A

torfa©

K

1

oitsmooiufcattdcbt

0d

Journal of Chromatography A, 1161 (2007) 207–213

Determination of urea using high-performance liquid chromatography withfluorescence detection after automated derivatisation with xanthydrol

Shona Clark, Paul S. Francis ∗, Xavier A. Conlan, Neil W. BarnettSchool of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3217, Australia

Received 30 April 2007; received in revised form 28 May 2007; accepted 29 May 2007Available online 2 June 2007

bstract

A high-performance liquid chromatography (HPLC) method for the determination of urea that incorporates automated derivatisation with xan-hydrol (9H-xanthen-9-ol) is described. Unlike the classic xanthydrol approach for the determination of urea, which involves the precipitationf dixanthylurea (N,N′-di-9H-xanthen-9-ylurea), the derivatisation procedure employed in this method produces N-9H-xanthen-9-ylurea, which

emains in solution and can be quantified using fluorescence detection (λex = 213 nm; λem = 308 nm) after chromatographic separation from inter-erences. The limit of detection for urea was 5 × 10−8 M (0.003 mg L−1). This method was applied to the determination of urea in human andnimal urine and in wine.

2007 Elsevier B.V. All rights reserved.

ometr

pdt

F9finrdUxtaapcd

eywords: HPLC; Urea; Xanthydrol; Fluorescence derivatisation; Mass spectr

. Introduction

Although the published methodology for the determinationf urea has mainly focussed on clinical applications [1,2], theres growing demand for sensitive and reliable procedures forhe determination of urea in other matrices, such as milk [3],oil extracts [4], seawater [5,6] and wine [7]. The most com-on approaches for both research and routine measurement

f urea involve (i) the enzyme (urease) catalysed hydrolysisf urea and subsequent detection of ammonia by colour form-ng reactions or electrochemistry or (ii) the direct reaction ofrea with butane-2,3-dione monoxime (or related reagents) toorm coloured products. Neither approach is ideal for all appli-ations. The non-enzymatic techniques require noxious reagentsnd produce an unpleasant odour, which make them unattrac-ive for clinical and oenological settings [1,7]. However, theseechniques are often favoured for environmental applicationsue to the presence of urease inhibitors and conditions that

onstrain the use of enzymes [4–6]. For many sample types,oth approaches involve time-consuming pre-treatment stepso remove interfering species [4,7]. Previously reported high-

∗ Corresponding author. Tel.: +61 3 52271294.E-mail address: psf@deakin.edu.au (P.S. Francis).

t

iwadi

021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.05.085

y

erformance liquid chromatography (HPLC) procedures for theetermination of urea [8–12] have not been widely adopted dueo their complexity or inadequate sensitivity.

The determination of urea with xanthydrol (9H-xanthen-9-ol;ig. 1a), based on the precipitation of dixanthylurea (N,N′-di-H-xanthen-9-ylurea; Fig. 1b) in concentrated acetic acid, wasrst described in the early 20th century [13,14]. The origi-al procedure involved weighing the dixanthylurea, but otheresearchers measured the absorbance of the yellow solution pro-uced when the precipitate was dissolved in 50% sulfuric acid.nfortunately, a similar colour was obtained from the unreactedanthydrol, which therefore had to be washed from the dixan-hylurea precipitate prior to the addition of the acid [15]. Thispproach suffered from poor reproducibility and was unsuit-ble for automation. Consequently, it was largely superseded byrocedures based on other colour-forming reactions or urease-atalysed hydrolysis of urea [1,2,16]. Nevertheless, the xanthy-rol test for urea is still included as part of the AOAC methodso confirm the presence of urine on foods and containers [17].

Xanthydrol also reacts with various primary amides [18–20],ndoles [21–23], phenols [19,24] and substituted ureas [19,25],

hich has been exploited for quantitative spectrophotometric

nalyses [20,23–25]. HPLC procedures with UV absorbanceetection have been developed for the determination of allantoinn plasma extracts [25] and N-acetyl-l-glutamine in rat urine

208 S. Clark et al. / J. Chromatogr.

Fig. 1. (a) xanthydrol (9H-xanthen-9-ol); (b) dixanthylurea (N,N′-di-9H-x9x

[tiuwaa

ditxhapd21a[

cdrti

ud

2

2

2

sflt5aTau

2

xw

2

wifrom the HPLC procedure was diluted 10-fold with acetonitrile.Slit widths of 5 nm were used for all experiments. Emissionspectra were corrected for the wavelength dependence of thedetector response and monochromator transmission.

Table 1Mobile phase gradients [solvent A: 20 mM sodium acetate (pH 7.2); solvent B:acetonitrile]

Gradient Time (min) Solvent (%B) Flow rate (mL min−1)

I 0 20 0.450.06 20 0.45

12.6 50 0.4513.6 100 0.4520.6 100 0.822.6 20 0.4523.6 20 0.45

II 0 20 1.00.06 20 1.0

12.6 50 1.013.6 100 1.020.6 100 1.022.6 20 1.023.6 20 1.0

III 0 50 1.03.3 50 1.04.0 100 1.07.0 100 1.0

anthen-9-ylurea); (c) N-9H-xanthen-9-ylurea; (d) N2-acetyl-N-9H-xanthen--yl-l-glutamine; (e) xanthylallantoin (N-(2,5-dioxo-4-imidazolidinyl)-N′-9H-anthen-9-ylurea); (f) xanthylurethane (N-9H-xanthen-9-ylurethane).

20]. Both involved manual derivatisation with xanthydrol andime-consuming steps to remove interfering species prior tonjection [20,25]. For example, as xanthydrol precipitates withrea much faster than N-acetyl-l-glutamine, the dixanthylureaas filtered from the reaction mixture approximately 1 h after the

ddition of the reagent, and the derivatised analyte was collectedfter standing overnight [20].

Herbert and co-workers reported HPLC procedures for theetermination of ethyl carbamate (urethane) [26] and urea [27]n alcoholic beverages, with faster manual methods to derivatisehe analytes. Samples were diluted with ethanol and mixed withanthydrol dissolved in 1-propanol. A small amount of 1.5 Mydrochloric acid was then added. Under these conditions, thenalyte was derivatised within 5 min (without the formation ofrecipitates) and could be injected directly onto the column. Theerivatised analyte was detected by fluorescence (excitation at33 nm and emission at 600 nm) [26,27]. Urea was determined in2 port wine samples and although the results appeared reason-ble, they were not validated against an established procedure27].

Based on these findings, we have developed a HPLC pro-edure for the determination of urea incorporating automated

erivatisation of the analyte with xanthydrol and compared theesults of this procedure with a conventional approach based onhe enzymatic breakdown of urea. Furthermore, we have exam-ned the nature of the fluorophore produced in the reaction of

A 1161 (2007) 207–213

rea and xanthydrol, to improve the analytical application of thiserivatisation reagent.

. Experimental

.1. Instrumentation and procedures

.1.1. High-performance liquid chromatographyUrea was determined using a 1200 series HPLC system with

olvent degasser system, quaternary pump, autosampler anduorescence detector (Agilent Technologies, Blackburn, Aus-

ralia). An Eclipse XBD RP-18 column (150 mm × 4.6 mm I.D.,�m) from Agilent Technologies was used for all separationst a constant temperature of 35 ◦C and the gradients shown inable 1. The derivatised analyte was detected with excitationnd emission wavelengths of 213 nm and 308 nm, respectively,sing slit widths of 20 nm.

.1.2. Automated sample preparationThe HPLC auto-sampler was programmed to perform the

anthydrol derivatisation (Table 2). The total injection volumeas 40.5 �L.

.1.3. Fluorescence spectraFluorescence excitation and emission spectra were collected

ith a Cary Eclipse fluorescence spectrophotometer from (Var-an, Mulgrave, Australia). The selected post-column fraction

7.5 20 1.010.0 20 1.010.5 50 1.012.0 50 1.0

S. Clark et al. / J. Chromatogr.

Table 2HPLC autosampler program for analyte derivatisation

Step Action Vial Volume (�L)

1 Draw 1 142 Draw 2 1.53 Mixa 15.54 Draw 3 05 Draw Sample 256 Mixa 40.57 Draw 3 08 Wait 5 min9 Inject

V3

2

w(lv

2

cyoddsaaaHt1fot2vwmt1WaltU

2

(B

euvtuwfla(0P(g((5gse

3

3

be1p5OaoWut1tr(Oe

mspdshmwo

ial 1: xanthydrol (0.02 M in 1-propanol), vial 2: hydrochloric acid (1.5 M), vial: acetonitrile for needle wash.a In air; maximum speed; three times.

.1.4. Mass spectraA 6210 MSDTOF mass spectrometer (Agilent Technologies)

as used with the following conditions: drying gas, nitrogen7 mL min−1, 350 ◦C); nebulizer gas, nitrogen (16 psi); capil-ary voltage, 4.0 kV; vaporizer temperature, 350 ◦C; and coneoltage, 60 V.

.1.5. Urease test kitA K-URAMR test kit (Megazyme, Bray, Ireland) was used for

omparison purposes. The test kit method involved urease catal-sed hydrolysis of urea to ammonia and the subsequent reactionf ammonia, 2-oxoglutarate and reduced nicotinamide–adenineinucleotide phosphate (NADPH) in the presence of glutamateehydrogenase to form glutamic acid and NADP+. The con-umption of NADPH is measured by the decrease in absorbancet 340 nm and is proportional to the original amount of urea overfinite range [28]. Tests were performed as described in the

ssay procedure. Urine samples were filtered through 0.45 �mT Tuffryn membranes (Acrodisc PSF syringe filters; Pall Aus-

ralia, Cheltenham, Australia) and diluted between 2500- and00,000-fold. A volume of 1.0 mL of diluted urine was usedor each test. White wine samples were filtered and a volumef 0.1 mL was used for each test. Where required, the pH ofhe wine sample was adjusted to approximately pH 8.0 usingM NaOH and then made up to double the original sampleolume with deionised water. Samples and reagent solutionsere dispensed with handheld autopipettes (Gilson Pipetmanodels P20, P200, P1000; John Morris Scientific, Balwyn, Aus-

ralia) into standard 10 mm disposable cuvettes (Kartell type939: UV grade, PMMA, 4.5 mL volume; Crown Scientific,antirna South, Australia) and mixed by repeated inversion

fter the cuvettes were sealed with either Para film or cuvetteids (Kartell type 1962; Crown Scientific). The absorbance ofhe solutions was measured at 340 nm using a Cary 300 BioV-visible spectrophotometer (Varian).

.2. Reagent and samples

Urea and xanthydrol were purchased from Sigma–AldrichCastle Hill, Australia). Sodium acetate was obtained fromDH (Poole, UK). Hydrochloric acid, propanol, acetonitrile,

tduT

A 1161 (2007) 207–213 209

thanol were obtained from Merck (Kilsyth, Australia). Animalrine samples were supplied by Ray C. Bartolo (Deakin Uni-ersity; Geelong, Australia) and diluted with deionised watero within the calibration range (1:5000 dilution for humanrine, 1:2500 for toad, 1:10,000 for rat and 1:100,000 forater deprived mouse. Two Australian wines were purchased

or analysis: Lindeman Bin 50 Shiraz (2005) and Queen Ade-aide Chardonnay (2005). Prior to derivatisation, all standardsnd samples (diluted urine and wine) were mixed with ethanol1 mL of standard or sample to 3 mL ethanol) and filtered through.45 �m HT Tuffryn membranes (Acrodisc PSF syringe filters;all Australia). Synthetic wine samples consisted of ethanol12%, v/v), tartaric acid (1200 mg L−1), potassium hydro-en tartrate (2110 mg L−1) malic acid (5000 mg L−1), catechinred wine, 1500 mg L−1; white wine, 30 mg L−1), caffeic acid100 mg L−1), gallic acid (100 mg L−1), quercetin (red wine,0 mg L−1; white wine, 10 mg L−1), proline (200 mg L−1),lutamic acid (200 mg L−1) and arginine (100 mg L−1). Theynthetic wine components were obtained from Sigma–Aldrich,xcept for ethanol, which was purchased from Merck.

. Results and discussion

.1. Sample preparation and chromatographic separation

In the manual pre-column derivatisation method describedy Herbert and co-workers, the samples were made up to 70%thanol. The diluted samples (500 �L) were then combined with00 �L of the derivatising reagent (0.02 M xanthydrol in 1-ropanol) and 50 �L of 1.5 M hydrochloric acid [26,27]. Aftermin, 30 �L of the reaction mixture was injected onto a HypersilDS column (200 mm × 2.1 mm, 5 �m particle size) and sep-

rated using gradient elution (Table 1; Gradient I). In the casef urea, the derivatised analyte was eluted within 7 min [27].e repeated this procedure using an Eclipse XBD RP-18 col-

mn (150 mm × 4.6 mm I.D., 5 �m particle size) at a constantemperature of 35 ◦C, and found a urea peak retention time of4.8 min. By increasing the flow rate and the proportion of ace-onitrile in the mobile phase (Table 1; Gradients II and III), theetention time was reduced to 6.0 min (Gradient II) or 2.1 minGradient III), without co-elution from the excess xanthydrol.verall run times (excluding derivatisation) were 23.6 min using

ither Gradient I or II and 12 min using Gradient III.To simplify the overall procedure, the derivatisation was auto-

ated using the HPLC autosampler. The maximum volume ofolution that can be drawn into the sample loop of the autosam-ler is 100 �L and therefore the volumes described in the manualerivatisation method by Herbert and co-workers [26,27] werecaled down by a factor of 20. Under these conditions, the peakeight and area for the derivatised urea using the automatedethod were slightly lower than when the manual procedureas used. The ratio of reagent and sample solutions was thenptimised to gain the highest response. The final settings for

he auto-sampler program are shown in Table 2. The repro-ucibility of the manual and automated methods was comparedsing 10 replicate injections of a urea standard (1 × 10−4 M).he automated derivation was more precise (RSD of 1.8%) than

210 S. Clark et al. / J. Chromatogr. A 1161 (2007) 207–213

Table 3Analytical figures of merit for the determination of urea using HPLC with automated analyte derivatisation

Gradient Retention time (min) LODa (M) LOQb (M) Equation (log–log)c R2 RSDd (%)

I 15.0 5 × 10−8 2.5 × 10−7 y = 1.04x + 7.14 0.9991 0.75II 6.0 5 × 10−8 2.5 × 10−7 y = 1.02x + 7.09 0.9995 1.26III 2.1 5 × 10−8 2.5 × 10−7 y = 1.02x + 7.13 0.9994 1.90

a Limit of detection (blank + 3σ).

tamdtcd

3

vtetc2wtatilpbcotswotod

tomutsdtwra

tb[

tmNisbacpt

3

tsitacdpehaD

wHwsswpchdt

b Limit of quantification (blank + 10σ).c Between 1 × 10−7 and 1 × 10−3 M.d Relative standard deviation (n = 10) for peak area.

he manual procedure (RSD of 2.6%), although both were at ancceptable level. The analytical figures of merit for the HPLCethod with automated derivatisation (and the three gradients

escribed above) are shown in Table 3. The limit of detec-ion for urea of 5 × 10−8 M (0.003 mg L−1) was superior to theoncentrations reported in previous HPLC procedures for theetermination of urea [4,7].

.2. Fluorophore characterisation

To examine the nature of the derivatised analyte, the rele-ant fraction of the column eluent (corresponding to a retentionime range of 14.5–15.5 min using Gradient I) was collected andxamined using a spectrofluorometer and mass spectrometer. Forhe fluorescence detection of the derivatised urea, Herbert ando-workers [27] used excitation and emission wavelengths of33 nm and 600 nm, respectively. Using a spectrofluorometer,e observed similar maxima at 211 nm and 240 nm in the exci-

ation spectrum and 592 nm in the emission spectrum. However,more intense peak at 298 nm was detected in the emission spec-

rum. Furthermore, both peaks (298 nm and 592 nm) remainedn the spectrum when an emission filter that only transmittedight between 250 nm and 395 nm was introduced and neithereak was detected when an emission filter that transmitted lightetween 550 nm and 1100 nm was used. It was therefore con-luded that the apparent peak at 592 nm was actually an artefactf the instrumentation (i.e. second-order diffraction with respecto the peak at 298 nm, arising from the grating within thepectrofluorometer). The excitation and emission wavelengthsere re-evaluated using the HPLC fluorescence detector and theptimum values were found to be 213 nm and 308 nm, respec-ively. The slight discrepancies between these values and thosebtained with the spectrofluorometer were due (in part) to theifferent slit widths used in each instrument.

The classic determination of urea by derivatisation with xan-hydrol in concentrated acetic acid is based on the formationf dixanthylurea (Fig. 1b), which precipitates from the reactionixture [15]. Coxon et al. [29] treated urea with xanthydrol

nder less acidic conditions (between 0.5 and 1.0 M acetic acid)o produce N-9H-xanthen-9-ylurea (Fig. 1c), which was moreoluble in polar solvents than dixanthylurea. The derivatisationescribed in this paper was performed in propanol and rela-

ively dilute hydrochloric acid and does not involve precipitation,hich suggests that N-9H-xanthen-9-ylurea is the dominant

eaction product. Other monoxanthyl derivatives (see Fig. 1dnd e) have been prepared for HPLC procedures [20,25] and

fmo

he xanthydrol derivative of ethyl carbamate, which is likely toe xanthylurethane (Fig. 1f), has similar fluorescence character26,27] to the product under investigation.

The formation of N-9H-xanthen-9-ylurea in the derivatisa-ion procedure was confirmed by examining the positive ion

ass spectrum of the relevant eluent fraction. The characteristic-9H-xanthen-9-ylurea [M + H]+ ion at m/z 241 and a fragment

on at m/z 196 were both observed. The base peak in the masspectrum was at m/z 181, which corresponds to the resonance sta-ilised xanthenyl ion. Characteristic ions for dixanthylurea werelso present (m/z 423 for [M + H]+ and m/z 239 for the parentompound minus a xanthenyl radical [M − 181]+ [29]), but theseeaks were relatively low in intensity (∼1%) in comparison tohe ions that were representative of the N-9H-xanthen-9-ylurea.

.3. Preliminary evaluation with real samples

The proposed HPLC procedure with automated derivatisa-ion for the determination of urea was applied to urine and wineamples. The determination of urea in urine is commonly usedn clinical laboratories to assess the nitrogen balance of hospi-alised patients on specialised nutritional support regimens [30]nd is important for research into the mechanism of urinary con-entrating ability and renal urea handling in mammals [31]. Theetermination of urea in wine has been used to establish theotential for ethyl carbamate formation. The concentration ofthyl carbamate (a group 2A carcinogen; IARC, 2007) in alco-olic beverages is regulated or monitored by organisations suchs the Canadian Health Protection Branch and the U.S. Food andrug Administration [7,32].Human and animal urine samples were prepared by dilution

ith deionised water to within the calibration range. For thePLC procedure, 1 mL of each diluted urine sample was mixedith 3 mL of ethanol. Typical chromatograms for human urine

amples using mobile phase Gradients I and III (Table 1) arehown in Fig. 2. In both cases, the peak for the derivatised analyteas resolved from all interferences. The results from the HPLCrocedure were compared to those obtained using a commer-ially available manual test kit, based on the enzyme-catalysedydrolysis of urea. Each test kit assay was performed using ailuted urine sample volume of 1.0 mL. As shown in Table 4,he results for the two procedures were in good agreement.

The concentration of urea in wine is far lower than the levelsound in urine and therefore sample preparation for the deter-ination of urea in wine using the HPLC procedure involved

nly dilution with ethanol (to avoid precipitation) and filtration.

S. Clark et al. / J. Chromatogr. A 1161 (2007) 207–213 211

FG

GTuwmbhIs(o

TUx

HHMTTR

FI

woflfh1

ig. 2. Determination of urea in human urine using (a) Gradient I and (b)radient III.

radient III could not be used due to co-eluting interferences.ypical chromatograms for the analysis of white and red wines,sing Gradients I and II are shown in Figs. 3 and 4. Samplesere spiked with urea to confirm the retention time in the wineatrix. For white wine samples, the peak for urea appeared to

e sufficiently resolved using either gradient (Fig. 3). There wasowever a more noticeable rise in the baseline when Gradient

I was used. For red wine samples, only Gradient I providedufficient separation of the derivatised urea from interferencesFig. 4). Furthermore, an examination of the main componentsf wine revealed that catechin co-eluted with the target analyte

able 4rea in human and animal urine determined using the HPLC procedure withanthydrol derivatisation and a commercially available test kit

Concentration of urea (M)

HPLCa HPLCb Test kit

uman 1 0.28 0.29 0.29uman 2 0.47 0.48 0.49ouse 1 1.49 1.64 1.30

oad 1 0.078 0.081 0.080oad 2 0.079 0.082 0.079at 1 0.29 0.30 0.30

a Gradient I.b Gradient III.

pnHmru

todutT(Tttu

ig. 3. Determination of urea in white wine using (a) Gradient I and (b) GradientI.

hen Gradient II was used (Fig. 5), which had a greater effectn the analysis of red wine due to the higher concentration ofavonoids such as catechin. Consequently, Gradient I was usedor all subsequent analysis of wines. Wine can contain relativelyigh concentrations of ammonia compared to urea. Therefore, a00 �L sample volume of undiluted wine was used in the test kitrocedure to place the total amount of ammonia (both endoge-ous and the product of urea hydrolysis) within the linear range.owever, this reduction in sample volume raises the theoreticalinimum error margin for the determination of the urea (cor-

esponding to the smallest differentiating absorbance of 0.005nits) to 0.62 mg L−1.

To examine the accuracy of the proposed approach, a syn-hetic wine sample was spiked with six different concentrationsf urea and analysed with both the HPLC and test kit proce-ures (Table 5). The percentage recoveries were close to 100%sing the HPLC procedure, but were lower than 100% usinghe test kit when the concentration of urea was below 6 mg L−1.he greatest difference occurred at the lowest urea concentration

2.0 mg L−1), at which the result for the test kit was 1.5 mg L−1.he addition of ammonium chloride (40 mg L−1) to the syn-

hetic wine matrix accentuated this discrepancy by lowering theest kit result for urea to 1.0 mg L−1. The HPLC procedure wasnaffected by the presence of ammonium chloride.

212 S. Clark et al. / J. Chromatogr. A 1161 (2007) 207–213

FI

osottddwtwt

TUt

U

A

11

Fig. 5. Separation of urea and catechin in synthetic wine using (a) Gradient Iand (b) Gradient II.

Table 6Urea in spiked white wine determined using the HPLC procedure with xanthy-drol derivatisation and a commercially available test kit

Urea (mg L−1) Recovery (%)

Spike HPLC Test kit HPLC Test kit

0 2.0 0.9 – –1 3.0 2.0 100 1102 3.9 2.6 95 854 5.9 4.5 98 90

ig. 4. Determination of urea in red wine using (a) Gradient I and (b) GradientI.

The two procedures were then applied to the determinationf urea in an Australian white wine and five samples of the winepiked with urea (Table 6). For each sample, the concentrationf urea determined using the HPLC procedure was higher thanhat of the test kit. Overall, the deviation from 100% recovery forhe test kit was greater than the deviation for the HPLC proce-ure. However, the percentage recovery for the HPLC procedureeclined (in general) as the concentration of urea was increased,

hich is indicative of matrix effects. These effects may arise due

o the consumption of the reagent by other sample components,hich reduce the rate that the target analyte is derivatised. When

he standard addition method was performed, the concentration

able 5rea in synthetic white wine determined using the HPLC procedure with xan-

hydrol derivatisation and a commercially available test kit

rea (mg L−1) Recovery (%)

ctual HPLC Test kit HPLC Test kit

2.0 2.0 1.5 100 753.0 3.0 2.6 100 874.0 3.9 3.5 98 886.0 6.3 6.5 105 1080.0 10.1 10.3 101 1032.0 11.9 12.1 99 101

1

ot

4

scdtomu

8 9.6 9.1 95 1030 11.2 11.1 92 102

f urea in the white wine was found to be 2.2 mg L−1, comparedo 2.0 mg L−1 using the external standard approach.

. Conclusion

The HPLC procedure described in this paper is a simple,ensitive, low-cost alternative for the determination of urea inomplex sample matrices. The automation of the xanthydrolerivatisation step reduced manual labour, sample preparation

ime and reagent consumption, and improved the reproducibilityf the analysis. For the determination of urea in human or ani-al urine, a rapid and robust separation with a retention time for

rea of approximately 2 min was developed. The results were in

atogr.

gkttdeamnw

R

[[[

[[

[[[

[[[

[[[[[

[

[

[

[

S. Clark et al. / J. Chrom

ood agreement with those using a commercially available testit. The relatively high concentrations of ammonia comparedo urea in wines can lower the accuracy of test kits based onhe enzyme catalysed hydrolysis of urea, but the HPLC proce-ure was unaffected by the presence of ammonia. Some matrixffects were observed and therefore calibration using standarddditions may be required for the analysis of wine. Further-ore, this approach has great potential for the development of

ew HPLC methods to determine other compounds that reactith xanthydrol, such as amides and substituted ureas.

eferences

[1] A.J. Taylor, P. Vadgama, Ann. Clin. Biochem. 29 (1992) 245.[2] P.S. Francis, S.W. Lewis, K.F. Lim, Trends Anal. Chem. 21 (2002) 389.[3] D. Lefier, Bull. Int. Dairy Fed. 315 (1996) 35.[4] D.F. Lambert, J.E. Sherwood, P.S. Francis, Aust. J. Soil Res. 42 (2004)

709.[5] N.M. Price, P.J. Harrison, Mar. Biol. 94 (1987) 307.[6] M. Revilla, J. Alexander, P.M. Glibert, Limnol. Oceanogr. Methods 3

(2005) 290.[7] P.S. Francis, Aust. J. Grape Wine Res. 12 (2006) 97.[8] R.B.H. Wills, J. Silalahi, M. Wootton, J. Liq. Chromatogr. 10 (1987) 3183.[9] A. Abshahi, S.S. Goyal, D.S. Mikkelsen, Soil Sci. Soc. Am. J. 52 (1988)

969.10] T. Matsudo, M. Sasaki, Biosci. Biotechnol. Biochem. 59 (1995) 827.11] S. Kodama, T. Suzuki, J. Food Sci. 60 (1995) 1097.12] P. Dallet, L. Labat, E. Kummer, J.P. Dubost, J. Chromatogr. B 742 (2000)

447.

[

[[

A 1161 (2007) 207–213 213

13] R. Fosse, Compt. Rend. 158 (1914) 1432.14] L. Hugounenq, A. Morel, C. R. Seances Soc. Biol. Ses. Fil. 74 (1913)

1055.15] R.G. Martinek, J. Am. Med. Technol. 31 (1969) 678.16] A.R. Butler, D. Walsh, Trends Anal. Chem. 1 (1982) 120.17] W. Horwitz (Ed.), Official Methods of Analysis of AOAC International,

17th ed., Association of Official Analytical Chemists (AOAC) Interna-tional, Gaithersburg, MD, 2000, p. 56, Ch. 16.

18] R.F. Phillips, B.M. Pitt, J. Am. Chem. Soc. 65 (1943) 1355.19] W. Lang, Arzneim.-Forsch. 1 (1951) 230.20] U. Hellmann, I. Luederwald, M. Neuhaeuser, Fresenius Z. Anal. Chem.

325 (1986) 290.21] W.R. Fearon, Analyst 69 (1944) 122.22] S.R. Dickman, W.L. Westcott, J. Biol. Chem. 210 (1954) 481.23] S.R. Dickman, A.L. Crockett, J. Biol. Chem. 220 (1956) 957.24] S. Mizukami, Y. Kanaya, Yakugaku Zasshi 83 (1963) 1152.25] K. Hirota, M. Kawase, S. Ohmori, T. Kishie, J. Chromatogr. 277 (1983)

165.26] P. Herbert, L. Santos, M. Bastos, P. Barros, A. Alves, J. Food Sci. 67 (2002)

1616.27] M.J. Veiga, P. Herbert, T. Simoes, A. Oliveira, A. Alves, Presented at the

4th International Conference on Instrumental Methods of Analysis: ModernTrends and Applications, Iraklion, Greece, October 2005, Poster P-III-16.

28] Urea/Ammonia (Rapid) Assay Procedure K-URAMR 11/05, MegazymeInternational, Bray, Ireland, 2004.

29] B. Coxon, A.J. Fatiadi, A. Cohen, H.S. Hertz, R. Schaffer, Org. Magn.

Reson. 13 (1980) 187.

30] R.N. Dickerson, A.C. Tidwell, G. Minard, M.A. Croce, R.O. Brown, Nutri-tion 21 (2005) 332.

31] B. Yang, L. Bankir, Am. J. Physiol. Renal Physiol. 288 (2005) F881.32] B. Zimmerli, J. Schlatter, Mutat. Res. 259 (1991) 325.