DART mass spectrometry: a rapid tool for the identification of feed additives containing coccidiostats as active substances

Figures & data

Table 1. Feed additives used in this study and information, the name and composition of these products containing the active substance as target and the unique identification number as specified in the corresponding legal acts available via the EU Register of Feed Additives (European Union 2021a), qs: quantum satis. In addition, the chemical formula, the accurate masses of the active substances measured with the DART-HRMS system on the feed additives, the ion assignments and the mass errors are shown. The legal act contains a reference to a feed additive specific EFSA opinion, which gives a full characterisation of the active substance

Feed additive, identification number	Main ingredients	Active substance(s)	Accurate mass	Ion assignment	Mass error (ppm)
Sacox 120, 5 1 766	Silicon dioxide 10–100 g kg^−1, calcium carbonate 500–700 g kg^−1,	Salinomycin sodium C42H69NaO11 114–132 g kg^−1	773.47806	[M+H]^+	−3.8
Sacox 200, 5 1 766	Silicon dioxide 50–150 g kg^−1, calcium carbonate 50–150 g kg^−1,	Salinomycin sodium C42H69NaO11 190–220 g kg^−1	773.47792	[M+H]^+	−4.0
Coxidin (CaCO3), 5 1 701	Perlite 150 to 200 g kg^−1, calcium carbonate qs.100 %	Monensin sodium, C36H61NaO11 equivalent to monensin activity: 25 %	693.41431	[M+H]^+	−5.9
Coxidin_(Wheat), 5 1 701	Perlite 150–200 g kg^−1, wheat bran 550–600 g kg^−1	Monensin sodium, C36H61NaO11 equivalent to monensin activity: 25 %	693.41421-	[M+H]^+-	−6.1 –
Stenerol, E 764	Povidone 10.0 g kg^−1, macrogolglycerol ricinoleate 20.0 g kg^−1, corn cobs 964 g kg^−1	Halofuginone 6 g kg^−1 C16H17BrClN3O3	414.02081	[M+H]^+	−1.6
Coxiril, 5 1 771	Starch 15 g kg^−1, wheat meal 700 g kg^−1. calcium carbonate 280 g kg^−1	Diclazuril 5 g kg^−1 C17H9Cl3N4O2	404.97295	[M-H]^−	2.7
Clinacox 0.5 %, 5 1 771	Protein-poor soybean meal 992,5 g kg^−1, polyvidone K30 2 g kg^−1, sodium hydroxide: 0,5 g kg^−1	Diclazuril 5 g kg^−1 C17H9Cl3N4O2	404.97307	[M-H]^−	3.1
Aviax 5 %, E 733	Sodium carbonate 40 g/kg, mineral oil 30–50 g/kg, sodium aluminosilicate 20 g/kg, soybean mill run 838,7–858,7 g/kg	Semduramicin sodium: 51,3 g kg^−1 C45H75O16Na	895.49804	[M+H]^+	−5.0
Elancoban, E 757	Mineral oil 10 g kg^−1 − 30 g kg^−1, limestone granular 130–230 g kg^−1, rice hulls or limestone granular qs 100 %	Monensin sodium, C36H61NaO11, dried fermentation product equivalent to Monensin activity 10 % w/w or 20 % w/w	693.41346	[M+H]^+	−7.2
Monteban, E 757	Soybean oil or mineral oil 10–30 g kg^−1, vermiculite 0–20 g kg^−1, soybean mill run or rice hulls qs 100 %	Narasin 100 g activity kg^−1, C43H72O11	787.49733	[M+ Na]^+	0.8
Maxiban G160, 5 1 772	Vegetal or mineral oil 10–30 g kg^−1 vermiculite: 0–20 g kg^−1, micro tracer red 11 g kg^−1, corn cob grits or rice hulls qs 1 kg	Narasin 80 g activity kg^−1, C43H72O11 Nicarbazin 80 g kg^−1 1,3-bis (4-nitrophenyl)urea 4,6-dimethylpyrimidin-2-(DNC), C13H10N4O5	787.49689 301.05686	[M+ Na]^+ [M-H]^−	0.3 –3.3
Cycostat 66 G, 5 1 758	Lignosulfonate 40 g kg^−1, calcium sulphate dehydrate 894 g kg^−1	Robenidine hydrochloride 66 g kg^−1 C15H13Cl2N5 (robenidine)	334.06176	[M+H]^+	−0.9
Avatec 15 0 G, 5 1 76 3	Calcium sulphate dehydrate 809 g kg^−1, calcium lignosulphonate 40 g kg^−1, ferric oxide 1 g kg^−1	Lasalocid A sodium 150 g kg^−1, C34H53NaO8	613.36685	[M+H]^+	−6.9
Cygro10 G, 5 1 770	Maduramicin ammonium alpha: 10 g kg^−1, carboxymethylcellulose sodium 20 g kg^−1, calcium sulphate dehydrate 970 g kg^−1	Maduramicin ammonium alpha, C47H80O17 (maduramicin) 10 g kg^−1	939.52736	[M-NH3+ Na]^+	−1.5
Deccox, E 756	Refined deodorised soya oil 28,5 g kg^−1, Wheat middling q.s. 1 kg	Decoquinate 60.6 g kg^−1 C24H35NO5	418.25770	[M+H]^+	−2.6

Figure 1. Isotope pattern of diclazuril based on nominal masses: Comparison of the average of the measured relative intensities (n = 180, plotted on logarithmic scale) with the corresponding calculated values. The standard deviation of the relative intensities are shown as error bars.

Figure 2. Mass spectra of polar solvent extracts from Coxiril measured in: (a) positive DART-MS mode and (b) negative DART-MS mode.

Figure 3. Mass spectra of extracts from Clinacox 0.5% measured in negative DART-MS mode: (a) extraction with the polar solvent and (b) extraction with n-hexane.

Figure 4. Between batch variation of each product expressed in terms of the scores of the first and the second principal component. Three batches with two sachets, where the letters A to C stand for the batch and the number 1 and 2 stand for the sachets: (a) Coxiril, polar solvent extracts measured in negative DART-MS mode and (b) Clinacox 0.5%, n-hexane extracts measured in positive DART-MS mode.

Figure 5. Variation between products: PCA score plots from the measurements of the polar solvent extracts. (a) Measurements in negative MS mode and (b) measurements in positive MS mode. Coxiril ‘1ʹ and Clinacox 0.5% ‘1ʹ are the samples to create the PCA model, while the Coxiril ‘2ʹ and Clinacox 0.5% ‘2ʹ samples are used for prediction purposes.

Figure 6. Classification of Coxiril and Clinacox 0.5 % samples: Presentation of the PLS-DA model obtained on the polar solvent extracts measured in negative MS mode: (a) response values of the model and prediction samples and the threshold. (b) Loading plot form the first latent variable.

[EC] European Commission. 2021a. European Union Register of Feed Additives [Internet]. [accessed 2021 Jan 11]. http://ec.europa.eu/food/safety/docs/animal-feed-eu-reg-comm_register_feed_additives_1831-03.pdf.

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