Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry

Copyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 1346–1353 (2008)DOI: 10.1002/bmc

1346 B. Crow et al.ORIGINAL RESEARCH ORIGINAL RESEARCH

Copyright © 2008 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 22: 1346–1353 (2008)Published online 25 July 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/bmc.1064

Analysis of urinary aromatic acids by liquidchromatography tandem mass spectrometry

Brian Crow,* Michael Bishop, Ekaterina Paliakov, Dean Norton, Joe George and J. Alexander Bralley

Metametrix Clinical Laboratory, 3425 Corporate Way, Duluth, GA 30096, USA

Received 1 February 2008; revised 12 March 2008; accepted 13 March 2008

ABSTRACT: The separation and detection of 11 urinary aromatic acids was developed using HPLC-MS/MS. The methodfeatures a simple sample preparation involving a single-step dilution with internal standard and a rapid 8 min chromatographicseparation. The accuracy was evaluated by the recovery of known spikes between 87 and 110%. Inter- and intra-assay precision(CV) was below 11% in all cases and the analytes were observed to be stable for up to 8 weeks when stored at −20°C.The method was validated based upon linearity, accuracy, precision and stability and was used to establish reference intervals forchildren and adults. Copyright © 2008 John Wiley & Sons, Ltd.

KEYWORDS: aromatic acids; LC-MS/MS; biomarkers

*Correspondence to: B. Crow, Metametrix Clinical Laboratory, 3425Corporate Way, Duluth, GA 30096, USA.E-mail: [email protected]

Abbreviations used: DHPP, 3,4-dihydroxyphenylpropionic acid;HIP, hippuric acid; HVA, homovanilic acid; IND, indican; KYN,kynurenic acid; PAA, phenylacetic acid; p-OH BA, p-hydroxybenzoicacid; p-OH PAA, p-hydroxyphenylacetic acid; p-OH PLA, p-hydroxyphenyllactic acid; VMA, vanilmandelic acid; XAN,xanthurenic acid.

INTRODUCTION

The analytical measurement of urinary vanilmandelic acid(VMA), homovanilic acid (HVA), hippuric acid (HIP),indican (IND), kynurenic acid (KYN), xanthurenic acid(XAN), p-hydroxyphenylacetic acid (p-OH PAA), p-hydroxyphenyllactic acid (p-OH PLA), phenylaceticacid (PAA), p-hydroxybenzoic acid (p-OH BA) and3,4-dihydroxyphenylpropionic acid (DHPP) can beuseful for the determination of a number of physio-logical issues and nutritional status. The characteristicsof these acids have been extensively studied and arewell represented in the literature. The urinary catecho-lamine metabolites VMA and HVA are good markersfor neuroblastoma, the most common solid tumor inchildren (Strenger et al., 2007; Gregianin et al., 1997;Tuchman et al., 1985). The measurement of urinaryHIP has been used to assess hazardous exposure totoluene (Tanaka et al., 2003; Wiwanitkit et al., 2002;Amorim and Alvarez-Leite, 1997) and the dietaryintake of benzoic acid (Christiani et al., 2000) andpolyphenols (Rios et al., 2003; Clifford et al., 2000).Urinary IND has been linked to oxidative stress inpatients with chronic renal failure (Dou et al., 2007)and is responsible for imparting the characteristic colorin purple urine bag syndrome (Ihama and Hokama,

2002). The tryptophan metabolites KYN and XANhave been reported to be elevated in the urine offebrile patients (Shaw and Feigin, 1971) and are com-monly used to evaluate vitamin B6 deficiency (Leklem,1990; el-Shawy et al., 1988; Lee and Leklem, 1985).Genetic tyrosinemias have been diagnosed by urinaryelevations of the tyrosine metabolites p-OH PAA andp-OH PLA (Scott, 2006). Urinary excretion of PAAhas been used to detect phenylketonuria (Goodwinet al., 1975; Blau, 1970) and has also been connectedto gut microflora overgrowth (Goodwin et al., 1994).The polyphenol urine metabolites p-OH BA andDHPP have been monitored for overgrowth of colonmicroflora and diet (Gonthier et al., 2003a).

The measurement of these compounds is routinelyperformed using a variety of techniques that includeHPLC, GC-MS and LC-MS (Shariati et al., 2007; Chenet al., 1996; Arin et al., 1992; Hommes, 1999; Gonthieret al., 2003b; Magera et al., 2003). However, in nearlyall of the published reports these methods only provideinformation regarding single compounds. In a few casesmore than one analyte can be determined, but no com-prehensive assessment has been developed on such abroad spectrum of urinary aromatic acids.

Presented in this report is a validated method for theanalysis of various aromatic acids that is both robustand rapid. The measurement of these urinary markerscan provide information regarding a number of im-portant health-related issues including disease states,nutrition and toxic exposure. The utility of this methodis found in the potential to examine these conditionswithout the need to perform multiple sample prepara-tions and analyses. The sample preparation required forthis method is only the addition of internal standardprior to analysis. The total chromatographic run time


Analysis of urinary aromatic acids by HPLC-MS/MS 1347ORIGINAL RESEARCH

for the developed method is 8 min, which includesthe time required to re-condition the analytical column.The method provides quantitative results for 11 aromaticacids and was validated based upon linearity, accuracy,precision and stability. The limit of detection (LOD)and the limit of quantification (LOQ) were also evalu-ated and found to be suitable for clinical evaluations.Reference intervals for children and adults were estab-lished for the method.

EXPERIMENTAL

Instrumentation. The chromatographic system consisted ofa Waters (Milford, MA, USA) 2695 high-performance liquidchromatograph featuring a refrigerated autosampling com-partment and column heater. This system was connected toWaters Quattro-micro tandem mass spectrometer equippedwith an electrospray ionization source. Data was collectedand processed using MassLynx v4.1.

Reagents and supplies. Formic acid, ACS-grade, waspurchased from Mallinckrodt Chemicals (Phillipsburg, NJ,USA) and HPLC-grade acetonitrile was obtained from EMDChemicals Inc. (Darmstadt, Germany). The deuteratedisotopes of vanilmandelic acid, p-hydroxyphenyllactic acidand hippuric acid were purchased from CDN Isotopes(Quebec, Canada) and deuterated benzoic acid fromCambridge Isotopes (Andover, MA, USA). Formic acidammonium salt, p-OH PLA, XAN, KYN, p-OH BA, DPHP,PAA, VMA, HVA, p-OH PAA, IND and HIP wereobtained from Sigma Chemical (St Louis, MO, USA).

Preparation of solutions. The internal standard was pre-pared in a urine matrix. In a 200 mL volumetric flask, d3-vanilmandelic acid (d3-VMA, 2.5 mg), d5-benzoic acid (d5-BA,2.5 mg), d4-p-hydroxybenzoic acid (d4-p-OH BA, 5.0 mg) andd2-hippuric acid (d2-HIP, 125 mg) were dissolved in pooledurine and stored at 4°C for up to one month without anydegradation. A standard stock solution with a concentration5 times greater than the highest calibrator was prepared bydissolving the reported amount of aromatic acids (Table 1),with a 95:5 water–acetonitrile solution in a 200 mL volumetric

flask. Working solutions of the calibrators were prepared byserial dilution with deionized water. All standard solutionswere stored at 4°C for up to one month.

Sample collection and preparation. Urine samples werecollected in tubes containing thymol as a preservative, andstored at −20°C until time for analysis. Samples wereprepared by addition of 350 μL of either calibrator or urine to650 μL of internal standard matrix solution in an autosamplervial.

Chromatographic separation. Separation was performed ona Phenomenex (Torrance, CA, USA) Synergi Polar-RP 4 μm80 Å, 2.0 × 50 mm, analytical column maintained at 40°Cthroughout the method. The mobile phase consisted of 10 mM

ammonium formate adjusted to a pH of 3.5 with formic acid(MPA) and acetonitrile with 0.15% v/v formic acid (MPB).Samples were injected (15 μL) on column with initial condi-tions set to 95% MPA and 5% MPB at a flow rate of 0.4 mL/min. The concentration of MPB was increased by the use ofWater’s concave gradient 7 to 50% over 4 min followed bya 1 min hold. The flow rate was increased to 0.5 mL/min at5 minutes and held until the start of the next injectioncycle. The mobile phase composition returned to the originalconditions at 6 min. The total run time, including column re-equilibration, was 8.0 min.

Evaluation of matrix effects. Matrix effects were evaluatedusing a simple matrix matching experiment (Bishop et al.,2007; Norton et al., 2007). Specifically, matrix effects wereevaluated by comparing the slopes of calibration curvesrepresenting standard solutions prepared in solvent (withoutmatrix) and prepared in urine (with matrix). For both calibra-tion curves, linear plots were made for the area under thecurve versus the standard concentration. The slopes for eachcurve, with and without matrix, were compared usingStudent’s t-test to determine the effects of the matrix onanalyte sensitivity.

Mass spectroscopy conditions. Detection of all analytes wascarried out in negative electrospray ionization mode. Massspectrometer conditions were obtained by a direct infusion ofindividual aromatic acid standard solutions in line with theHPLC set to initial mobile phase conditions. The desolvation

Table 1. Analyte stock concentration and calibration levels

Analyte Amount (mg) Stock (μg/mL) Calibration range (μg/mL)a

p-Hydroxyphenyllactic acid 5.00 25.0 0.313–5.00Xanthurenic acid 10.0 50.0 0.625–10.0Kynurenic acid 10.0 50.0 0.625–10.0p-Hydroxybenzoic acid 10.0 50.0 0.625–10.0Dihydroxyphenylpropionic acid 10.0 50.0 0.625–10.0Phenylacetic acid 10.0 50.0 0.625–10.0Vanilmandelic acid 20.0 100 1.25–20.0Homovanillic acid 30.0 150 1.88–30.0p-Hydroxyphenylacetic acid 100 500 6.25–100Indican 400 2000 25.0–400Hippuric acid 2000 10000 125–2000

a Blank not included.


1348 B. Crow et al.ORIGINAL RESEARCH

and nebulizer nitrogen gasses were maintained at 650 and50 L/h, respectively. The capillary voltage was set to 2.0 kV,with source and desolvation temperatures at 140 and 350°C,respectively. The cone and collision settings were establishedindividually for each analyte and internal standard for multiplereaction monitoring (MRM) detection (Table 2). The dwelltime for each mass was 0.1 s, and each mass was collected atunit mass resolution.

Measurement of urinary creatinine. Urinary creatinineconcentration was measured on a Cobas Mira Plus using acreatinine assay kit purchased from Roche (Quebec, Canada)following a modified picric acid method (Slot, 1965).

Method validation. The method was validated based uponlinearity, accuracy, precision, and analyte stability (long-termstorage and prepared sample; CLSI, 2007; CLIA, 1988). Theanalytes were quantified from a standard curve, in matrix.The six point calibration curve was fitted by linear regression,including the intercept for blank subtraction (y = mx + b) andweighted by 1/x. The peak area for each acid was collectedand the response was calculated from the ratio of analytepeak area to internal standard peak area. The concentrationvalue was obtained by blank subtraction followed by divisionby the slope from the standard curve. The internal standardd3-VMA was used for VMA and HVA, d5-BA was used forPAA, d4-p-OH BA was used for p-OH BA, p-OH PLA,p-OH PAA, XAN, KYN and DHPP, and d2-HIP was used asan internal standard for HIP and IND.

The linear range was established individually for eachanalyte, in matrix, to encompass the clinically significantregion of the patient distribution and to be above the LOQof each acid. A standard curve consisting of a blank andsix calibrators was constructed while another blank andsix calibrators were prepared and analyzed as unknowns.The relative standard deviation (%RSD) and residual ofeach analyte’s concentration from their expected values wascalculated and used to evaluate linearity over the calibrationrange. Values ≤15% were considered acceptable.

Accuracy was evaluated by a spike recovery experiment.Three spiking solutions of concentrations covering the entirecalibration range were prepared from the calibration stock

by serial dilution with water. Spiked samples were preparedby the addition of 100 μL of corresponding spike solution(water was used for the base spike) to 900 μL of pooledurine. Each sample was prepared five times and quantifiedfrom a standard curve. The average of five readings was usedin all calculations. Recovery of the spike was calculated as apercentage of the difference between the measured spikedsample concentration and base sample concentration dividedby the concentration of the added spike. Recoveries between85 and 115% were considered acceptable.

Precision was measured by the variation of analyte con-centration from two urine pools over a 10 day period. Thefirst pool was spiked with a standard solution to produceanalyte concentrations in the middle of the calibration curvewhile the second pool was spiked to produce concentrationsat the upper end of the curve. The intra-assay precision wascalculated from 10 samples of each medium and high concen-tration level measured in a single day. Two samples of eachpool level were run once a day for 10 days to assess the inter-assay precision. Variation of ≤15% was considered acceptablefor precision.

Analyte stability was evaluated in matrix at three differentconcentrations stored at room temperature (25°C), refriger-ated (4°C) and frozen (−20°C). Additionally, stability wascalculated during three freeze–thaw cycles, one cycle per dayfor three days, to simulate shipping and re-sampling condi-tions. The samples were analyzed on the date of collection tobe used for the initial reading and were stored accordingly.The percentage difference from the initial reading was calcu-lated for each day at each condition tested. Suitable stabilitywas taken to be a ≤20% difference.

Stability of a prepared sample was monitored over a periodof five days. Three samples of urine were prepared andmeasured once per day, in duplicate, starting with an initialmeasurement and evaluated at 24 h intervals through theremainder of the experiment. During the course of the evalu-ation, the samples were stored in the instrument’s refrigeratedsample compartment at 5°C. The preparation stability foranalyte and internal standard was evaluated based upon thepercentage deviation of the concentration of each from theirrespective initial values. Suitable stability was taken to be a≤20% difference.

Table 2. Mass spectroscopy conditions for select aromatic acids

Analyte Precursor ion (m/z) Product ion (m/z) Cone potential (V) Collision energy (eV)

p-Hydroxyphenyllactic acid 181 135 25 15Xanthurenic acid 204 160 30 15Kynurenic acid 188 144 25 13p-Hydroxybenzoic acid 137 93 25 12Dihydroxyphenylpropionic acid 181 59 30 15Phenylacetic acid 135 91 20 8Vanilmandelic acid 197 138 30 12Homovanillic acid 181 137 23 7p-Hydroxyphenylacetic acid 151 107 22 6Indican 212 80 30 19Hippuric acid 178 134 30 10d3-Vanilmandelic acid 200 138 30 13d5-Benzoic acid 126 82 30 13d4-p-Hydroxybenzoic acid 141 97 25 15d2-Hippuric acid 180 136 30 11



Limits of detection and quantitation. The LOD and LOQwere determined from the average of five replicate calibrationstandard dilutions covering at least two orders of magnitudeand starting at the lowest possible concentration value withan average value of ≤15%RSD. The average concentrationvalues were plotted vs the theoretical concentration valuesand the data was fit to a linear curve. The LOD and LOQvalues were calculated using the equations: LOD = 3 ×(standard error for the y estimate)/slope and LOQ = 10 ×(standard error for the y estimate)/slope (Mocak et al., 1997).

Reference intervals. Urinary concentration of the listed aro-matic acids was measured in over 300 intra-laboratory patientsamples following method validation. In total, urine samplesfrom 127 children (0–13 years) and 200 adults (14+ years),representing a normal population, were analyzed. The valueswere normalized by the sample’s creatinine concentrationand reported as μg/mg creatinine. The reference intervals foreach age group were established by the use of non-parametricstatistics (Solberg, 1987) calculated by MedCalc v9.3.8.0 soft-ware. The data was evaluated to determine any differencesbetween the two age ranges and to establish working intra-laboratory reference intervals to identify abnormal results.

RESULTS AND DISCUSSION

LC–MS/MS

An ether-linked phenyl column with polar endcappingwas chosen to provide the best retention and separationof the aromatic acids. Using this stationary phase thechromatographic method for the separation of 11 acidsin under 5 min was developed. The total run time of8 min allowed for column re-equilibration and stabiliza-tion between sample injections. No co-eluting inter-ferences or cross-talk was observed in any MRM channel.A typical set of standard and patient chromatogramsare shown in Fig. 1. Identification of each peak wascarried out by a comparison of ion mass formation andretention times with those of authentic samples.

Negative electrospray ionization was used to chargethe analytes prior to sample introduction. Despite severalheteroatom containing analytes (XAN, KYN, IND,HIP and d2-HIP) displaying ion abundance in both thepositive and negative electrospray ionization modes,the latter was chosen to be used for all analytes so thatthe best signal for the remaining aromatic acids in thegroup could be achieved. Optimization of the detectorparameters was carried out on solutions of the purecompound, in initial mobile phase conditions, directlyinfused into the mass spectrometer. With this configura-tion, individual cone and collision settings were obtainedfor MRM detection, Table 2. General settings such asthe capillary voltage, gas flow rates and interface tem-perature settings were established separately to give thebest overall sensitivity for the method. The precursorion m/z for each analyte was observed at (M − H)−. In

nearly every case, the most abundant product ion m/zwas observed to be generated from the expectedneutral loss of 44 amu, corresponding to the loss ofCO2. However, several analytes produced more inter-esting high abundance fragments.

Fragmentation of VMA resulted in a net loss of mass59 corresponding to the loss of the carboxylic CO2 anda methyl group from the anisole methoxy group. Theanalogous fragmentation pattern was seen for d3-VMAwith loss of mass 62 corresponding to loss of CO2 andthe isotopically labeled anisole d3-methoxy group. Aloss of mass 46, consistent with loss of CO and water,was observed during the fragmentation of p-OH PLA.Indican experienced a loss of mass 132 consistent withloss of 3-oxyindole. Fragmentation of DHPP showedcleavage between the α- and β-carbon bond resulting inloss of dihydroxybenzyl, mass 122, in agreement with aprevious report (Gonthier et al., 2003b).

Matrix effects

The initial results of this experiment suggest significantmatrix effects on analyte sensitivity; a noticeable differ-ence between the slope of the calibration curve withoutmatrix and the curve with matrix suggests the presenceof a matrix effect on analyte response. Two approacheswere taken in order to correct for the changes inanalyte response caused by the matrix. First, a matrix-matched procedure, as mentioned above, was deve-loped to correct for the matrix effects by the additionof a pre-screened matrix to all of the test samples. Ineffect all samples were exposed to the matrix com-ponents allowing for the differences in sensitivity to bemimicked in the calibration standards. This allowed foraccurate quantification of analyte values without notice-able effects of the matrix. It is understood that the pre-screened matrix is subject to change from one batch toanother and therefore selected internal standards wereadded as a second measure to ensure the precision ofthe assay. With both measures in place to correct forthe observed matrix effect, an analytical method wasdeveloped that was both accurate and reproducible.

Method validation

The calibration curves produced slopes with goodcorrelation, r ≥ 0.995 in all cases. Deviations from thestandard values, based upon recovery, were less than15% for all analytes. Residuals about the line of regres-sion were less than 15% of the target value for allanalytes.

The accuracy of each analyte was evaluated basedon the percentage recovery for three levels of spikedsamples compared with a baseline of pooled urine. Thepercentage deviation from the theoretical value forthe recovered spike was less than 15% for all spikes.



Figure 1. Representative chromatograms for the aromatic acids and internal standards. The chromatograms on the left show acalibration standard. The chromatograms on the right show a typical patient. Time in minutes. This figure is available in colouronline at www.interscience.wiley.com/journal/bmc

Table 3. Recovery data for spiked aromatic acids in human urine

Base Low spike Mid spike High spike

Analyte (μg/mL) Observeda Addeda %b Observeda Addeda %b Observeda Addeda %b

p-OH PLA 0.296 1.22 0.99 93 2.53 2.22 101 5.46 5.00 103XAN 0.669 2.39 1.98 87 5.19 4.44 102 11.2 10.0 105KYN 1.95 3.72 1.98 89 6.26 4.44 97 10.8 10.0 88p-OH BA 0.448 2.52 1.98 105 4.89 4.44 100 10.8 10.0 104DHPP 5.85 8.02 1.98 110 10.3 4.44 100 15.2 10.0 94PAA 0.007 1.89 1.98 95 4.27 4.44 96 10.3 10.0 103VMA 4.36 8.18 3.95 97 12.3 8.89 89 24.2 20.0 99HVA 2.88 8.85 5.93 101 15.1 13.3 92 30.4 30.0 92p-OH PAA 12.5 31.1 19.8 94 56.8 44.4 100 105 100 92IND 65.1 145 79.0 102 256 178 108 474 400 102HIP 685 1093 395 103 1560 889 98 2633 2000 97

a The observed and added analyte concentrations are listed in μg/mL. b The percentage recovery is calculated by [(observed − base)/added] × 100.

The average recoveries from the three levels of spikeare reported in Table 3.

The %CVs for the inter- and intra-assay precisionare displayed in Table 4. The intra-assay precision was

less than or equal to 7% for the mid levels and nogreater than 10% for the high samples. The inter-assayprecision was less than or equal to 11% for the midlevels and no greater than 8% for the high samples.

www.interscience.wiley.com/journal/bmc



Table 4. Intra- and inter-assay precision for aromatic acids in human urine

Intra-daya Inter-dayb

Mid CV High CV Mid CV High CVAnalyte (μg/mL) (%) (μg/mL) (%) (μg/mL) (%) (μg/mL) (%)

p-Hydroxyphenyllactic acid 1.25 6 2.15 7 1.18 9 2.25 6Xanthurenic acid 2.49 5 4.61 4 2.31 6 4.46 5Kynurenic acid 2.54 5 4.77 3 2.45 7 4.53 5p-Hydroxybenzoic acid 2.81 6 5.05 7 2.74 6 5.17 5Dihydroxyphenylpropionic acid 2.57 7 4.91 10 2.60 11 4.91 7Phenylacetic acid 2.59 6 4.53 7 2.40 8 4.53 8Vanilmandelic acid 6.27 4 11.1 7 6.30 6 10.9 8Homovanillic acid 9.37 3 16.3 5 8.63 8 15.3 5p-Hydroxyphenylacetic acid 27.9 5 48.5 7 26.1 7 49.0 5Indican 117 2 211 5 104 5 197 5Hippuric acid 598 2 1075 2 576 4 1033 4

a n = 10. b Samples were prepared and analyzed in duplicate over a period of 10 days.

Figure 1. (Continued)



The %CVs for the inter-assay precision were higherthan the intra- for each analyte at the mid level whilethe variations were similar for the high samplesbetween inter- and intra-assay precision.

The long-term stability was monitored at three differentconcentrations in urine stored at room temperature(25°C), in the refrigerator (4°C) and in the freezer (−20°C).By examining the percentage difference between initialanalyte concentration and any changes over time, weobserved that all of the analytes were stable for up to8 weeks when stored at −20°C. Additionally, all ana-lytes were stable for three freeze and thaw cycles. Theconcentration of DHPP dropped dramatically afterone week when stored at room temperature while themajority of the remaining analytes were stable at roomtemperature for a period of 6 weeks. After this time,the observed concentrations of p-OH PLA, p-OHPAA, PAA, XAN and KYN deviated by more than20% of the initial value.

The stability of the acids in prepared urine sampleswas carried out on three individual urine samples overa period of five days. The samples were stored in theinstrument’s refrigerated sample compartment betweenanalyses. All analytes were observed to yield reproduciblevalues for the duration of the study.

Limits of detection and quantitation

The LOD (S/N = 3) and LOQ (S/N = 10) were measuredfor each analyte based upon the linear regression ofcalibration from the established linear range. Thelimits of detection and quantitation for the acids arereported in Table 5. The method provides amplesensitivity for the detection of abnormal values for eachanalyte given that the clinically significant results aremuch higher than the LOQ.

Reference intervals

The 95% reference intervals for children and adultsare listed in Table 5. The values were calculated froma right-sided distribution. Therefore, any value foundto be above the corresponding interval reported inTable 5 was outside of the normal population distribu-tion and considered to be abnormal. When the meanvalues of each acid from both populations were com-pared, we found a significant difference (p < 0.05)between the results for VMA, XAN, KYN, p-OH BA,p-OH PAA and HVA. In all cases, the reference inter-vals and population means for the assayed urinaryaromatic acids were higher in children than in adults.The ranges for urinary aromatic acids in μg/mg creatininewere observed to be: p-OH PLA (adults: <LOQ to4.74; children: <LOQ to 15.2), XAN (adults: <LOQ to2.16; children: <LOQ to 6.18), KYN (adults: 0.217–7.74;children: 0.668–10.5), p-OH BA (adults: <LOQ to15.9; children: 0.221–44.4), DHPP (adults: <LOQ to 4.45;children: <LOQ to 3.82), PAA (adults: <LOQ to74.9; children: <LOQ to 78.9), VMA (adults: <LOQ to7.34; children: 1.23–16.3), HVA (adults: <LOQ to 35.8;children: <LOQ to 251), p-OH PAA (adults: <LOQto 101; children: 1.56–477), IND (adults: 5.11–219;children: 5.95–695) and HIP (adults: <LOQ to 3880;children: <LOQ to 6370).

CONCLUSION

An analytical method for the quantification of 11urinary aromatic acids using HPLC with tandem MSdetection was developed and validated. The methodrequires only sample dilution with internal standardsolution prior to analysis. The separation was performed

Table 5. Limits of detection and quantitation and the 95% reference intervals for urinary aromatic acids in children and adults

Reference interval(μg/mg creatinine)a

Analyte LOD (μg/mL) LOQ (μg/mL) Children Adult

p-Hydroxyphenyllactic acid 0.008 0.027 <1.42 <1.41Xanthurenic acid 0.071 0.235 <1.53 <1.18Kynurenic acid 0.017 0.058 <4.59 <2.53p-Hydroxybenzoic acid 0.019 0.066 <12.8 <2.88Dihydroxyphenylpropionic acid 0.121 0.403 <0.861 <0.608Phenylacetic acid 0.043 0.142 <1.69 <0.364Vanilmandelic acid 0.094 0.313 <7.85 <4.68Homovanillic acid 0.077 0.256 <18.3 <10.4p-Hydroxyphenylacetic acid 0.119 0.396 <79.7 <35.7Indican 0.729 2.43 <154 <126Hippuric acid 0.820 2.73 <2150 <1110

a Calculated using non-parametric statistics.



in less than 5 min with a total run time of 8 minrequired to re-equilibrate the analytical column. Themethod displayed excellent linearity, accuracy andprecision while providing ample sensitivity to quantitatelow concentrations and accurately assess clinical abnor-mal concentrations of the urinary aromatic acids. Threefreeze and thaw cycles had no effect on analyte stabilityin urine and all analytes and prepared samples werefound to be stable for a period of 8 weeks at −20°C and5 days at 4°C, respectively. The validated method wasused to establish unique reference intervals for childrenand adult populations and offers a good approachfor screening urine for a wide variety of metabolicdisorders and toxic exposure.

REFERENCES

Amorim LC and Alvarez-Leite EM. Determination of o-cresol by gaschromatography and comparison with hippuric acid levels in urinesamples of individuals exposed to toluene. Journal of Toxicologyand Environmental Health 1997; 50: 401–407.

Arin MJ, Diez MT and Resines JA. Rapid and simple method forthe determination of urinary benzoic and phenylacetic acidsand their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography. Journal of Chromatography1992; 582: 13–18.

Bishop MJ, Crow BS, Kovalcik KD, George J and Bralley JA. Quan-tification of urinary zwitterionic organic acids using weak-anionexchange chromatography with tandem MS detection. Journal ofChromatography B Biomedical Applications 2007; 848: 303–310.

Blau K. Aromatic acid excretion in phenylketonuria. Analysis of theunconjugated aromatic acids derived from phenylalanine. ClinicaChimica Acta 1970; 27: 5–18.

Chen XB, Pagella JH, Bakker ML and Parra O. Determination ofaromatic metabolites in ruminant urine by high-performance liquidchromatography. Journal of Chromatography B Biomedical Appli-cations 1996; 682: 201–208.

Christiani DC, Chang SH, Chun BC and Lee WJ. Urinary excretionof hippuric acid after consumption of nonalcoholic beverages.International Journal of Occupational and Environmental Health2000; 6: 238–242.

CLIA. Clinical Laboratory Improvement Amendments GuidelinesSubpart K—Quality Systems for Nonwaived Testing—Section493.1253(b)(2) Standard: Establishment and Verification ofPerformance Specifications, 1988.

Clifford MN, Copeland EL, Bloxsidge JP and Mitchell LA. Hippuricacid as a major excretion product associated with black teaconsumption. Xenobiotica 2000; 30: 317–326.

CLSI. Clinical and Laboratory Standards Institute. Mass Spectrometryin the Clinical Laboratory: General Principles and Guidance;Proposed Guideline. CLSI document C50-P. Clinical and Labora-tory Standards Institute, Wayne, PA, USA, 2007.

Dou L, Jourde-Chiche N, Faure V, Cerini C, Berland Y,Dignat-George F and Brunet P. The uremic solute indoxyl sulfateinduces oxidative stress in endothelial cells. Journal of Thrombosisand Haemostasis 2007; 5: 1302–1308.

el-Shawy S, Osman M, el-Tabakh S, Khamis Y and Amine AK.Effect of administration of vitamin B6 at two levels of intake onxanthurenic acid excretion among oral contraceptive pill users.Journal of the Egyptian Public Health Association 1988; 63: 393–405.

Gonthier MP, Cheynier V, Donovan JL, Manach C, Morand C andMila I. Microbial aromatic acid metabolites formed in the gutaccount for a major fraction of the polyphenols excreted in urineof rats fed red wine polyphenols. Journal of Nutrition 2003a; 133:461–467.

Gonthier MP, Rios LY, Verny M-A, Remesy C and Scalbert A.Novel liquid chromatography–electrospray ionization mass spectro-metry method for the quantification in human urine of microbial

aromatic acid metabolites derived from dietary polyphenols.Journal of Chromatography B Biomedical Applications 2003b; 789:247–255.

Goodwin BL, Ruthven CRJ and Sandler M. Gas chromatographicassay of phenylacetic acid in biological fluids. Clinica Chimica Acta1975; 62: 443–446.

Goodwin BL, Ruthven CRJ and Sandler M. Gut flora and theorigin of some urinary aromatic phenolic compounds. BiochemicalPharmacology 1994; 47: 2294–2297.

Gregianin LJ, McGill AC, Pinheiro CM and Brunetto AL.Vanilmandelic acid and homovanillic acid levels in patients withneural crest tumor: 24-hour urine collection versus random sample.Pediatric Hematology and Oncololgy 1997; 14: 259–265.

Hommes FA. The assay of phenylacetic acid and 4-phenylbutyric acidin physiological fluids. Clinica Chimica Acta 1999; 284: 109–111.

Ihama Y and Hokama A. Purple urine bag syndrome. Urology 2002;60: 910.

Lee CM and Leklem JE. Differences in vitamin B6 status indicatorresponses between young and middle-aged women fed constantdiets with two levels of vitamin B6. American Journal of ClincialNutrition 1985; 42: 226–234.

Leklem JE. Vitamin B-6, a status report. Journal of Nutrition 1990;11: 1503–1507.

Magera MJ, Thompson AL, Matern D and Rinaldo P. Liquidchromatography–tandem mass spectrometry method for the deter-mination of vanillylmandelic acid in urine. Clinical Chemistry 2003;49: 825–826.

Mocak J, Bond AM, Mitchell S and Scollary G. A statistical overviewof standard (IUPAC and ACS) and new procedures for determin-ing the limits of detection and quantification: application to voltam-metric and stripping techniques. Pure and Applied Chemistry 1997;69: 297–328.

Norton D, Crow B, Bishop M, Kovalcik K, George J and Bralley JA.High performance liquid chromatography-tandem mass spectro-metry (HPLC/MS/MS) assay for chiral separation of lactic acidenantiomers in urine using a teicoplanin based stationary phase.Journal of Chromatography B Biomedical Applications 2007; 850:190–198.

Rios LY, Gonthier MP, Remesy C, Mila I, Lapierre C, Lazarus SA,Williamson G and Scalbert A. Chocolate intake increases urinaryexcretion of polyphenol-derived phenolic acids in healthy humansubjects. American Journal of Clinical Nutrition 2003; 77: 912–918.

Scott CR. The genetic tyrosinemias. American Journal of MedicalGenetics Part C (Seminars in Medical Genetics) 2006; 142C: 121–126.

Shariati S, Yamini Y, Darabi M and Ammi M. Three phase liquidphase microextraction of phenylacetic acid and phenylpropionicacid from biological fluids. Journal of Chromatography B BiomedicalApplications 2007; 855: 228–225.

Shaw RC and Feigin RD. Excretion of kynurenic acid andxanthurenic acid during infection. Peadiatrics 1971; 47: 47–56.

Slot C. Plasma creatinine determination. A new and specific Jaffereaction method. Scandinavian Journal of Clinical and LaboratoryInvestigation 1965; 17: 381–387.

Solberg HE. Approved recommendation on the theory of referencevalues part 5. Statistical treatment of collected reference values.Determination of reference limits. Journal of Clinical Chemistryand Clinical Biochemistry 1987; 25: 645–656.

Strenger V, Kerbl R, Dornbusch HJ, Ladenstein R, Ambros PF,Ambros IM and Urban C. Diagnostic and prognostic impact ofurinary catecholamines in neuroblastoma patients. Pediatric Bloodand Cancer 2007; 48: 504–509.

Tanaka K, Maeda T, Kobayashi T, Tanaka M and Fukushima T.A survey of urinary hippuric acid and subjective symptoms amongoccupational low toluene exposed workers. Fukushima Journal ofMedical Science 2003; 49: 129–139.

Tuchman M, Morris CL, Ramnaraine ML, Bowers LD and Krivit W.Value of random urinary homovanillic acid and vanillylmandelicacid levels in the diagnosis and management of patients withneuroblastoma: comparison with 24-hour urine collections.Pediatrics 1985; 75: 324–328.

Wiwanitkit V, Suwansaksri J, Srita S and Fongsoongnern A. Highlevels of hippuric acid in the urine of Thai press workers. SoutheastAsian Journal of Tropical Medicine and Public Health 2002; 33:624–627.

Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry

Documents

Transcript of Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry