Determination of urea in pet feed: assessing the suitability of different analytical techniques using proficiency test data

Figures & data

Table 1. HPLC gradient elution program.

Time (min)	Mobile phase A (%)	Mobile phase B (%)
0.2	5	95
5.0	40	60
6.0	40	60
6.1	5	95
10	5	95

Mobile phase A: Milli Q water + formic acid 0.1% (v/v), mobile phase B: acetonitrile + formic acid 0.1 % (v/v).

Table 2. Mass spectrometry acquisition settings.

Compounds	Time (min)	Precursor ion [M + H]^+ (m/z)	Product ion (m/z)	DP (V)	EP (V)	CE (V)	CXP (V)
Urea	3.0	60.96	44.1	60	9	21	20
			61.0	60	9	5	10
^13C urea		61.95	45.0	61	9	21	20
^13C^15N2 urea		63.92	46.0	56	10	21	20

DP: declustering potential; EP: entrance potential; CE: collision energy; CXP; exit potential, acquisition time in scheduled MRM – 120 s. MRM transitions in bold are used for quantification.

Figure 1. Extracted ion chromatograms of (a) urea (m/z 61 > 44) in test material C containing 258.9 mg kg⁻¹ of urea and (b) internal standard ¹³C¹⁵N₂ urea (m/z 64 > 46) at a mass fraction of 526 mg kg⁻¹.

Table 3. Reference values of urea and moisture contents of the test materials accompanied by the respective uncertainty values (k = 2).

Test materials	[Urea] ± U (mg kg^−1)	Moisture ± U (%, m/m)
Material J	18.9 ± 0.98	9.31 ± 1.32
Material C	258.9 ± 5.9	8.58 ± 1.22
Material G	977.4 ± 21.7	8.01 ± 1.14

Material J was not spiked and contained only background levels of urea, Material C was additionally spiked with 240.0 mg kg⁻¹ of urea and Material G was spiked with 958.5 mg kg⁻¹ of urea.

Table 4. Results (mg kg⁻¹) of urea in the test materials J, C and G, as submitted by the interlaboratory study participants.

		Test materials
Laboratory	Method applied	Code J	Code C	Code G
L002	Spectrophotometry	2554	<2500	2714
L003	Spectrophotometry	2288	1472	2280
L004	Spectrophotometry	2000	1900	2200
L005	Spectrophotometry	5994	4993	4940
L007	Spectrophotometry	1456	1184	1911
L008	Spectrophotometry	4680	3352	4145
L009	Spectrophotometry	6353	2402	3740
L010	Spectrophotometry	3677	2270	3010
L014	Spectrophotometry	2700	1900	3300
L015	Spectrophotometry	4195.18	4508.22	6516.3
L017	Spectrophotometry	2815	<2500	3165
L024	Spectrophotometry	3810	3570	4030
L025	Spectrophotometry	2400	1000	2200
L030	Spectrophotometry	1350	1012.5	1762.5
L011	LC-MS/MS	17.8	325	990
L012	LC-MS/MS	<50	257	946
L013	LC-MS/MS	<100	310	1300
L016	LC-MS/MS	43	58	375
L019	LC-MS/MS	21.2	256	1011
L020	LC-MS/MS	<50	269	920
L028	LC-MS/MS	<10	1320	2850
L018	Enzymatic	<1500	<1500	<1500
L021	Enzymatic	130	227	1061
L022	Enzymatic	<1700	<1700	<1700
L023	Enzymatic	170	2400	8600
L029	Enzymatic	704	856	980
L031	Enzymatic	<500	673	1345
L006	HPLC-UV	3946.38	4564.81	5274.39
L026	HPLC-UV	341.81	478.43	1012.6
L027	in-house photometric	71.2	267.5	891.2
L001	Gravimetry	<1500	<1500	<1500

Figure 2. Distribution of the results submitted by the PT participants for the test materials J, C and G organized by analytical technique. Note: The horizontal line represents the reference mass fraction of urea in each test material. The uncertainties of the reference values are indicated in Table 3.

Table 5. Statistical parameters of the interlaboratory test: robust mean (ISO 5725-5) and reproducibility (RSD_R).

Test item	Analytical technique	N	Robust mean (mg kg^−1)	RSDR (%)
Code J	LC-MS/MS	3	–	–
	In-house photometric	1	–	–
	Enzymatic	3	–	–
	HPLC-UV	2	–	–
	Spectrophotometry	14	3221	49.2
Code C	LC-MS/MS	7	283	47.8
	In-house Photometric	1	–	–
	Enzymatic	4	–	–
	HPLC-UV	2	–	–
	Spectrophotometry	12	2434	60.3
Code G	LC-MS/MS	7	1041	45.3
	In-house Photometric	1	–	–
	Enzymatic	4	–	–
	HPLC-UV	2	–	–
	Spectrophotometry	14	3171	38.4

(–) insufficient number of results for statistical calculation.

Table 6. Performance parameters of the methods used by the participants.

Lab Code	Method	LOQ (mg kg^−1)	Intermediate precision (%)	Recoveries (%)	Working range (mg kg^−1)
L002	Spectrophotometry	2500	6.45	98	2500–25,000
L003	Spectrophotometry	1200	10	95	1200–17,800
L004	Spectrophotometry
L005	Spectrophotometry	3000	10		3000–1000
L007	Spectrophotometry	100	5		100–500,000
L008	Spectrophotometry
L009	Spectrophotometry	200	9	97	10–100
L010	Spectrophotometry	2500	5–10		2500–25,000
L014	Spectrophotometry	100	4.85	99	>100
L015	Spectrophotometry
L017	Spectrophotometry	2500			>2500
L024	Spectrophotometry	2500		112	2500–25,000
L025	Spectrophotometry	2000	6	102.1	2000–400,000
L030	Spectrophotometry	50	10	90–110	50–1,000,000
L011	LC-MS/MS
L012	LC-MS/MS	50	5–10	90–110	50–1100
L013	LC-MS/MS
L016	LC-MS/MS	1	42		1–500 (2000)
L019	LC-MS/MS	10	13	90	10–100
L020	LC-MS/MS	50			53–2310
L028	LC-MS/MS	10		100	5–220
L018	Enzymatic	1500	10	103	1500–14,000
L021	Enzymatic	0.15		95	140
L022	Enzymatic	1700	6.72	102	1000–7000
L023	Enzymatic
L029	Enzymatic	500		>95	500–1,000,000
L031	Enzymatic	500	5–12		>500
L006	HPLC-UV	1000		90	1000–50,000
L026	HPLC-UV	39.25	4.73		60–2575
L027	In-house photometric	50		>95	50–1,000,000
L001	Gravimetry	1500			1500–15,000