Casetta, B

La Marca, Giancarlo

Zammarchi, Enrico

English

Florence Research

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom. 2003; 17: 788–793Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.980Rapid determination of orotic acid in urine by a fastliquid chromatography/tandem mass spectrometricmethodGiancarlo la Marca1*, Bruno Casetta2 and Enrico Zammarchi11Metabolic and Neuromuscolar Unit, 1st Pediatric Clinic, A. Meyer Children’s Hospital, University of Florence, 50132 Florence, Italy2Applied Biosystems, 20052 Monza, Milan, ItalyReceived 9 December 2002; Revised 10 February 2003; Accepted 10 February 2003Quantification of orotic acid (uracil-6-carboxylic acid) in urine is an important tool to diagnose someinherited diseases, such as urea cycle disorder (OTCD) and hereditary orotic aciduria. New rapidanalytical methods are necessary to provide high-throughput orotic acid analyses. A new analyticalmethod has been developed for the rapid analysis of orotic acid in urine by liquid chromatographycoupled with ion spray tandem mass spectrometry (LC/MS/MS). After a sample dilution 1:20, theanalysis was performed in the selected reaction monitoring mode in which orotic acid was detectedthrough the transition m/z 155 to 111. The retention time was 3.9 min in a 4.5-min analysis. Dailycalibration between 0.5–5.0mmol/L of orotic acid, corresponding to 10–100mmol/L in urine beforethe 1:20 dilution, offered consistent linearity and reproducibility. Interassay coefficient of variance(c.v.) was 4.97% at a mean concentration of 10.99mmol/L. The sensitivity and specificity of tandemmass spectrometry permitted a high volume of analyses of orotic acid. The sample preparation issimple, inexpensive and not time demanding. Copyright # 2003 John Wiley & Sons, Ltd.The analysis of orotic acid (OA) is used in clinical chemistry todiagnose some diseases associated with urea cycle disorders;ornithine transcarbamylase deficiency (OTCD)1 is the mostcommon, and also hereditary orotic aciduria.2 The urea cyclehas three roles: it produces urea as the waste product incor-porating nitrogen not used for biosynthetic purposes, it isinvolved in the biochemical reactions that degrade andsynthesise arginine de novo, and it plays a fundamental rolein pH homeostasis.3Ornithine transcarbamylase, a mitochondrial matrixenzyme, catalyses the synthesis of citrulline from carbamylphosphate and ornithine. OTCD male patients show classicalclinical symptoms such as vomiting, lethargy, and confusion;clinical laboratory indications are high values of glutamineand ammonia, low citrulline, and high urinary excretion oforotic acid. Also, female manifesting carriers generally havehigher than normal values of urinary orotic acid excretiondue to X-inactivation.Hereditary orotic aciduria is a pyrimidine synthesis defectcaused by uridine-50-monophosphate (UMP) synthase defi-ciency.4 This multienzyme catalyses the synthesis of UMPstarting from orotic acid via orotidine-50-monophosphate.Some characteristic clinical features are anaemia, hematuria,pallor, diarrhoea, retarded and poor development, whilelaboratory indications include very high urinary orotic acidexcretion.Therefore, determination of orotic acid levels in urineis becoming an assay routinely performed in clinicalanalysis.5–13 To date, orotic acid has been determined mostlyby liquid chromatography (HPLC) with UV detection at275 nm.14 This approach is quite cumbersome in thesubsequent integration and interpretation process. Signalsfrom a ‘normal’ specimen are quite difficult to quantify sincethe LC-UV peaks are very close to the baseline (lack ofadequate sensitivity) and are sometimes not sufficientlyresolved from other endogenous components owing to thenonspecific wavelength chosen for detection. In addition thechromatographic run is time demanding.In this paper we evaluate the possibility to take advantageof tandem mass spectrometry15 (MS/MS) as the basis for arapid method for determination of OA in urine with verysimple preparation, which could be a candidate for high-throughput routine analyses. The main criteria in developinga robust high-throughput analytical method center aroundminimal sample preparation, no derivatization, high sensi-tivity and specificity, high throughput capability, andminimal instrument maintenance. It was hoped that such amethod would provide a suitable tool for a large-scaleinvestigation.EXPERIMENTALMaterialsAn orotic acid standard was purchased from Sigma-Aldrich(Steinheim, Germany), and a stock solution was made inCopyright # 2003 John Wiley & Sons, Ltd.*Correspondence to: G. la Marca, Metabolic and NeuromuscolarUnit, 1st Pediatric Clinic, A. Meyer Children’s Hospital, Uni-versity of Florence, 50132 Florence, Italy.E-mail: giancalm@yahoo.comwater (corresponding to 1 mg/mL OA). Successive dilutionswere made using a 50% aqueous solution of acetonitrile con-taining 5 mmol/L ammonium acetate. All chemicals and sol-vents were of the highest purity available from commercialsources and were used without further purification.Sample preparationUrine samples were prepared by dilution (1 vol. urineþ19 vol. water containing 0.1% formic acid). For the purposesof this study, urine samples with different creatinine concen-trations were spiked with known amounts of orotic acid. 2 mLof the diluted samples were injected for the LC/MS/MSexperiments.MethodsAn Applied Biosystems-Sciex (Toronto, Canada) API 2000bench-top triple-quadrupole mass spectrometer, equippedwith a TurboIonSpray source, was employed for this study.The TurboIonSpray source was operated in negative ionmode with a needle potential of 4800 V and with a ‘turbo’gas flow of 10 L/min of air heated at 3008C (nominal heating-gun temperature). Mass calibration and resolution adjust-ments on the resolving quadrupoles were performed auto-matically by using a 104 mol/L PPG solution introducedvia the built-in infusion pump. The peak width was set onboth resolving quadrupoles at 0.7 Th (measured at halfheight) for all MS and MS/MS experiments.Collision-activated dissociation (CAD) MS/MS was per-formed in the LINAC Q2 collision cell, operating with10 mTorr pressure of nitrogen as collision gas. The decluster-ing potential (DP) and collision energy (CE) were automati-cally optimized for orotic acid using the instrument software.The resulting DP was10 V, and optimal CE was found to be12 eV (laboratory frame).MS and MS/MS spectra were collected in continuous flowmode by connecting the infusion pump directly to theTurboIonSpray source. A standard solution of 10 ng/mL ofOA in an aqueous solution of 50% acetonitrile containing5 mmol/L ammonium acetate was infused at 10mL/min. Thequantitation experiments were performed using a series 1100Agilent Technologies (Waldbronn, Germany) CapPumpcoupled to an Agilent Micro ALS autosampler, both fullycontrolled from the API 2000 datasystem. Liquid chromato-graphy was performed using a Phenomenex Luna C18 5mm,3 150 mm HPLC column (Chemtek-Analitica, AnzolaEmilia, Italy). Column flow rate was 0.45 mL/min using anaqueous solution of 60% acetonitrile containing 0.1% formicacid. The eluent from the column was directed to theTurboIonSpray probe with a split ratio of 1:5.The acquired data were processed using the Analyst 1.1proprietary software including the ‘Explore’ option (forchromatographic and spectral interpretation) and the ‘Quan-titate’ option (for quantitative information generation).RESULTS AND DISCUSSIONFigure 1 shows the structure of orotic acid. Figure 2 shows themass spectrum collected via infusion through the TurboIon-Spray probe at 10 mL/min of a solution of 10 ng/mL of oroticacid. The [M–H] ion is clearly observed atm/z 155.1. Figure 3shows the MS/MS spectrum obtained by fragmenting the[M–H] precursor ion of OA using the conditions describedabove. From these experiments, the MS/MS transition cho-sen for the quantitative experiments (selected reaction mon-itoring, SRM) is m/z 155.1! 111.1. Figure 4 displays a typicalcalibration curve obtained in the concentration range 0.5–5.0 mmol/L (10–100 mmol/L before the 1:20 dilution).Figure 5 shows the SRM chromatograms obtained for a stan-dard solution (1.0mmol/L, left) and a urine sample (right)containing 1.6 mmol/L diluted 20-fold. Chromatographicconditions were chosen in order to minimise the run time(less than 4.5 min) since the outstanding specificity is pro-vided by the MS/MS detection. Comparison of the two panesof Fig. 5 indicates that the bulk of the matrix does not lead toany extra chromatographic peaks by this method.The sample dilution ratio (20:1) and injection volume (2 mL)were selected in order to not overload the chromatographiccolumn even after a considerable number of sample injec-tions (the equivalent of 0.1 mL of original urine is introducedthrough each single injection); the injection sample volumewas selected after several trials. The dilution was made withwater and no organic solvent, resulting in a solvent withweaker elution capabilities than the LC eluent used forreversed-phase LC; this was intended to pack the injectedsample plug at the front end of the column. The outcome isthat the column shows robust performance as long as only0.1 mL of the original urine is loaded (2 mL of 20dilutedsample), whatever the salt concentration in the specimen(expressed by the creatinine concentration in the presentapplication).Concerning calibration, it was considered that externalcalibration with aqueous standards was sufficient for thepresent purpose. To test this, a range of urine samples withvery different densities and creatinine concentrations werespiked with 10mmol/L of OA. Samples were processed andquantitated by comparison with aqueous standards asdescribed in the Experimental section (results are reportedwithout normalisation for the creatinine content). As anexample, Table 1 reports the data obtained for three samples,with low (0.58 mmol/L), medium (2.3 mmol/L) and high(3.74 mmol/L) creatinine concentrations.Including all the other samples examined, discrepanciesbetween spiked values and measured values using externalcalibration with aqueous standards were within 6%. Thesedata lead to the conclusion that a simple external calibrationwith aqueous standard should be viable in these experi-mental conditions, for present purposes.In order to assess the robustness of the method, severalurine samples with different creatinine contents wereprocessed several times, resulting an intra-day reproduci-Figure 1. Structure of uracil-6-carboxylic acid (orotic acid).Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 788–793Fast LC/MS/MS analysis of orotic acid in urine 789Figure 2. Mass spectrum in negative-ion mode recorded by infusing a solution of 10 ng/mL of orotic acid.Figure 3. MS/MS spectrum obtained by fragmenting the [M–H] precursor ion (m/z 155.1) of OA innegative-ion mode with a collision energy of 12 eV.790 G. la. Marca, B. Casetta and E. ZammarchiCopyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 788–793bility below 6.4% for values above 5mmol/L (see Table 2). Inan inter-day reproducibility test, reproducibility valuesbetter than 5% were obtained (see Table 3).With the proposed experimental parameters, the estimatedlimits of detection (signal-to-noise (S/N)¼ 3) in urine were0.15 mmol/L. With the same experimental parameters, theFigure 4. Typical calibration curve obtained in the concen-tration range 0.5–5.0mmol/L of orotic acid, corresponding to10–100mmol/L in urine before the 1:20 dilution.Figure 5. SRM chromatograms obtained for a standard solution (1.0 mmol/L, left) and a urine sample(right) containing 1.6mmol/L OA, 20 times diluted.Table 1. Determination of OA in urinewith different creatinineconcentrations, against aqueous calibration standards withoutinternal calibrationOA concentrationmmol/LCreatinineconcentrationmmol/LDetermina-tion againstaq. standard(no IS) mmol/L (%)Ratio Read/Calculated1.02 0.58 1.00 98.011.02 0.58 10.44 94.70.97 2.3 0.97 100.010.97 2.3 10.3 93.91.36 3.74 1.4 102.911.36 3.74 11.52 101.4Fast LC/MS/MS analysis of orotic acid in urine 791Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 788–793limits of quantitation in urine (S/N¼ 10) were 0.383 mmol/L.No deterioration in column efficiency was observed afteranalysis of 200 urine samples.CONCLUSIONSThe proposed method is characterised by a fast and very sim-ple sample preparation. Calibration is made externally withaqueous standards with no need for expensive isotopicallylabeled standards. Instrumental analysis time is less than4.5 min. Fast chromatography, combined with the specificityprovided by tandem mass spectrometric detection, allows fora fast, robust and specific procedure for OA determination.Several factors have made it possible to develop this simpleapplication. These include the good MS/MS sensitivity innegative-ion mode even with the presence of formic acid(0.1%) in the chromatographic eluent, the MS/MS specificityguaranteed even at such low m/z values by the resolution setfor both quadrupoles (peak widths 0.7 Th, FWHM), and therobustness of the TurboIonSpray interface, which makespossible the injection of crude samples after a simple dilution,even without the need for an internal standard. The latteraspect is possible provided that the chromatographic reten-tion factor (k-factor) is large enough relative to the columnvoid volume.The detection limit of 0.15 mmol/L of OA in urine shouldnot be regarded as the ultimate figure since an increase ofsensitivity could be expected by downsizing the LC columndiameter. However, the value achieved here seems to fulfillthe sensitivity requirements of the present application, and ithas been decided to stay with the 3.0-mm column since it ismore robust for several hundreds of injections. Switching to asmaller column, e.g., 1 mm i.d., should increase the sensitiv-ity by a factor of about one order of magnitude.REFERENCES1. Pelet A, Rotig A, Bonaiti-Pellie C, et al. Hum. Genet. 1990; 84:167.2. Simmonds HA, Webster DR, Becroft DM, Potter CF. Eur. J.Clin. Invest. 1980; 10: 333.Table 2. Intra-day reproducibility for OA-spiked urine with different creatinine concentrations. (Quantitation against aqueouscalibration standards)Urine with 21 mmol/L, low creatinine Urine with 20.03mmol/L, medium creatinine20.18 20.5820.01 21.1220.83 21.1221.49 19.7021.16 20.7621.00 18.80Average 20.78 Accuracy 98.65 Average 20.35 Accuracy 101.58St. Dev. 0.57 St. Dev. 0.92c.v. % 2.77 c.v. % 4.52Urine with 11 mmol/L, low creatinine Urine with 10.03mmol/L, medium creatinine11.75 9.1611.47 8.5811.78 9.3611.35 8.8210.27 9.7612.04 10.16Average 11.44 Accuracy 104.02 Average 9.31 Accuracy 92.79St. Dev. 0.62 St. Dev. 0.59c.v. % 5.46 c.v. % 6.31Urine with 0.6 mmol/L, low creatinine Urine with 21.36mmol/L, high creatinine0.85 20.370.39 20.180.65 19.390.56 21.430.51 20.570.66 21.64Average 0.60 Accuracy 100.61 Average 20.59 Accuracy 96.42St. Dev. 0.16 St. Dev. 0.83c.v. % 25.68 c.v. % 4.04Table 3. Example of Intraday reproducibility. Urine with11 mmol/L orotic acid. (Quantitation against calibrationaqueous standard)Day 1 11.41Day 2 11.62Day 3 11.22Day 4 10.19Day 5 10.54Day 6 10.94Average 10.99 Accuracy 99.92St. Dev. 0.55CV % 4.95792 G. la. Marca, B. Casetta and E. ZammarchiCopyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 788–7933. Bean ES, Atkinson DE. J. Biol. Chem. 1984; 259: 1552.4. Scriver CR, Beaudet, Valle, Sly, Childs, Kinzler, Vogelstein.The Metabolic and Molecular Bases of Inherited Disease (VIIIthedn). McGraw-Hill: New York, (2) 2667.5. Rogers LE, Porter F. Pediatrics 1968; 42: 423.6. Jeevanandam M, Shoemaker J, Horowitz G, Lowry S,Brennan M. Metabolism 1985; 34: 325.7. Glasgow A. Am. J. Clin. Pathol. 1982; 77: 452.8. Van Gennip A, Van Bree-Blom E, Grift J, De Bree P,Wadman S. Clin. Chim. Acta 1980; 104: 227.9. Kidouchi K, Ohba S, Nakamura C, Katoh T, Kibe T, Wada Y.Int. J. Pur. Pyr. Res. 1992; 2(Suppl. 1): 58.10. Jakobs C, Sweetman L, Nyhan W, Gruenke L, Craig J,Wadman S. Clin. Chim. Acta 1984; 143: 123.11. McCann M, Thompson M, Gueron I, Tuchman M. Clin.Chem. 1995; 41: 739.12. Carpenter K, Potter M, Hammond J, Wilcken B. J. Inherit.Metab. Dis. 1997; 20: 354.13. Sevcik J, Adam T, Sazel V. Clin. Chim. Acta 1997; 259: 73.14. Banditt P. J. Chromatogr. B Biomed. Appl. 1994; 660: 176.15. Niessen WMA. J. Chromatogr. A 1998; 828: 401.Fast LC/MS/MS analysis of orotic acid in urine 793Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 788–793

Rapid determination of orotic acid in urine by a fast liquid chromatography/tandem mass spectrometric method.

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Rapid determination of orotic acid in urine by a fast liquid chromatography/tandem mass spectrometric method.

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