Showing papers in "Journal of AOAC International in 1993"

Liquid chromatographic determination of biogenic amines in dry sausages

Abstract: A liquid chromatographic method is described for the determination of biogenic amines found in dry sausages: tryptamine, phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermidine, and spermine. Amines were extracted with perchloric acid solution and derivatized with dansyl chloride. After derivatization, ammonia was added to remove an interfering peak near cadaverine. Liquid chromatographic separations were performed by using a Spherisorb ODS2 column and an ammonium acetate-acetonitrile gradient elution program. The limits of determination of the individual amines were 1-5 mg/kg. This method is also applicable to detection of amines in other food samples.

Ochratoxin A in cow's milk and in human milk with corresponding human blood samples.

Abstract: A method for determining ochratoxin A in milk has been elaborated in which the sample was subjected to a liquid-liquid extraction step and then purified on a silica gel column packed in a Pasteur pipet. The purified samples were analyzed by ion-pair liquid chromatography with fluorescence detection. The detection and quantitation limits for determination of ochratoxin A in cow's milk were 10 and 40 ng ochratoxin A/L milk, respectively. The same limits were valid for the analysis of human milk. A total of 36 cow's milk and 40 human milk samples were analyzed. All samples were collected in Sweden. Ochratoxin A was found in 5 (14%) of the cow's milk samples (range 10-40 ng/mL) and in 23 (58%) of the human milk samples (range 10-40 ng/L). Blood samples were collected from the mothers who gave milk samples. A total of 39 samples were analyzed. All blood samples contained ochratoxin A in concentrations exceeding the quantitation limit (60 ng/L blood). The mean concentration of ochratoxin A in the samples was 167 ng/L blood (range 90-940 ng/L). The concentration of ochratoxin A in human milk was < or = 0.1 of that in the human blood.

Survey of arsenic in total diet food composites and estimation of the dietary intake of arsenic by Canadian adults and children

Abstract: During a comprehensive total diet study extending from 1985 to 1988, foods were collected in 6 Canadian cities (in one of them, a pilot study was conducted twice). For each of the 7 collections, foods were processed into 112 composites (105 in the initial pilot trial). Total arsenic was determined in all samples. The mean, median, and range of arsenic concentrations in all samples were 73.2, 5.1, and < 0.1-4830 ng/g, respectively. Food groups containing the highest mean arsenic levels were fish (1662 ng/g), meat and poultry (24.3 ng/g), bakery goods and cereals (24.5 ng/g), and fats and oils (19.0 ng/g). The estimated daily dietary ingestion of total arsenic by the average Canadian was 38.1 micrograms and varied from 14.9 micrograms for the 1- to 4-year-old group to 59.2 micrograms for 20- to 39-year-old males.

The Chinese total diet study in 1990. Part I. Chemical contaminants.

Abstract: The Chinese total diet study in 1990 estimated the dietary intake of 24 chemical contaminants and 72 nutrients from 4 market baskets collected and prepared in 12 provinces. Twelve food group composites were made for each regional market basket. The overall dietary Pb, Cd, Hg, hexachlorocyclohexane HCH, and DDT intakes were well below their corresponding acceptable daily intakes. However, the Pb content of eggs from the 2 southern regions exceeded the tolerance limit. The Hg content of legumes just reached the tolerance limit, and Hg in eggs from the North 1 region exceeded the tolerance limit. The dietary HCH intake has decreased significantly since the 1980s, but dietary DDT intake has decreased rather slowly. Five organophosphorus pesticides were detected out of a total of 12 organophosphorus pesticides analyzed. Among them, methamidophos was the most outstanding. The intake of total committed dose equivalents (CDEs) of the 6 radionuclides was 0.24 mSv/a; only 1.5% was accounted for by 90Sr and 137Cs. 210Pb, 210Po, 226Ra, and 228Ra accounted for 98.5% of the total CDEs. The main food sources of those radionuclides were cereals, vegetables, and aquatic foods. Aflatoxin B1 was detected at very low levels only in the cereal composite of the North 1 region. Aflatoxin M1 was not detected in any of the milk and milk products. The overall results show that there is no significant environmental contamination of the average Chinese diet.

Determination of metals in foodstuffs by atomic absorption spectrophotometry after dry ashing: NMKL interlaboratory study of lead, cadmium, zinc, copper, iron, chromium, and nickel.

Abstract: An interlaboratory study of a method for determination of lead, cadmium, zinc, copper, iron, chromium, and nickel in foodstuffs by atomic absorption spectrophotometry (AAS) after dry ashing at 450 degrees C was conducted in 16 laboratories. The study was preceded by a practice round of familiarization samples and another round in which solutions were distributed and the metals were determined directly by AAS. The study included 5 different foods (liver paste, apple sauce, minced fish, wheat bran, and milk powder) and 2 composite diets. A single analysis was carried out on each sample. Suitable sample combinations were used as split level combinations for determination of the repeatability standard deviation. The relative reproducibility standard deviation for each of the elements ranged as follows: lead, 74-18% at 0.025-0.28 mg/kg; cadmium, 80-11% (0.002-0.51 mg/kg); zinc, 12-7% (44-72 mg/kg); copper, 47-10% (0.48-41 mg/kg); iron, 35-9% (2-228 mg/kg); chromium, 48-21% (0.008-0.22 mg/kg); nickel, 64-39% (0.025-0.39 mg/kg).

Rapid gas chromatographic method for the multiresidue screening of fruits and vegetables for organochlorine and organophosphate pesticides.

Abstract: A rapid and reliable method was developed for the determination of pesticides in fruits and vegetables. A 100 g sample is extracted with a mixture of 200 mL petroleum ether and 100 mL 2-propanol. The extract is backwashed 4 times, twice with aqueous sodium sulfate and twice with 350 mL distilled water, and then dried over 15 g sodium sulfate. The dried extract is analyzed by capillary gas chromatography with selective organochlorine and organophosphorus detection. The method determines primarily nonpolar pesticides, with recoveries ranging from 81 to 114%, and has an average limit of detection of 10 ppb for both detectors.

Aflatoxin contamination in foods and foodstuffs in Tokyo: 1986-1990.

Abstract: Aflatoxins were determined in 3054 samples of foods or foodstuffs, including cereals, nuts, beans, spices, dairy products, dry fruits, and edible oil. Samples were collected in Tokyo from 1986 to 1990. Aflatoxins were found in rice products, adlay, corn, crude sugar, peanut products, pistachio nuts, brazil nuts, sesame products, butter beans, white pepper, red pepper, paprika, nutmeg, and mixed spices. The highest incidence of aflatoxin contamination was observed in nutmeg (80%), and the highest level of aflatoxin B1 was observed in pistachio nuts (1382 ppb).

Survey of benzene in foods by using headspace concentration techniques and capillary gas chromatography.

Abstract: Recently, the combination of sodium or potassium benzoate with ascorbic acid was shown to produce low levels (ng/g) of benzene in fruit-flavored soft drinks. The presence of benzene also was reported in butter, eggs, meat, and certain fruits; levels of these findings ranged from 0.5 ng/g in butter to 500-1900 ng/g in eggs. Because benzoates are widely used as food preservatives, a limited survey of other foods containing added benzoate salts was conducted to investigate the potential for benzene formation. Selected foods that did not contain added benzoates but were previously reported to contain benzene were analyzed for comparison. More than 50 foods were analyzed by purge-and-trap or static headspace concentration and capillary gas chromatography. Benzene was quantitated by using the method of standard additions, and its identity was confirmed by mass selective detection. Results of this limited survey show that foods without added benzoates (including eggs) contained benzene at levels equal to or less than 2 ng/g. Slightly higher levels were present in some foods and beverages containing both ascorbic acid and sodium benzoate.

Rapid determination of cholesterol in single and multicomponent prepared foods

Abstract: A rapid method has been developed for cholesterol determination in single and multicomponent foods. The method involves alcoholic KOH saponification of the samples, extraction of the nonsaponifiable fraction with hexane, and injection of concentrated extract into the gas chromatograph without derivatizations. It has been applied to a wide variety of frozen and refrigerated foods. More than 300 samples were analyzed with a coefficient of variation (CV) ranging from 0.5 to 8.6%. The average recoveries of cholesterol from spiked oil and tomato vegetable soup samples were 100 +/- 1.5% and 99.7 +/- 1.6% and the CVs were 1.5 and 1.6%, respectively. This method reduces labor by > 70%, eliminates dangerous chemicals, and minimizes solvent use, compared to the AOAC method and other methods cited in the manuscript. The method was used successfully on a wide variety of multicomponent foods. We recommend this method for collaborative study under the AOAC guidelines for method approval.

Organochlorine pesticides and polychlorinated biphenyl congeners in commercial fish from the Great Lakes.

Abstract: Fillets from 11 species of commercial fish from the Great Lakes were analyzed for residues of 39 polychlorinated biphenyl (PCB) congeners and 24 other organochlorine compounds. Eel and trout contained the highest amount of PCB (753 and 633 ppb wet weight, respectively) and other organochlorines (607 and 1404 ppb, respectively); perch and carp contained the lowest residues. The penta- and hexachlorinated PCBs were the major congeners in all species except whitefish, in which the tetrachlorinated congeners predominated. Toxaphene was the most abundant organochlorine pesticide in trout; p,p'-DDE was the major component of this class in eel. Residue concentrations in commercial carp were compared with residues present in carp from a fishery closed to commercial operation. Although both PCB and organochlorine pesticide levels in carp were among the lowest for all commercial fish samples, levels from the contaminated area were among the highest.

The Chinese Total Diet Study in 1990. Part II. Nutrients

Abstract: This paper reports the intakes of 72 nutrients and their dietary sources obtained from the Chinese total diet study in 1990 Most of the nutrient intakes are close or equal to their corresponding recommended daily allowances (RDAs) Both the total energy intake (2203 kcal) and the proportions contributed by protein, fat, and carbohydrate meet the current Chinese RDAs and the World Health Organization (WHO) nutrient goals The average protein intake was 64 g/day The intake of essential amino acids all exceeded the Chinese RDA, and their proportions were generally consistent with the WHO recommended pattern The average fat intake was 512 g/day (212% of the total energy intake) However, the dietary fat intake has been increasing significantly in the Chinese diet and the proportion of animal fat has reached 53% of the total fat intake The total saturated:total monounsaturated: total polyunsaturated fatty acid ratio was 10:15:10 Although the average cholesterol intake was only 179 mg/day, it was 248 mg/day in the South 1 region The intakes of thiamine and riboflavin were below the RDA Retinol intakes in all the 4 regions were low Most (80%) of the retinol (equivalent) intakes were from carotenoids The average intake of total tocopherol was 89% of the RDA, and among the 4 regions, only the South 2 region has relatively low intake The intakes of iron, copper, manganese, sodium, and phosphorus were adequate The intakes of calcium, zinc, and potassium were insufficient, and intakes of selenium and magnesium were a little low High sodium and low potassium intake is a traditional problem in the Chinese diet

Determination of aspartame and its major decomposition products in foods

Abstract: A liquid chromatographic procedure already evaluated in a preceding study for the analysis of acesulfam-K is also suitable for the determination of the intense sweetener aspartame in tabletop sweetener, candy, fruit beverage, fruit pulp, soft drink, yogurt, cream, cheese, and chocolate preparations. The method also allows the determination of aspartame's major decomposition products: diketopiperazine, aspartyl-phenylalanine, and phenylalanine. Samples are extracted or diluted with water and filtered. Complex matrixes are centrifuged or clarified with Carrez solutions. An aliquot of the extract is analyzed on a reversed-phase muBondapak C18 column using 0.0125M KH2PO4 (pH 3.5)-acetonitrile ([85 + 15] or [98 + 2]) as mobile phase. Detection is performed by UV absorbance at 214 nm. Recoveries ranged from 96.1 to 105.0%. Decomposition of the sweetener was observed in most food samples. However, the total aspartame values (measured aspartame + breakdown products) were within -10% and +5% of the declared levels. The repeatabilities and the repeatability coefficients of variation were, respectively, 1.00 mg/100 g and 1.34% for products containing less than 45 mg/100 g aspartame and 4.11 mg/100 g and 0.91% for other products. The technique is precise and sensitive. It enables the detection of many food additives or natural constituents, such as other intense sweeteners, organic acids, and alkaloids, in the same run without interfering with aspartame or its decomposition products. The method is consequently suitable for quality control or monitoring.

Simultaneous liquid chromatographic determination of thiamine and riboflavin in selected foods.

Abstract: A reliable, improved liquid chromatographic (LC) method has been developed for the measurement of thiamine and riboflavin in foods. The major improvement in the method is the chromatographic separation achieved. The method is also very reproducible and extremely sensitive. After autoclave extraction, samples are derivatized to form thiochrome (a highly fluorescent oxidation product of thiamine). Riboflavin is naturally fluorescent. Interferences are removed on a C18 cartridge and chromatographed by using a reversed-phase separation. The mobile phase used is 72% 0.005M NH4OAc (pH 5.0)-28% MeOH. Fluorescence detection using wavelength switching, 370-435 for thiamine and 370-520 for riboflavin, allows determination of each vitamin at its optimum wavelength for maximum sensitivity. Detection limits were 0.05 ng for both thiamine and riboflavin. The method can also be performed by using a fluorescence detector at a single wavelength, but with a sacrifice of sensitivity. Data comparisons between AOAC fluorometric and LC results were excellent for routine samples, as well as for American Association of Cereal Chemists (AACC) check samples. All LC results from AACC samples were within 2 standard deviations of the mean. Reproducibility was 1.9% for thiamine and 1.6% for riboflavin.

U.S. Food and Drug Administration monitoring of pesticide residues in infant foods and adult foods eaten by infants/children.

Abstract: The U.S. Food and Drug Administration uses 3 approaches to monitor pesticide residues in foods: regulatory monitoring, incidence/level monitoring, and the Total Diet Study. The results of monitoring infant foods and adult foods that may be eaten by infants/children under these 3 approaches are presented. Under regulatory monitoring, which is performed to enforce tolerances set by the U.S. Environmental Protection Agency (EPA), during fiscal years 1985-1991, over 10,000 such domestic and imported food samples were collected and analyzed, and under the Total Diet Study, in which pesticide residue intakes are estimated in foods prepared for consumption, the food items in 27 market baskets were analyzed. Under incidence/level monitoring, which is complementary to regulatory monitoring, over 4000 analyses were performed on infant foods and adult foods eaten by children. Fewer than 50 of the 10,000 regulatory samples had violative residues; nearly all of those were residues for which there was no tolerance for the particular commodity/pesticide combination. Under incidence/level monitoring and the Total Diet Study, the levels of pesticide residues found in infant foods and adult foods eaten by children were well below tolerances set by EPA.

U.S. Food and Drug Administration survey of methyl mercury in canned tuna.

Abstract: Methyl mercury was determined by the US Food and Drug Administration (FDA) in 220 samples of canned tuna collected in 1991. Samples were chosen to represent different styles, colors, and packs as available. Emphasis was placed on water-packed tuna, small can size, and the highest-volume brand names. The average methyl mercury (expressed as Hg) found for the 220 samples was 0.17 ppm; the range was <0.10-0.75 ppm. Statistically, a significantly higher level of methyl mercury was found in solid white and chunk tuna. Methyl mercury level was not related to can size. None of the 220 samples had methyl mercury levels that exceeded the 1 ppm FDA action level. 11 refs., 1 tab.

Combustion method for determination of crude protein in meat and meat products: collaborative study.

Abstract: Twelve laboratories participated in a collaborative study to compare a combustion method with the AOAC mercury catalyst Kjeldahl method (92808) for the determination of crude protein in meat and meat products Three different combustion instruments were used; consequently, the combustion method for this study is written in generic terms describing the principle, the apparatus specifications, and the performance requirements needed Fifteen sample pairs were used for the study; each pair consisted of the same commercial meat product from each of 2 different manufacturers Protein content of all samples ranged from about 10 to 20% In addition, nicotinic acid and lysine monohydrochloride were used as standards to assess combustion equipment performance All laboratories and all instruments performed the combustion method satisfactorily on the basis of results for the standards For the meat samples, repeatability standard deviations (Sr) ranged from 011 to 040 for the Kjeldahl method and from 012 to 041 for the combustion method; the repeatability relative standard deviations (RSDr) ranged from 082 to 241% and from 060 to 223% for the Kjeldahl and combustion methods, respectively Reproducibility standard deviations (SR) ranged from 020 to 049 for the Kjeldahl method and from 018 to 046 for the combustion method, whereas the reproducibility relative standard deviations (RSDR) ranged from 159 to 284% for the Kjeldahl method and from 132 to 335% for the combustion method Overall grand means were 1559% protein for the Kjeldahl method and 1575% protein for the combustion method The combustion method was adopted first action by AOAC International

Reliability of mycotoxin assays--an update.

Abstract: The precision parameters of the method-performance (collaborative) studies for mycotoxins published in the literature through 1991 have been recalculated on a uniform basis by following the International Union of Pure and Applied Chemistry protocol. About 80% of the 793 accepted assays for mycotoxins, almost all of which have been conducted by thin-layer chromatography (TLC), liquid chromatography (LC), and enzyme-linked immunosorbent assays (ELISA), exhibit relative standard deviations among laboratories (RSDR) that are less than 2 times the values predicted from the Horwitz equation: RSDR, % = 2(1-0.5log10C) where C is the concentration expressed as a decimal fraction. The precision of TLC and LC methods is about the same, but that of ELISA is somewhat poorer. For those commodities for which sufficient data exist to provide a meaningful comparison, the methods applied to cottonseed products have the best precision and corn the worst, with peanuts intermediate. Overall, however, the primary factor affecting RSDR is concentration, more or less independent of analyte, method, matrix, and age of the study. If it is assumed that the test results are normally distributed and that an RSDR of 50% is the point where effective control of the results begins to be lost (a value equivalent to the production of 2% false-negative values), then relying on the Horwitz curve, the limit of quantitative measurement is the single digit, i.e., 5, micrograms/kg (10(-9); ppb) concentration for solid food commodities. Such a value must be considered as a limit applicable to a single analyte, aflatoxin B1, and not as a mean, and not applicable to the sum of the individual components, each of whose associated standard deviation would lie in the unacceptable region. Enforcement of a 5 micrograms aflatoxin B1/kg limit, under the assumptions made, requires that a responsible manufacturer and a prudent regulator operate at opposite extremes of tolerance limits: e.g., the producer at 2 micrograms/kg and the consumer at 10. A proposed Codex "maximum level" of 0.05 micrograms aflatoxin M1/kg milk cannot be supported by the available data applied in an interlaboratory enforcement environment. These conclusions are also supported by an examination of the reported data from the ongoing, large-scale proficiency studies routinely performed by the American Oil Chemists' Society and the International Agency for Research on Cancer.

A Simplified Alternative to the AOAC Official Method for Cholesterol in Multicomponent Foods

Abstract: An AOAC official method for quantitating cholesterol in multicomponent foods, which was first published in the 13th edition of the Official Methods of Analysis, is rarely used. The method includes so many operations and manipulations--all described in excruciating detail--that most laboratories shun it altogether. Intent on finding an alternative, laboratories have developed their own methods for specific foods. As a result, new methods have proliferated, but still no practical method has been developed for the broader categories of multicomponent foods. The aim of AOAC, which is to promote greater accuracy and uniformity of analytical results primarily through collaborative testing, has not been well served under these circumstances. A different approach guided the work reported in the present paper. This approach was directed toward updating and dramatically simplifying the existing AOAC official method. The method's chloroform-methanol-water mixed-solvent extraction is preserved; however, all the remaining steps have been streamlined, updated, or eliminated by using newer technology. Cholesterol is quantitated with highly specific capillary gas-liquid chromatography using the internal standardization technique. The lipid extract is prepared for the chromatography step by a brief saponification carried out in a culture tube. The resulting method has been validated by using Standard Reference Materials and the standard addition method. Because a simplified method is now available for quantitating cholesterol in the lipid extracts, the expectation is that more attention can be given to the development of improved and efficient extraction methods. This step remains as the central difficulty in any number of methods of analysis for lipid analytes.

Organochlorine pesticide residues in dairy milk in and around Delhi.

Abstract: A survey of dairy milk and milk products in and around Delhi, India, was undertaken to monitor the levels of DDT and HCH. The survey revealed that pesticide residue levels in milk often exceeded the limits recommended by the Food and Agriculture Organization/World Health Organization Expert Committee. Maximum residue levels of total HCH in milk (fat/whole milk basis) has not yet been prescribed. The 15 samples of dairy milk and 4 samples of milk products analyzed had residue levels ranging from 0.022 to 0.166 micrograms/g for HCH and from 0.042 to 0.382 micrograms/g for DDT.

Determination of pesticides in indoor air and dust.

Abstract: Improved analytical and sampling methods were developed for the multiresidue determination of pesticides in indoor air. Air analysis consists of adsorption of the pesticides in 1 m(3) of air onto Tenax TA via an air sampling pump, desorption with acetone and determination and quantitation by gas chromatography/mass spectrometry (GC/MS) with chemical ionization on an ion trap mass spectrometer. Limits of detection for the 23 pesticides studied ranged from 0.5 ng/m(3) for chlorpyrifos and diazinon to 30 ng/m(3) for o-phenylphenol (approximately 0.5-30 parts per trillion on a w/w basis). A simple method for the detection of pesticides in dust was also developed. This method involves emptying the contents of a vacuum cleaner bag into a standard household food processor and extracting 1 g homogenized dust with acetone before GC/MS. Limits of detection were 25-100 ppb because of interferences by common household chemicals. However, pesticide concentrations were higher in dust than in air, therefore, analysis of dust is a better indicator of indoor pesticide exposure.

Total organochlorine content of fish from the Great Lakes.

Abstract: Residues of polychlorinated biphenyls (PCBs) and organochlorine pesticides were determined in several species of commercial fish from the Great Lakes and compared to the total organic chlorine determined by neutron activation analysis. The mean organochlorine contents ranged from 44 to 138 ppm (lipid basis) and were 5 to 72 times higher than the contents of PCBs and organochlorine pesticides. Marine fish also contained a large proportion of unidentified organic chlorine. The unknown material in the Great Lakes fish was found to chromatograph with the high molecular weight lipid fraction by gel permeation chromatography.

Enzymatic-gravimetric determination in foods of dietary fiber as sum of insoluble and soluble fiber fractions: summary of collaborative study.

Abstract: A collaborative study was conducted on an enzymatic-gravimetric method for determination of total dietary fiber in foods, in which soluble fiber and insoluble fiber are determined separately Ten collaborators analyzed blind duplicate test samples from 5 food products: turnip, wheat bran, beans canned with tomato sauce, rice, and whole wheat bread Repeatability and reproducibility relative standard deviations ranged from 148 to 1473% and from 413 to 1794%, respectively The method was adopted first action by AOAC International

Evaluation and application of a bioluminescent bacterial genotoxicity test.

Abstract: The Mutatox test (commercial name for the bioluminescent bacterial genotoxicity test) has been shown to be a good alternative to the Ames test The test uses dark mutants of luminous bacteria (Vibrio fischeri) and determines the ability of various genotoxic agents to restore the luminescence by inducing mutation It provides a rapid screening test which can be used to assay the genotoxicity of large numbers of pure and complex compounds The test is completed in 1 day, and by serially diluting the compound, dose response data plus toxicity data can be generated for a number of samples simultaneously For the direct assay (without exogenous metabolic activation), the positive controls selected were 3,6-diaminoacridine (proflavine) and N-methyl-N-nitro-nitrosoguanidine For the S-9 assay, which incorporated the microsome fraction (S-9) from rat liver as an exogenous metabolic activation system, the positive controls selected were aflatoxin B1 and benzo(a)pyrene This study also indicated that methyl-imidazo-quinoline and tryptophan pyrolysates were genotoxic in the presence of S-9 activation, aflatoxin B1 epoxide and fumonisin B1 showed direct genotoxic activity, and aflatoxin B2 and ochratoxin A were not genotoxic

Liquid chromatographic determination of aflatoxin M1 in milk powder using immunoaffinity columns for cleanup: interlaboratory study.

Abstract: A liquid chromatographic method for determining low aflatoxin M1 concentrations in milk was evaluated in an International Dairy Federation (IDF) interlaboratory study. The study involved 16 participants from 11 countries. The method, chosen after a comparison of several methods by a preparatory group, uses an immunoaffinity column for cleanup. As the sample passes through the column, antibodies selectively bind with aflatoxin M1 (antigen) present and form an antibody-antigen complex. All other components of the sample matrix are washed off the column with water. Then, aflatoxin M1 is eluted from the column with acetonitrile, which is collected. Final determination is carried out by reversed-phase liquid chromatography with fluorescence detection. Over the tested range (80-600 ng aflatoxin M1/kg milk powder), an RSDR ranging from 11 to 23% was obtained by analyzing 24 samples (blind duplicates), 2 samples of which were blanks.