire range of aqueous and organic solvents, with the exception of strong mineral acids (hydrochloric, nitric, sul- furic), which will attack the stainless steel flow path of the system. In those cases in which an acidic pH is required, small amounts (1–5%) of acetic, phosphoric, or other weak acids can be used. The choice of solvent should also be considered in light of the postextraction analysis technique to be utilized. Solvents such as methanol and ace- tonitrile are suitable for direct HPLC injection, whereas solvents such as hexane, meth- ylene chloride, or acetone are more suitable for complete evaporation, or concentration and GC analysis. If the target compounds are easily oxidized, solvents should be degassed before use. It has been observed that solvents that perform only marginally Copyright © 2004 AOCS Press well at ambient temperature will often perform quite well at elevated temperature. This increases the range of solvent choices available to the analyst considering ASE because the use of more than one solvent may result in good recovery of target ana- lytes. The selection of the appropriate solvent can then be made on the bases of selec- tivity of extraction, solvent cost, safety and exposure factors, and compatibility with postextraction processing steps. Solvent mixtures should also be considered in cases in which minor adjustments to polarity are desired. Automated sequential extraction of the same sample with multiple solvents is also possible. This approach can be used for fractionation of analytes based on polarity from the same sample matrix. This approach is very valuable for isolation and separation of bioactives from different nat- ural sources. Extraction Temperature. ASE extraction can be performed from ambient tempera- ture to 200°C. Increased temperature will increase the efficiency of the extraction process, and this should be optimized short of the point at which analyte degradation or excessive co-extraction of matrix components occurs. Many applications are per- formed in the 75–150°C range, with 100°C as the recommended starting point for new methods development. In this temperature range, significant increases in extrac- tion efficiency are observed without breakdown of target compounds. If an extraction is to be performed on a compound with a known thermal degradation point, then the method should be developed to operate below that point. Extractions performed at low (40–70°C) or ambient temperatures may be sufficient for analytes that are weakly or only surface-bound to the matrix. The extracts generated using ASE will be similar in composition to those produced by other techniques using the same solvents. If a pos- textraction clean-up step is required after a Soxhlet extraction, the same process will most likely have to be performed after ASE. Extraction Pressure. Although essential to the process, pressure is not generally con- sidered a critical parameter. Normal operating pressures of 1500–2000 psi (10.3–13.8 MPa) are well above the threshold pressures required to maintain the solvents in their liquid states at ASE operating temperatures. The main purpose of using pressures in the ranges indicated is to provide rapid filling and flushing of the extraction cells. Typical extractions are performed in 12–20 min, although this time can be extended for difficult samples. In addition, multiple static cycles can be used to periodically introduce aliquots of fresh solvent during the extraction process. Method Development and Optimization When developing a new method, the following approach has proven useful. A rep- resentative sample should be prepared as outlined above. Select an extraction cell size that most closely matches the desired sample size. The extraction cells do not have to be filled completely; however, a full cell will use less solvent in the extrac- tion process than a partially filled one. Select the extraction solvent using the con- siderations listed above, although normally the same solvent or solvent mixture Copyright © 2004 AOCS Press used in a traditional liquid extraction method is used. Extract the sample starting with the following standard conditions: pressure, 1500 psi (10.3 MPa); tempera- ture, 100°C; heat time, 5 min; static time, 5 min; flush volume, 60% of cell vol- ume; purge time, 60 s; static cycles, 1. Extract the same sample multiple times to assess the efficiency of the method. If there is significant analyte present in the second or third extracts, adjust the following parameters (one at a time), and repeat the validation process: (i) Increase the temperature (use 20°C steps). (ii) Add a sec- ond or third static cycle. (iii) Increase the static time (use 5-min increments). If these steps do not result in a complete extraction, reexamine the sample prepara- tion steps and/or the choice of extraction solvent. Applications ASE extraction technology has been used extensively in various industries. Some of the applications of ASE extraction technologies are summarized briefly below. Extraction of Crude Fat (Oil) from Soybean Seeds Ground corn and soybean seeds were placed in the extraction cell and crude fat was extracted with ASE. Details of extraction parameters and solvent used are list- ed in Table 3.1. The total crude fat content was determined by collecting the extracts in preweighed vials followed by evaporation of the solvent under a nitro- gen stream. The percentage crude fat content was determined gravimetrically. Comparison of Crude Fat Content Determination by ASE with Standard Butt- tube and Soxtec Procedures. The total crude fat content (dry matter basis) was determined in three soybean samples by three different extraction methods. Each sample was ground with a ball grinder mill and the ground sample was mixed to ensure homogeneity. The same ground sample was used for analyses to reduce the effect of particle size on extraction efficiency. Results are reported on a dry matter basis (DMB) to eliminate the effect of moisture content on crude fat analyses. Six replicate analyses were performed on each sample for each of the three methods: ASE , Soxtec TM (Foss North America, Eden Prairie, MN), and Butt-tube. Table 3.2 compares the percentage of crude fat, standard deviation (SD) and the relative stan- TABLE 3.1 Operating Conditions for Accelerated Solvent Extraction Preheat 0 min Pressure 1000 psi Heat 6 min Temperature 105°C Static 5 min Solvent compartment A Flush 50% volume Petroleum ether 100% Purge 90 s Average run time/sample 30 min Cycles 3 Copyright © 2004 AOCS Press TABLE 3.2 Comparison of Three Crude Fat Extraction Procedures from Three Soybean Samples a Soxtec Soxtec Soxtec ASE ASE ASE Butt-tube Butt-tube Butt-tube 1 g/2 g 1 g/2 g 1 g/2 g 2 g 2 g 2 g 2 g 2 g 2 g Sample ID Average SD RSD Average SD RSD Average SD RSD SOY 1 19.68 0.20 1.02 21.15 0.07 0.32 19.54 0.08 0.41 SOY 2 21.86 0.23 1.05 22.66 0.05 0.21 21.22 0.11 0.54 SOY 3 23.70 0.18 0.74 24.61 0.12 0.50 23.32 0.11 0.46 Soy average 21.75 0.20 0.94 22.81 0.08 0.34 21.36 0.10 0.47 a Results are average of six replicate analyses. Copyright © 2004 AOCS Press dard deviation (RSD) from the three soy samples by three different methods. The results in Table 3.2 indicate that the percentage crude fat extracted by the ASE pro- cedure for soybean samples were 1.1 and 1.4% higher, than the Soxtec and Butt- tube methods, respectively. The higher extraction yield may be due to differences in extraction conditions or passage of very fine particles through the frit, or pas- sage of moisture during the flush cycle because analyses were carried out on “as is” basis and the results were converted to DMB after analysis. Effect of Sample Size. To evaluate the effect of sample size on crude fat determi- nation, a single ground soybean sample was extracted in six replicates with sample sizes varying from 20 mg to 2 g. The average crude fat extracted from the various sample sizes ranged from 20.3 to 21.3% (Table 3.3). The SD and % RSD gradually decreased as sample size increased from 20 mg to 2 g. Reproducibility. Table 3.4 depicts the ruggedness data of the percentage of crude fat extracted from 125 soy samples with two ASE instruments by multiple operators over a period of time. The SD and % RSD from 125 replicate analyses of crude fat extract- ed from the soybean samples were 0.32 and 1.56%, respectively. The results indicate that single seed or partial seed analysis is feasible using ASE. In particular, this technique should be very helpful for the analysis in F1 and F2 stages of plant breeding and for screening rare and elite germplasm lines in which sample amounts available are always limited. Extraction of Tocopherols from Soy and Corn Analysis of tocopherols in soy and corn is of considerable importance from the nutritional perspective. Although there are several HPLC methods reported in the literature, few reliable sample preparation/extraction techniques exist that ensure the integrity and stability of tocopherols with quantitative recoveries. Addition of an antioxidant such as pyrogallol or ascorbic acid to the extraction solvent usually helps in achieving quantitative recoveries. However, no such antioxidant need be used if the ASE technique is used because the extraction is performed under nitro- gen atmosphere and samples are collected in sealed vials. Figure 3.2 illustrates the comparison between the manual tissue grinder extraction with ethanol containing pyrogallol and ASE extraction with EtOH alone (ASE conditions are the same as those stated in Table 3.1). Defatting of Soy Samples for Isolating Soy Protein–Enriched Fractions The soy protein–enriched fraction is currently used for different nutraceutical for- mulations. Preparation of soy protein isolate is usually accomplished by the stirring and/or soaking approach with hexane. ASE extraction offers a much better alterna- tive because similar results are obtained more quickly, with reduced solvent usage (Table 3.5). Copyright © 2004 AOCS Press TABLE 3.3 Effect of Variation of Sample Size on the Percentage of Oil Recovery from Ground Soybeans Vial Vial Expected Actual sample Average number weight sample size size (mg) Vial + Oil Oil % Oil % oil SD RSD (%) 1 21900.58 20 20.12 21904.67 4.09 20.33 20.92 0.32 6.6944 2 21751.64 20 20.06 21755.86 4.22 21.04 3 22114.1 20 20.45 22118.39 4.29 20.98 4 21936.47 20 20.22 21940.73 4.26 21.07 5 21901.18 20 20.73 21909.59 4.41 21.27 6 21852.14 20 20.48 21856.41 4.27 20.85 13 21879.49 50 50.42 21889.76 10.27 20.37 20.29 0.53 10.7537 14 21877.48 50 50.4 21887.62 10.14 20.12 15 21993.32 50 50.56 22003.76 10.44 20.65 16 22170.74 50 50.43 22181.61 10.87 21.55 17 21847.3 50 50.26 21857.48 10.18 20.25 18 21892.28 50 50.24 21902.47 10.19 20.28 1 22043.77 75 75.5 22059.1 15.33 20.3 20.23 0.2 4.046 2 21982.11 75 75.31 21997.59 15.48 20.56 3 21654.63 75 74.95 21669.62 14.99 20 4 21922.27 75 75.26 21937.41 15.14 20.12 5 22017 75 75.24 22032.14 15.14 20.12 6 21646.67 75 75.23 21661.94 15.27 20.3 7 21799.92 100 100.67 21819.99 20.07 19.94 20.11 0.11 2.2121 8 22152.1 100 100.02 22172.21 20.11 20.11 9 21916.85 100 100.16 21937.03 20.18 20.15 10 21838.45 100 100.77 21858.88 20.43 20.27 11 21769.81 100 100.82 21790.12 20.31 20.14 12 21932.01 100 100.5 21952.14 20.13 20.03 (Continued) Copyright © 2004 AOCS Press TABLE 3.3 (Cont.) Vial Vial Expected Actual sample Average number weight sample size size (mg) Vial + Oil Oil % Oil % oil SD RSD (%) 13 21848.53 250 250.32 21900.57 52.04 20.79 20.86 0.08 1.6688 14 21847.81 250 250.51 21900.02 52.21 20.84 15 21732.02 250 250.24 21784.43 52.41 20.94 16 22029.23 250 250.52 22081.57 52.34 20.89 17 21855.07 250 250.45 21907.02 51.95 20.74 18 21984.08 250 250.32 22036.51 52.43 20.95 19 21956.02 500 500.17 22062.05 106.03 21.2 21.13 0.04 0.8452 20 21887.51 500 500.62 21993.18 105.67 21.11 21 22427.7 500 500.68 22533.39 105.69 21.11 22 21715.06 500 500.53 21820.67 105.61 21.1 23 22307.78 500 500.08 22413.38 105.6 21.12 24 21969.67 500 500.35 22075.50 105.83 21.15 25 21750.43 1000 1000.26 21964.14 213.71 21.37 21.23 0.11 2.3353 26 21799.76 1000 1000.17 22013.21 213.45 21.34 27 21771.48 1000 1000.81 21983.71 212.23 21.21 28 22017.33 1000 1000.55 22229.52 212.19 21.21 29 21919.8 1000 1000.28 22130.69 210.89 21.08 30 21796.91 1000 1000.38 22008.87 211.96 21.19 31 22122.96 2000 2000.62 22550.82 427.86 21.39 21.32 0.05 1.066 32 21917.83 2000 2000.51 22344.12 426.29 21.31 33 21956.1 2000 2000.05 22380.74 424.64 21.23 34 22179.94 2000 2000.76 22607.11 427.17 21.35 35 21554.21 2000 2000.2 21980.69 426.48 21.32 36 21686.5 2000 2000.09 22112.87 426.37 21.32 Copyright © 2004 AOCS Press [...]... petroleum ether (AOAC Method 90 .39 ) Results for a variety of samples are shown in Table 3. 6 The ASE method used here was shown to be useful for both low- and high-fat meat samples and saved considerable time compared with the traditional approach 35 000 30 000 Control ASE 25000 Amount 20000 15000 10000 5000 0 Stigmasterol Stigmasterol Sitosterol Fig 3. 3 Extraction and GC analysis of fecal sterols Copyright... Accelerated Solvent Extraction: A Technique for Sample Preparation, Anal Chem 68: 1 033 –1 039 Schäfer, K (1998) Accelerated Solvent Extraction of Lipids for Determining the Fatty Acid Composition of Biological Material, Anal Chim Acta 35 8: 69–77 Luthria, D., and Cantrill, R (2002) Evaluation of Five Different Methodologies for Determination of Oil Content in Ground Corn and Soybean Seeds, Inform 13: 8 93 894 Official... Ezzell, J.L (1999) Extraction Methods in Organic Analysis: Pressurised Fluid Extraction (PFE) in Organic Analysis, (Handley, A.J., ed.), pp 146–164, Sheffield Academic Press, Sheffield, England Richter, B.E (1999) The Extraction of Analytes from Solid Samples Using Accelerated Solvent Extraction, LC/GC 17: 6S 32 S Richter, B.E., Jones, B.A., Ezzell J.L., Porter, N.L., Avdalovic, N., and Pohl, C (1996)... Tortilla chips Snack chips 34 .0 32 .8 33 .3 21.5 19.2 0.11 0.08 0.17 0.07 0.10 0 .33 0.25 0 .34 0 .34 0. 53 aConditions: 3- g samples, 125°C, 1000 psi, 6 min heat up, 10 min static, 100% flush, 60 s purge, chloroform, 3 static cycles Copyright © 2004 AOCS Press TABLE 3. 8 Extraction of Fat from Dog Biscuits: Comparison of Results by Soxhlet and ASEa Method Soxhlet ASEa aConditions: Solvent Petroleum ether Petroleum... Press Sitosterol TABLE 3. 6 Extraction of Fat from Meat Samples by ASEa Sample (n = 3) Beef Chicken Ham Bacon Sausage Average % fat weight SD Soxhlet 2.85 0.82 1.82 46.66 33 .80 0.046 0.025 0.069 0.820 0.280 2.81 0.75 1.72 46. 83 33. 54 aConditions: 1-g samples, 125°C, 1500 psi, 6 min heat up, 10 min static, 60% flush, 60 s purge, hexane, 2 static cycles Other Applications Table 3. 7 depicts the percentage... pharmaceutical and nutraceutical products used for cholesterol reduction The most common approach for quantifying the amount of fecal sterol is the saponification of the fecal matter followed by extraction with dichloromethane Figure 3. 3 shows the comparison of extraction of fecal sterol by a standard procedure vs ASE procedure The extracted sterols were isolated and analyzed by GC Extraction of Fat... 15 6 90 60 2 10 1 10 10 2 ASE extraction conditions were the same as those in Table 3. 1 except for the number of cycles and static time ent formula types, including a certified reference material (SRM 1846) available from the National Institute of Standards and Testing The fat content was determined gravimetrically, and verified by fatty acid methyl ester analysis Fat extraction from a variety of meat... traditional extraction techniques, the recoveries generated by ASE normally equal or slightly exceed the comparative method The ability to use the same liquid solvents used in traditional methods allows for rapid converTABLE 3. 7 Extraction of Fat from Snack Food by ASEa Sample (n = 5) Average % fat weight SD RSD (%) Potato chips Corn chips Cheese snacks Tortilla chips Snack chips 34 .0 32 .8 33 .3 21.5 19.2... Tissue Grind 100 ASE 50 0 Alpha Gamma Delta Fig 3. 2 Correlation of ASE and tissue grinder extractions for tocopherols in soybeans Copyright © 2004 AOCS Press TABLE 3. 5 Defatting of Soy Samples for Isolation of Soy Protein–Enriched Fractions Classical stirring/soaking Amount of solvent per extraction, mL Number of extractions, n Total amount of solvent, mL Total extraction time, min Amount of fat left over,... with wet Hydromatrix (3 g + 0.4 g water) and extracted at 100°C ASE extraction of these samples can be performed without the aggressive alkaline pretreatments required by some methods Extraction results were compared directly with results obtained using alkaline pretreatment followed by Mojonnier extraction with a mixture of petroleum ether, diethyl ether, and ethanol (AOAC Method 932 .06) The results . 500 500 .35 22075.50 105. 83 21.15 25 21750. 43 1000 1000.26 21964.14 2 13. 71 21 .37 21. 23 0.11 2 .33 53 26 21799.76 1000 1000.17 220 13. 21 2 13. 45 21 .34 27 21771.48 1000 1000.81 219 83. 71 212. 23 21.21 28. 1.066 32 21917. 83 2000 2000.51 2 234 4.12 426.29 21 .31 33 21956.1 2000 2000.05 2 238 0.74 424.64 21. 23 34 22179.94 2000 2000.76 22607.11 427.17 21 .35 35 21554.21 2000 2000.2 21980.69 426.48 21 .32 36 . 106. 03 21.2 21. 13 0.04 0.8452 20 21887.51 500 500.62 219 93. 18 105.67 21.11 21 22427.7 500 500.68 22 533 .39 105.69 21.11 22 21715.06 500 500. 53 21820.67 105.61 21.1 23 2 230 7.78 500 500.08 224 13. 38