Gluten contamination of canned and dry grain-free commercial pet foods determined by HPLC-HRMS

Figures & data

Table 1. Pet food samples and their composition.

Code	Formulation	Main ingredient	Type	Animal	Dry matter	Crude protein	Lipid	Ash	Crude fibre
1	Control with gluten	Cereals	Dry food	Dog	92.0	22.0	10.0	8.5	3.5
2	Control with gluten	Cereals	Dry food	Dog	93.1	22.0	10.0	8.5	3.5
3	Grain-free	Fresh turkey meat	Dry food	Dog	93.2	28.0	18.0	8.4	2.4
4	Grain-free	Fresh fish	Dry food	Dog	93.8	30.0	19.0	8.0	2.6
5	Grain-free	Fresh mackerel	Dry food	Dog	92.6	30.0	18.0	8.5	2.4
6	Grain-free	Fresh tuna	Dry food	Dog	93.8	30.0	19.0	8.3	2.4
7	Grain-free	Fresh rabbit meat	Dry food	Cat	94.1	31.0	17.4	7.4	3.6
8	Grain-free	Fresh chicken meat	Dry food	Cat	95.1	30.0	17.8	7.3	3.5
9	Grain-free	Fresh salmon	Dry food	Cat	94.8	30.8	17.8	7.8	3.3
10	Grain-free	Beef	Canned	Dog	19.2	10.0	4.5	1.8	0.2
11	Grain-free	Lamb meat	Canned	Dog	17.5	9.0	4.2	2.6	0.3
12	Grain-free	Salmon	Canned	Dog	17.5	8.0	5.8	2.7	1.0
13	Grain-free	Pork	Canned	Dog	19.0	8.6	4.5	2.2	0.3
14	Grain-free	Chicken meat	Canned	Cat	20.3	8.1	5.1	2.4	0.3
15	Grain-free	Salmon	Canned	Cat	20.2	7.5	5.3	2.5	0.4
16	Grain-free	White fish	Canned	Cat	19.4	8.0	3.8	2.5	0.3
17	Grain-free	Beef	Canned	Cat	18.8	8.9	4.8	1.9	0.4

Table 2. Peptides selected for quantitative determination of wheat gluten in pet food samples (the letters denote the following amino acids: A: alanine; I: isoleucine; Y: tyrosine; S: serine; V: valine; L: leucine; Q: glutamine; E: glutamic acid; R: arginine; C: cysteine; G: glycine; M: methionine; K: lysine; H: histidine; T: treonin; F: phenylalanine; P: proline).

Peptide	m/z
AIIYSIVLQEQQQVR (protein code in UNIPROT: Q18NR2_WHEAT, LMW glutenin subunit,  Triticum aestivum),	894.5043 (z = 2^+) (for quantification)
CGALYSMLDSMYKEHGAQEGQAGTGAFPR (protein code in in UNIPROT: IAA1_WHEAT, Alpha-amylase inhibitor 0.19 Triticum aestivum)	1045.1384 (z = 2^+) (for confirmation)

Figure 1. Calibration curve using grams of wheat flour type ‘00’ as the reference standard.

Table 3. m/z and type of fragment ions derived from the MS/MS of the quantifier ion m/z = 894.5043, z = 2. The MS/MS spectrum is shown in Figure 1.

m/z fragment ion (relative intensity)	Type of fragment
1028.5819 (100)	Y8
1127.6538 (99)	Y9
915.4941 (67)	Y7
1327.7778 (49)	Y11
787.4313 (41)	Y6
658.3843 (33)	Y5
1490.8429 (24)	Y12

Table 4. Analysis of pet food samples expressed as mg (±standard deviation) of wheat flour type ‘00’ present in 1 g of food, first column, or as mg of protein per g of food (ND, not detectable, <LOQ).

Code	Feed	mg wheat flour/g feed	mg protein/g feed
1	Dry food	50.3 ± 3.52	4.5
2	Dry food	53.4 ± 3.74	4.8
3	Dry food	ND	ND
4	Dry food	ND	ND
5	Dry food	ND	ND
6	Dry food	ND	ND
7	Dry food	ND	ND
8	Dry food	10.0 ± 1.40	0.9
9	Dry food	ND	ND
10	Canned	ND	ND
11	Canned	ND	ND
12	Canned	ND	ND
13	Canned	ND	ND
14	Canned	ND	ND
15	Canned	ND	ND
16	Canned	ND	ND
17	Canned	ND	ND

Figure 2. Chromatogram and MS/MS spectrum of ion m/z 894.5043, z = 2, in standard of wheat flour type 00, weight 0.3 grams.

Figure 3. Chromatogram and MS/MS spectrum of ion m/z 894.5043, z = 2, in sample feed code 1 (feed based on chicken for dog).

Figure 4. Chromatogram and MS/MS spectrum of ion m/z 894.5043, z = 2, in sample feed code 8 (feed based on chicken with potatoes for cat).