Development and validation of two analytical methods for urea determination in compound feed, including pet food, and yeast using high-performance liquid chromatography coupled with fluorescence detection and tandem mass spectrometry

Figures & data

Figure 1. Overlay of LC-MS/MS chromatograms of urea (m/z 61→44) as solid line and¹³C¹⁵N₂-urea (m/z 64→46) as dotted line of (a) a water blank without internal standard, (b) a water blank with internal standard and (c) a standard solution with 0.4 mg l^–1 urea in water

Table 1. Coefficient of variation (CV) and recovery rate of a 6-fold analysis of fortified samples with different fortification levels of urea using LC-MS/MS on two different LC-MS/MS systems and HPLC-FLD on two different HPLC-FLD systems

Sample	Fortification urea [mg kg^−1]	Urea detected [mg kg^−1] LC-MS/MS-1	Coefficient of variation [%] LC-MS/MS-1	Recovery [%]LC-MS/MS-1	Urea detected [mg kg^−1] LC-MS/MS-2	Coefficient of variation [%] LC-MS/MS-2	recovery [%] LC-MS/MS-2	urea detected [mg kg^−1] HPLC-FLD-1	Coefficient of variation [%]HPLC-FLD-1	recovery [%]HPLC-FLD-1	urea detected [mg kg^−1]HPLC-FLD-2	Coefficient of variation [%]HPLC-FLD-2	recovery [%]HPLC-FLD-2
Complete feed for laying hens	100	98	6.9	98	/	/	/	99	4.7	99	/	/	/
	50 000	48 968	3.7	98	47 537	4.4	95	47 146	2.7	94	47 350	2.7	95
Dry complete pet food for dogs	20	21	7.2	105	/	/	/	17	3.2	86	/	/	/
	10 000	10 036	4.1	100	9 855	3.8	99	9 660	1.9	97	9 753	1.4	98
Wet complete pet food for cats	100	96	2.6	96	101	3.6	101	98	1.4	98	99	1.4	99
	5 000	4 928	3.7	99	/	/	/	4 803	3.4	96	/	/	/
Fresh yeast	20	18	3.0	91	18	5.0	91	18	2.0	89	17	2.8	86
	20 000	18 561	2.3	93	/	/	/	18 577	1.6	93	/	/	/

/- not analysed on this instrument.

Figure 2. HPLC-FLD chromatograms of (a) a water blank with xanthydrol-derivatisation, (b) a cat feed sample without xanthydrol-derivatisation and (c) a standard solution with 0.4 mg l^–1 urea with xanthydrol-derivatisation

Table 2. Results of a 6-fold analysis by LC-MS/MS and HPLC-FLD of ruminant feed from proficiency tests (PT) by the Association of German Agricultural Analytic and Research Institutes

Sample	Assigned concentration [%]spectro-colorimetric	Assigned concentration range [%] spectro-colorimetric	Urea detected [%]LC-MS/MS	Coefficient of variation [%] LC-MS/MS	Urea detected [%] HPLC-FLD	Coefficient of variation [%]HPLC-FLD
Complementary feed for dairy cows (PT 449)	0.83	0.71–0.95	0.84	5.1	0.79	3.4
Complementary feed for dairy cows (PT 458)	0.98	0.79–1.19	0.96	4.6	0.89	1.6
Complementary feed for fattening cattle (PT 433)	1.66	1.40–1.94	1.64	3.7	1.51	1.1

Table 3. Coefficient of variation (CV) and recovery rate of a 6-fold analysis of fortified samples with different fortification levels of urea using an enzyme kit

Sample	Fortification urea [mg kg^−1]	Urea detected [mg kg^−1]	Coefficient of variation [%]	Recovery [%]
Complete feed for laying hens	100	100	5.6	100
	50 000	49 150	4.1	98
Dry complete pet food for dogs	10 000	10 270	1.8	103
Wet complete pet food for cats	5 000	4 825	6.1	97
Fresh yeast	20	12	7.3	62
	20 000	18 535	1.6	93

Table 4. Results of a one-fold analysis of different matrices fortified with five different urea concentrations using an enzyme kit

		Fortification level [mg kg^−1]
		100	200	300	400	500
% recovery	Complete feed for laying hens	103	97	98	96	95
	Dry complete pet food for dogs	472	473	453	383	326
	Wet complete pet food for cats	30	15	10	0	0
	Fresh yeast	100	93	99	79	73

Figure 3. Selected chromatograms for different matrices. Upper row: HPLC-chromatograms; Lower row: LC-MS/MS chromatogram overlays of urea (m/z 61→44) as solid line and¹³C¹⁵N₂-urea (m/z 64→46) as dotted line. (A1/A2) 2 g dry complete feed for dogs (approx. 128 mg kg^–1 urea) (B1/B2) 10 g wet complete feed for cats (approx. 173 mg kg^–1 urea); (C1/C2) 2 g complete feed for turkeys (approx. 10 mg kg^–1 urea); Approx. urea contents are given relative to a feed with a moisture content of 12 %

Figure 4. Comparison of LC-MS/MS and HPLC-FLD single measurement results for urea content of different feed and yeast matrices in mg kg^–1 relative to a feed with a moisture content of 12 %; Horizontally striped bar: First LC-MS/MS analysis; black dotted bar: second LC-MS/MS analysis; white bar: first HPLC-FLD analysis; diagonally striped bar: second HPLC-FLD analysis;  LOD_MS: 3 mg kg^–1; LOQ_MS: 8 mg kg^–1; LOD_FLD: 2 mg kg^–1; LOQ_FLD: 7 mg kg^–1