J Sei Food Agrie 1997, 73, 39-45 Could the Dumas Method Replace the Kjeldahl Digestion for Nitrogen and Crude Protein Determinations in Foods?” AH Simonne,‘} E H Simonne,’ R R Eitenmiller,* H A Mills‘ and C P Cresman III* * Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA * Department of Horticulture, 101 Funchess Hall, Auburn University, AL 36849-5408, USA Athens, GA 30602, USA (Received 19 January 1996; rovised version received 25 May 1996; accepted 9 August 1996) © Department of Horticulture, University of Georgi Abstract: Increased demand for determinations of nitrogen (N), and hence erude protein (CP), has led to wider use of the Dumas method in glace of the tradi- tional Kjeldahl methods. Although Kjeldahl N (KN) and Dumas N (DN) rep- resent diferent N fractions, published studies on infant forma, animal feed and meat products have indicated that DNV could replace KN with litle practical ‘impact on the coliabiiy of the N values obtained. This study was conducted to cstablish whether DN determination could replace that of KN in a broader range of foods for CP calculation. Statistical analysis was performed om in-house assayed KN and DN yaluss together with published KN and DN values for selected food products. Inthe range 00S-6:8% N, KN may be estimated from DN with the equation: KN = 1-001" DN ~ 009° (n= 101, R? = 098, P-regression < 0.01) Because N levels in individual groups of food dic ot span the entre range of N contents, KN : DN ratios were calculated for each food group. KN: DN ratios differed significantly (R* = 0-25, P <0-01) from group to group. Ratios of 1-01 for dairy, 1-00 for oilseeds, 0-99 for feed, 0-98 for infant formulas, 095 for eoteals, 094 for meats, 089 for vegetables, 0-80 for fish and 0-73 for fuits were valid for the estimation of KN and CP using DN data, CP. was independently calculated as CPL = Hx KN CP2=H x KN:DN x DN, where H is the nitrogen to protein conversi factor for the food group. Mean differences between CPI and CP2 values were 0% for dairy, oilseeds, feed, infant formulas and baby foods, cereals, meat and meat products, vegetables and vegetable products and vit, and 1% for fish ‘These results suggest that DN may replace KN for the determination of N and CCP in selected food groups when appropriate coeficients are use Key words: Kjeldahl nitrogen, Dumas nitrogen, crude protein, INTRODUCTION Crude protein (CP) is among the mandatory nutrients of the Nutrition Labeling and Education Act of 1990 and is usually estimated by multiplying nitrogen (N) content by a nitrogen to protein conversion factor. In most foods, amino-N accounts for approximately 16% * This paper was presented at the 1995 annual meeting of the Institute of Food Technologists 3-7 June, 1995 in Anabeins, CA, USA. To whom correspondence should be addressed at: Depart: tment of Nutrition and Food Science, 328 Spidle Hall, Auburn University, AL 36849, USA. 39 of the protein weight. Hence, the nitrogen to protein conversion factor is usually 6:25, but specific foods have smaller (cereals) or higher (milk) conversion factors depending on the proportions of N in their proteins. Direct _methods for protein determination include biuret, Lowry, bicinchoninic acid (BCA), ultraviolet (UV) absorption (280 nm), dye binding, Bradford, ninhydrin and turbidimetry. These methods are based fon properties of specific proteins, specific amino acid residues in proteins or peptide bonds present in proteins or peptides and involve extraction, isolation and some- times purification (Chang 1994; Pomeranz and Meloan 1994). ‘These methods are often used in biochemical 1 Sel Food Agric 0022-5142/9T/S09.00 ©) 1997 SCI. Printed in Great Britain40 investigations but they are completely impractical for routine food analysis because foods contain a complex tnixture of proteins (Chang 1994; Pomeranz and Meloan, 1994). Moreover, only dye binding methods (official methods 957-12 and 975-17) are approved for direct determination of protein in milk (AOAC 1995). Official methods for N- determinations in foods include modifications of the Kjeldahl method (981-10, 955:04, 977-02, 978-04, 920+152, 920-87, 945-188, 95036, 930-33, 930-28, 928-08, 977-14, 95048 and 93539 with a Hg catalyst; and 991-20, 991-22 and 991-23 with a Cu catalyst) (Sullivan and Carpenter 1993), AOAC Offical Metiod 984-13 “Protein (Crude) in Animal Feed’ also utilises a Cu catalyst (AOAC 1995), The Kjeldahl method consists of (a) & digestion step whore N is con- verted into ammonium (NH@), and (b) un analytical step where NH’ is quantified by ttrimeiry, colorimetry of using an ion-specilie electrode. This method assumes that N recovered during digestion is mainly smino-N from proteins (total organic N) and that the contribur tion of inorgenic N (nitrate, nitrite, ammonium) or ‘other organic N (nucleotides, nuceic acs) is nepisibe. Presently, methods used for determining the total protein (refered to as erude protein) content of foods involve (a) determination of N, and (b) calculation where the N value is converted to protein by a food- ‘specific nitrogen to protein conversion factor, which will be refer to as H’ throughout this manuscript. ‘The main advantage of Kjeldahl methods is its widely established use in CP estimation, However, Kjeldahl ‘methods require wet chemistry, the handling of concen- trated sulphuric acid and a heavy-metal catalyst. In consequence, for routine analysis the Kjeldahl method is a tedious’ and time-consuming procedure requiring disposal of hezardous wastes. For these reasons, an- iytical laboratories tend to adopt automated and easy- to-use equipment (such as the LECO FP-428, LECO CHN 600 or Carlo Era NA-1500) for routine, unat- tended N determination, These instruments use the Dumas method which consists of (a) converting all the forms of N into gaseous nitrogen oxides (NO,) by com plete combustion in an induetion furnace, (b} reducing the NO, gases to Ny and (e) quantifying N, by thermal conductivity (Sweeney and Rexroad 1987; Jones 1991). ‘Thus, for the Dumas method (also referred to as the — Kjeldant N after salieytic acid pre-treatment = Nitrate AH Simonne et al combustion method) wet chemistry is not involved and time for analysis is reduced to approximately 6 min per sample, furthermore liquid, semi-solid or solid sample can be analysed. Moreover, its accuracy and repeatabil- ity may be superior to that of the Kjeldahl method (Schmitter and Ribs 1989). Because of its nature, Dumas N (DN) is a true measure of total N. In food products (Minagawa et al 1984) and in plants (Sweeney and Rexroad 1987; ‘Simonne et al 1994), DN and Kjeldahl N (KN) recov- cred different fractions of N (Fig 1), During the Kjeldahl digestion, reducing conditions caused by the release of free carbon favour the conversion of nitrate to NHJ and losses of nitrate as N, (Bradstreet 1960). Therefore, KN may include some non-amino N from nitrates, nitrites, nucleotides or nucleic acids (Nelson and Sommers 1980; Pace et al 1982; du Preez and Bate 1989a,b; Goyal and Hafez 1990; Barbano et af (991), The teduction of non-amino N during Kjeldahl digestion depends on the chemical form of the non- amino N and sample matrix. Henes, the recovery of non-amino N is non-quantitative. The combustion method was approved for CP determination in animal feed (Sweeney 1989), meat and meat products (King: Brink and Sebranek’ 1993), and cereal grains and oil seeds (Bicsak 1993). An in-depth study was also con- ducted with infants foods (Bellomonte et al 1987) and two collaborative studies carried out on dairy products| {Bradley 1995); one on fruit and vegetable products (Huang et al 1995) is currently being prepared. Although the approach of systematically evaluating the effect of analytical method in each food group is very thorough, itis slow and expensive. In addition, itis sometimes difficult to classify food products into a single category. For example, a food designated for infant food and containing turkey is classified as a infant food, while a meat or blood meal designated for ‘animal consumption was part of a study on animal feed. Matrix differences between these two samples may not be different enough to justify the distinction implied by the two categories. Another limitation to this classi- fication is that compound foods may belong to two dif ferent food groups. For instance, soups containing milk, chicken and fruits may be regarded as a modified dairy product or infant formula, Therefore, the present study —— Kjeldahl N— Dumas N——— Nucleic acids Nitrate ‘Amino acids Proteins Fig 1, Expected recovery of main nitrogen fractions in plant tissues for selected lytical methods for N determination. The size of the cells is not proportional to the mean sample content, Levels of free ammonium (NH) and nitite (NOz) are negligible in Plants because oftheir toxicity (from Simonne etal 1994),The Dumas method for nitrogen and erude protein determinations 41 TABLE 1 ‘Samples, their categories and their N contents (% sample as is) Category Sample names and ‘Mean Mean ——_-‘KjeldaW Reference deseription Kjeldah! Dumas N= Dumas _ N N Ratio Cereals Gold medal all-purpose flour 1-680 198 bas In-house Gold medal bread flour 2290 252 091 Inshouse Long grain vice L320 16 097 In-house Pillsbury all-purpose flour 1960 22 092 In-house Pillsbury bread flour 2130 232 092 Biesak (1993) Barley 2.00 205 099 Biesak (1993) Corn 1410 143 099 Bicsak (1993) Sorghum 1-400 142 099 Biesak (1993) Wheat 1 2360 231 100 Biesak (1993) Wheat 2 3010 3.04 099 Bicsak (1993) Chemicals EDTA 9570 938 100 Sweeney and Rexroad (1987) Ammonium sulphate 1 2140 2130 098 Minagava eta! (1984) ‘Ammonium sulphate 2 21-20 2160 098 Minagawa et a! (1984) Histidine 2040 2000 102 Minagawa eal (1984) Lysine HCL 1536 1535 100 ‘Sweeney and Rexroad (1987) Lysine HCl 1536 1527 tol Biesak (1993) Nicotinic acid 106s 132 094 Biesak (1993) Thiowrea 3740 3650 10 Minagawa eta! (1984) Tryptophan 1368 130 100 ‘Sweeney and Rexroad (1987) Valine 1210 1210 100 Minagava etal (1984) Dairy products Chocolate milkshake S60 O56 tot In-house Cottage eheese 1 1820 201 090 In-house Cottage cheese 2 2070 197 Los Inchouse Low-moisture Kroger mozzarella cheese 4.090 364 112 ‘Skim-mill 05008 105, 1% milk 1 oso 049 106 In-house ‘Skiee-ilk 2 0500 O80 101 In-house 1% milk 2 0500049 102 In-house Sargento Mozzarella 4050 453 089 In-house Day mile 5-540 5:58 099 Sweeney and Rexroad (1987) Animal feeds Alfalfa pellets 2760 271 100 ‘Sweeney and Rexroad (1987) Blood meal 13.04 13.06 100 ‘Sweeney and Rexroad (1987) Broiler finisher 3420 342 100 ‘Sweeney and Rexroad (1987) Cattle concentrate oe 669 099 ‘Sweeney and Rexroad (1987) Feather meal 1356 B61 100 ‘Sweeney and Rextoad (1987) High nitrate grass 230 262 093 ‘Sweeney and Rexrond (1987) Hog feed 3380 339 100 ‘Sweeney and Rexroad (1987) Meat meal 8730 B75, 100 ‘Sweeney and Rexrond (1987) Soya protein concentrate 1398 14.02 100 ‘Sweeney and Rexroad (1987) Soya bean meal 7980 8:00 100 ‘Sweeney and Rextoad (1987) Fish Bumble boo tuna in it 3370 46 on In-house Bumble bee tuna in oil 2 4520 5:62 080 In-house Bumble been tuna in water 4310 5565, 06 In-house Flounder 3-000 285, 195 In-house Flat fish (uncooked) 2.610 280 093 In-house Star kist tuna in oi! 1 3790 473, 079 In-house Star kist tuna in ofl 2 3.780 58, 073 In-house White tuna in oit 4700 651 072 Inchouse White tuna in water 3810 5.80 065 In-house Fruit Guava om = 06? 02s In-house Nectarines (with skin) 190022 083 In-house Peaches 1 O40 On 089 In-house Peaches 2 9200 (Ot 091 Inchouse Peas 007 = 007 088 In-house Plum 1 010 OS 095 Inshouse2 TABLE 1 Coninued AH Simonne et al Category ‘Sample names and Mean Mean Kjeldahl Reference description Kjeldaht = Dumas Nz Dunas N y N Retio Fruit Plum 2 0.120 Di 093 Tmhouse Sapodilla 0.010 008 ous Tochouse Baby foods and Beef 1560 159 098 Bellomonte etal (1987) infant formula, Beef and ham 1650 161 099 Bellomonte etal (1987) Biscuits 1 L810 187 097 Bellomonte eta! (1987) Biscuits 2 1-360 139 098 Bellomonte etal (1987) Chicken L770 17s bot Bellomonte etal (1987) Chicken carrots and potatoes L010 103 098 Bollomoate etal (1987) Creme of cereal L640 167 098 ellomonte etal (1987) Crome of rice Leo 169 097 Bellomonte etal (1987) Formula | 2480 252 098 Bollomonte eral (1987) Formula 2 2850 292 098 Bellamonte etal (1987) Formula 3 2:500 2.62 095 Bellomonte etal (1987) Formula 4 1830 192 095 Bellomonte eta! (1989) Formula $ 2160 220 0.98 Bellomonte eta! (1987) Formula 6 2000 208 096 Bellomonte etal (1987) Formala 7 2-560 289 099 Rellomonte etal (1987) Ham and eggs 7400 137 098 Bollomonte etal (1987) Milk soup with cereal and fruits 2180 223 098 Bellomonte et uf (1987) Milk soup with cereal and apples, = 1-190 tat 098 Bellomonte etal (1987) Semolina 1270 132 096 Bellomonte et al (1987) ‘Turkey 1-920 199 096 Bellomonte et al (1987) Veal 9260 946 098 Bellomonte etal (1987), Veal and brain 1320 132 1-00 Bellomonte er al (1987) Wheat four with milk and cat 2.050 2.08 099 Bellomonte etal (1987) Meats and meat Bekrich bologna 1620 175 092 In-house products Heinz beef gravy (in jar) 0130 043 Lol Inshouse Heine chicken gravy oto 013 086 In-house Oscar myer bologna 1950 184. 095 In-house Oilseeds Canola 1 3330 334 100 Biceak (1993) Canola 2 3-710 373 099 Bicsak (1993) Soya bean 1 5-640 564 100 Bicsak (1993) Soya bean 2 6540 657 100 Bicsak (1993) Sunflower 2970 297 100 Bicsak (1993) Vegetables Bush red kidney beans (canned) 0830 os 098 In-house and vegetable Campbell tomato soup. 0260 027 097 In-house products Cooked broccoli 1 oto 047 086 Inchouse Cooked broccoli 2 0410 08 os In-house Cooked cabbage 0180 O42 140 nshouse CCucurnber with skin 007m = OL 064 Inhouse ‘Cucumber without skin 0060 0.07 082 inhouse Heinz catsup 0230 (023 097 Inhouse Hunts catsup 024 Os 133 In-house Feeberg lettuce O10 6 0-68 In-house Kroger red kidney beans (canned) 0-910 105 087 Invhouse Kroger tomato soup 0280 035 078 Inhouse Raw broccoli 1 0560 049 13 In-house Raw broceoli 2 9210 064 033 Inchouse Raw cabbage 180 09 095 In-house Tomato (raw with O10 o2s 042 Toshouse Tomato (raw with kin) 2 0.220 019 120. In-houseThe Dumas method for nitrogen and erude protein determinations a TABLE 2 Influence of food group on lowest, highest and mean KN:DN ratio Food type ‘Number of Lowest Highest = Mean Standard obsereations ratio. — ratio. —ratia®—devauion Dairy products 10 0892 Ola 0.07 Oilseeds 5 099 100 10a 000. Chemicals 0 094 1020994 0.02 Animal feed 0 093100099 0.02 Tafant formulas and baby foods B 095 1010980 Cereal 10 085 = 100095205, Meats and meat products 4 08 101 (094a 006, Vegetables and vegetable products 7 033 1408 OD Fish 9 065 105 RYE OZ Fruit 8 O15 095 «Oe = 03 = Mean followed by diferent leters are significantly diflerent according to Duncan's multiple range test (alpha = 5%) aimed to (1) compare the Kjeldahl and Dumas methods ‘over several food types and (2) evaluate the effect of replacing KN by DN on the calculation of CP. MATERIALS AND METHODS Food samples from grocery stores of the Athens (GA, US) area and from the Georgia School Lunch Program monitoring study were selected to cover a wide range of ‘matrices and expected N values. Published data from ‘comparative studies using food and chemical samples ‘were also included (Table 1). Kjeldahl N and DN were determined in duplicate on in-house samples. The mean, of duplicates was used as the estimate of N content. KN was determined with a macro-Kjeldahl appar- ‘tus using copper sulphate as a catalyst by Method 984-13 (AOAC 1995), For DN determination, 20 g of fresh sample was weighed in a tin foil and placed in a drying oven at 70°C for 12 h. Samples were pre-dried so that differences in sample moisture would not influence the results. Afler cooling at room temperature, samples were analysed (FP-428, Leco Corp, St Joseph, MI, USA). KN and DN were expressed as g N per 100 g of the sample before drying (%N). CP was independently calculated as CPi = H x KN or CP2 =H x mean KN: DN ratio x DN. CP was also caleulated with DN and the single lowest (CP3) ‘and highest (CP4) calculated KN: DN ratios. Dilfer- ences between CP] and CP2 were used to evaluate the ‘effect of N from different analyticsl method on CP. CP3 and CP4 provided extreme CP estimates. Nitrogen to protein conversion factors (H) of 57 for the cereal ‘group, 6-38 for the milk group and 6:25 for the veget= able, fruit and meat groups were used to estimate CP. Regression analysis was performed on in-house and compiled values, For each food type, KN: DN ratios were calculated and differences were evaluated by ANOVA (SAS 1987). Coefficients of variation (CV) were TABLE 3 Predicted erude protein value using Kjeldahl N or Dumas N data Food type ‘Nitrogen to protein conversion Crude protein estates (g per 100 g asi) factors (2) CPi cP? Ps Ps (Cereal 5-70 1 a 0 2 Dairy products 638 B B un a Animal Feed 625, a a “4 8 Fish 625, B 2m 20 32 Fruit 635 1 1 0 1 Infant formulas and baby foods 625 1s 18 15 6 “Meat and meat products 625 6 6 5 6 Vegetables and vegetable products 625 2 2 ' 3 Oilseeds 625 B *% 26 8 "Pt, Kjeldaht N x H; CP2, Dumas N x Mean KN: DN ratio x H; CP3, Dumas N x Lowest KN: DN ratio x H; CP4, Dumas N x Highest KN: DN ratio x44 calculated as group standard deviation divided by the group mean multiplied by 100. Published results on chemical samples were statistically analysed separately from food samples RESULTS AND DISCUSSION Chemical samples The mean KN: DN ratio calculated from published data on chemical samples was 0:99 (CY = 2%) and ranged between 0:94 and 1-02 (n= 10 observations} Mean KN: DN ratios wore [00 for valine, 0:98 for NBS Nel and NBS N-2- ammonium suiphate (Minagawa et al 1984), 093 for EDTA and 1-00 for tryptophan and lysine HCI (Sweeney and Resroad 1987), 0-94 for nicotinic acid (Biowak 1993), 099 for nicotinic acid and 1-00 for iysine HCl (King-Brink and Sebranek 1993), 1-01 for thiourea, 1-02 for histidine hydrochloride (Minagawa etal 1984) and 1-0 for lysine HCI (Bicsak 1993), In these chemicals, N was involved in ~C-N- (EDTA), H-N- (ammonium sulphate, lysine, thiourea, valine) or heterocyclic (tryptophan, histidine, nicotinic acid) linkages. Because KN : DN ratios were close to 1-00, N recovery by either method can be con- sidered identical Food samples Regression analysis on in-house samples (r= 52 observations, R? = 0.96, P-regression < 0-01) over the 0:1-6:6% N range and all data combined (n= 101 observations, R? = 0-98, P-regression < 0-01) showed a close relationship between KN and DN. For all data KN may be estimated from = DN with KN = 1-00"<°% x DN —009"<"8° in the 0-05 68% N range. Because different food matrices were included and because N levels within food types did not span the entire N range, KN: DN ratios were caleu- lated for each food type. KN:DN ratios were significantly (P< 001; R? = 025) affected by food type (Table 2). KN may be estimated from DN using the appropriate corrective ratio, The numerical value of KN: DN ratios suggest that for dairy, feed, infant formula, cereal and meat, DN may replace KN without adjustment. However, adjust- ments of 0-89, 0-80 and 0-73 should be used for voget- able, fish and fruit samples, respectively For the dairy, feed, infant formula, cereal and meat types, CV of the mean KN : DN ratios ranged between J and 5%, Values of 15, 31 and 45% were observed for the fish, vegetable and fruit types, respectively, The highest ‘ratio found was 1-40 for cooked cabbage (ample no 85). This variation in mean KN: DN ratios may be attributed to differences in nitrate contents in AH Simonne et al vegetable and fruit samples and to the low level of N. However, tis difference may not be of practical impor- tance since N levels in fruits and vegetables are usually low (<1% N). The high CV obsorved for the fish food group may be attributed to the relatively high non amino nitrogenous compounds in fish, Crude protein Mean CPL and CP2 values were 2, 0, 0, 1, 0, 0 and (0% and mean CP4 and CP3 values ware 7, 3, 4 12,1, 1 and 2 for the cereal, dairy, feed, fish, fruit, infant formula, meat and vegetable food type, respectively (Fable 3), These results suguest that differences between CP estimates using KN or DN are of little practical importanee in CP calculation, CONCLUSIONS Dumas N could replace Kjeldahl N in food analysis when the numerical differences betwoen KN and DN are small (KN:DN ratios > 0:90) without. practical effect on CP calculation (CP! and CP2 < 2%), When the KN: DN <090, replacing KN by DN without adjustment will result in an overestimation of N and CP. However, DN may be used along with the appro- priate KN: DN ratio. This adjustment may be easily included in computer programs that handle nutrient data, KN: DN ratios for the fruit and vegetable groups suggested the need for adjustment. However, expected NN levels are low in these food groups; the error intro- duced by replacing KN with DN will be of little practi cal importance. For complex foods which are difficult to classify in a single food group, CP may be estimated by adding the weighed partial values of cach component. Partial values may be calculated with the appropriate KN : DN ratio and DN. CP may also be estimated by a fit-all KN: DN ratio, The mean KN: DN ratio calculated in this study was 0:93, However, a larger database would provide a better estimate ofthe ft-all ratio. DN wonld be less appropriate for CP calculations in cases such as shelf-life studies where changes of proteis or amino-N to other non-amino N compounds occur, ACKNOWLEDGEMENTS ‘The authors wish to thank Mrs Wonda Rogers (Food Science and Technology Department, University of Georgia) and Mr Mark Couvillon (Micro Macro International) for their technical assistance,The Dumas method for nitrogen and crude protein determinations 45 REFERENCES AOAC 1995 Official Methods of Anulysis (16th edn), AOAC Tnternational, Aclington, VA, USA. Barbano D M, Lynch J M, Fleming J R 1991 Divect and indi- ‘ect determination of true protein content of milk by Kjel- ‘dahl analysis: Collaborative study. J AOAC 74 281-288. Bellomonte ©, Constantin’ A. Giammaroli S 1987 Compari- son of mouifed automatic Dumas method and traditions Kjeldahl method for nitrogen determination in infant food. 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