Determination of hydrazine in maleic hydrazide technical and pesticide formulations by gas chromatography: collaborative study

Twelve collaborating laboratories assayed hydrazine in technical maleic hydrazide (MH), 6-hydroxy-2H-pyridazin-3-one, and 2 formulated products, a liquid concentrate and a soluble granule, using gas chromatography (GC) with electron capture detection. The hydrazine content in the samples ranged from 0.03 ppm, in the liquid concentrate, to 0.26 ppm, in MH technical. Hydrazine and MH are dissolved in an aqueous solution. The MH is then precipitated out of solution by acidification. The solution containing hydrazine is treated with excess pentafluorobenzaldehyde (PFB) to form pentafluorobenzaldehyde azine (PFBA). The PFBA is extracted with hexane for analysis by GC using an electron capture detector. Peak area responses of PFBA are measured and quantified by external standardization. Hydrazine concentration is calculated from the PFBA determination. The laboratories weighed each test sample in duplicate with duplicate analysis for each weighing. Data from these laboratories were statistically analyzed. The average relative repeatability was determined to be 5.34% and the average relative reproducibility was 27.99%.     

Determination of sulfamethazine in swine and cattle feed by reversed-phase liquid chromatography with post-column derivatization: collaborative study

A liquid chromatographic (LC) method for the analysis of sulfamethazine (SMT) in complete swine and cattle feed was collaboratively studied. The method uses post-column derivatization with dimethylaminobenzaldehyde and detection at 450 nm. To 5g finely ground feed, extractant (0.2N HCl + 1.5% diethylamine in 25% methanol), and internal standard solutions are added, and the SMT is extracted by shaking for 1 h. Clarified extract (high-level sample extract diluted to a target concentration of ca 5.5 microg/mL) is chromatographed on a Cla reversed-phase LC column with acetonitrile-2% acetic acid (17 + 83) mobile phase. Sulfamerazine is used as an internal, or surrogate standard to correct for variable recovery of sulfamethazine from a variety of feed matrixes. Six Youden matched-pair samples were sent to 10 collaborators in Korea, Canada, and the United States. Label claims on the commercial feeds ranged from 0.0077 to 0.22% SMT. The SMT mean recovery as determined from the 5 samples with known analyte content was 99.8%. The within-laboratory relative standard deviation (repeatability) ranged from 0.28 to 4.72%. Among-laboratory (including within-laboratory) relative standard deviation (reproducibility) ranged from 1.26 to 4.87%. The authors recommend the method for AOAC INTERNATIONAL Official First Action status.     

Determination of phytase activity in feed by a colorimetric enzymatic method: collaborative interlaboratory study.

Fourteen laboratories participated in a collaborative study (coded fyt9404) and 13 laboratories participated in a study (coded fyt9410) to validate a colorimetric assay for determination of microbial phytase activity in feed. For each study, all laboratories received 6 laboratory samples provided by one commercial supplier (phytase activity levels within the range of 200-400 per kg) to be analyzed in duplicate. Method performance was calculated and statistical calculations were executed according to AOAC guidelines. Results from 3 laboratories for study fyt9404 and from one laboratory for study fyt9410 were excluded from statistical analysis because of invalid data determined during initial review by Youden pair, value versus laboratory. For study fyt9404, repeatability relative standard deviation (RSDr) values ranged from 6.2 to 8.6%, and reproducibility relative standard deviation (RSDR) values ranged from 14.1 to 27.6%. No outliers were identified. For study fyt9410, RSDr values ranged from 3.9 to 7.9%, and RSDR values ranged from 14.0 to 20.5%. With outliers excluded, RSDr values ranged from 2.5 to 7.9%, and RSDR values ranged from 14.0 to 20.5%.     

Determination of trans-galactooligosaccharides in selected food products by ion-exchange chromatography: collaborative study

Eleven collaborating laboratories assayed 7 blind duplicate pairs of food and feed products for tans-galactooligosaccharides. The 7 laboratory sample pairs ranged from low (2%) to high levels (15%). Following the proposed method, the test samples were treated with beta-galactosidase and the released galactose was determined by ion-exchange chromatography. Repeatability standard deviation ranged from 2.9 to 11.6%; reproducibility standard deviation ranged from 4.6 to 11.6%.     

Determination of neutral lactase activity in industrial enzyme preparations by a colorimetric enzymatic method: collaborative study

Thirteen laboratories participated in a collaborative study to validate a colorimetric assay for determining neutral lactase activity in industrial enzyme preparations. Each laboratory received 5 duplicate samples with activity levels of 2000 and 5000 neutral lactase units provided by 4 commercial suppliers. Two laboratories did not return results. Method performance was calculated according to AOAC guidelines. From the 11 remaining laboratories, 3 were excluded from statistical analysis because of invalid data determined during initial review by Youden pair, value versus laboratory. Repeatability relative standard deviation (RSDr) values ranged from 3.20 to 8.62%, and reproducibility relative standard deviation (RSDR) values ranged from 8.77 to 16.35%. With outliers excluded, RSDr values ranged from 2.94 to 5.01%, and RSDR values ranged from 7.50 to 13.84%. The colorimetric enzymatic method for determining neutral lactase activity in industrial enzyme preparations has been adopted first action by AOAC INTERNATIONAL.     

Determination of phenols and phenates in disinfectant formulations by liquid chromatography with UV detection: collaborative study

Fourteen collaborating laboratories assayed o-phenylphenol (OPP), p-t-amylphenol (PTAP), and o-benzyl-p-chlorophenol (OBPCP) in formulated products, both ready-to-use and concentrates, by RP-HPLC. The actives in the samples ranged from 0.03 to 11% OPP, 0.06 to 4% PTAP, and 0.07 to 10% OBPCP either in free forms or as salts. Seven blind duplicates were analyzed. The samples were diluted/extracted with acidified methanol, filtered, and analyzed by LC on a C18 column using gradient elution and UV detection at 285 nm. The concentration of the active ingredients was calculated from a standard curve. Each laboratory weighed each test sample twice within a single analytical run. The data were analyzed using all 14 laboratory results, with appropriate statistical tests to detect outliers. The repeatability RSDs ranged from 0.98 to 3.40% for the free phenols, and 1.26 to 2.51% for the salts. The reproducibility RSDs ranged from 5.31 to 7.80% for the free phenols, and 5.50 to 8.67% for the salts. The HorRat ranged from 0.86 to 2.17 for the free phenols, and 1.54 to 2.72 for the salts.     

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.     

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.     

Determination of docosahexaenoic acid and n-3 fatty acids in refined fish oils by 1H-NMR spectroscopy: IUPAC interlaboratory study

A high-resolution proton nuclear magnetic resonance (NMR) method for determining the concentration (mg/g) of docosahexaenoic acid (DHA), the molar proportion (mol%) of DHA, and the molar proportion of total n-3 fatty acids in fish oils was validated by an IUPAC interlaboratory study (the Commission VI-6 on Oils, Fats, and Derivatives WG 3/98). Thirteen laboratories from 5 countries tested 6 pairs of blind duplicate fish oils: a refined tuna oil, 2 extracted tuna oils, an extracted bonito oil, an extracted salmon oil, and an extracted sardine oil ranging from 9 to 30 mol% DHA and from 20 to 35 mol% n-3 fatty acids. Before 1 D-proton NMR measurements with 300-500 MHz instruments, oil samples were weighed and diluted with deuterochloroform solution containing ethylene glycol dimethyl ether as internal standard. To achieve precise performance, a detailed procedure for signal area measurement was described in the protocol, and all participants were instructed about the critical importance of following the protocol. Statistical performances with invalid and outlier data removed were as follows: repeatability relative standard deviations (RSDr) ranged from 0.91 to 2.62% and reproducibility relative standard deviation (RSDR) ranged from 1.73 to 4.27% for DHA concentration (mg/g); RSDr ranged from 0.39 to 2.06%, and RSDR ranged from 0.59 to 3.46% for mol% DHA; RSDr ranged from 0.23 to 0.90% and RSDR ranged from 0.85 to 2.01 % for mol% total n-3 fatty acids. The method is expected to be recommended by IUPAC.     

Proficiency testing of eight French laboratories in using the AOAC mouse bioassay for paralytic shellfish poisoning: interlaboratory collaborative study

In an interlaboratory study, 8 French laboratories were tested for their proficiency in using the AOAC mouse bioassay for paralytic shellfish poisoning (PSP). Each laboratory received 1 saxitoxin (STX) standard solution, 1 STX acidified water solution for determination of the titer, 1 noncontaminated shellfish sample, 1 naturally contaminated shellfish sample, and 2 shellfish samples spiked, respectively, at low (152.8 microg STX/100 g meat) and moderate (334.7 microg STX/100 g meat) levels. All samples were analyzed in duplicate. Mean recoveries were 35.1% for the low level and 46.6% for the moderate level. Relative standard deviations (RSD) for within-laboratory variations (repeatability) ranged from 5.4 to 9.8%; RSD for between-laboratory variations (reproducibility) varied from 7.8 to 39.6%, depending on STX level. On the basis of overall performance, all 8 participating laboratories were proficient in their use of the AOAC mouse bioassay.     

Determination of zearalenone in barley, maize and wheat flour, polenta, and maize-based baby food by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatography (LC) method for the determination of zearalenone (ZON) in a variety of cereals and cereal products at proposed European regulatory limits. The test portion is extracted with acetonitrile:water. The sample extract is filtered, diluted, and applied to an affinity column. The column is washed, and ZON is eluted with acetonitrile. ZON is quantified by reversed-phase LC with fluorescence detection. Barley, wheat and maize flours, polenta, and a maize-based baby food naturally contaminated, spiked, and blank (very low level) were sent to 28 collaborators in 9 European countries and 1 collaborator in New Zealand. Participants were asked to spike test portions of all samples at a ZON concentration equivalent to 100 microg/kg. Average recoveries ranged from 91-111%. Based on results for 4 artificially contaminated samples (blind duplicates) and 1 naturally contaminated sample (blind duplicate), the relative standard deviation for repeatability (RSDr) ranged from 6.9-35.8%, and the relative standard deviation for reproducibility (RSDR) ranged from 16.4-38.2%. The method showed acceptable within- and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values <1.7.     

Determination of total taurine in pet foods by liquid chromatography of the dansyl derivative: collaborative study

A liquid chromatographic (LC) method for the determination of total taurine in pet foods was evaluated in a collaborative study. Ten laboratories assayed 6 blind duplicate pairs of wet and dry pet foods. The taurine in the 6 sample pairs ranged from low (170 mg/kg) to high (2250 mg/kg) concentrations as is. Collaborators also assayed a sample of known taurine concentration for familiarization purposes. Samples were hydrolyzed to release bound taurine, which was subsequently converted to the dansyl derivative and quantitated by gradient-elution LC with fluorescence detection. Repeatability relative standard deviations, RSDr, ranged from 3.2 to 10.0%; reproducibility relative standard deviations, RSDR, ranged from 6.1 to 16.1%. The method has been adopted Official First Action status by AOAC INTERNATIONAL.     

Determination of semicarbazide in baby food by liquid chromatography/tandem mass spectrometry: interlaboratory validation study

An interlaboratory validation study funded by the European Commission, Directorate General for Health and Consumer Protection (DG SANCO), was conducted to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in different types of baby food at a possible future European regulatory limit (10 ng/g). The test portion of the sample was extracted with hydrochloric acid, and the analyte was derivatized with 2-nitrobenzaldehyde, with 1,2-[15N2, 13C] SEM as an internal standard. The extract was neutralized and then purified on a solid-phase extraction cartridge. The SEM was determined by reversed-phase LC with detection by MS/MS. Apple puree, rice pudding, and meat/vegetable meal baby food materials, spiked with SEM at levels of about 3, 10, and 30 ng/g, respectively, were sent to 20 laboratories in 12 different European countries, which submitted results from 17 participants. Recoveries ranged from 88.8 to 106.1%. Based on results for spiked samples (blind pairs at 3 levels), the relative standard deviations for repeatability (RSDr) ranged from 4.2 to 6.9% and the relative standard deviations for reproducibility (RSDR) ranged from 16.6 to 24.3%. The method showed acceptable within- and between-laboratory precision for all 3 matrixes, as evidenced by HorRat values, at the target levels for the determination of SEM.     

Determination of fumonisins B1 and B2 in corn and corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative study.

A liquid chromatographic (LC) method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn and corn flakes was collaboratively studied by 23 laboratories, which analyzed 5 blind duplicate pairs of each matrix to establish the accuracy, repeatability, and reproducibility characteristics of the method. Fumonisin levels in the corn ranged from <0.05 (blank) to 1.41 microg/g for FB1 and from <0.05 to 0.56 microg/g for FB2, whereas in the corn flakes they ranged from <0.05 to 1.05 microg/g for FB1 and from <0.05 to 0.46 microg/g for FB2. The method involved double extraction with acetonitrile-methanol-water (25 + 25 + 50), cleanup through an immunoaffinity column, and LC determination of the fumonisins after derivatization with o-phthaldialdehyde. Relative standard deviations for the within-laboratory repeatability (RSDr) of the corn analyses ranged from 19 to 24% for FB1 and from 19 to 27% for FB2; for the corn flakes analyses, RSDr ranged from 9 to 21 % for FB1 and from 8 to 22% for FB2. Relative standard deviations for the between-laboratories reproducibility (RSDR) of the corn analyses ranged from 22 to 28% for FB1 and from 22 to 30% for the FB2; for corn flakes analyses, RSDR ranged from 27 to 32% for FB1 and from 26 to 35% for FB2. Mean recoveries of FB1 and FB2 from corn spiked with FB1 at 0.80 microg/g and with FB2 at 0.40 microg/g were 76 and 72%, respectively; for corn flakes spiked at the same levels recoveries were 110 and 97% for FB1 and FB2, respectively. HORRAT ratios for the analyses of corn ranged from 1.44 to 1.53 for FB1 and from 0.96 to 1.48 for FB2, whereas for corn flakes they ranged from 1.60 to 1.82 for FB1 and from 1.39 to 1.68 for FB2.     

Determination of semicarbazide in fresh egg and whole egg powder by liquid chromatography/tandem mass spectrometry: interlaboratory validation study

An interlaboratory validation study was conducted according to harmonized protocols to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in fresh whole egg and in an industrially processed whole egg powder. The sample was extracted with hydrochloric acid and derivatized with 2-nitrobenzaldehyde, with 1,2-[(15)N2(13)C]SEM as the internal standard. The extract was neutralized and purified on a solid-phase extraction cartridge. SEM was determined by reversed-phase LC with detection by MS/MS. Five fresh egg samples, of which 3 were obtained from hens fed nitrofurazone (NFZ), one was spiked with SEM at 50 microg/kg and one was a blank sample, and 5 industrial whole egg powder samples, of which 3 were spiked with fresh whole egg from hens fed NFZ, one was spiked with SEM at 350 microg/kg, and one was a blank sample, were sent to 15 laboratories in 10 different European countries. Results were obtained from 12 participants. Average recoveries of SEM from the fresh egg and the egg powder samples were 105.3 and 121.3%, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.9 to 9.3%, and the relative standard deviation for reproducibility (RSDR) ranged from 22.5 to 38.1%. The method showed acceptable within- and between-laboratory precision for both matrixes, as evidenced by the HorRat values, at the target levels for the determination of SEM.     

Determination of aristolochic acid I in botanicals and dietary supplements potentially contaminated with aristolochic acid I using LC-UV with confirmation by LC/MS: collaborative study

An interlaboratory study was conducted to evaluate a method for the determination of aristolochic acid I, also known as aristolochic acid A, at levels > 2.00 microg/g in botanical species and dietary supplements potentially contaminated with aristolochic acid I. Aristolochic acid I was extracted from various matrixes with aqueous acetonitrile. The amount of aristolochic acid I present was determined by liquid chromatography (LC) using an ultraviolet (UV) detector with confirmation by LC/mass spectrometry (MS). Thirteen blind duplicates were successfully analyzed by 10 collaborators, and aristolochic acid I was successfully confirmed in 1 blind duplicate by 8 collaborators. For repeatability, the relative standard deviation (RSD(r)) ranged from 1.72 to 16.3% and for reproducibility, the RSDR ranged from 5.42 to 19.8%. HorRat values were not applicable for 2 materials but varied from 0.7 to 1.8 for 11 materials. Each collaborating laboratory had calibration curves with correlation coefficients > 0.998. In addition, all of the collaborators that conducted the confirmation were able to verify the identity of aristolochic acid I using LC/MS/MS (using either ion trap or triple quad).     

Determination of aflatoxins B1, B2, G1, and G2 and ochratoxin A in ginseng and ginger by multitoxin immunoaffinity column cleanup and liquid chromatographic quantitation: collaborative study

The accuracy, repeatability, and reproducibility characteristics of a method using multitoxin immunoaffinity column cleanup with liquid chromatography (LC) for determination of aflatoxins (AF; sum of aflatoxins B1, B2, G1, and G2) and ochratoxin A (OTA) in powdered ginseng and ginger have been established in a collaborative study involving 13 laboratories from 7 countries. Blind duplicate samples of blank, spiked (AF and OTA added) at levels ranging from 0.25 to 16.0 microg/kg for AF and 0.25 to 8.0 microg/kg for OTA were analyzed. A naturally contaminated powdered ginger sample was also included. Test samples were extracted with methanol and 0.5% aqueous sodium hydrogen carbonate solution (700 + 300, v/v). The extract was centrifuged, diluted with phosphate buffer (PB), filtered, and applied to an immunoaffinity column containing antibodies specific for AF and OTA. After washing the column with water, the toxins were eluted from the column with methanol, and quantified by high-performance LC with fluorescence detection. Average recoveries of AF from ginseng and ginger ranged from 70 to 87% (at spiking levels ranging from 2 to 16 microg/kg), and of OTA, from 86 to 113% (at spiking levels ranging from 1 to 8 microg/kg). Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 2.6 to 8.3% for AF, and from 2.5 to 10.7% for OTA. Relative standard deviations for between-laboratory reproducibility (RSDR) ranged from 5.7 to 28.6% for AF, and from 5.5 to 10.7% for OTA. HorRat values were < or = 2 for the multi-analytes in the 2 matrixes.     

Determination of the total nitrogen content of hard, semihard, and processed cheese by the Kjeldahl method: collaborative study

The objective of this collaborative study was to determine interlaboratory performance statistics for a modified and optimized version of AOAC Method 920.123 for the determination of the total nitrogen content of hard, semihard, and processed cheese by Kjeldahl analysis. Details included addressing the issues of material homogeneity, test portion size (1 g), quantitative transfer (weighing on to filter paper), ensuring system suitability (nitrogen recoveries), and using AOAC Method 991.20 as the basis for nitrogen analysis. Fifteen laboratories tested 18 pairs of blind duplicate cheese materials with a crude protein content between 18 and 36%. Materials represented hard, semihard, and processed commercial cheeses with a wide range of composition. Statistical performance parameters expressed as crude protein (nitrogen x 6.38), g/100 g, with invalid and outlier data removed were mean = 26.461, repeatability standard deviation (Sr) 0.111, reproducibility standard deviation (S(R)) = 0.153, repeatability relative standard deviation (RSDr) = 0.42%, reproducibility relative standard deviation (RSDR) = 0.58%, repeatability (r) = 0.312, and reproducibility (R) = 0.428. The interlaboratory study results were acceptable and comparable to those for the milk Kjeldahl nitrogen method on a relative nitrogen basis. The Study Directors recommend that this modified method for the determination of total nitrogen in hard, semihard, and processed cheese by Kjeldahl analysis be adopted First Action as an improved method to replace Method 920.123.     

Determination of cholecalciferol (vitamin D3) in selected foods by liquid chromatography: NMKL collaborative study

Results are presented from an NMKL (Nordic Committee on Food Analysis) collaborative study of a method for the determination of cholecalciferol (vitamin D3) in foods. The method is based on the addition of an internal standard (vitamin D2), followed by saponification and extraction with n-heptane. The fraction that contains vitamin D2/D3 is separated by preparative normal-phase liquid chromatography (LC), and the analytes are determined by reversed-phase LC with UV detection at 265 nm. The method was tested by 8 participating laboratories. In this study 6 different matrixes were analyzed for cholecalciferol content: milk, liquid infant formula (gruel), cooking oil, margarine, infant formula, and fish oil. The contents varied from 0.4 to 12 microg/100 g. Three matrixes (milk, gruel, and margarine) were fortified with vitamin D3. In the other matrixes, vitamin D3 was added at 3 different levels at the Swedish National Food Administration. The milk was analyzed as a blind duplicate, whereas the other matrixes were analyzed as split-level pairs. The recoveries from the samples with vitamin D3 added varied from 93 to 102%. The repeatability relative standard deviation (RSDr) values for accepted results varied between 2.2% (fish oil) and 7.4% (cooking oil), whereas the reproducibility relative standard deviation (RSD(R)) values varied between 6.8% (margarine) and 24% (cooking oil).     

Determination of beta-carotene in supplements and raw materials by reversed-phase high pressure liquid chromatography: collaborative study

Twelve laboratories representing 4 countries participated in an interlaboratory study conducted to determine all-trans-veta-carotene and total beta-carotene in dietary supplements and raw materials. Thirteen samples were sent as blind duplicates to the collaborators. Results obtained from 11 laboratories are reported. For products composed as softgels and tablets that were analyzed for total beta-carotene, the reproducibility relative standard deviation (RSDR) ranged from 3.35 to 23.09% and the HorRat values ranged from 1.06 to 3.72. For these products analyzed for trans beta-carotene, the reproducibility relative standard deviation (RSDR) ranged from 4.28 to 22.76% and the HorRat values ranged from 0.92 to 3.37. The RSDr and HorRat values in the analysis of a beadlet raw material were substantial and it is believed that the variability within the material itself introduced significant variation in subsampling. The method uses high pressure liquid chromatography (LC) in the reversed-phase mode with visible light absorbance for detection and quantitation. If high levels of alpha-carotenes are present, a second LC system is used for additional separation and quantitation of the carotene species. It is recommended that the method be adopted as an AOAC Official Method.