Analysis of Urea in Petfood Matrices: Comparison of Spectro-Colorimetric, Enzymatic and Liquid Chromatography Electrospray Ionization High Resolution Mass Spectrometry Methods

doi:10.4236/ajac.2012.39080

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American Journal of Analytical Chemistry, 2012, 3, 613-621

doi:10.4236/ajac.2012.39080 Published Online September 2012 (http://www.SciRP.org/journal/ajac)

Analysis of Urea in Petfood Matrices: Comparison of

Spectro-Colorimetric, Enzymatic and Liquid

Chromatography Electrospray Ionization High Resolution

Mass Spectrometry Methods

Patrick Pibarot

, Serge Pilard

Nestlé Research and Development Centers, Aubigny, France

Analytical Platform, University of Picardie Jules Verne, Amiens, France

Email: [email protected]

Received July 4, 2012; revised August 12, 2012; accepted August 19, 2012

ABSTRACT

Adulteration may consist in non authorized source of nitrogen addition to increase the protein content of some raw ma-

terials. Urea which is authorized for feed is a non nutritional source of nitrogen in food and pet food. Adulteration of

food or pet food raw material by urea is thus monitored by manufacturer and governmental authorities with official

methods which are either enzymatic (Association of Official Agricultural Chemists, AOAC) or spectro-colorimetric

(European Community, EC). Each method gives results which are not comparable and spectro-colorimetric methods

may result in false-positive urea detection. Liquid chromatographic (LC/UV-DAD) analysis of extracts from spectro-

colorimetric method indicates that presence of free amino-acid may interfere with colorimetric detection of urea in the

EC method with pet food samples. Liquid chromatography electrospray ionization high resolution mass spectrometry

(LC/ESI-HRMS) has allowed to quantify low content (<0.01%) of urea in pet food water extracts for samples which

resulted in significant urea detection with colorimetric method and in content below the detection threshold with enzy-

matic method. This study demonstrates the EC colorimetric method is not applicable to pet food and also food samples

which have a complex composition with significant levels of free amino acids. On the other hand we clearly evidenced

by means of the LC/ESI-HRMS results that the AOC Enzymatic method is applicable to urea quantification in pet food

samples and gives reliable results.

Keywords: Pet Food; Food; Urea; Adulteration; Enzymatic Method; Spectro-Colorimetry; LC/UV-DAD;

LC/ESI-HRMS

1. Introduction

Non authorized food ingredient can be used to adulterate

ingredients, raw materials (e.g. Gluten) or finished prod-

uct (e.g. milk products) to artificially increase the protein

content. Melamine, for instance, has been used as the food

additive to increase the apparent protein content in food

products [1].

Ruminant can convert non protein nitrogen into protein

and urea may be used to increase the nutritional nitrogen

content of feed, hay or silage. Urea is authorized as a com-

ponent of animal feeds to provide nutritional source of

nitrogen. Urea is accepted as a “Generally Recognized as

Safe” food additive and is also used as a fermentation aid

in foods and beverages [2].

Urea is produced in the liver of mammals as an end-

product of protein metabolism and excreted in urine. Hu-

mans excrete up to 30 grams of urea per day in the urine [2].

Avian species (e.g. poultry) metabolize mainly uric acid

(55% - 72%) and some urea in lower proportion (2% -

11%), the rest of protein metabolism product is ammonia

(11% - 21%) [3,4].

Therefore, poultry meat and by products should not

present significant amount of urea while mammal meat

by-product may contain some urea in more significant

amounts.

Kjeldahl or Dumas methods measure the protein con-

tent by analyzing total nitrogen content, without identi-

fying its sources. The research of non-protein nitrogen

source is complementary to protein analysis to check the

absence of adulteration in raw materials and finished pro-

ducts.

Urea is one possible molecule which may be used to

adulterate (increase nitrogen content) food and petfood or

one of the ingredients used to manufacture food or pet-

food.

P. PIBAROT, S. PILARD

614

Therefore, even if toxicology is not an issue, the analy-

sis of urea in food ingredients and in finished products

may be of importance to prevent the use of non author-

ized nitrogen sources in food and petfood industries.

Various analytical methods including colorimetry, urea

enzymatic conversion to ammonia, liquid or gas chroma-

tography or near infrared spectrometry are described for

urea analysis in feed, silage, fertilizers, blood, serum, urine

and other matrices [5].

The two main analytical methods used in food and feed

areas are:

 Spectro-colorimetric methods [6,7];

 Enzymatic (urease) methods [8].

These methods are either based:

 On the reaction of urea with a specific molecule to

create a chromophore which content is analyzed by spe-

ctro-colorimetry;

 Or on the enzymatic degradation of urea in presence

of urease to ammonia and on ammonia analysis by the

Kjeldahl method (acidic titration) or other various met-

hods.

Their main advantage is rapid and easy to use protocol.

Other more sophisticated methods based on gas chro-

matography coupled to isotope ratio [9] or isotope dilu-

tion [10,11] mass spectrometry and liquid chromatogra-

phy hyphenated to tandem mass spectrometry [1] have been

developed.

Several cases of interference in colorimetric method or

enzyme activity disturbance are reported [5,11,12].

Co-elution problem leading to result variations in chr-

omatography methods are also reported [13].

The objective of the current study is:

 To compare the results of spectro-colorimetric method

and enzymatic method reliability for the routine analy-

sis of urea dry petfood products;

 To define which method is the most suitable for urea

analysis in dry petfood by comparing the results ob-

tained by the routine methods to the results obtained

with liquid chromatography electrospray ionization high

resolution mass spectrometry (LC/ESI-HRMS).

Finally the conclusion of the study should allow to rec-

ommend an official method for analyzing urea in dry pet-

food products and in pet food raw materials.

2. Experimental Sections

2.1. Spectro-Colorimetric Method (European

Regulation)

This method corresponds to the method recommended by

the European Union and is described in European Com-

mission Regulation (EC) No. 152/2009 laying down the

methods of sampling and analysis for the official control

of feed [6]. This method is also described as a AOAC

method [7].

The sample is suspended in water with a clarifying agent

(active carbon). The suspension is filtered. The urea con-

tent of the filtrate is determined after the addition of 4-

dimethylaminobenzaldehyde (4-DMAB) by measuring the

optical density at a wavelength of 420 nm. If the sample

contains simple nitrogenous compounds such as amino

acids, the optical density shall be measured at 435 nm.

2.2. Enzymatic Method

The enzymatic method is the official method of urea ana-

lysis in feed of AOAC international (No. 941.04) [8].

The method consists in digesting 1 g of samples in urease

enzyme solution at pH = 7 in order to convert the urea in

carbon dioxide and ammonia. After alkalization, ammo-

nia vapor is released and distillated in a known amount

of acid and the distillate titrated with standard base solu-

tion (Kjedhal method).

It should be noticed that ammonia is analyzed together

with urea and that some heavy metal may inhibit the ac-

tivity of urease.

2.3. Liquid Chromatography Ultraviolet-Visible

Diode Array Detection (LC/UV-DAD)

The analyses were run on AS DX 500 HPLC (Dionex,

Sunnyvale CA, USA) equipment with UV-Vis Diode Ar-

ray detector UVD 340 S.

After several trials with several columns and elution

conditions (ion exchange, pH adjustment) the following

chromatographic conditions were selected. The reverse

phase column was an Acclaim PA2 C18, 4.6 × 150 mm,

5 µm supplied by Dionex France.

The chromatographic conditions were:

 Isocratic elution with 100% purified water at 1 mL/min

and 30˚C;

 Sample injection 250 µL (auto-sampler AS 50);

 Detection 200 - 595 nm (resolution 1nm). Urea quan-

tification is performed at 435 nm as indicated in the

EC protocol [6,7].

The samples of petfood were ground and extracted in

the same conditions than for the DMAB spectro-colori-

metric analysis (except active carbon treatment). The ex-

tract reacted with DMAB were directly injected on the

HPLC column. The separation and detection of the mole-

cules produced by reaction between urea and DMAB has

been chosen with two objectives:

 To improve the selectivity and sensibility of the urea

detection by UV absorbance;

 To check the presence of other molecules than urea in

dry pet food which may react with DMAB and absorb

at the wavelength used by the EC colorimetric method.

2.4. Liquid Chromatography Electrospray

Ionization High Resolution Mass

Spectrometry (LC/ESI-HRMS)

The LC/ESI-HRMS experiments were performed on a Q-

P. PIBAROT, S. PILARD

615

TOF Ultima Global hybrid quadrupole time-of-flight inst-

rument (Waters-Micromass, Manchester, UK), coupled

with an Ultra-Fast Liquid Chromatograph (UFLC) system

(Shimadzu, Duisburg, Germany).

The separation was obtained on a Nucleodur Pyramid

2.0 mm i.d. × 250 mm, 3 m C

reversed phase column

(Macherey-Nagel, Duren, Germany) at 25˚C, using an iso-

cratic elution with 100% water. The flow-rate and the

sample injection volume were set to 0.2 mL/min and 10

µL, respectively.

The effluent from the LC column was directly intro-

duced in the electrospray ion source (Z-spray) and the

ESI-HRMS data were recorded in the positive ion mode

with capillary and cone voltages of 3.7 kV and 184 V,

respectively. Nitrogen was used as a nebulizing (100 L/h)

and a drying (450 L/h) gas. The source and desolvatation

temperatures were kept at 120˚C and 250˚C, respectively.

The mass range was 100 - 200 Da and spectra were re-

corded in the profile mode at a scan rate of 0.1 s/scan

(interscan time: 0.1 s) and at a resolution of 5,000

(FWHM). Accurate mass calibration was achieved using

the cluster ions of an orthophosphoric acid solution (0.2%

in H

O/CH

CN 50/50 v/v). Data acquisition and proc-

essing were performed with MassLynx 4.0 SP4 software.

The ground pet food samples were extracted by mix-

ing with water (2 g in 500 mL, 30˚ C, 30 min) and sam-

pling of the supernatant after decantation. Urea quantifi-

cation was performed using the integration of the recon-

structed ionic current (RIC) of the [di-Urea+Na]

ion

Na, exact mass: 143.0545) selected with a 150

ppm (0.02 Da) mass window. For each set of samples,

urea calibration curves were obtained after injection of

standard solutions prepared in water.

3. Results and Discussion

3.1. Analyses of Dry Pet Food Finished Products

by EC and AOAC Methods

The DMAB spectro-colorimetric method is the European

official method for analyzing urea in feed materials while

in the American zone, for instance, the enzymatic method

is the official one. The comparison of the two methods

with a range of dry pet-food sample (from various man-

ufacturers) was therefore an interesting exercise.

The results of this comparison are given in Table 1.

The result demonstrate the two methods do not give

similar results and the spectro-colorimetric method gives

systematically urea concentration between 0.14 and 0.27

g/100g while the enzymatic method gives non detectable

amount of urea whatever is the analyzed sample (except

one sample at 0.01 g/100g).

Therefore either the enzymatic method is not able to

detect urea in petfood samples (e.g. urease inhibition) or

the spectro-colorimetric method results in false positive

by detecting other interfering molecules than urea.

Table 1. Comparison of urea content (g/100g) obtained in

dry pet food samples with spectro-colorimetric method and

enzymatic method.

Samples EC colorimetric method AOAC enzymatic method

Dog food 10.18 <0.01

Dog food 20.14 <0.01

Dog food 30.26 <0.01

Dog food 40.25 <0.01

Cat food 10.26 <0.01

Cat food 20.25 0.01

Cat food 30.27 <0.01

Cat food 40.17 <0.01

3.2. Analysis of Raw Materials by EC and

AOAC Methods

If the finished product contains some non acceptable level

of urea, some urea at higher level of concentration should

be found in the raw materials used in the formulation of

the finished products.

A wide range of raw materials have been analyzed.

Significant concentrations of urea are found by the spec-

tro-colorimetric method for animal and fish meals, vis-

cera and liver digest, colostrum and pure-amino acids.

All these colorimetric method positive samples are rich

in free amino acids.

The highest concentrations are found with pure amino-

acids which are already identified as molecules able to

react with DMAB and to be detected by UV absorption

even if the wavelength of 435 nm is selected by method

authors to minimize the interference of amino-acids.

A selection of the ingredients with significant urea con-

centration determined by spectro-colorimetry was con-

troled by enzymatic method. The results are shown in the

following tables.

Two batches of methionine, which may be added in

relatively significant amount in the recipe for palatability

purpose, were analyzed and the results of two different

samples are presented on Table 2.

Methionine is detected by the EC colorimetric method

as urea even if obviously the quantification is far from

the true value. Enzymatic method is not able to produce

ammonia with methionine as a substrate.

Analysis results comparison by both methods was also

performed with chicken viscera digest and pork liver digest

(See Table 3).

Chicken viscera digest contains free amino acid (because

of the enzymatic digestion) but is unlikely to contain sig-

nificant amount of urea as chicken metabolism produces

very limited amount of urea and mainly produces uric acid.

As matter of fact, the colorimetric method gives a pos-

itive answer while the enzymatic method does not detect

any urea.

Pork liver digest contain higher level of free amino acid

P. PIBAROT, S. PILARD

616

Table 2. Comparison of urea contents (g/100g) obtained

with methionine ingredient samples by spectro-colorimetric

(measured at 435 nm) and enzymatic methods.



Colorimetric method

(g/100g)

Enzymatic method

(g/100g)

Methionin 1 5.56 <0.01

Methionin 2 4.67 <0.01

Table 3. Comparison of urea content (g/100g) obtained in

chicken viscera digest with spectro-colorimetric and enzy-

matic methods.

Colorimetric method

(g/100g)

Enzymatic method

(g/100g)

Chicken viscera digest 0.13 <0.01

Pork liver digest 0.95 0.12

and may contain urea as the pork metabolism (mammal-

ian) produce urea. Again as a matter of fact, the colori-

metric method results are higher than the enzymatic one

because of the amino acid interferences and the enzymatic

method is able to detect the natural low level of urea

which is expected.

To make a further proof of the adequacy of the enzy-

matic method for the analysis of urea in petfood, pet food

samples were spiked with 3 different levels of pure urea.

Results are given in Table 4.

For the non spiked sample, the enzymatic method gave

non detectable result. Sample spiked with known level of

urea were correctly analyzed by the enzymatic method and

this prove the enzymatic method is able to detect correctly

urea when it is present in the pet food products.

This comparison of urea content results obtained by

EC colorimetric method and AOAC enzymatic method,

gives first indication that some interfering amino com-

pound may result in false positive with the EC colorimet-

ric method. This should be confirmed by analyzing the

pet food samples with other analytical methods.

3.3. LC/UV-DAD Analyses of DMAB Product

Extracts

The objective of the HPLC analysis of samples extracted

and treated with DMAB in similar way than with the EC

colorimetric method is to chromatographically separate

the interfering compounds from urea in order to demon-

strate that other molecules than urea are analyzed in pet

food samples when EC colorimetric method is used.

Urea molecule presents low retention time with reverse

or normal chromatography and is not easily separated from

the free amino-acid or bio-amines [14]. After several trials

the Acclaim PA2 C18 (Dionex) was selected as the most

able to separate urea and free amino-acids. Four dry prod-

ucts from the super-premium cat and dog products range

were selected for their significant contents in urea as ana-

lyzed with EC colorimetric method. These products were

analyzed by HPLC after water extraction and reaction with

DMAB as described in EC colorimetric protocol. Figure

1 gives an example of HPLC analysis with pet food sam-

ple spiked with 0.1% urea.

Analyses results of dog and cat pet food samples by

HPLC method are given in Table 5.

Free amino acid peaks detected after the peak of urea

(see Figure 1) were arbitrary quantified in order to rank

them. It should be noticed that the detection was at 435

nm and only molecules able to react with DMAB were

detected by the HPLC analysis.

The first fact is that the levels of urea quantified by the

HPLC method are lower of a magnitude of about 100 to

the content of urea quantified by the EC colorimetric

method.

Nevertheless, no clear correlation is observed between

either the levels of urea quantified by both methods nei-

ther between amino-acid arbitrary ranking and the urea

levels.

Table 4. Results of analysis of spiked dry pet food by enzy-

matic method.

Dry PF with spiked Urea (g/100g,

spike levels)

Enzymatic method

(g/100g)

0<0.01

0.50.57

11.01

1.51.38

–

0.53 –0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.02

142

125

113

100

–4

SEM 12 17 MARS 2009 #54 [modified by RDLab05AUR]

Ech 4 + 0.1 %+ DMAB

Ech 4 + 0.1%+ DMAB

WVL:435 nm

mAU

min

1 - 0.083

2 - DMAB - 1.775

3 - Urea - 2.150

Amino acids

Figure 1. Example of chromatogram obtained by LC/UV-

DAD analysis of pet food sample spiked with 0.1% of urea

(Acclaim C18, detection at 435 nm, urea peak at 2.1 min,

DMAB at 1.7 min)

Table 5. Results of analysis of dry pet food by LC/UV-DAD,

comparison with spectro colorimetric results.

Colorimetric method LC/UV-DAD analysis

Samples Urea (g/100g) Urea (g/100g)

Amino acids

(arbitrary unit)

Dog food 5 0.32 0.004 3.61

Cat food 1 0.26 0.0058 5.99

Cat food 5 0.60 0.0062 2.75

Cat food 6 0.75 0.0038 3.99

P. PIBAROT, S. PILARD

617

Therefore the LC/UV-DAD analyses have proven that

other molecules than urea can react with DMAB and in-

terfere in the colorimetric analysis to give overestimated

results.

Even if some interesting facts are deduced from this

HPLC analysis, the proof of the overestimation and the

true level of urea in pet food samples is still to be done as

the urea and amino-acid peaks separation is not enough

to validate the quantification of urea. This lack of reten-

tion of urea and difficulty to separate urea from amino-acid

in HPLC has been already observed by some authors [14].

The selectivity and the effective separation of urea with

the other molecules derivatized by DMAB is not enough

to seriously quantify the true level of urea in dry pet food.

3.4. LC/ESI-HRMS of Pet Food Samples

The objective of the LC/ESI-HRMS analysis was to ac-

cess to urea content in pet food samples using a simple

water extraction without any further reaction (e.g. DMAB)

and a highly specific detection method. In order to sepa-

rate as far as possible urea from the interfering molecules

(e.g. free amino acids) we have used a liquid chromatog-

raphy method developed by Macherey Nagel on a Nu-

cleodur Pyramid C

column with water (100%) as the

eluent [15], in these conditions urea was detected at a

retention time of 2.86 min.

Electrospray and atmospheric pressure chemical ioni-

zations coupled to tandem mass spectrometry (ESI-MS/MS

and APCI-MS/MS) were applied to determine the con-

centration of urea present in the water of swimming pool

[16] but, to our knowledge, these methods were never

used for urea quantification in complex samples, such as

pet food. High resolution mass spectrometry, allowing the

determination of molecule elemental composition, is a

very selective and sensitive method to identify and quan-

tify a specific compound in mixtures. Consequently, in

these work we have investigated online LC/ESI-HRMS

using the high resolution capabilities of a quadrupole or-

thogonal time-of-flight (Q-TFOF) mass spectrometer [17].

The exact mass which was used for urea characteri-

zation and quantification was 143.0545, corresponding

to the elemental composition of the [di-Urea+Na]

ion

Na). The mass selection window was set to

150 ppm (0.02 Da) for reconstructed ion chromatogram

(RIC of m/z 143.05 or 143.07 according to the mass spe-

ctrometer calibration).

Pure urea was diluted in water to prepare a range of

standard solutions. A stock solution was prepared by dis-

solution of 40 mg of pure urea in 1000 mL of de-ionized

water to obtain a urea solution at 4 mg/100mL. The stock

solution was diluted in water to prepare standard solutions

at 0.2, 0.4, 0.8, 1.2, 1.6 and 2 mg/100mL, in the range of

the urea concentration expected in pet food samples. These

standard solutions were injected in the LC/MS system to

build a calibration curve before each analyses session. The

chromatograms acquired at the exact mass of 143.05 (m/z)

and peak areas (retention time 2.86 min.) obtained are pre-

sented on Figure 2.

The corresponding calibration curve was obtained from

the least-squares linear regression presented with its cor-

relation coefficient (R

= 0.9927) on Figure 3.

The linearity of the calibration curve was demonstrated

for urea concentrations up to 4 mg/100mL. For each dry

pet food analysis 2 g of pellets were extracted in 500 mL

pure water during 30 minutes at 30˚C. For each series of

pet food samples new dilutions of urea stock solution at 4

mg/100mL were prepared to build a dedicated calibration

curve.

3.5. Pet Food Matrices Effect on LC/ESI-HRMS

To validate the eventual effect of the pellet matrix on the

quantification of urea by LC/ ESI-HRMS, the extraction

of a urea doped dry pet food sample, which was previ-

ously analyzed by EC spectro-colorimetric method (re-

sult: 0.46 g/100g), was performed. A sample of 4 grams

of ground pellets was doped with respectively 40 and 20

milligrams of pure urea powder and the resulting samples

analysis at 1% urea (Chromatogram A) and 0.5% (Chro-

matogram B) are shown Figure 4.

Time

2.202.402.602.803.003.203.403.603.80

100

2.202.402.602.803.003.203.403.603.80

100

2.202.402.602.803.003.203.403.603.80

100

2.202.402.602.803.003.203.403.603.80

100

2.202.402.602.803.003.203.403.603.80

100

2.202.402.602.803.003.203.403.603.80

100

UREE-TEMOIN-005-070710-b Sm (SG, 3x20)TOF MS ES+

143.05 150PPM

1.76

Area

2.89;0.14

UREE-TEMOIN-01-070710-b Sm (SG, 3x20)TOF MS ES+

143.05 150PPM

3.25

Area

2.86;0.32

UREE-TEMOIN-02-070710-a Sm (SG, 3x20)TOF MS ES+

143.05 150PPM

4.54

Area

2.86;0.50

UREE-TEMOIN-03-070710-a Sm (SG, 3x20)TOF MS ES+

143.05 150PPM

5.58

Area

2.86;0.71

UREE-TEMOIN-04-070710-a Sm (SG, 3x20)TOF MS ES+

143.05 150PPM

6.99

Area

2.87;0.98

UREE-TEMOIN-05-070710-b Sm (SG, 3x20)TOF MS ES+

143.05 150PPM

9.84

Area

2.87;1.25

Figure 2. LC/ESI-HRMS analysis of urea standard solu-

tions at 0.2, 0.4, 0.8, 1.2, 1.6 and 2 mg/100mL (respectively

chromatogram from top to bottom; integration of urea

peak at 143.05 (m/z) and 2.86 min (Rt)).

P. PIBAROT, S. PILARD

618

y = 1.6111x

R² = 0.9927

0.5

1.5

2.5

0 0.2 0.4 0.60.8 1 1.21.4

Solution

concentration

(mg/100 ml)

Peak area at m/z 143.05

Figure 3. LC/ESI-HRMS urea analysis calibration curve bet-

ween 0.2 and 2 mg/100mL.

Time

2.252.502.753.003.253.503.75

100

2.252.502.753.003.253.503.75

100

UREE-046-20-b Sm (SG, 3x20)TOF MS ES+

143.055 150PPM

11.8

Area

2.86

1.71

UREE-046-10-b Sm (SG, 3x20)TOF MS ES+

143.055 150PPM

9.04

Area

2.85

0.82

Figure 4. LC/ESI-HRMS urea analysis of dry dog food ex-

tracted with a urea solution at 1% (chromatogram A) and

at 0.5% (chromatogram B).

The results of this doped samples analyses are shown

on Table 6.

The pellet matrix appears to suppress around 35% the

MS signal.

The EC spectro-colorimetric has determined a content

of 0.46 g/100g in this pet food sample which was doped

with urea. In order to take into account the matrix effect

on the LC/ESI-HRMS quantitative analysis, the correla-

tion between the urea content in the dry pellets concen-

tration of the doped samples and the LC/ESI-HRMS peak

area was established. The extrapolation of the curve to

zero added urea gives directly the urea concentration in

the analyzed sample (see Figure 5).

It can be determined from the curve the value of 0.05

g/100g for the real urea content of this sample. This value

is significantly below the EC spectro-colorimetric result

Table 6. Results of analysis by LC/ESI-HRMS (m/z 143.05)

of dry pet food pellets doped at 0.5% and 1% with pure

urea powder.

Sample

Area m/z

143.05

Measured

concentration

(mg/100mL)

Theoretical

concentration

(mg/100mL)

Extraction

yields (%)

Doped at

0.5%

0.82 1.32 2 66

Doped at

1.71 2.75 4 69

Figure 5. Extrapolation of the correlation between peak area

and urea concentration in doped pellet sample (g/100g) to

determine the real content of urea in the analyzed sample.

of 0.46 g/100g. Spiking method, being less accurate than

calibration method, further analyses are necessary to vali-

date this result.

3.6. LC/ESI-HRMS and Spectro-Colorimetric or

Enzymatic Methods Comparison

Further analyses were performed to compare the EC

spectro-colorimetric method, the enzymatic method and

LC/ESI-HRMS. The selected samples are described in

Table 7.

A new calibration file was established and results are

shown on Figures 6 and 7.

The chromatograms and peak areas obtained by analy-

sis of samples listed in Table 7 are presented on Figure 8

(the chromatogram at the top corresponds to a standard

solution at 0.2 mg/100mL urea).

The peaks at retention time between 2.70 and 3.05 min.

were taken as urea peak to keep the worst case hypothe-

sis. The results for the five analyzed samples are presented

on Table 8.

The results obtained by LC/EIS HRMS are significantly

below the results obtained by EC colorimetric method and

in the same magnitude than the results of the enzymatic

analysis (<0.01 g/100g, Table 7) even if the corrective

factor to take into account the 65% yield is not applied

(see Table 9).

P. PIBAROT, S. PILARD

619

Table 7. Selection of a range of petfood samples for analysis

by LC-ESI-HRMS.

Samples

Colorimetry

(g/100g)

Enzymatic

(g/100g)

Pet food 1 0.69 <0.01

Pet food 2 0.31 <0.01

Pet food 3 0.17 <0.01

Pet food 4 1.09 <0.01

Pet food 5 0.26 <0.01

Time

2.502.602.702.802.903.003.103.203.30

100

2.502.602.702.802.903.003.103.203.30

100

2.502.602.702.802.903.003.103.203.30

100

UREE-TEMOIN-01-070710-c Sm (SG, 3x20)TOF MS ES+

143.07 150PPM

19.8

Area

2.87;2.85

UREE-TEMOIN-005-070710-c Sm (SG, 3x20)TOF MS ES+

143.07 150PPM

14.4

Area

2.86;1.64

UREE-TEMOIN-0-070710-c Sm (SG, 3x20)TOF MS ES+

143.07 150PPM

1.43

Area

Figure 6. LC/ESI-HRMS analysis of urea standard solution

at 0, 0.2 and 0.4 mg/100mL concentrations (respectively

chromatogram from top to bottom; integration of urea peak

at 143.07 (m/z) and 2.86 min (Rt)).

y=0.1358x

R²=0.9914

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

00.511.522.53

Ureaconcentration(m

g/100ml)

Peakareaatm/z143.07

Figure 7. LC/ESI-HRMS urea analysis calibration curve at

0, 0.2 and 0.4 mg/100mL.

The concentrations analyzed by LC/ESI HRMS for this

set of dry pet food samples are in the magnitude order of

Figure 8. LC/ESI-HRMS urea analysis of dry dog food wa-

ter extracts. (Pet food 1: chromatogram at the bottom and

respectively pet food 2, 3, 4, 5 from bottom to top of the

figure, chromatogram at the top corresponds to a standard

solution at 0.2 mg/100mL urea).

Table 8. Results of analysis of dry pet food samples by

LC/ESI-HRMS (m/z 143.07) (2 g of samples extracted in 500

mL of water).

Peak area m/z

143.04

Water extract

concentration (mg/100mL)

Pellet content

(g/100g)

Standard (0.2

mg/100mL)

1.64 0.223 na

Pet food 1 0.11 0.015 0.004

Pet food 2 0.03 0.004 0.001

Pet food 3 0.16 0.022 0.005

Pet food 4 0.08 0.011 0.003

Pet food 5 0.3 0.041 0.010

na: not applicable.

Table 9. Comparison of urea content in dry pet food pel-

lets obtained by EC colorimetric method, enzymatic method

and LC/ESI-HRMS.

Colorimetric methodEnzymatic method

LC/ESI-HRMS

method

Samples

(g/100g) (g/100g) (g/100g)

Pet food 10.69 <0.01 0.004

Pet food 20.31 <0.01 0.001

Pet food 30.17 <0.01 0.005

Pet food 41.09 <0.01 0.003

Pet food 50.26 <0.01 0.010

P. PIBAROT, S. PILARD

620

the enzymatic method (<0.01%) when the EC colorimet-

ric method resulted in urea concentration between 0.1%

to 1%.

4. Conclusions

As a conclusion, the EC colorimetric method for urea

analysis is not applicable to dry petfood products as in-

terfering molecules result in significant urea concentra-

tion over dosing.

The interfering molecules may be free amino-acids as

they react with DMBA and absorb at 435 nm as shown

by the LC/UV-DAD analyses. As a matter of fact ingre-

dients known to contain significant level of free amino

acid present some content of urea when analyzed by the

EC colorimetric method.

The enzymatic method gives results <0.01 g/100g for

all petfood samples and is proven to be able to detect urea

in petfood samples when urea is spiked and also by analy-

sis of some ingredients like pork liver which may natu-

rally contain urea.

After several trials and studies, the analysis of a range

of dry petfood by LC/ESI-HRMS has allowed the valida-

tion of the results obtained using the enzymatic method.

The recommendation would be to propose a modifica-

tion of the EC Commission Regulation No. 152/2009 and

to recommend the enzymatic method for monitoring urea

in pet food products and in food product with complex

matrices and significant level of free amino acids. This

would also harmonize the regulation between Europe and

America zones as the AOAC already recommends the

enzymatic method for urea analysis in pet food.

The complete validation of the LC/ESI-HRMS would

require further work to validate the extraction method

and to study the stability of extracted petfood water solu-

tions doped with urea. This method is also quite sophis-

ticated and expensive for routine control in analytical labo-

ratories. A promising approach using a benchtop Orbi-

trap mass spectrometer for mass accuracy improvement

(mass selection window less than 3 ppm) [18] may also

be considered.

As a general comment the methods developed and vali-

dated for feed are often not suitable to petfood as the two

categories of products present very different compositions.

5. Acknowledgements

The authors are thankfull to the Eurofins laboratory who

have conducted the colorimetric and enzymatic analyses

of pet food samples.

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