Introduction
In livestock production, quantitative and qualitative nutrition constitutes one of the fundamental pillars, since its composition and nutritional content can directly affect the pathophysiological state of the individual and, therefore, their productive performance indexes. Additionally, in the field of pets, especially in dogs and cats, diet exerts crucial epigenetic and metabolic effects on development and longevity.
Consequently, ration formulation must be strictly adjusted based on breed, productive phase, and physiological stage, optimizing nutritional parameters to maximize animal performance.
From an analytical perspective, a pellet should not be considered just a mixture of ingredients; it is one of the most complex and heterogeneous matrices. The coexistence of cereals, plant protein sources, fats, industrial by-products, macrominerals and microminerals, enzymes, and encapsulated synthetic additives represents an analytical challenge for the reproducibility and accuracy of assays (Steiner et al., 2020).
This intrinsic compositional variability constitutes the main limiting factor in analytical control, due to the presence of biochemical and physicochemical phenomena such as:
- The presence of soluble non-starch polysaccharides (NSPs), which alter extract viscosity.
- Covalent interactions resulting from Maillard reactions (non-enzymatic glycation between proteins and reducing sugars) induced by thermal extrusion or pelleting processing.
- The heterogeneity of trace analytes, such as mycotoxins and both fat-soluble and water-soluble vitamins.
These variables critically interfere with the operational phases of sampling, extraction, and instrumental quantification. Consequently, standard reference methods frequently show severe limitations due to matrix effects, which induce analytical signal suppression or enhancement phenomena (signal suppression or enhancement), compromising the representativeness of the results (Steiner et al., 2020).
Composition of pellets
The composition of pellets exhibits significant variability, determined by the evolution of the gastrointestinal tract morphology of each species, whose anatomical characteristics condition their digestive capacity.
Gallinas ponedoras
In the case of laying hens, a relationship has been demonstrated between nutrition, egg production, shell quality, and the hen’s health.
The diet must include calcium and phosphorus to ensure the proper formation of the eggshell; additionally, protein content is essential to guarantee an adequate egg size and a nutritious yolk. For their part, carbohydrates are incorporated as an energy source, while fiber contributes to the regulation of intestinal transit (Instituto de Estudios del Huevo, n.d.).
Table 1 shows an example of the ingredients and the typical formulation found in a laying hen pellet.
| Ingredient | Amount (%) |
| Corn | 48 |
| Soy | 15 |
| Fish meal or meat meal | 6 |
| Wheat bran or Middings | 20 |
| Seashell | 7 |
| Bone meal | 3.2 |
| Methionine | 0.125 |
| Lysine | 0.125 |
| Layer premix | 0.25 |
| Salt | 0.3 |
Table 1. Pellet composition for laying hen (Gallinasponedoras.org, s.f.).
Finishing Pigs
In finishing pigs, diet composition is a key factor, as it determines growth rate, meat quality, economic efficiency, and environmental impact in production.
The efficiency of lean tissue deposition is closely related to protein supplementation. This approach requires an optimal profile of essential amino acids. Synchronizing these monomers with protein sources boosts carcass yield (Cincaporc, 2024).
On the other hand, the carbohydrate fraction represents the main substrate to satisfy energy requirements (Cincaporc, 2024).
Finally, immunological homeostasis and qualitative carcass characteristics are regulated by the micromineral and vitamin supply (Cincaporc, 2024).
Table 2 details the percentage inclusion of ingredients for the diet formulated for pigs in the finishing phase.
| Ingredient | Amount (%) |
| Corn | 30 |
| Wheat bran | 9.5 |
| Meat and bone meal | 6 |
| Herbal meal | 5 |
| Peas | 5 |
| Soybean meal or Sunflower meal | 3 |
| Chalk | 1 |
| Salt | 0.5 |
Table 2. Pellet composition for finishing pigs (Garden Time, s.f.).
Ruminants
Unlike monogastric species such as poultry and swine, ruminants possess a specialized digestive system characterized by a complex, multi-compartment stomach. The largest compartment is the reticulo-rumen, which functions as a continuous anaerobic fermentation chamber. In this ecosystem, bacteria, protozoa, and fungi hydrolyze the structural polymers of the plant cell wall, transforming them into nutrients.
Ruminal dynamics strictly depend on the fiber supply. This structural fraction is essential for maintaining pH balance because it stimulates chewing and rumination, which trigger increased saliva production. This saliva acts as a natural buffer, neutralizing the acids generated by fermentation and maintaining a pH above 5.7 (Garza, 2017).
To complement the energy density of the diet and sustain production levels (meat or milk), rations incorporate carbohydrates from cereal grains, which are responsible for providing energy (Garcisan, n.d.).
Table 3 shows the percentage inclusion of ingredients in a balanced ration for ruminants.
| Ingredient | Amount (%) |
| Wheat straw | 20 |
| Cottonseed meal | 9 |
| Molasses | 16 |
| Urea | 1 |
| Sorghum | 35 |
| Alfalfa hay | 17 |
| Mineral premix | 2 |
Table 3. Pellet composition for ruminants (Lascano, 2018).
Aquaculture
The formulation of diets for aquatic organisms presents high heterogeneity due to the diverse feeding habits of the species (carnivorous, herbivorous, and omnivorous).
Consequently, aquaculture is the zootechnical sector with the greatest variability in the nutritional profiles of its balanced feeds. Optimizing the nutritional value of these diets is a critical factor for maximizing the feed conversion ratio, preserving the health status of the animals, and ensuring the environmental sustainability of the industry (Acuicultura de España, 2021).
In the field of aquaculture, nutritional requirements demand high crude protein levels as well as optimal fatty acid profiles, specifically of the ω-3 fatty acids, which are essential for physiological development (Acuicultura de España, 2021).
Regarding raw materials of plant origin, their inclusion must be strictly adjusted to the production stage and the enzymatic capacity of the animal. The digestibility of complex carbohydrates varies significantly among species; therefore, an inappropriate inclusion rate can induce gastrointestinal pathologies.
Furthermore, due to the restrictions of the aquatic environment, the physical properties of the pellet are decisive. Specific binders and technological additives are incorporated to modulate hydrostability and buoyancy through extrusion processes, thereby optimizing the feed intake and minimizing nutrient loss (Acuicultura de España, 2021).
Table 4 details the composition of the pellet intended for the gilthead seabream, a species classified as carnivorous.
| Ingredient | Amount (%) |
| Fish meal | 33 |
| Squid meal | 30.6 |
| Fish oil | 7.65 |
| Corn starch | 24.75 |
| Vitamin premix | 2 |
| Mineral premix | 2 |
Table 4. Composition of aquaculture pellets for carnivorous species (Universidad de Las Palmas de Gran Canaria, n.d.).
Pets
The formulation of balanced feeds for companion animals is intrinsically linked to their physiological homeostasis, metabolic performance, and longevity.
Diets intended for canines and felines require a predominant base of protein of high biological value (frequently incorporated as dehydrated meat meal or fresh fish muscle tissue) to provide the essential amino acids necessary for muscle development and the maintenance of lean mass. Likewise, omega-3 fatty acids are included because they play essential structural and metabolic roles, contributing to the maintenance of skin barrier integrity and hair follicle health (Santo Ángel, n.d.).
The carbohydrate fraction is restricted to moderate or low proportions. High-digestibility sources are prioritized, such as rice, potato, or peas, while the use of grains like corn and wheat is limited because of their association with adverse food reactions.
Table 5 shows the composition of a reference diet for dogs.
| Ingredient | Amount (%) |
| Salmon | 50 |
| Chicken fat | 15 |
| Salmon oil | 3 |
| Potato | 26 |
| Others | 6 |
Table 5. Composition of dog pellets (Zooplus, n.d.).
Pellet analysis
The heterogeneity in the chemical composition of pellets requires analytical strategies must be specifically adapted to optimize the extraction efficiency of the analytes of interest. Despite the existence of various analytical protocols applicable to the same matrix, the complexity and interference of the pellet’s composition present a common analytical challenge.
The primary methodological obstacle is the matrix effect. Certain macromolecules and compounds, such as phospholipids, inorganic salts, and proteins, are recognized as universal interferents. Consequently, pellets with high percentages of these components require extremely rigorous and selective sample preparation protocols for the effective removal of interferences (Panuwet et al., 2016; Valenzuela et al., 2006).
When comparatively evaluating the different matrices, aquaculture pellets present the highest analytical complexity due to their high fish oil content. This ingredient is critical due to its high concentration of free fatty acids and triglycerides. Furthermore, these formulations have a high protein fraction; the sum of both macronutrients represents 70% to 80% of the total mass of the pellet, thereby conferring an extremely complex nature on the matrix.
Secondly, pet food pellets show intermediate complexity due to a protein and lipid contents that ranges between 30% and 50% of the total composition, likewise requiring rigorous purification methodologies to mitigate the matrix effect.
In contrast, pellets intended for ruminants, pigs, and poultry (laying hens) are predominantly made up of plant-derived ingredients. However, their analytical behavior differs depending on the plant tissue used:
- Ruminants: A large part of the pellet is composed of grass, hay, and sorghum, which provide a high content of insoluble fiber (cellulose, hemicellulose, and lignin), thereby decreasing analyte extraction yields. Likewise, their filamentous macroscopic structure offers high mechanical resistance, hindering grinding processes and the subsequent diffusion of the solvent for analyte desorption.
- Pigs and laying hens: They present analogous compositional profiles, meaning their matrices exhibit homogeneous behavior in the laboratory. Generally, they contain lower levels of complex interfering compounds, allowing for conventional sample preparation treatments with low operational complexity.
Strategies to reduce matrix effects in pellets
Mitigating the matrix effect is a critical requirement to ensure accuracy and precision in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The choice of strategy intrinsically depends on the physicochemical nature of the interferent. The most established methodologies are described below (Guialab, n.d.):
- Sample clean-up procedures: These techniques aim to selectively remove of matrix components before injection into the system. Although highly effective at reducing interferences, they present limitations such as a high workload, increased analytical costs, and the risk of analyte loss during fractionation. The most widely used techniques include liquid-liquid extraction, solid-phase extraction, and the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method.
- Sample Dilution: This strategy consists of reducing the concentration of co-extracts through dilution, which proportionally decreases the impact of the matrix effect. However, this approach leads to a reduction in the analyte signal, thereby compromising the limit of quantification. Consequently, its feasibility is conditioned on the availability of high-sensitivity instrumentation that can maintain an adequate signal-to-noise ratio at high dilution factors.
- Matrix-Matched Calibration: This approach involves constructing a calibration curve by spiking aliquots of the sample with known concentrations of the analyte. This technique compensates for the matrix effect by ensuring that the calibration curve experiences the same interferences as the test sample. Although considered a highly reliable standard due to its precision, its implementation significantly increases analysis times and the consumption of analytical costs.
- Isotopically Labeled Standards: The incorporation of an isotopically labeled internal standard can be performed at two stages of the analytical procedure. Adding it immediately before injection into the LC-MS/MS system compensates for instrumental drift and fluctuations in injection volume. Conversely, adding it at the initial stage, before sample preparation, corrects for losses incurred during this step, thereby optimizing the recovery percentage of the extraction process. Regarding the mitigation of the matrix effect in the ionization source, both addition points are analytically valid to correct for signal suppression or enhancement, provided that the standard co-elutes with the native analyte.
Conclusions
The magnitude of the matrix effect is directly related to the chemical composition of the pellet. Matrices characterized by high fractions of phospholipids, inorganic salts, and proteins require the implementation of more rigorous and selective sample preparation protocols (such as thorough extraction and clean-up processes) to minimize the co-extraction of interferents and mitigate the suppression or enhancement of the analytical signal.
Consequently, prior characterization or prior knowledge of the pellet’s composition is an indispensable requirement before method development, as it unequivocally determines the design and selectivity of the sample preparation.
In scenarios where the residual matrix effect remains critically high after physicochemical processing, the complementary application of instrumental or methodological compensation strategies must be utilized to guarantee analytical accuracy.
