Compound Feedingstuffs Research Articles

Influence of the Season on the Nutritive Value and Gas Production of Opuntia ficus-indica and Agave americana L. in Ruminant Feed.

Using invasive plants in animal production can provide an economical and eco-sustainable competitive advantage in a globalized market. The Opuntia ficus-indica and the Agave americana L. are invasive plants historically used by Azorean farmers as an alternative ruminant water source in the summer. This study aims to better understand their properties and how they vary throughout the year, so their use to complement animal diets can be optimised. Six samples of each species were collected on the Terceira Island during 2 growth seasons: winter (January 2021) and summer (September 2021), and their chemical composition, in vitro digestibility, and gas production, were determined. Significant (p < 0.05) differences were found in all parameters between the summer and the winter, with larger variations in both fibre (NDF and ADF) and digestibility parameters found between the Opuntia and the Agave. Gas production was greater in the summer for Opuntia and in the winter for Agave. Even though the digestibility was lower in the summer, we found that both plants offer a viable ruminant feed complement in both seasons, providing a cheap and eco-sustainable alternative water source, that can be associated to dry forage and compound feeding stuff with a high crude protein content when designing ruminant diets.

Studying the Thermophysical Characteristics of the Muscle Mass of the Black Soldier Fly Larvaes (Hermetia Illucens) as a Drying Object

Introduction. The main component of the compound feedstuff is fish meal, which has unstable quality and high price. Fish and meat-and-bone meals are replaced with protein concentrates and higher quality larvae proteins. The source of feed protein is the biomass of the black soldier flies (Hermetia illucens), which have a rich amino acid composition and also process food waste. The aim of the work is to study the thermal-physical characteristics of the muscle mass of the black soldier fly larvaes (Hermetia illucens). Материалы и методы. The study focused on the muscle mass of black soldier fly larvaes (Hermetia illucens). The subject of the study is thermal-physical regularities during the drying process. The studies were conducted on the basis of Don State Technical University. The article describes determination of thermal-physical characteristics such as specific heat, thermal conductivity, moisture of the raw material, and oiliness. Results. Heat conductivity coefficient of water 0.555 W/(m∙K) for food and feed products from 0.25 to 0.40 W/(m∙K) black Soldier Fly larvae have a heat conductivity equal to 0.144 W/(m∙K), which is lower than conventional feedstuff components. The humidity of the examined raw material is 45% or higher while the heat conductivity remains linear and practically does not increase. Discussion and Conclusion. The results obtained during the work can be used for parameter determination and design of various types of dryers, and for mathematical description of the dynamics and kinetics of drying.

Контроль качества и безопасности комбинированных кормов для сельскохозяйственных животных и птиц

The article presents materials for the assessment quality and safety of compound feedstuff for farm animals and poultry. In the structure of the cost of livestock products, a significant share about 70 % is occupied by feed. The efficiency of the livestock sector, further growth rates of the industry are largely determined by the quality of feed components, their availability for domestic agricultural enterprises. The compound feedstuff industry is inextricably linked with the production of livestock and poultry products. Therefore, the prevailing trends in these sectors are directly reflected in the compound feedstuff industry. All feed for farm animals must be subjected to laboratory control for compliance with quality and safety. Feed quality and safety control is carried out in accordance with regulatory documents that establish mandatory requirements aimed at preventing animal diseases, producing complete and veterinary safe livestock products and protecting the population from diseases common to humans and animals. According to organoleptic and physicochemical indicators, combined feeds must comply with GOST R 51899-2002 Granulated compound feedstuff. General specifications. The most important indicators in determining feed safety are the content of mycotoxins (aflatoxin B1, patulin, deoxynivalenol, ochratoxin, T-2 toxin), nitrates, nitrites, toxic elements (mercury, cadmium, lead, arsenic).

Determination of urea in compound feed for ruminants: interlaboratory comparison for the validation of the spectrophotometric method in Commission Regulation (EC) No 152/2009

Description of the subject. Urea is a feed additive authorized in ruminant feedingstuff with a maximum content in compound feed of 8,800 mg·kg-1. Commission Regulation (EC) No 152/2009 displays the official method for the determination of urea in feed but does not specify the type of feed for which the method is applicable. Following reports from Member States stating that the official spectrophotometric method is not fit for purpose for other feed than ruminant feed and given the fact that no method performance characteristics are set, the Joint Research Centre organized an interlaboratory exercise for the validation of this method. Objectives. The objective was to carry out an interlaboratory ring trial for the validation of the official spectrophotometric method for the determination of urea in compound feed for ruminants, under specific request from DG SANTE of the European Commission. Method. A collaborative study was carried out on six different test materials prepared from ruminant compound feeds containing urea at various mass fractions and a blank test material. Nineteen laboratories enrolled for the study and eighteen laboratories from ten EU Member States submitted results. Results. The performance characteristics were established, namely, the validation criterion used in this study (RSDR) was in all cases, except in sheep feed at 3,000 mg·kg-1 measured at 420 nm, achieved (i.e. lower than 20%). Slightly better results in terms of precision and trueness were obtained at 435 nm. Conclusions. Commission Regulation (EC) No 152/2009 should be revised as regards the official spectrophotometric method with a restriction of the scope, which is the determination of urea in ruminant compound feedingstuff at authorized level. Additionally, the selected measurement wavelength, which is either 420 nm or 435 nm, has a minor impact on the performance profile of the method.

Validation of a multi-analyte HPLC method for the determination of carotenoids used as feed additives in fish and poultry feed: results of an interlaboratory study

ABSTRACT The performance characteristics of a multi-analyte method for the determination of all 10 carotenoids authorised as feed additives within the EU were assessed via an interlaboratory study. The analytical method is based on reversed phase high performance liquid chromatography (RP-HPLC) coupled to an optical detector set at 410 nm. The analysis is particularly challenging due to the presence of various stereoisomers of each carotenoid, and the use of these compounds via natural or synthetic formulations, requiring a special sample preparation. EU regulations specifying the conditions of use set legal limits for these substances in compound feedingstuffs ranging from 6 mg kg−1 to 138 mg kg−1, depending on the individual carotenoid and the target animal for which the feed is supplemented with this carotenoid. The purpose of the multi-analyte method validated in this paper is to facilitate the monitoring of carotenoids at relevant levels when used as feed additives in compound feedingstuffs and pre-mixtures. The interlaboratory study delivered precision data for 43 different analyte/mass fraction/matrix combinations, covering a mass fraction range of the target analytes from 2.6 mg kg−1 to 3861 mg kg−1. The relative standard deviations for repeatability (RSDr) varied from 2.2 to 16.2 % with a mean value of 6 %, while the relative standard deviations for reproducibility (RSDR) varied from 6.8 to 39 % with a mean value of 21 %. Given the broad scope of the method covering 10 carotenoids added to compound feedingstuffs and pre-mixtures via different formulations, this multi-analyte method is considered fit for the intended purpose.

Analysis of disperse composition of the dust in air of working zone of feed mills

In the compound feedstuff production, one of the main problems is the dust emission at all stages of production. The most significant dust emission occurs at the raw materials acceptance stage, where the concentration of dust can be many times higher than the maximum permissible values. The impact of dusts and their capture depends on the dispersed dust composition, and since the raw materials come in different types (bran, wheat, corn, sunflower meal, etc.), the dispersed dust composition is complex. For this reason, it becomes necessary to study their dispersed composition. The article presents the analysis results on some types of the dispersed dust, which generated in the air of the working area on feed mill reception center.

DETERMINANTS OF THE SELECTION OF RAW MATERIALS USED IN ANIMAL FEED PRODUCTION

The main purpose of this paper was to identify the factors affecting the selection of raw materials used by feed operators in feed production. An attempt was also made to indicate the barriers to increasing the use of native protein plants by feed operators in feed production. Today, many EU countries (including Poland) primarily rely on vegetable protein derived from genetically modified soya bean meal (mainly imported from South America and the U.S.) in addressing their needs for protein raw material used in animal feed. For many years now, Poland has taken steps to increase the production and use of native protein raw material to partially replace soya bean meal imports. The use of mixes of diverse domestic protein sources derived from grain legumes (peas, field beans, lupine) can provide an advantageous alternative to compound feeding stuff based on imported post-extraction soya bean meal. To meet the objective defined above, this paper relied on the results of a 2018 survey conducted with a sample of 29 feed operators located across the country. More than half (55%) of the enterprises surveyed did not use legumes in feed production; the use of legumes was above 10% in only 3% of respondents. When asked about the key factors affecting the selection of raw materials used in production processes, the respondents declared to be interested in buying large batches of homogeneous raw materials that meet specific quality parameters (33% replied “rather yes” and 67% replied “definitely yes”). The protein content of plant seeds used in feed production was identified as another aspect of extreme importance (55% replied “rather yes” and 24% replied “definitely yes”).

Greenhouse Gas Emissions in Dairy Goat Farming Systems: Abatement Potential and Cost.

Simple SummaryAgriculture and particularly livestock farming is associated with the production of certain gases that contribute to global warming, commonly referred to as greenhouse gases. These gases are the result of the use of machinery and other inputs such as fertilizers and pesticides or are associated with the digestion process of animals. In this work, we have analyzed data from dairy goat farms in Greece to estimate the amount of greenhouse gases per kilogram of milk produced and identify farming practices that can result in their reduction. We found that greenhouse gases per kilogram of milk are fewer in farms that are characterized by higher milk production per goat. Furthermore, certain practices like the use of homegrown feed instead of purchased feed and the use of compound feedstuffs or oil-rich feedstuffs like cottonseed cake can result in lower greenhouse gases in goat farms. Also, the analysis suggests that the reduction of greenhouse gases can lead to a reduction of farm income, especially in the case of intensive farms. This finding has to be taken into consideration by policy makers and possible measures to compensate for this income loss have to be explored.Dairy goat farming is an important agricultural activity in the Mediterranean region. In Greece the activity offers occupation and income to thousands of families mainly located in mountainous and semi-mountainous areas of the country where it utilizes low productivity pastures and shrub lands. Furthermore, goats are more resilient to climate changes compared to other species, and are often characterized as ideal for keeping in drought areas. However, there is still limited evidence on total greenhouse gases (GHG) emitted from goat farms and their mitigation potential. In this context, this study aims to estimate GHG emissions of goat farms in Greece and explore their abatement options using an economic optimization model. Three case studies are explored i.e., an extensive, a semi-intensive and an intensive goat farm that correspond to the main goat production systems identified in Greece. The analysis aims to assess total GHGs as well as the impact of abatement on the structures, gross margins and labor inputs of the farms under investigation. The issue of the marginal abatement cost is also addressed. The results indicate that the extensive farm causes higher emissions/kg of milk produced (4.08 kg CO2-eq) compared to the semi-intensive and intensive farms (2.04 kg and 1.82 kg of CO2-equivelants, respectively). The results also emphasize the higher marginal abatement cost of the intensive farm. In all farm types, abatement is achieved primarily through the reduction of the livestock capital and secondarily by other appropriate farming practices, like substitution of purchased feed with homegrown feed.

Conformity assessment of information on crushing equipment for agricultural raw materials: consequences of the lack of appropriate standards

The paper reviews the analysis of information about hammer (rotary) crushers for the production of compound feedstuff. It is shown that for the majority of crushers studied, the information is incomplete or unreliable, which is misleading for consumers purchasing this equipment. Measures to improve the quality of information on crushing equipment are proposed.

Revision of a European Union Official Method to Enforce Legal Provisions: The Case of Diclazuril.

Background: Diclazuril is a coccidiostat currently authorized as feed additive in the European Union (EU), with a legal limit set at 1 mg/kg. For official control, an official EU method based on reversed-phase HPLC coupled with UV detection at 280 nm needs to be applied. Recently, the EU Reference Laboratory for feed additives was informed that the recovery rate for diclazuril was very low when implementing this method and performed experiments demonstrating that the indicated sorbent mass of the solid-phase extraction (SPE) was too low. Objective: Therefore, the paper presents a modified method protocol and the results of an interlaboratory study, performed on two compound feedingstuffs containing diclazuril around the legal limit. Methods: The official method was modified by using a higher SPE sorbent mass and was further subjected to validation. Results: The obtained values for the relative standard deviation for repeatability were 4.5 and 11.2%, and the corresponding values for the relative standard deviation for reproducibility were 14.3 and 18.1%; the calculated HorRat values were 0.95 and 1.14. Furthermore, acceptable mean recovery values of 98 and 111% were obtained for the two test materials, respectively. Conclusions: Based on the obtained performance profile, it was concluded that the modified official method was fit for purpose. In consequence, the official EU method will be corrected accordingly. Highlights: The highlights of this work are reflected by the following terms, namely Diclazuril, correction of EU official method, and interlaboratory study.

РАСПРОСТРАНЕННОСТЬ ГРИБОВ РОДА PENICILLIUM LINK В СЫРЬЕ ДЛЯ ПРОИЗВОДСТВА КОМБИКОРМОВ

The article presents the results of the study that investigated the degree of infection of various types of raw materials used for the production of compound feedstuff (oil cakes, oil meals, glutens, animal meal, protein-vitamin-mineral supplements, skimmed milk substitute) by fungi of genus Penicillium. According to the data, 43 out of 48 samples examined were contaminated with these fungi, the dominating species being P. cyclopium, P. palitans and P. chrysogenum.

Development and validation of a multi-analyte method for the regulatory control of carotenoids used as feed additives in fish and poultry feed

ABSTRACTCarotenoids are used in animal nutrition mainly as sensory additives that favourably affect the colour of fish, birds and food of animal origin. Various analytical methods exist for their quantification in compound feed, reflecting the different physico-chemical characteristics of the carotenoid and the corresponding feed additives. They may be natural products or specific formulations containing the target carotenoids produced by chemical synthesis. In this study a multi-analyte method was developed that can be applied to the determination of all 10 carotenoids currently authorised within the European Union for compound feedingstuffs. The method functions regardless of whether the carotenoids have been added to the compound feed via natural products or specific formulations. It is comprised of three steps: (1) digestion of the feed sample with an enzyme; (2) pressurised liquid extraction; and (3) quantification of the analytes by reversed-phase HPLC coupled to a photodiode array detector in the visible range. The method was single-laboratory validated for poultry and fish feed covering a mass fraction range of the target analyte from 2.5 to 300 mg kg–1. The following method performance characteristics were obtained: the recovery rate varied from 82% to 129% and precision expressed as the relative standard deviation of intermediate precision varied from 1.6% to 15%. Based on the acceptable performance obtained in the validation study, the multi-analyte method is considered fit for the intended purpose.

Влияние использования препаратов витаминов С и Вс в комбикорме на продуктивность и качество яиц кур-несушек

Влияние использования препаратов витаминов С и Вс в комбикорме на продуктивность и качество яиц кур-несушек

Scientific Opinion on the safety and efficacy of citric acid when used as a technological additive (acidity regulator) for all animal species

An application has been made for the authorisation of citric acid (anhydrous and monohydrate) when used as an acidity regulator in feed and in water for drinking for all animal species. Citric acid (anhydrous and monohydrate) is already authorised for use in food following the quantum satis principle. There is evidence from published studies that citric acid is safe for the target species when used up to 30 000 mg citric acid/kg complete feedingstuffs and the corresponding concentration in water for drinking (10 000 mg citric acid/L). The additive is consequently also safe for the target species at the proposed conditions of use of 15 000 mg citric acid/kg complete feedingstuffs and 5 000 mg citric acid/L in water for drinking. The quality of the available data does not allow a margin of safety to be derived. The use of citric acid in animal nutrition is safe for the consumer. It is prudent to regard citric acid as potentially hazardous to workers by exposure of the skin, eyes or mucous membranes or by inhalation. The use of citric acid in animal nutrition would not pose a risk for the environment. The addition of citric acid reduces the pH of aqueous suspensions of compound feedingstuffs and feed materials. However, the relevance of pH and buffer capacity measurements made with aqueous suspensions to the effects of citric acid in dry feed (< 12 % moisture) is open to question. Citric acid might have the potential to act as an acidity regulator in feedingstuffs.

Surveillance of aflatoxin content in dairy cow feedstuff from Navarra (Spain)

Aflatoxins (B1, B2, G1 and G2) are produced by the fungi Aspergillus (A. flavus and A. parasiticus) in substrates used in cattle feed manufacturing. Aflatoxin M1 (AFM1) is a major metabolite of Aflatoxin B1 (AFB1) which may be present in milk from animals that consume contaminated feed. Levels of aflatoxins in 78 dairy cow feedstuff samples from 40 farms located in Navarra were determined by HPLC-FLD (High Performance Liquid Chromatography with fluorescence detection) and post-column derivatization. The influence of geographical location, season and type of feeding system on aflatoxin content was studied. The climatic profile of AFB1 pointed to spring as the season with the highest aflatoxin level (0.086μg/kg), followed by winter and summer (0.075 and 0.030μg/kg, respectively), and to a lesser degree, autumn (0.017μg/kg). Moreover, wet and dry TMR (Total Mixed Ration) feeding systems (i.e. AFB1: 0.076 and 0.068μg/kg; Aflatoxin G1 (AFG1): 0.050 and 0.011μg/kg, respectively) showed a greater content of the analyzed aflatoxins in comparison with compound feed (i.e. AFB1: 0.039μg/kg; AFG1: 0.007μg/kg). The fact that the majority of the samples collected were based on compound feed shows that this type was preferred by most dairy farmers. The undetectable levels of aflatoxins in the organic homemade compound feedstuff are also worth mentioning. While none of the feedstuff samples contained amounts over those permitted under European legislation (5μg/kg), the theoretical extrapolation of the carryover rate suggested in previously published experiments of AFB1 to AFM1 in secreted cow's milk predicts that only one of the feed samples studied had a positive aflatoxin level (53.4ng/kg) higher than the legal limit for raw cow's milk.

Scientific Opinion on the safety and efficacy of fumaric acid as a feed additive for all animal species

Fumaric acid is currently authorised as a preservative in feedingstuffs for all animal species. Fumaric acid is considered safe for pigs and poultry provided that an expected use level of 20 000 mg/kg in complete diets for pigs and poultry is not exceeded. The margin of safety for pigs and poultry is approximately two. The tolerated fumaric acid concentration in complete diets for ruminants is higher. The margin of safety for pigs, poultry and ruminants is sufficiently high that setting a maximum content is not necessary. In contrast, the maximum safe level in milk replacer for veal calves (and probably other young mammals) is provisionally estimated to be 10 000 mg/kg milk replacer, with no margin of safety. Fumaric acid is rapidly metabolised by well-recognised pathways, and neither fumarate nor its metabolites are expected to accumulate in animal tissues. Consequently, human exposure is not expected to be increased by the use of fumaric acid in animal nutrition. Fumaric acid is a severe irritant to eyes. Given the potential for eye irritation, the particle size and the dusting potential, exposure to other mucous membranes such as the respiratory tract may also be a concern and it is advisable to minimise exposure by inhalation. The use of fumaric acid in animal nutrition will not pose a risk to the environment. Fumaric acid has value as a food preservative only in the presence of significant moisture. Comparable situations in feed are limited to some feedingstuffs, feed processing and feeding techniques. In such situations, its function in feed is essentially the same as that in food and no further demonstration of efficacy is considered necessary. However, the FEEDAP Panel has reservations about the effectiveness of any preservative, including fumaric acid, in raw materials and compound feedingstuffs with a typical moisture content of ≤ 12 %.

Effect of Silage from Maize and Strip-Cropped Sorghum and Maize on Dairy Cow's Yield and Milk Composition

Effect of Silage from Maize and Strip-Cropped Sorghum and Maize on Dairy Cow's Yield and Milk CompositionIn an experiment conducted on 34 mid-lactation dairy cows of the Red-and-White and Black-and-White breed, in a random square design, the effect of maize or sorghum-maize silage present in partly mixed ration (PMR) on milk yield, milk composition and blood serum parameters was investigated. The PMR diet contained maize silage, which compared with PMR diet contained sorghum-maize silage made from strip-cropped plants. Both fodder plants were harvested with a 4-row precision chopper, which cut 2 rows of maize and 2 rows of sorghum, giving mixed maize/sorghum forage. The feeding experiment lasted 84 days and consisted of four sub-periods, each 21 days in length to record milk yield, feed and milk chemical composition, and blood parameters. In addition to PMR diets containing part of ration compound feedingstuffs, the cows received part of compound feedingstuffs given at feed stations to meet their nutritional requirement. The compound feedingstuffs in station were controlled by an electronic system related to actual cow's milk yield. Chemical composition of both silages and milk production efficiency were compared. The average dry matter intake in both groups was 18.80 vs 20.4±1.95 kg/day, but compound feedingstuff intake from station was 3.61 vs 4.56 (P&gt;0.01). Milk yield was 21.8 and 20.5±0.51 kg/day, respectively (P&gt;0.01). The amount of standardized fat and protein content of milk (FPCM) was 21.0 and 20.2 kg/g ±0.48 kg/day (P≥0.01). No significant differences were found in the fat, protein, casein, lactose, urea, total solids and solids not fat percentage of milk or in milk traits (acidity, renneting time, density) among groups (P≥0.01). Feeding cows PMR ration with maize silage significantly elevated total cholesterol (P&lt;0.05), but decreased urea levels in blood plasma (P&lt;0.01). It is concluded that strip cropping of sorghum and maize could be an alternative to maize grown as a pure stand in maize high-risk areas for dairy cows in mid-lactation.

Improving NIRS predictions of ingredient composition in compound feedingstuffs using Bayesian non-parametric calibrations

The selection of an appropriate algorithm for modelling the relationship between spectral and reference data is one of the factors that influence the accuracy of predictions using near infrared (NIR) spectroscopy. For the prediction of ingredient composition in compound feeds, several modelling approaches, both linear and non-linear, have shown the possibilities of NIR technology although with some limitations. The present work evaluates the use of Bayesian non-parametric calibrations for the prediction of 26 ingredients, with different nature, distribution and behaviour. One advantage of this approach is that it avoids the bias towards the mean of the reference data that is not appropriate for the non-Gaussian data distributions of the ingredients of compound feeds. Another is the provision of confidence intervals that quantify the uncertainty in individual predictions. These confidence intervals were used in the tuning process, to help avoid over fitting. The results obtained with the Bayes method for most of the ingredients were similar to those obtained by local regression, both methods improving on the linear approach by around 50%. However, unlike the local regression, the Bayes approach predicted all samples and moreover provided prediction intervals that appear to be very realistic for this dataset.

Scientific Opinion on modification of the terms of authorisation of VevoVitall® (Benzoic acid) as a feed additive for weaned piglets

VevoVitall® is a feed additive consisting of benzoic acid. It is currently authorised as zootechnical additive for weaned piglets and pigs for fattening. The applicant is now seeking for the modification of the terms of authorisation for weaned piglets, laid down in Commission Regulation (EC) No 1730/2006. The applicant proposes to modify the text of the “Other provisions” in the authorisation by deleting the sentences “Complementary feedingstuffs for piglets may not contain more than 10000 mg/kg of benzoic acid” and “The additive shall be incorporated in compound feedingstuffs in form of a premixture” and replacing them with “Complementary feedingstuffs for piglets should be thoroughly mixed with other feed materials of the daily ration”. Based on the similar coefficients of variation achieved for benzoic acid concentration after direct incorporation or incorporation via premixtures, the FEEDAP Panel concludes that there is no reason to restrict the inclusion of benzoic acid to compound feed via premixtures. In the view of the FEEDAP Panel, the restrictions on the use of the additive in complementary feed established for pigs for fattening by Commission Regulation (EC) No 1138/2007, are considered sufficient in respect to the narrow margin of safety of the compound and should be applied to weaned piglets.

Key parameters for the development of a NIR microscopic method for the quantification of processed by-products of animal origin in compound feedingstuffs

The aim of this work is to show new advances in the analytical methods developed in the frame of the ban of processed animal by-products in compound feed that is currently applied within the European Union. With this aim, studies to develop a quantitative near infrared microscopy (NIRM) approach have been undertaken in order to fulfil future requirements of European legislation like the introduction of tolerance levels that would require for official control purposes the availability of specific quantitative methods. The capabilities of the NIRM method have been improved; no sample preparation is required and the acquisition parameters are optimised. Both the gross and the fine fractions of the samples are considered; the reflexion mode was used to analyse the gross raw fraction and the transmission mode was chosen to analyse the fine raw fraction. Parameters for reflexion analyses were already fixed in our previous studies while those of transmission mode have been determined in the present study. Because particles are too small, it is difficult to mark them; spectra were collected using the mapping technique. Quantitative analyses have been carried out for different percentages of adulteration (0.5, 1, 2 and 5%). Results were depending on the particle size distribution of the feed and of the fish meal which led to experimental values of adulteration varying between 0.13-0.92%, 0.93-3.7%, 2.42-5.83% and 1.95-9.39% for theoretical percentages of adulteration equal to 0.5, 1, 2 and 5%, respectively. The established protocol with the key parameters proposed has to be considered for the development of an accurate method of quantification.