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

Figures & data

Table 1. Composition of the nine test materials (MATs) of the study, classified in terms of the target species, i.e. fish versus poultry and type of additive, i.e. by synthesis versus natural: The target mass fractions of the carotenoids in the different test materials are shown in Tables 2,Tables 3 and Tables 4. AXN DMDS: Astaxanthin dimethyldisuccinate; BACARE: Ethyl ester of beta-apo-8ʹ-carotenoic acid. Materials used in the study: Complete feedingstuffs: For fish: MAT 1–3; For poultry: Grower feed: MAT 5, Laying hens: MAT 6 and 7. Premixtures: For fish: MAT 8, For poultry: MAT 9

Target animal of compound feed	Fish		Poultry
Source of feed additive	By synthesis	Natural	By synthesis	Natural
Astaxanthin	MAT 3,8	MAT 1,2
AXN DMDS	MAT 1,2,3,8
Adonirubin		MAT 1,2
Capsanthin				MAT 5,6,7,9
Citranaxanthin			MAT 5,6,7,9
BACARE			MAT 5,6,7,9
Lutein				MAT 5,6,7,9
Zeaxanthin				MAT 5,6,7,9
Canthaxanthin	MAT 3,8	MAT 1,2	MAT 5,6,7,9
beta-carotene	MAT 2,8		MAT 5,6,7,9

Table 2. Results from the collaborative trial: Performance of the method for the determination of carotenoids exclusively used in fish feedingstuffs. AXN DMDS: Astaxanthin dimethyldisuccinate; Calculated value: Mass fraction of the carotenoids in the test material based on the certificate of analysis of the specific feed additive used; Average: Average obtained from the statistical assessment of the results from the laboratories. RSD_r: relative standard deviation of repeatability; RSD_R: relative standard deviation of reproducibility. No. of laboratories indicates the number of laboratories retained after removal of outliers identified by Cochran and Grubbs tests. The number in brackets is the number of laboratories identified as outliers

		MAT 1	MAT 2	MAT 3	MAT 8
Astaxanthin
	No. of laboratories	16(1)	16(0)	16(0)	14(0)
	Calculated value (mg kg^−1)	52.8	26.4	100	4485
	Average (mg kg^−1)	42.8	21.0	92.3	3861
	RSDr (%)	4.5	6.6	6.9	16.2
	RSDR (%)	24.3	30.3	20.8	22.7
AXN DMDS
	No. of laboratories	8(2)	9(1)	9(1)	8(1)
	Calculated value (mg kg^−1)	140	99.9	69.7	2012
	Average (mg kg^−1)	180	121	87.4	2671
	RSDr (%)	2.2	2.6	5.8	2.5
	RSDR (%)	11.3	14.4	16.2	6.8
Adonirubin
	No. of laboratories	15(2)	17(2)
	Calculated value (mg kg^−1)	20.4	10.2
	Average (mg kg^−1)	14.8	7.1
	RSDr (%)	5.4	8.3
	RSDR (%)	20.6	24.7

Table 3. Results from the collaborative trial: Performance of the method for the determination of carotenoids exclusively used in poultry feedingstuffs. BACARE: Ethyl ester of beta-apo-8ʹ-carotenoic acid; Calculated value: Mass fraction of the carotenoids in the test material based on certificate of analysis of the specific feed additive used. Average: Average obtained from the statistical assessment of the results from the laboratories. RSD_r: relative standard deviation of repeatability; RSD_R: relative standard deviation of reproducibility. No. of laboratories indicates the number of laboratories retained after removal of outliers identified by Cochran and Grubbs tests. The number in brackets indicates the number of laboratories identified as outliers

		MAT 5	MAT 6	MAT 7	MAT 9
Capsanthin
	No of laboratories	12(0)	13(0)	16(0)	11(1)
	Calculated value (mg kg^−1)	49.3	78.9	24.7	1479
	Average (mg kg^−1)	34.1	54.9	17.5	1148
	RSDr (%)	3.9	3.4	13.7	3.7
	RSDR (%)	27.6	24.2	35.7	26.3
Citranaxanthin
	No of laboratories	9(1)	16(0)	16(1)	12(1)
	Calculated value (mg kg^−1)	25.0	50.0	80.3	2497
	Average (mg kg^−1)	28.9	49.1	80.1	2306
	RSDr (%)	8.0	4.8	6.6	8.9
	RSDR (%)	18.0	26.1	25.3	19.4
BACARE
	No of laboratories	11(2)	13(0)	12(0)	14(0)
	Calculated value (mg kg^−1)	80.2	24.8	50.2	3009
	Average (mg kg^−1)	75.1	25.2	55.6	2853
	RSDr (%)	3.8	6.4	5.9	4.5
	RSDR (%)	14.7	26.3	13.8	10.5
Lutein
	No of laboratories	14(1)	12(2)	16(0)	14(0)
	Calculated value (mg kg^−1)	26.8	52.9	80.9	853
	Average (mg kg^−1)	21.3	41.2	63.9	784
	RSDr (%)	5.3	2.3	4.8	3.6
	RSDR (%)	18.4	12.9	17.9	18.3
Zeaxanthin
	No of laboratories	13(0)	15(0)	16(0)	15(0)
	Calculated value (mg kg^−1)	19.1	34.5	35.6	593
	Average (mg kg^−1)	14.1	24.4	27.2	475
	RSDr (%)	4.8	6.2	5.7	2.9
	RSDR (%)	13.8	16.0	15.4	16.2

Table 4. Results from the collaborative trial: Performance of the method for the determination of carotenoids used in fish feedingstuffs (MAT 1-MAT 3, MAT 8) and poultry feedingstuffs (MAT 5-MAT 7, MAT 9). Calculated value: Mass fraction of the carotenoids in the test material based on certificate of analysis of the specific feed additive used, *: Average from the homogeneity study due to a strong discrepancy with the calculated value probably caused by a problem with the preparation of the test material. Average: Average obtained from the statistical assessment of the results from the laboratories. RSD_r: relative standard deviation of repeatability; RSD_R: relative standard deviation of reproducibility. No. of laboratories indicates the number of laboratories retained after removal of outliers identified by Cochran and Grubbs tests. The number in brackets is the number of laboratories identified as outliers

		MAT 1	MAT 2	MAT 3	MAT 8	MAT 5	MAT 6	MAT 7	MAT 9
Canthaxanthin
	No of laboratories	8(2)	9(0)	14(1)	10(0)	15(1)	10(1)	10(1)	12(2)
	Calculated value (mg kg^−1)	5.3	2.6	99.7	1031	24.8	19.4*	10.8*	1031
	Average (mg kg^−1)	4.35	2.04	84.2	827	27.1	19.7	9.4	1103
	RSDr (%)	5.3	10.3	4.0	8.2	4.9	2.8	3.7	5.6
	RSDR (%)	11.2	15	17.6	15.9	24.8	39	32	19.3
Beta-carotene
	No of laboratories		9(0)		8(1)		7(0)	7(1)	11(0)
	Calculated value (mg kg^−1)		30.1		975		37.1	35.2	1290
	Average (mg kg^−1)		19.6		877		29.3	28.7	1122
	RSDr (%)		8.1		4.5		9.5	13.3	6.8
	RSDR (%)		29.9		17.8		38.0	13.3	25.8

Figure 1. Two chromatograms of a solution containing exclusively the astaxanthin dimethyldisuccinate standard substance, namely the chromatograms prior to the isomerisation experiment (blue line) and after this experiment (red line). While the chromatogram of the standard prior to the experiment exclusively shows a single peak labelled “1” corresponding to the all-trans isomer, the chromatogram shows after the experiment two additional peaks labelled “2”, which are two main cis isomers

Figure 2. Chromatographic profile for some isomerised carotenoids obtained by analysis of Mat 6. The peaks of the same colour are different cis-trans isomers of the same anlayte labelled with the following numbers: (1) canthaxanthin, (2) capsanthin, (3) citranaxanthin, (4) ethyl ester of beta-apo-8ʹ-carotenoic acid, (5) lutein (6) zeaxanthin and (7) beta-carotene

Figure 3. Comparison of the results from two laboratories having applied PLE with the results of all laboratories shown by the mass fraction ratio. This parameter is obtained by dividing separately for each analyte/matrix combination the average mass fraction reported by two laboratories having applied PLE by the corresponding average value of the mass fractions of all laboratories. AXN: astaxanthin. AXN DMDS: astaxanthin dimethyldisuccinate. ADR: adonirubin. CSN: capsanthin. CIXN: citranaxanthin. BACARE: ethyl ester of beta-apo-8ʹ-carotenoic acid. LUT: lutein. ZEA: zeaxanthin. CXN: canthaxanthin. BCAR: beta-carotene

Figure 4. Precision expressed in terms of relative standard deviation for reproducibility RSD (%) of the method obtained from the 43 statistical assessments versus the mass fraction of the 10 carotenoids. AXN: astaxanthin. DMDS: astaxanthin dimethyldisuccinate. ADR: adonirubin. CSN: capsanthin. CIXN: citranaxanthin. BACARE: ethyl ester of beta-apo-8ʹ-carotenoic acid. LUT: lutein. ZEA: zeaxanthin. CXN: canthaxanthin. BCAR: beta-carotene