CLIN.CHEM.33/4, 473-480 (1987)

CLINICAl..CHEMISTRY, Vol. 33, No. 4, 1987 473

UrinaryOrganic Acids: Isolationand Quantificationfor Routine MetabolicScreeningJoachlm Greter and Carl-Eric Jacobson

A method for isolatingorganic acids from acidifiedurine onan equivolume mixture of Porapak 0 and Porapak T isdescribed, and results are compared with extraction withethyl acetate and ion exchange on DEAE-Sephadex. Aver-age recoveriesof 14C-labeledoxalicacid, lacticacid,succinicacid, a-ketoglutancacid, citric acid, and cinnamicacid wereequal to or better than those obtained with the solvent-extraction method. The ion-exchange method gave higherrecoveries for oxalic acid, lactic acid, and citric acid. Thequantification of separated acids from reconstructed massspectrometric ion traces is compared with quantification fromthe simultaneously recorded flame ionization detector re-sponse signals. A good correlation was obtained. With thepresent routine metabolic screening method we have detect-ed several patientswith inbornerrorsof metabolism.

Additional Keyphrases:column chromatography automa-tion - gas chromatography-mass spectrometry inborn er-rors of metabolism - heritable disorders - anion-exchange

Analysis for organic acids in human urine by gas chroma-tography-mass spectrometry (GC-MS) is becoming quitecommon in clinical laboratories. By this technique it ispossible to diagnose many inborn errors of lipid, amino acid,and carbohydrate metabolism and to monitor dietary andother forms of therapy (1-4).

Routine metabolic screening for inborn errors of metabo-lism requires access to a method that makes it practicable toprocess many samples and that at the same time fulfilsreasonable criteria of specificity and reproducibility.

The compounds of interest cover a wide range of polarity,which makes the initial isolation step very critical. Atpresent, extraction with ethyl acetate or diethyl ether, orboth, is widely used. This type of liquid extraction yieldspoor analytical recoveries of the more-polar compounds (5-8) and, like all liquid-extraction methods, it is inconvenientfor use with large numbers of samples. Initial isolation of an“organic acid fraction” by use of anion-exchange resinsbased on organic polymers or cellulose is an approach takenby several groups (2,3). The more-polar compounds are wellrecovered, but the large amounts of sulfate and phosphatethat are present in the isolated fraction cause problems inthe chromatographic separation that follows (5,6). It is alsoa disadvantage that a final aqueous solution is obtained,because removal of water is more cumbersome than evapo-ration of a solution containing mainly an organic solvent.

We report here our use of columns of ethylvinylbenzene-dimethylbenzene copolymers (Porapak#{174})’for the initial iso-lation of an “organic acid fraction,” followed by separationand identification by use of capillary gas chromatography-mass spectrometry. We have compared the isolation methodon Porapak material with solvent extraction and with ion-exchange chromatography. The clinical usefulness of this

Department of Clinical Chemistry, Gothenburg University, Sahl-gren’s Ho8pital, S-413 45 Gothenburg, Sweden.

‘“Porapak” is a trademark of Waters Associates, Milford, MA.Received January 27, 1986; accepted December 30, 1986.

new isolation method has been validated by analysis of morethan 4000 samples sent to the laboratory for diagnostic andfollow-up purposes.

Materials and Methods

Reagents and Instrumentation

All reagents were of analytical or “HPLC” grade. Urinesamples were stored at -20 #{176}C,with no additives.

For liquid-scintillation counting we used a Packard Tn-Carb Model 3320 instrument (Packard Instrument Co.,Downers Grove, IL). As counting solution we used a 1:2 (byvol) mixture of Triton X-100 surfactant and a toluenesolution containing 2,5-diphenyloxazole (PPO), 5 g/L, and1,4-bis(5-phenyloxazol-2-yl)benzene (POPOP), 100 mg/L. To10 mL of counting solution, 500 uL of the radioactiveaqueous sample was added after adjustment to pH 9 with0.1 mol!L sodium hydroxide. [2,3-’4C]Succinic acid (98%),and [U-’4C12-oxoglutaric acid (99%) were from NEN (NewEngland Nuclear Chemicals, Dreieich, F.R.G.). [“CjOxalicacid (97%), [side-chain-3-’4C]cinnamic acid (97%), [1,5-‘4C]citric acid (98%), and [U-’4C]sodium-i..-lactate (98%)were from Amersham, Buckinghamshire, England. Theradioactive materials were used as supplied, and the valuesfor the radiochemical purities given in parentheses after thenames of the compounds are those stated by the supplier forthe batch delivered. For quench correction ‘4C-standardpills were used (LKB Wallac, Stockholm, Sweden).

Porapak Q and Porapak T, mesh size 100-200, were fromWaters Associates, Milford, MA. The material was wettedovernight with acetone and thoroughly washed with demin-eralized water before use.

For capillary gas chromatography-mass spectrometry weused a MAT 445 quadrupole mass spectrometer-data sys-tem (Finnigan MAT, Bremen, F.R.G.). The mass spectrome-

ter was operated in the electron impact mode at an ionsource temperature of 220 #{176}Cand an electron voltage of 70eV. The mass-spectrometric data were collected and evalu-ated with the SS200 Data System (software releases 4, 5,and 6).

The quadrupole analyzer was mass calibrated with his-(perfiuoroheptyl)-S-triazine up to miz 1166 to yield relativepeak intensities comparable to the mass spectrum obtainedfrom a sector-field instrument. As a final test, we comparedthe mass spectrum of persilylated citric acid with thatobtained from a sector-field instrument.

The mass spectrometer was interfaced through a slightlymodified open split coupling (9) with solvent diverter to aModel 3700 gas chromatograph equipped with a Model 8000autosampler (both from Varian, Palo Alto, CA) modified towork in conjunction with a previously described septumlessinjection port (10). The gas-chromatographic columns usedwere 25 m long, 0.5 mm i.d. open tubular Pyrex columns,statically wall-coated with SE-54 in our laboratory accord-ing to Grob (11) to yield a film thickness of 1.2 .tm. At anaverage sample throughput of 10 samples a day the columnshave a useful lifetime of about two months. The carrier gaswas helium, used at the rate of 15 mLi’min during injection

474 CLINICAL CHEMISTRY, Vol. 33, No. 4, 1987

(constant pressure regulation) and 3 mL/min during thetemperature program (constant flow regulation). The tem-perature was programmed from 50 to 280 #{176}C,at 7 #{176}C/min.The initial temperature was held for 2 mm, the finaltemperature for 8 mm. At the column outlet the flow wasdiverted to the flame ionization detector (63%) and to themass spectrometer (37%) through a glass-coated stainless-steel T-piece (Scientific Glass Engineering, Melbourne, Aus-tralia).

The flame-ionization detector-response signals were re-corded and evaluated on a C-R2A integrator (Shimadzu Co.,Kyoto, Japan).

The sample extracts derived from the solvent-extractionand Porapak methods were dried in a centrifugal evaporator(Savant Instrument, Hicksville, NY) modified to hold amaximum temperature of 32#{176}C.

Isolation of Organic Acids

Stock solutions for each of the radioactively labeled organ-ic acids were made from 30 mL of pooled urine by adding0.01 MBq of the labeled compound diluted with unlabeledmaterial to give an additional 10 mg of the compound perliter in the stock solution.

From these stock solutions 2-mL portions were taken forthe ethyl acetate extraction, ion-exchange separation, andthe separation on Porapak.

Ion-exchange chromatography of urinary acids was per-formed according to Chalmers and Watts (12), except thatwe used methoxylamine hydrochloride instead of ethoxyla-mine hydrochloride.

We extracted 2 mL of urine with ethyl acetate afteradding an equal amount of water saturated with sodiumchloride and acidifying the sample with concentrated hydro-chloric acid to pH <1. The sample was extracted three timeswith 3-mL portions of ethyl acetate, centrifuging to speed upphase separation. The combined extracts were taken todryness in a centrifugal evaporator, followed by two repeat-ed additions and evaporations of 1 mL of methylene chlo-ride.

To prepare the adsorption columns for the isolation ofurinary organic acids, we poured an equivolume slurry ofPorapak Q and Porapak T in acetone into 15 x 0.8 cmcolumns plugged with glass wool, to yield columns 10 cm inheight and containing about 1.5 g of material. About 700 mgof the Porapak mixture sufficed for adsorption of the organicacids from 2 mL of urine containing up to 500 mg ofcreatinine per liter. Before the acidified (pH <1) urinesample was applied, the column was washed with threecolumn volumes of 0.1 molJL hydrochloric acid. After 2 mLof acidified urine was applied to the column, it was washedtwice with 1-mL portions of 0.1 molIL hydrochloric acid. Theorganic acids were then eluted with about 4 mL of acetoni-true. The eluate was collected, accompanied by as littlewater as possible, and dried in the centrifugal evaporator.To facilitate the removal of collected water, 1 mL of methyl-ene chloride was added twice during the evaporation step.

Before either solvent or column extraction, 200 pL of aninternal standard solution of 3-chlorobenzoic acid and 5-pentynoic acid in water was added to each urine sample togive a concentration of 50 mg/L for each. When urinesamples for routine metabolic screening are processed theamount of urine applied to the Porapak column depends onthe creatinine value. Thus 10 mL of urine is applied whenthe creatinine value is <150 mg/L, 4 mL is applied whenthere is between 150 and 300 mg of creatinine per liter, and2 mL is applied when the creatinine exceeds 300 mg/L.

Liquid-Scintillation Counting

All the dried samples from the solvent-extraction method,the ion-exchange method, and the Porapak separationmethod were dissolved in 2 mL of demineralized water, andthe solutions were adjusted to pH 9 with sodium hydroxide.A 500-FL aliquot was then added to 10 mL of the liquid-scintillation cocktail. Radioactivity in all samples wascounted twice the same day as prepared and again one daylater, to check for chemiluminescence. The same procedurewas followed after addition of the radioactive standard forquench correction.

Derivatization for Gas Chromatography-Mass Spectrometry

For gas chromatography-mass spectrometry the sampleswere derivatized with 200 .uL of methoxylamine hydrochlo-ride dissolved in redistilled pyridine (30 g/L, including 100mg of tetracosanoic acid per liter as external standard) for45 mm at 40 #{176}Cand with 200 pL of N,Obis(trimethylsilyl)-trifluoroacetamide (BarrA) for 30 mm at 80#{176}Cto yieldmethoxime-trimethylsilyl derivatives.

Standard Curves for Method Comparisons

We prepared standard curves for 14 organic acids normal-ly present in urine, using the Porapak and the solvent-extraction methods. Mixtures of all 14 acids were preparedin water containing, per liter, 200 mg of urea, 200 mg ofhippuric acid, and 10, 20, 50, 100, or 200 mg of each of theorganic acids. Twice, we took 2 mL of each standard mixturethrough the procedure described for use with urine samples.The signal from the flame ionization detector and the wholemass spectra in the mass range 50-650 amu (1.4-s cycletime) were recorded simultaneously, with a flame ionizationdetector:mass spectrometer split ratio of 2:1. All componentsin the standard mixture were resolved on the capillarycolumn, and calculations were based on the peak areas fromthe signal from the flame ionization detector and from thereconstructed specific-ion traces for each component (13-15).

Results

Comparison of Chromatograms after Initial Isolation byPorapak Adsorption, Extraction with Ethyl Acetate, and Ion-Exchangeand SubsequentDerivitization

Figure 1 shows chromatograms of the derivatives oforganic acids isolated from pooled urine by the three meth-ods. The good agreement of the simultaneously recordedsignals from the flame ionization detector and the massspectrometer shows the good performance of the effluentsplitter, and also that the mass-spectrometric cycle time isadequate to resolve the eluting gas-chromatographic peaks.The flame-ionization detector chromatogram from the ion-exchange isolation method was recorded with the attenua-tion increased by a factor of two as compared with the otherchromatograms, because of the larger amounts of citric andhippuric acid recovered. A prominent feature of Figure 1 isthat qualitatively and quantitatively more of the polarcompounds (notably peaks 18,22,33, and 36 in Figure 1) butless urea and phosphoric acid (peaks 12 and 13 in Figure 1)are recovered with adsorption on Porapak than with solventextraction with ethyl acetate. Another significant feature isthe dramatic decrease in sulfuric acid and phosphoric acidrecovered (peaks 8 and 13 in Figure 1) with the Porapak-adsorption method as compared with ion-exchange chroma-tography, although at the expense of some of the very polarpolyhydroxycarboxylic acids that, together with urea and

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identitiesof selected peaks: 1, lactic acid; Z 2-hydroxyisobutync acid; 3, glycolicacid; 4, oxahcacid; 5, 3-hydroxypropsonucacid + reagentartetact;6, p.cresol; 7,3-hydroxyisobutyricacid; 8, sulfuricacid; 9, 3-hydroxy-2-methylbutyric acid; 10, 3-hydroxyisovalericacid; 11, 2ethyihydracryiicacid; 12, urea; 13, ethylmalonicacid +

phosphoncacid; 14, unidentifiedcompound; 15, succunicacid; 16, methylsuccinic acid; 17. uracul;18,enjthro-4-deoxytetronic acid; 19, glutaric acid; 20, 2-deoxytetronicacid;21, adipicacid; 22, 3-methytadipicacid + pyroglutamicacid; 23, unidentified compound; 24, furane-5-hydroxymethyl-2-cathoxylic acid; 25, erythronicacid; 26,2-oxoglutaricacid;27, 3,4-methylenehexaneduoic acid; 28, 3-hyduuxy-3-methylglutaric acid; 29, 4-hydroxyphenylacetucacid + 2,5-furanedicarboxylic acid; 30, glycylfurane-2-carboxylic acid; 31, frans-aconfticacid; 32. citricacid lactone; 33, citric acid; 34, hippunc acid + 3-hydroxyphenythydracrylic acid; 35, 3-deoxyarabinohexonic acid; 36.tyrosine;37, unidentified compound; 38, ghiconic acid; 39, 3-hydroxyhippunc acid; 40, 4-hydroxyhuppuncacid;A, 4-pentynoic acid (internalstandard);B. 3-chlorobenzoicacid (internalstandard);C, tetracosanoic acid (externalstandard;not addedto DEAE extracts); G, glucuronides and glutamic acid-conjugated compounds

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CLINICALCHEMISTRY, Vol. 33, No. 4, 1987 475

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phosphoric acid, are found in the wash fraction precedingthe elution with acetonitrile.

Analytical Recovery of 14C-Labeled and Unlabeled Acidsfrom Porapak Columns

Table 1 shows the radioactivity accounted for on use of thethree isolation methods with pooled normal urine supple-mented with six ‘4C-labeled organic acids. Recovery ofsuccinic acid, 2-oxoglutaric acid, and cinnamic acid fromPorapak compares well with that by the ion-exchangemethod, whereas the more polar compounds-oxalic acid,lactic acid, and citric acid-are less well recovered from thePorapak material. Recovery of oxalic and cinnamic acidfrom Porapak is equal to the recovery obtained with thesolvent-extraction method. The other radiolabeled corn-

pounds we studied are better recovered with adsorption onPorapak than with the ethyl acetate extraction method,notably 66% of citric acid recovered vs <10%, and 97% of 2-

Table 1. Recovery of Selected Organic Acids as aPercent of Initial Radioactivity (Mean ± SD)

Porapak Solvent extractionn=1O n=1O

Compounds(50 mg/L)

Table 2. Peak Areas (Mean of Five Determinations, SD <10%) of Selected Organic Acids as a Percent of internalStandard Area (3-Chlorobenzolc acid) Measured with a Flame Ionization Detector (FID) and by Mass Spectrometry

(MS)Standard Solvent extraction Porapak adsorption

miza FID MS RD %R6 MS %R FID %R MS %R

1 Lactic acid 219 150 6 24 16 1 17 42 28 2 332 Oxalic acid 190 122 12 26 21 3 25 17 14 2 173 3-Hydroxybutyric acid 233 87 9 24 28 2 22 45 52 4 444 2-Hydroxyisovaleric acid 219 177 20 89 50 12 60 123 69 14 705 Benzoic acid 179 184 217 97 53 127 59 113 61 138 646 Succinicacid 172 173 17 92 53 10 59 105 61 10 597 Adipicacid 275 178 18 109 61 10 56 113 63 11 618 3-Methyladipicacid 125 186 93 111 60 60 65 115 62 62 679 2-Oxoglutaric acid 198 160 45 10 6 2 4 101 63 31 69

10 4-Hydroxyphenylacetic acid 296 224 25 132 59 17 68 122 55 16 6411 frans-Aconitic acid 229 171 65 44 26 20 31 104 61 46 7112 Homovanillic acid 326 180 36 115 64 24 67 126 70 25 6913 Citric acid 273 182 118 12 7 9 8 89 49 62 5314 Vanillylmandelicacid 297 193 256 116 60 159 62 133 69 162 6315 Cinnamicacid 205 198 110 116 59 69 63 116 59 68 6216 3-Chlorobenzoic acid 213 100 100 100 - 100 - 100 - 100 -

49800 19314 50909 102 19056 99 51070 103 19193 99B Mass-to-chargeratios of the ionsreconstructedforquantification.6Recoveriesas measuredagainstthe relativeareasfromthe derivatizedstandardsdissolvedin pyridine.

476 CLINICALCHEMISTRY, Vol. 33, No.4, 1987

oxoglutaric acid recovered vs 58%. If the columns werewashed with twice the amount of 0.1 mol(L hydrochloricacid we have recornmended here, nearly all lactic acid andall oxalic acid were found in the wash fraction.

No radioactivity was recovered from the Porapak columnsafter either an additional elution with acetonitrile or afterthe acetone wash. After all the recovery experiments hadbeen run, no radioactivity could be detected in the columnmaterial when it was examined as a slurry in the scintilla-tion cocktail.

We made a further comparison between recovery by thesolvent-extraction method and Porapak-adsorption methodby simultaneous quantification of 14 unlabeled organicacids with the flame ionization detector and the massspectrometer (13-15), using reconstructed specific ion tracesafter establishing that standard curves were linear by eithermethod (see below). For this quantitative comparison,pooled urine could not be taken as for the recovery experi-ments with radiolabeled acids and the routinely preparedstandard curves, because interfering compounds would havemade quantification with the flame ionization detectorimpossible. Therefore we used an “artificial urine” contain-ing urea and hippuric acid to compare the two methods.Table 2 gives the results obtained from the analysis of anaqueous standard solution containing 50 mg of each com-pound per liter. The measured areas from the flame ioniza-tion detector and from the reconstructed specific ion tracesare given in relation to the area under the internal standardpeak (recovered to the same extent by both extractionmethods). No correction for detector response differenceswas made. Recoveries were calculated relative to the areaobtained from directly derivatized standards dissolved inpyridine (50 mg of each compound per liter) with no urea orhippuric acid added (cf Table 2). Overall recoveries obtainedwith the Porapak method as assessed by this approach wereequal to or better for all compounds, except oxalic acid, thanthose with the solvent-extraction method. The Porapakmethod gives much better overall recoveries for (e.g.) citricacid (53% vs 8%) and 2-oxoglutaric acid (69% vs 4%).

The Porapak columns can be reused at least 50 times with

IS 3-Chlorobenzoicacidrawarea

no deterioration in performance. To regenerate the columns,they are washed with four colunm volumes of acetone,followed by at least 10 column volumes of demineralizedwater. We could detect no compound in blank runs from thewashed columns.

Quantification of Compounds in the Isolated Organic-AcidFraction

Compounds in the isolated organic-acid fraction can inprinciple be quantified by measuring the response from theflame ionization detector, from the total ion current, or fromthe area under a specific ion in a reconstructed ion chro-matogram (13-15) as well as from single or multiple ionmonitoring (16-18). For 14 compounds we prepared stan-dard curves based on five different concentrations. Theaqueous standard solutions were taken through the isola-tion procedure, in duplicate. Both flame ionization detectionand measurement from reconstructed mass-spectrometricion traces gave linear standard curves in the range of 0-200mg/L (Figure 2, panels F and M). None of the calculatedregression coefficients were less than 0.98. The interceptwith they-axis gives an estimate of the detection limit of thetwo isolation procedures.

The sensitivity of the mass-spectrometric detection de-pends on the intensity of the ion selected for quantification.1f as here, a flame ionization detector is used in parallel, italso depends on the split ratio used.

We also calculated linear-regression lines to correlate themethod based on reconstructed ion chromatograms fromwhole mass spectra with the flame ionization detectorresponse-based quantification reference method (Figure 2,panel M/F) and to correlate the Porapak method with thesolvent-extraction method (Figure 2, panel P/E). The twoquantification and isolation methods correlated well, asshown by the calculated regression coefficients, which allexceed 0.98. Calculated coefficients of variation from differ-ent runs were in the range of 1-7% for all compounds exceptlactic acid and oxalic acid, for which CVs >10% wereoccasionally found.

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Fig. 2. Linear regression lines for 14 components of normal urine (1 to14asinTable2) from thestandardcurves(left side; duplicatevaluesat10, 20, 50, 100, and 200 mg/L) and the corresponding correlations ofthe differentdetectionand isolation methods (right side)Abbreviations are as fellows: M, mass-spectmmetric quantitation; F. flame-ionization detector quantitabon; P. Porapak adsoption method; E, eth acetateextraction method; PM correlation between mass-spectromebic method andsimultaneous quantification with the flame-ionization detector; PIE, correlationofthe Porapak-isolationmethodagainst the eth)l acetateextraction method

Validationof ClinicalUsefulness

Figure 3 shows examples of chromatograms obtained withthe Porapak method for urines of patients with differentinborn errors of metabolism. The compounds most charac-teristic of each disease are clearly visible, and minor com-pounds of interest can also be discerned. Thus, in a case ofmaple-syrup urine disease, the largest peak is produced by2-hydroxyisovaleric acid (peak 50, Figure 3, panel MSUD,about 80 mgfL), but even 2-oxoisovaleric acid, 2-oxo-3-methylvaleric acid, 2-oxoisocaproic acid, and 2-hydroxyiso-caproic acid in the range of 10 to 40 mg/L (peaks 45, 50, 52,and 53, respectively, Figure 3, panel MSUD), characteristicof maple-syrup urine disease, can be seen. In the case ofmethylmalonic aciduria and in the case of methylcrotonyl-glycinuria due to biotinidase (EC 3.5.1.12) deficiency (19)the characteristic presence of methylcitric acid (peak 67,Figure 3, panels MM and MCG, >60 and 200 mgfL, respec-tively) can be noted in addition to the main diagnosticmetabolites, methylmalonic acid (peak 51, Figure 3, panelMM, >400 mg/L) and 3-hydroxyisovaleric acid and 3-meth-ylcrotonylglycin (peaks 10 and 60, respectively, Figure 3,panel MCG, >200 and 40 mg/L, respectively). Detection ofthe huge amount of lactic acid (peak 1, Figure 3, panel PC,

>300 mg/L) in a case of pyruvate carboxylase (EC 6.4.1.1)deficiency (20) poses no problem, and even in the heavilycontaminated sample of a case of ornithine carbamoyltrans-ferase (EC 2.1.3.3) deficiency, orotic acid (peak 64, Figure 3,panel OCT, 95 mg/L) is easily detected by our screeningmethod.

The urine from an untreated patient with hereditarytyrosinemia shows the presence of succinylacetone andsuccinylacetoacetate (peak multiplets 59 and 72, Figure 3,panel TYR, both above 50 mg/L), which are required asdiagnostic criteria for this disease (21) but are easy tooverlook (22).

Discussion

Non-ionic polymers have been used to isolate (e.g.) ste-roids and water pollutants before their analysis with gaschromatography-mass spectrometry (23). DressIer (23) stat-ed that for acidic compounds neither of the sorbents issuitable, but we tested several commercially available ad-sorbents such as Tenax, Amberlite XAD resins, SephadexLH-20, Lipidex 1000 and 5000 (24), and octadecyl-modifiedsilica (Sep-pak#{174})but found them unsatisfactory becauseconsiderably more water or fewer components were recov-ered than with the solvent-extraction method. The bestresults were obtained with Porapak Q, but very polarcompounds such as oxalic acid were not recovered as well aswith the ethyl acetate method. We therefore tested themore-polar Porapak T material, but here less-polar com-pounds such as decanedioic acid were not recovered as wellas with the solvent-extraction method. We found a one-to-one mixture of Porapak Q and Porapak T to be a goodcompromise in polarity between the nonpolar Porapak Qand the more-polar Porapak T.

Except for lactic acid, oxalic acid, and citric acid, recover-ies from the Porapak columns exceeded 80% for the com-pounds studied by the radioactive tracer method (cf. Table1). Table 2 summarizes an attempt to assess and compareoverall recoveries by the solvent-extraction method and thePorapak adsorption method by simultaneous flame ioniza-tion detection and mass spectrometry. To get resolved peaksfor quantification with the flame ionization detector, we hadto use an artificial urine, although, owing to matrix effects,overall recoveries were lower for all compounds as comparedwith supplemented urine (Table 1). In Table 2, internal-standard-corrected values for area response were calculatedand compared. The values obtained for “artificial urine”were compared with the values obtained for directly deriva-tized standards dissolved in pyridine.

Except for oxalic acid, all compounds studied were recov-ered equally well or better with adsorption on Porapak thanby solvent extraction. For 2-oxoglutaric acid and citric acid,recoveries were more than fivefold greater with Porapak ascompared with solvent extraction.

Lactic acid and oxalic acid are not well recovered by eithertechnique, but one does not run the risk of overlooking anysignificant increase, as for instance of lactic acid in cases oflactic acidosis (e.g., Figure 3, panel PC). If one wishes toquanti1y these acids more accurately, one should use specificmethods based on enzymic reactions.

Sulfuric acid and phosphoric acid are recovered with theion-exchange isolation method, leading to problems in thefollowing gas-chromatographic separation. The quality ofthe chromatograms obtained with Porapak adsorption orextraction with ethyl acetate is better than with the ion-exchange technique (cf. Figure 1). Reportedly, sulfuric acid

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478 CLINICALCHEMISTRY,Vol.33, No.4, 1987

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Fig. 3. Total ion-current chromatograms of methoximated and silylated compounds isolated from the urine of different patients by thePorapakmethodandseparatedby capillary gas chromatographyBecause of an error in the acquisitionprogramthe elapsedtimeshownincludes theoven cool-downtimewiththeexceptionof the MM and OCTpanels,wheretheclockwas staried at injectiontime. Dtterences in spectrumnumbers wise becausedifferentcolumnshave beenusedthroughoutthe years.Abbreviationsusedare: MSUD,maple syrup urine disease (treated);MM, methylmaJonicaciduna(treated);MCG, 3-methyicrotonyiglycinuria;PC, pyruvate cartooxylase deficiency; OCT. omithinecarbamyltransferasedeficiency; TYR, hereditarytyrosinemia.In additiontothepeakidentitiesgiveninthelegendto Fig.1,thefollowingpeaksare identified:41, ethyleneglycol; 42, tigbc acid; 43, pyruvic acid; 44, phenol; 45, 2-oxo-isovalenc acid; 46 acetoacetic acid; 47. 2-hydroxybutyric acid; 48, 3-hydroxybutyric acid; 49, 2-hydroxyisovalenc acid; 50, 2-oxo-3-rnethylvalericacid; 51. methylmalonic acid: 52, 2-oxo-isocaproic acid; 53, 2-hydroxyisocaproicacid; 54, thymol;55,fumancacid; 56,2-hydroxy-2-methylsuccinic acid; 57, 5-oxo-2-tetrahydrofuraneacelic acid; 58, glutamic acid; 59, succinylacetone; 60, 3-methyicrotonylglydne; 61, 4-oxo-6-hydroxyhep-tanoic acid; 62, phenylalanine (mono- and ditrimethylsilyl derivatives, respectively); 63, octanedioic acid; 64, orotic acid; 65, vanillicacid; 66, homovanillicacid;67,methylcitric acid; 68, vanillylmandelicacid; 69, 4-hydroxyphenyllactic acid; 70, 4-hydroxyphenylpyruvic acid; 71, glucose; 72, succinylacetoacetate; 73, palmitic acid; 74,3-hydroxydecanedioic acid;75, uricacid;76, oleicacid; 7Z N.phenytacetylglutamine; U, uncertain identification: 0. sample contaminant; R, reagent arlefact

and phosphoric acid can be removed by precipitation withbarium hydroxide, but this would introduce a further stepand also the potential risk of coprecipitation of organic acids(5, 25, 26). More different compounds (including severalneutral compounds) are recovered with the Porapak proce-dure than with ethyl acetate extraction. For example, 2-oxoproline at concentrations of interest for the diagnosis of2-oxoprolinuria is detected after Porapak adsorption.

The three isolation procedures give reproducible yields,which makes it possible to quantify excretions with the aidof an internal standard-at least if recovery exceeds 30%.Irrespective of which isolation procedure is used, a very

complex mixture is obtained. Capillary gas chromatographywith use of a flame ionization detector suffices to detect thelarge abnormal patterns that are seen in many classicalcases of inborn errors, but this technique cannot be general-ly used for quantification or even identification, because ofoverlapping components (13, 15, 16, 27). With the mass-spectrometric technique as used in our laboratory, a massspectrum is recorded every 1.4 s. Thus it is possible toinspect any peak of interest to establish its purity. Forexample, orotic acid is usually eluted close to or togetherwith aconitic acid, but a mass spectrum recorded from thispeak reveals the presence of orotic acid, which is important

CLINICALCHEMISTRY,Vol.33, No.4, 1987 479

in order not to overlook cases of ornithine carbamoyltrans-ferase deficiency (Figure 3, panel OCT). Most compoundshave at least one specific ion within a given retention-timewindow and a reconstructed ion chromatogram can then beused for quantification (13-15).

Because quantification of resolved peaks -with the flameionization detector is well established (28, 29), we simulta-neously recorded the signals from the detector and from themass spectrometer. The mass-spectrometric quantificationgives results that correlate well (r >0.99) with those ob-tained from the simultaneously recorded signal from theflame ionization detector (Figure 2, panel M/F). For complexmixtures with unresolved components the mass-spectromet-nc quantification is clearly preferable, and therefore it isroutinely used in our laboratory. In conjunction with alibrary search program and standard curves from urinesupplemented with the compounds of interest, quantitativedata are obtained automatically (15, 30).

The described isolation procedure has now been in routineuse for five years in our laboratory, with up to 20 samples aday, and was run in parallel to the solvent-extractionmethod for half a year in the beginning. The usefulness ofthe method has been shown by the fact that we detecteddifferent cases of metabolic disorders such as 3-methylcro-tonylglycinuria (19), pyruvate carboxylase deficiency (20),tyrosinemia, methylmalonic aciduria, and three cases oforotic aciduria (ornithine carbamoyltransferase deficiency)that had gone undetected with the solvent-extraction meth-od (unpublished). Because of the mild isolation conditionsand good reproducibility of our method it has been possibleto follow the quantitative variation in excretion of (e.g.)succinylacetoacetate and succinylacetone in tyrosinemic pa-tients during treatment (31). The mild separation attainedthrough adsorption on Porapak has also been found usefulin studies of mercapturic acids in our laboratory (32,33) andby others (34).

Even with high-resolution capillary chromatography andthe use of a good mass-spectrometric system, one willencounter peaks that cannot be properly identified or quan-tified. In these cases, one must resort to subfractionation ofthe initial isolate by chromatographic techniques and (or) tothe use of different methods of derivatization (35-39). Quan-tification from reconstructed ion traces is practicable inscreening programs and sufficiently sensitive for theamounts in question. For accurate quantification of traceamounts-for example, in amniotic fluid-the addition oflabeled internal standards and analysis by selected ionmonitoring is more appropriate.

In a review article, Gates and Sweeley (40) stated: “Bar-ring a major breakthrough in GC-MS design, it seems likelythat repetitive scanning CC-MS will be restricted to theexhaustive quantitative analysis of a relatively small num-ber of samples, and will not be used widely for broad-scalescreening programs” and “Few of the extraction procedurestypically used for metabolic proffling are completely satis-factory; most will need considerable modification beforethey can be considered routine, easy-to-use procedures.” Webelieve that, with the presently described approach andconsidering the availability of fully computerized massspectrometers at acceptable prices, metabolic screeninganalysis of an “organic acid fraction” can now be morewidely made routine in clinical laboratories.

This study was supported by grants from the Swedish MedicalResearch Council (03X-585) and from Torsten and Ragnar S#{246}der-bergs Stiftelse. -

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