Within-laboratory Relative Standard Deviation Research Articles

Chiral determination of nornicotine, anatabine and anabasine in tobacco by achiral gas chromatography with (1S)-(-)-camphanic chloride derivatization: Application to enantiomeric profiling of cultivars and curing processes.

The alkaloid enantiomers are well-known to have different physiological and pharmacological effects, and to play an important role in enantioselectivity metabolism with enzymes catalysis in tobacco plants. Here, we developed an improved method for simultaneous and high-precision determination of the individual enantiomers of nornicotine, anatabine and anabasine in four tobacco matrices, based on an achiral gas chromatography-nitrogen phosphorus detector (GCNPD) with commonly available Rtx-200 column using (1S)-(-)-camphanic chloride derivatization. The method development consists of the optimization of extraction and derivatization, screening of achiral column, analysis of the fragmentation mechanisms and evaluation of matrix effect (ME). Under the optimized experimental conditions, the current method exhibited excellent detection capability for the alkaloid enantiomers, with coefficients of determination (R2) > 0.9989 and normality test of residuals P > 0.05 in linear regression parameters. The ME can be neglected for the camphanic derivatives. The limit of detection (LOD) and limit of quantitation (LOQ) ranged from 0.087 to 0.24 μg g-1 and 0.29 to 0.81 μg g-1, respectively. The recoveries and within-laboratory relative standard deviations (RSDR) were 94.3%~104.2% and 0.51%~3.89%, respectively. The developed method was successfully applied to determine the enantiomeric profiling of cultivars and curing processes. Tobacco cultivars had a significant impact on the nornicotine, anatabine, anabasine concentration and enantiomeric fraction (EF) of (R)-nornicotine, whereas the only significant change induced by the curing processes was an increase in the EF of (R)-anabasine.

Graphene Adsorbent-Based Solid-Phase Extraction for Aflatoxins Clean-Up in Food Samples

In this research, graphene nanoparticles were used as an adsorbent for the extraction and pre-concentration of the aflatoxins (AFs) in rice and wheat samples. The graphene was synthesized according to the Hummer’s method, packed in empty SPE cartridges and different parameters affecting the efficiency of the process for the extraction of AFs were thoroughly investigated. The extracts were subsequently analyzed using high-performance liquid chromatography and fluorescence detection (HPLC–FLD). Under the optimized conditions, limits of detection of 0.63, 0.47, 0.62 and 0.83 ng g−1 and limits of quantification of 1.92, 2.65, 1.88 and 2.83 ng g−1 were obtained for AFs B1, B2, G1 and G2, respectively. The accuracy was estimated by determination of the relative recoveries (RR%) and acceptable values obtained. The RR% values of 75.88–113.30 and 70.61–110.75 were obtained in cereal samples and at the spiked level of 2 and 5 ng g−1, respectively. Within-laboratory relative standard deviations (%RSDs) for repeatability (n = 6) were in the range of 2.14–3.17. The proposed new method can be applied for the separation and pre-concentration of the mentioned AFs in food samples with satisfactory results.

Development and validation of LC-MS/MS method for the determination of Ochratoxin A and its metabolite Ochratoxin α in poultry tissues and eggs

ABSTRACTThe objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Ochratoxin A (OTA) and Ochratoxin α (OTα) in poultry tissues and eggs. The two toxins were extracted by a mixture of acetonitrile/water, purified with a reversed phase C18 solid phase extraction column (SPE) and determined by LC-MS/MS. The LC-MS/MS method performances were evaluated in terms of linearity in solvent and in matrix (ranged from 0.5 to 15.10 µg L−1 for OTA and from 0.60 to 17.85 µg L−1 for OTα), limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy and precision in repeatability conditions. Recovery experiments were performed by spiking poultry liver, kidney, muscle and eggs around 1 µg kg−1 and 10 µg kg−1. LODs were 0.27 and 0.26 µg kg−1 while LOQs were fixed at 1.0 and 1.2 µg kg−1 for OTA and OTα, respectively. Main recoveries for OTA ranged from 82 to 109% and for OTα ranged from 55 to 89%. The values of within-laboratory relative standard deviation (RSDr) were equal to or below 20%. Considering the results obtained and that all analytical performance criteria were fulfilled, the new extraction and purification method developed for OTA and OTα determination in animal tissues and eggs was found appropriate for control laboratories and research activities designed to ensure food safety.

AACCI Approved Methods Technical Committee Report: Collaborative Study on a Method for Determining the Water Holding Capacity of Pulse Flours and Their Protein Materials (AACCI Method 56-37.01)

A method for determining the water holding capacity of pulse flours and their protein materials has been developed and subjected to an interlaboratory study. Eleven participants analyzed twelve blind duplicates of six different samples in a collaborative study to evaluate the repeatability and reproducibility of the method. Statistical analysis of the collaborative data determined that the within-laboratory repeatability standard deviation (sr) ranged from 0.018 to 0.083, and the among-laboratory reproducibility standard deviation (sR) ranged from 0.039 to 0.18. The within-laboratory relative standard deviation (RSDr) of samples ranged from 2.08 to 5.49%, and the among-laboratory relative standard deviation (RSDR) ranged from 4.99 to 7.49%. Results indicated that the method has good repeatability and reproducibility.

Rapid determination of residues of pesticides in honey by µGC-ECD and GC-MS/MS: Method validation and estimation of measurement uncertainty according to document No. SANCO/12571/2013

ABSTRACTA simple and straightforward method for simultaneous determination of residues of 13 pesticides in honey samples (acrinathrin, bifenthrin, bromopropylate, cyhalothrin-lambda, cypermethrin, chlorfenvinphos, chlorpyrifos, coumaphos, deltamethrin, fluvalinate-tau, malathion, permethrin and tetradifon) from different pesticide classes has been developed and validated. The analytical method provides dissolution of honey in water and an extraction of pesticide residues by n-Hexane followed by clean-up on a Florisil SPE column. The extract was evaporated and taken up by a solution of an injection internal standard (I-IS), ethion, and finally analyzed by capillary gas chromatography with electron capture detection (GC-µECD). Identification for qualitative purpose was conducted by gas chromatography with triple quadrupole mass spectrometer (GC-MS/MS). A matrix-matched calibration curve was performed for quantitative purposes by plotting the area ratio (analyte/I-IS) against concentration using a GC-µECD instrument. According to document No. SANCO/12571/2013, the method was validated by testing the following parameters: linearity, matrix effect, specificity, precision, trueness (bias) and measurement uncertainty. The analytical process was validated analyzing blank honey samples spiked at levels equal to and greater than 0.010 mg/kg (limit of quantification). All parameters were satisfactorily compared with the values established by document No. SANCO/12571/2013. The analytical performance was verified by participating in eight multi-residue proficiency tests organized by BIPEA, obtaining satisfactory z-scores in all 70 determinations. Measurement uncertainty was estimated according to the top-down approaches described in Appendix C of the SANCO document using the within-laboratory reproducibility relative standard deviation combined with laboratory bias using the proficiency test data.

AACCI Approved Methods Technical Committee Report: Collaborative Study on Bread Volume Determination by Laser Topography Using a Bread Volume Meter

Volume is an important measure of fermented bread quality that has traditionally been determined by seed displacement methods. These methods are cumbersome; poorly suited to compressible, sticky, or creased products; prone to error; and, to date, lack standardization. A collaborative study was conducted by the AACC International (AACCI) Physical Testing Methods Technical Committee in 2005 to assess the performance of a new method that uses laser topography to determine the volume of baked bread products. Eleven laboratories analyzed six Youden pairs of polyurethane bread replicas using bread volume meter (BVM) laser volumeters. Samples ranged in volume from 200 to 2,800 cm3 and included round, elongated, and rectangular shapes. Averaged laboratory results ranged from 98.7 to 100.3% of the corresponding reference water displacement volume for each sample. Within-laboratory relative standard deviations (RSDr) ranged from 0.2 to 1.5%, and between-laboratory relative standard deviations (RSDR) ranged from 0.8...

On the heterogeneity of biochar and consequences for its representative sampling

Representativeness of sampling has often been neglected for heterogeneous discrete materials such as biomass or products there from, potentially leading to inaccurate and non-reproducible/imprecise results. Here, we investigate how elemental and PAH concentrations in specimen non-probabilistically selected from a 750kg biochar lot by grab sampling after initial mixing differ from those obtained from presumably probabilistically gathered samples by iterative mass reduction with fractional shoveling and repeated mixing. To assess reproducibility/precision, the concentration dependent relative standard deviations (RSD) are compared with the empirical Horwitz function, describing the within-laboratory RSD for multiple analyses, and by an F-test. While the precision was generally better for the samples, it was still satisfactory (RSD within 10%) for elemental concentrations in the specimen. However, replicates of specimen varied by 37% for PAHs. This number could be reduced to 4% in presumably representative samples. Accuracy as assessed by t-tests of specimen and samples revealed significant, but overall minor differences for some elements.

AACCI Approved Methods Technical Committee Report: Collaborative Study on a Method for Determining Firmness of Cooked Pulses (AACCI Method 56-36.01)

A method based on compression through a Kramer shear cell was developed for determining the firmness of cooked pulses. Ten laboratories analyzed twenty-six blind duplicates of thirteen different samples in a collaborative study to evaluate the repeatability and reproducibility of the method. Statistical analysis of the collaborative data indicated that within-laboratory repeatability standard deviation (sr) was in the range of 0.53 to 1.43, and among-laboratory reproducibility standard deviation (sR) varied from 0.74 to 1.94. The within-laboratory relative standard deviation (RSDr) of samples ranged from 2.45 to 7.24%, and the among-laboratory relative standard deviation (RSDR) ranged from 4.23 to 8.80%.

Method for the Rapid Determination of Protein in Meats Using the CEM Sprint™ Protein Analyzer: Collaborative Study

A collaborative study was conducted to determine the protein content of raw and processed meat products by a protein-tagging and colorimetric technique. Meat products were prepared following AOAC Official Method 983.18 and analyzed using CEM Corporation's Sprint Rapid Protein Analyzer. Sprint provides protein results by combining an accurately weighed test portion with a known amount of dye-binding agent. The dye-binding agent binds with the lysine, histidine, and arginine, as well as the n-terminus of the proteins commonly found in raw meat and processed meat products. Results are displayed and reported by the Sprint as a percentage (g/100 g) of protein. Ten blind duplicate study samples were sent to 10 collaborating laboratories in the United States. The within-laboratory (repeatability) relative standard deviation (RSD(r)) ranged from 0.91 to 3.04%, and between-laboratories (reproducibility) relative standard deviation (RSDR) ranged from 1.50 to 3.41% for protein. The method is recommended for Official First Action.

Method for the Rapid Determination of Protein in Meats Using the CEM Sprint™ Protein Analyzer: Collaborative Study

Abstract A collaborative study was conducted to determine the protein content of raw and processed meat products by a protein-tagging and colorimetric technique. Meat products were prepared following AOAC Official MethodSM 983.18 and analyzed using CEM Corporation's Sprint Rapid Protein Analyzer. Sprint provides protein results by combining an accurately weighed test portion with a known amount of dye-binding agent. The dye-binding agent binds with the lysine, histidine, and arginine, as well as the n-terminus of the proteins commonly found in raw meat and processed meat products. Results are displayed and reported by the Sprint as a percentage (g/100 g) of protein. Ten blind duplicate study samples were sent to 10 collaborating laboratories in the United States. The within-laboratory (repeatability) relative standard deviation (RSDr) ranged from 0.91 to 3.04%, and between-laboratories (reproducibility) relative standard deviation (RSDR) ranged from 1.50 to 3.41% for protein. The method is recommended for Official First Action.

Survey of T-2 and HT-2 toxins in soybean and soy meal from Argentina using immunoaffinity clean-up and high performance liquid chromatography

Soybean and soy meal samples collected during the harvest season 2008-2009 in the soybean-growing area of Córdoba Province in Argentina were analysed for T-2 and HT-2 toxins occurrence. These mycotoxins were detected using HPLC analysis with fluorescence detection after derivatisation with 1-anthronylnitrile and immunoaffinity column clean-up. Characteristics of in-house validated method such as accuracy, precision, detection and quantification limits were defined by means of recovery test with spiked soybean and soy meal samples. Mean recoveries for T-2 within the spiking range 125-500 µg/kg, were 90.9 and 81.3% for soybean and soy meal, respectively with a withinlaboratory relative standard deviation <10%. Analysis of samples spiked with HT-2 in the same range gave a mean recovery of 70.2 and 77.5% for soybean and soy meal, respectively, with relative standard deviations <12%. The limit of detection for the method was 25 µg/kg for T-2 and HT-2, based on a signal-to-noise ratio 3:1 and the limit of quantification was established as three times the detection limit. Out of 64 samples, only two soybean samples showed contamination with A-type trichothecenes evaluated. Confirmatory analyses of the contaminated samples were performed by LC-MS/MS. This study demonstrated low incidences and levels of T-2 and HT-2 in soybean harvested among the areas in the Cordoba Province.

Evaluation of the state-of-the-art measurement capabilities for selected PBDEs and decaBB in plastic by the international intercomparison CCQM-P114

An international intercomparison involving eight national metrology institutes (NMIs) was conducted to establish their current measurement capabilities for determining five selected congeners from the brominated flame retardant classes polybrominated diphenyl ethers and polybrominated biphenyls. A candidate reference material consisting of polypropylene fortified with technical mixtures of penta-, octa- and decabromo diphenyl ether and decabromo biphenyl, which was thoroughly assessed for material homogeneity and stability, was used as study material. The analytical procedures applied by the participants differed with regard to sample pre-treatment, extraction, clean-up, employed calibrants and type of calibration procedure as well as regarding analytical methods used for separation, identification and quantification of the flame retardant congeners (gas chromatography coupled to an electron capture detector (GC-ECD), gas chromatography-mass spectrometry in the electron ionisation mode (GC-EI-MS), gas chromatography-mass spectrometry in the electron capture negative ionisation mode (GC-ECNI-MS), and liquid chromatography-inductive coupled plasma-mass spectrometry (LC-ICP-MS)). The laboratory means agreed well with relative standard deviations of the mean of means of 1.9%, 4.8%, 5.5% and 5.4% for brominated diphenyl ether (BDE) 47, 183 and 209 and for the brominated biphenyl (BB) congener 209, respectively. For BDE 206, a relative standard deviation of 28.5% was obtained. For all five congeners, within-laboratory relative standard deviations of six measurements obtained under intermediate precision conditions were between 1% and 10%, and reported expanded measurements uncertainties typically ranged from 4% to 10% (8% to 14% for BDE 206). Furthermore, the results are in good agreement with those obtained in the characterization exercise for determining certified values for the flame retardant congeners in the same material. The results demonstrate the state-of-the-art measurement capabilities of NMIs for quantifying representative BDE congeners and BB 209 in a polymer. The outcome of this intercomparison (pilot study) in conjunction with possible improvements for employing exclusively calibrants with thoroughly assessed purity suggests that a key comparison aiming at underpinning calibration and measurement capability (CMC) claims of NMIs can be conducted.

Influence of roasting and different brewing processes on the ochratoxin A content in coffee determined by high-performance liquid chromatography-fluorescence detection (HPLC-FLD)

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in green and roasted coffee. The method consists of extraction of the sample with methanol–5% aqueous sodium hydrogen carbonate/1% PEG8000 (20:80), followed by immunoaffinity column (IAC) clean-up and, finally, high-performance liquid chromatography (HPLC) determination with fluorimetric detection. Mean recoveries for green and roasted coffee spiked at different levels ranging from 94 and 105% were obtained. The limit of determination (S/N = 3) was 0.032 ng g−1 and the precision (within-laboratory relative standard deviation) was 6%. The method described has been used to assess the influence of roasting and different brewing processes on OTA content in commercial lots of green and roasted coffee. The results provided evidence that roasting led to a significant drop on OTA levels (65–100%). Also, the way coffee is prepared affects the OTA content: brewing using a Moka Express (Italian coffee) led to a significant reduction of OTA concentration (50–75%) since hot water stays in contact with coffee for a short time. On the contrary, Turkish coffee-making (infusion for about 10 min) cause poor reduction in OTA.

Determination of Fat,Moisture, and Protein in Meat and Meat Products by Using the FOSS FoodScan Near-Infrared Spectrophotometer with FOSS Artificial Neural Network Calibration Model and Associated Database: Collaborative Study

A collaborative study was conducted to evaluate the repeatability and reproducibility of the FOSS FoodScan near-infrared spectrophotometer with artificial neural network calibration model and database for the determination of fat, moisture, and protein in meat and meat products. Representative samples were homogenized by grinding according to AOAC Official Method 983.18. Approximately 180 g ground sample was placed in a 140 mm round sample dish, and the dish was placed in the FoodScan. The operator ID was entered, the meat product profile within the software was selected, and the scanning process was initiated by pressing the "start" button. Results were displayed for percent (g/100 g) fat, moisture, and protein. Ten blind duplicate samples were sent to 15 collaborators in the United States. The within-laboratory (repeatability) relative standard deviation (RSD(r)) ranged from 0.22 to 2.67% for fat, 0.23 to 0.92% for moisture, and 0.35 to 2.13% for protein. The between-laboratories (reproducibility) relative standard deviation (RSD(R)) ranged from 0.52 to 6.89% for fat, 0.39 to 1.55% for moisture, and 0.54 to 5.23% for protein. The method is recommended for Official First Action.

Immunoaffinity Column Cleanup with Liquid Chromatography Using Post-Column Bromination for Aflatoxins in Medicinal Herbs and Plant Extracts

A new and accurate method to quantitate aflatoxins in medicinal herbs is developed. This method consists of extraction of the sample with MeOH-H2O (70:30) followed by clean-up of the extracts with immunoaffinity columns and, finally, high-performance liquid chromatographic determination with fluorescence detection. Aflatoxins B1 and G1 are determined as their bromine derivatives, produced in an online post-column derivatization system. The overall average recoveries for three different medicinal herbs spiked at levels of 1.3 and 2.6 ng/g of total aflatoxins range from 93% to 97%. The detection limit is 0.15 ng/g for both G2 and B2 and 0.20 ng/g for both G1 and B1, based on a signal-to-noise ratio of 3:1 and a precision (within-laboratory relative standard deviation) ranging from 0.8% to 1.4%. The use of immunoaffinity columns provides excellent clean-up of these particular extracts, which are generally difficult to analyze. The method is applied successfully to 96 samples of natural drugs.

Determination of the mycotoxin fumonisin B1 in maize by reversed-phase thin-layer chromatography: a collaborative study

A simple and cost-effective method using thin-layer chromatography for the determination of the mycotoxin fumonisin B1 in maize is described. The analytical method consisted of the extraction of ground maize by shaking with methanol/water (75:25) for 60 min and clean-up of the resultant extract by means of strong anion exchange solid-phase extraction. The purified residue, formed by evaporation of the elution solvent, was reacted with fluorescamine and the fumonisin B1-derivative was separated by reversed-phase thin-layer chromatography using a developing solution of methanol/aqueous 4% potassium chloride (70:30). The derivatized FB1 was readily visualized as a greenish-yellow spot under long wavelength ultraviolet light and quantified by visual comparison with a set of similarly derivatized standards in the range 20–300 ng FB1 spotted on plate. Based on visual comparison, levels down to 0.5 mg kg−1 were successfully estimated. The method was collaboratively studied in 14 laboratories using four duplicate maize meal samples (including a blank) and a spiked sample for determination of recovery. No significant difference was observed between mean FB1 levels by high-performance liquid chromatography or thin-layer chromatography. Based on within-laboratory relative standard deviations of 27.1–41.7% and between-laboratory relative standard deviations of 35.0–63.3%, the method can be considered semiquantitative. The mean recovery achieved by participants at a spiking level of 2.00 mg kg−1 was 74.5%.

Crude Fat, Hexanes Extraction, in Feed, Cereal Grain, and Forage (Randall/Soxtec/Submersion Method): Collaborative Study

Abstract A method for determining crude fat in animal feed, cereal grain, and forage (plant tissue) was collaboratively studied. Crude fat was extracted from the animal feed, cereal grain, or forage material with hexanes by the Randall method, also called the Soxtec method or the submersion method. The use of hexanes provides for an alternative to diethyl ether for fat extractions. The proposed submersion method considerably decreases the extraction time required to complete a batch of samples compared to Soxhlet. The increase in throughput is very desirable in the quest for faster turnaround times and the greater efficiency in the use of labor. In addition, this method provides for reclamation of the solvent as a step of the method. The submersion method for fat extraction was previously studied for meat and meat products and was accepted as AOAC Official Method 991.36. Fourteen blind samples were sent to 14 collaborators in the United States, Sweden, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.23 to 5.80% for crude fat. Among-laboratory (including within) relative standard deviation (reproducibility) ranged from 1.88 to 14.1%. The method is recommended for Official First Action.

Crude Fat, Diethyl Ether Extraction, in Feed, Cereal Grain, and Forage (Randall/Soxtec/Submersion Method): Collaborative Study

A method for determining crude fat in animal feed, cereal grain, and forage (plant tissue) was collaboratively studied. Crude fat was extracted from the animal feed, cereal grain, or forage material with hexanes by the Randall method, also called the Soxtec method or the submersion method. The use of hexanes provides for an alternative to diethyl ether for fat extractions. The proposed submersion method considerably decreases the extraction time required to complete a batch of samples compared to Soxhlet. The increase in throughput is very desirable in the quest for faster turnaround times and the greater efficiency in the use of labor. In addition, this method provides for reclamation of the solvent as a step of the method. The submersion method for fat extraction was previously studied for meat and meat products and was accepted as AOAC Official Method 991.36. Fourteen blind samples were sent to 14 collaborators in the United States, Sweden, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.23 to 5.80% for crude fat. Among-laboratory (including within) relative standard deviation (reproducibility) ranged from 1.88 to 14.1%. The method is recommended for Official First Action.

Development of methods for the determination of vitamins A, E and β-carotene in processed foods based on supercritical fluid extraction: a collaborative study

New methodologies based on supercritical fluid extraction (SFE) have been developed for the determination of fat-soluble vitamins in processed foods. The results obtained so far indicate that SFE is well suited to extraction of fat-soluble vitamins from food products, although validation work is required to establish accuracy and precision. The vitamins investigated were A, E and β -carotene, and the processed foods were UHT milk, milk powder, minced meat, liver paste, infant formula, canned baby food and margarine. Extraction equipment employed analyte collection on either a solid-phase trap or in a solvent. After extraction, the samples were saponified and the vitamins determined using reversed-phase liquid chromatography with ultraviolet or fluorescence detection. Sample throughput was at least 12 samples day-1, i.e. at least twice the number achievable with a conventional extraction methodology. The detection limits for the vitamins in different processed foods were well below 0.1 μg g -1. Recoveries (in comparison with vitamin levels obtained using conventional solvent extraction) were close to 100% for experienced personal with access to modern automatic equipment. To reach this level, it was necessary to protect the vitamins with an antioxidant during the different steps of the analysis procedure, to add methanol or ethanol to the extraction cell to facilitate the analyte extraction from the food matrix, and when using a solid-phase trap, to employ a fractionated extraction-elution procedure to prevent breakthrough losses. The developed methods were tested in a validation exercise between five laboratories, which had taken part in the method development, and in an intercomparison between 10 laboratories including laboratories with less experience of vitamin determination. The within-laboratory RSD was generally ≤ 11%. The average of the between-laboratory relative standard deviation (RSD) was about 23% in the validation, and increased to about 40% in the intercomparison. Ruggedness tests performed at different steps of the project showed that different types and models of equipment did not give large differences in recoveries. Thus, the increasing RSD can largely be ascribed to differences in experience in vitamin analysis of the participants.

Determination of Water (Moisture) and Dry Matter in Animal Feed, Grain, and Forage (Plant Tissue) by Karl Fischer Titration: Collaborative Study

A Karl Fischer method for determining water (dry matter) in animal feed and forages was collaboratively studied. Water was extracted from animal feed or forage material into methanol-formamide (1 + 1) directly in the Karl Fischer titration vessel by high-speed homogenization. The water was titrated at 50 degrees C with one-component Karl Fischer reagent based on imidazole. Ten blind samples were sent to 9 collaborators in the United States, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.14 to 6.99% for water or from 0.09 to 0.56% for dry matter. Among-laboratory (including within-) relative standard deviation (reproducibility) ranged from 5.35 to 10.73%, or from 0.44 to 0.77% for dry matter. The authors recommend that the method be adopted as Official First Action by AOAC INTERNATIONAL. A comparable alternative extraction procedure using boiling methanol is also recommended for Official First Action.