Liquid Chromatography Analysis of Common Nutritional Components, in Feed and Food

Abstract

Food and feed laboratories share several similarities when facing the implementation of liquid-chromatographic analysis. Using the experience acquired over the years, through application chemistry in food and feed research, selected analytes of relevance for both areas were discussed. This review focused on the common obstacles and peculiarities that each analyte offers (during the sample treatment or the chromatographic separation) throughout the implementation of said methods. A brief description of the techniques which we considered to be more pertinent, commonly used to assay such analytes is provided, including approaches using commonly available detectors (especially in starter labs) as well as mass detection. This manuscript consists of three sections: feed analysis (as the start of the food chain); food destined for human consumption determinations (the end of the food chain); and finally, assays shared by either matrices or laboratories. Analytes discussed consist of both those considered undesirable substances, contaminants, additives, and those related to nutritional quality. Our review is comprised of the examination of polyphenols, capsaicinoids, theobromine and caffeine, cholesterol, mycotoxins, antibiotics, amino acids, triphenylmethane dyes, nitrates/nitrites, ethanol soluble carbohydrates/sugars, organic acids, carotenoids, hydro and liposoluble vitamins. All analytes are currently assayed in our laboratories.

Keywords: additives; challenges; contaminants; food and feed analysis; liquid chromatography; nutritional analysis.

Figure 1

Polyphenols structure and classification [97]. Highly functionalized structures account for the molecules radical scavenging, metal ion chelating, and enzyme inhibition. Hydrogen bonding can stabilize phenoxyl radicals.

Figure 2

Chemical structures for (A) capsaicin (8-methyl-N-vanillylamide) and (B) dihydrocapsaicin (8-methyl-N-vanillylnonamide), the aromatic vanillyl radical is shown in red.

Figure 3

Chemical structures for (A) caffeine (1,3,7-trimethylxanthine), (B) theobromine (3,7-dimethylxanthine), (C) theophylline (1,3-dimethylxanthine), (D) paraxanthine (1,7-dimethylxanthine), and (E) antipyrine (2,3-Dimethyl-1-phenyl-3-pyrazoline-5-one or phenazone). (F) Caffeine biotransformation pathway is dependent on the CYP1A2 and CYP2A6 enzyme system. 1. 1,3,7-trimethylxanthine 2. 1,7-dimethylxanthine 3. 7-methylxanthine 4. 7-methyluric acid 5. 1-mthyluric acid 6. 5-acetylamino-6-formylamino-3-methyluracil 7. 1,7-dimethyluric acid 8. 5-acetylamino-6-amino-3-methyluracil [145].

Figure 4

Chemical structures for (A) ochratoxin A, (B) ochratoxin B, (C) ochratoxin C, blue colored circles represent changes in the structure between ochratoxins, loss of Cl and OH in ochratoxin B and C respectively render a more lipophilic molecule. Et = C₂H₅, and (D) are the general backbone of Fumonisins. FB₁ = 721.83 g mol⁻¹ R₁: H R₂: OH R₃: OH; FB₂ = 705.84 g mol⁻¹ R₁: OH R₂: H R₃: OH; FB₃ = 705.84 g mol⁻¹ R₁: H R₂: H R₃: OH; FB₄ = 689.84 g mol⁻¹ R₁: H R₂: H R₃: H. Functional groups colored in green and red represent a positively and negatively ionizable moiety, respectively.

Figure 5

Chemical structures for three triphenylmethane dyes which are sharing a common phenyl backbone sharing a methylidyne. Each molecule has extended π-delocalized electrons justifying their crystal coloration and visible light absorption (ca. 621 nm for malachite green).

Figure 6

Schematic representation for the interaction of nitrite ion with (A) a cation exchange stationary phase or (B) interaction with TBAHS present in the mobile phase and stationary phase C₁₈.

Figure 7

Chromatograph of (A) an aqueous 10 mg L⁻¹ nitrite (4.95 min) and nitrate (6.26 min) standard (B) hay sample after extraction with hot water, SPE cleanup, and micropore filtration presence of nitrite (4.91 min) and nitrate (6.23 min) is evident.

Figure 8

Chromatographs of (A) 2 g/100 mL standard mixture of four sugars including fructose (5.24 min), glucose (6.26 min), sucrose (9.12 min), and lactose (13.09 min) separated using amino column (Zorbax Carbohydrate, 0.7 mL min⁻¹, 80 ACN: 20 H₂O). (B) Sugar content of a molasses sample after hot water extraction, fructose (5.18 min) and glucose (6.31 min) signals are evident. (C) 1 g/100 mL standard solution for arabinose (3.89 min) (D) 1 g/100 mL standard solution for xylose (4.30 min) (E) 1 g/100 mL standard solution for ribose (4.76 min), and (F) 1 g/100 mL standard solution for mannose (5.42 min). Signal at ca. 1.80 min corresponds to the solvent front; constant in all injections.

Figure 9

Schematic representation of sugar interaction mechanism using (A) amine based (B) calcium ion-based ligand exchange column.

Figure 10

Chromatographs of (A) Mix of organic acid standards malic acid (9.24 min) methanoic acid (formic acid, 10.92 min), ethanoic acid (acetic acid, 11.65 min), propanoic acid (propionic acid, 12.62 min), lactic acid (14.92 min), 2-methylpropanoic acid (isobutyric acid, 17.22 min), butanoic acid (butyric acid, 18.52 min). (B) A silage sample after extraction with acid 0.01 mol L⁻¹ H₂SO₄. Fermentation products identified at 18.499 min, 14.903 min, 12.606 min. The signal at ca. 5.70 min corresponds to the solvent front.

Figure 11

Single quadrupole LC/MS ESI⁺ chromatographs of (A) Total ion chromatogram α-tocopherol (a 1 mg L⁻¹ solution in butanol) signal positively identified at 11.75 min (B) Mass spectra for α-tocopherol (a 1 mg L⁻¹ solution in butanol) using a cone energy of 120 V extracted from a signal with a retention time of 11.71 min (C) α-tocopherol (retention time 11.77 min) identified in a chicken plasma sample after extraction with chloroform and butanol (D) α-tocopherol in selected ion monitoring (SIM) mode using a cone energy of 120 V extracted from signal with a retention time of 11.82 min (E). α-tocopherol acetate in an injectable vitamin E solution for veterinary use using a “dilute and shoot” approach (16.32 min), and (F) α-tocopherol acetate in SIM mode using a cone energy of 60 V extracted from signal with a retention time of 16.34 min.

Figure 12

Hydrosoluble vitamin analysis based on ion pairing [383]. (A) Successful separation of 7 complex B vitamins including niacin (nicotinic acid, B₃, 6.67 min), FMN (B₂, 14.12 min), pyridoxal (B₆, 17.007 min), pyridoxamine (B₆, 18.607 min), pyridoxine (B6, 19.963 min), folic acid (B9, 20.630 min), and thiamine (B₁, 25.074 min). (B) Analysis of a vitamin premix destined for feed formulation. Another advantage presented is that the separation can be performed using a reverse phase C₁₈ column.

Figure 13

Chromatographs for vitamin A standards mixtures separated with C₈ column at 325 nm and 50 °C, of (A) retinyl acetate (3.11 min) and retinyl palmitate (17.63 min) using MeOH/H₂O (90:10) and (B) retinyl acetate (2.89 min) and retinyl palmitate (13.30 min) using MeOH/2-propanol/acetonitrile (95:1.5:3.5).

Figure 14

Separation for vitamin D₂+D₃ standards at 264 nm using (A) a C₈ column and (B) a C₁₈ column D₂ (16.47 min) y D₃ (17.24 min). Analysis performed at 30 °C using MeOH/2-propanol/ACN (90:3:7) (C) Superposed chromatograms for vitamin D₂ + D₃ (blue line) and δ/γ/α-tocopherol standards using a C₁₈ column and MeOH/H₂O (90:10), 30 °C.