Journal of Chromatography, 562 (1991) 125 138 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam CHROMBIO. 5528

Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease a JAMES D. SHOEMAKER* and WILLIAM H. ELLIOTT E. A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Medical Center, 1402 S. Grand, St. Louis, MO 63104 (U.S.A.)

ABSTRACT Eighty-five clinical urine samples and nineteen urine samples previously found by other laboratories to suggest genetic metabolic defects were prepared for trimethylsilylation by treatment with urease, followed by azeotropic dehydration. The "Target Analyte Search" program provided with the VG Trio 2 gas chromatograph-mass spectrometer required 6 min to quantify 103 compounds relative to endogenous urinary creatinine. This technique has been used to confirm diagnoses including cystinuria, lysinuria, medium-chain acyldehydrogenase deficiency, ornithine transcarbamylase deficiency, aspartylglucosaminuria, methylmalonic, propionic and glutaric acidurias.

INTRODUCTION

Silylating agents including trimethylsilyl (TMS) and tert.-butyldimethylsilyl donors can stabilize a wide variety of compounds for volatilization during gas chromatography (GC) [1]. However, organic extraction of biological fluids is generally directed to a single hydrophilic or hydrophobic moiety: carboxylic acids, amines or hydrophobic groups, for example [2]. Column procedures using various solid phases [3,4] have been used to clean-up urine and amniotic fluid prior to derivatization. Solvent extraction is cumbersome and time-consuming and column clean-up procedures cannot be used if the chemical nature of the compounds of interest is unknown or unanticipated. We now describe a method for preparing metabolites of urine or amniotic fluid for derivitization without solvent extraction or column clean-up. The method depends on the use of the enzyme urease to remove urea, the major organic constituent from the samples, which then renders the minor components accessible to the derivatizing agent. The number of metabolites detectable by this method is large enough to require Some of the data in this paper appeared as Abstract No. RPA 79 at the American Society for Mass Spectrometry Annual Meeting, Miami, FL, May 1989 and in Abstract No. 1160 at the Second International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, CA, August 1989. 0378-4347/91/$03.50

©

1991 Elsevier Science Publishers B.V.

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J . D . S H O E M A K E R , W. H. E L L I O T T

highly automated interpretation of the mass spectral data and depends on methods developed by Horning and Horning [5] and Jellum et al. [6]. Single ion chromatograms [7] must be used to resolve overlapping peaks. Rapid confirmation of peak identities requires a condensed library of compounds of interest such as that published by Markey et al. [8]. Our procedure included two steps: first, during the GC run, the mass spectra of total ion current (TIC) peaks were matched against a library of 1700 mass spectra (TMS derivatives from the 42 000-entry NBS library plus compounds we have derivatized). Second, after the GC run, 130 target peaks (from 103 compounds) were identified by retention time and their eight major mass spectral peaks, then quantified by single ion chromatograms. This program required approximately 6 min after the GC run of 67 min or 1900 scans. EXPERIMENTAL

Materials

Authentic standards of the compounds listed in Table I, crystalline urease (Type C-3) and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) were purchased from Sigma (St. Louis, MO, U.S.A.). Carbon dioxide, ultra-purity helium and nitrogen gasses were purchased from Matheson (Joliet, IL, U.S.A.). Trideuterated creatine [(methyl)-d3-creatine] was purchased from MSD Isotopes (Montreal, Canada). All solvents were purchased from Fisher (Fairlawn, N J, U.S.A.) and were HPLC grade. Hydrogen sulfide gas was produced from sodium sulfide by addition of concentrated sulfuric acid. Unassembled disposable syringes were purchased from Crown Distributing (Bridgeton, MO, U.S.A.). Anotop 0.2-#m filters were purchased from Alltech Assoc. (Deerfield, IL, U.S.A.). Urine samples

Over a six-month period, 85 urine samples were received from a local pediatric hospital. Prior to this, urine samples from 19 previously confirmed cases of genetic metabolic abnormalities were provided by other laboratories. The diagnoses, confirmed by our procedure, included: methylmalonic aciduria (three), dicarboxylic aciduria (five) two of which were due to medium-chain acyl dehydrogenase deficiency, propionic, glutaric (two) and hydroxymethylglutaric aciduria, ornithine transcarbamylase deficiency, lysinuric protein intolerance (two), glycinuria, maple syrup urine disease, isovaleric aciduria and aspartyl-glucosaminuria. Urine was kept at - 2 0 ° C until processed. Urease treatment

After defrosting the urine sample at 37°C, an aliquot of 0.5-1.0 ml was transferred to a 2-ml vial by passage through a 0.20-#m Anotop filter using a glycerolfree disposable syringe. To this were added 2 #mol/ml trideuterated creatine as a 0.1 M aqueous solution. The vial was capped with a siliconized rubber septum. Carbon dioxide gas was injected into the vial with a needle through the septum

A U T O M A T E D SCREENING OF URINE SAMPLES

127

and the cap was tightened maintaining positive CO2 pressure. The gas was allowed to equilibrate for 5 min, then the vial was uncapped. Approximately 125 U/ml of urine (1-2 crystals per sample) of urease were added and mixed by vortexing. The vial was immediately recapped, COz-positive pressure again established and the vial maintained at 37°C for 30 min. If concavity of the septum indicated negative internal pressure, C O / w a s again added as needed. After 30 min, another 125 U of urease per ml of urine were added, the vial was recapped and the CO2 pressure established. After 15 min, the vial was uncapped, acetone was added to a concentration of 50% and the vial was chilled in an ice bath for 15 min to precipitate urease and other proteins. The sample was again passed through a 0.20 #m filter into a 2.0-ml vial. Acetonitrile was added to the top of the vial and 40 /~1 of triethylammonium trifluoroacetate, a clear liquid, were added. The solution was concentrated under a nitrogen stream at 70°C. When the volume reached 50-70% of starting volume, acetonitrile was again used to 'top-off" the samples. As precipitate began to appear, methylene chloride was added to remove the last traces of water as an azeotropic mixture under the nitrogen stream at 700C. A volume of MSTFA equivalent to 25-100% of the starting volume of the urine sample was added to the residue. Derivatization proceeded under nitrogen at 70°C for 1 h. A 1-2/~t volume of supernatant was injected into the gas chromatograph, using splitless mode and a valve time of 2.5 min. The temperature was held at 80°C for 1 min, then programmed to increase from 80 to 130°C at 2°C/ min, 130 to 200°C at 3°C/min and 200 to 280°C at 6°C/min, then held at 280°C for 10 min. The column was a 30 m × 0.32 mm I.D. OV-5 capillary, with a coating thickness 0.5 #m, purchased from Ohio Valley Specialty Chemical (Marietta, OH, U.S.A.). The carrier gas was ultra-purity helium kept at a head pressure of 1 bar.

Mass spectrometry and automated searching The VG Trio 2 gas chromatograph-mass spectrometer-computer (VG Masslab, Altrincham, U.K.) was used for the analyses. Spectra were obtained in positive electron-impact (EI +) mode by scanning from 50 to 800 m/z in 1.95 s with an interscan interval of 0.05 s. A custom program started the GC run and after 4 min began to identify scan numbers associated with TIC peaks, calculate their areas and find the best match for the scan within a custom library of 1700 TMS derivatives. The library was comprised of TMS derivatives from the 42000-entry NBS library of mass spectra supplied by VG Masslab, plus 200 additional compounds we have derivatized. When the program had found the best library match for the first batch of compounds, the rest of the TIC chromatogram acquired up to that point was analyzed and the areas and identification data were added to the previous list. Generally, the 50-60 largest peaks were identified by the end of the 67 min GC run. A proprietary program supplied by the manufacturer was then used to search for 'target' compounds and quantify them. The data used by the pro-

128

J . D . SHOEMAKER, W. H. ELLIOTT

TABLE I I N F O R M A T I O N R E Q U I R E D F O R A U T O M A T E D Q U A N T I F I C A T I O N OF 103 C O M P O U N D S F R O M U R I N E SAMPLES Missing row numbers are due to removal of duplicate information supplied in the program for cases in which double peaks may occur. The quantification ion shown is not necessarily the ion of highest intensity, rather it is a fragment ion unique to the target compound within the vicinity of its elution. It is the ion used to create the single ion chromatogram for quantification, shown graphically in Fig. 2B. RF = Response factor (molar) of the quantification ion relative to mass 118 in d3-creatinine, the internal standard. RRT = Relative retention time compared to retention time standard 1 (d3-creatinine) or 2 (pseudouridine). Row No. 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Target compound a

RRT

RF

Mass:intensity (versus 1000) of quantification ion

da-Creatinine TMS 3 Lactic acid TMS 2 Hexanoic acid TMS Glycolic acid TMS 2 Pyruvic acid TMS 2 Alanine TMS z Glycine TMS 2 Oxalic acid TMS 2 Sarcosine TMS z fl-Hydroxybutyric TMS 2 fl-Alanine TMS 2 fl-Aminoisobutyric TMS 2 Methylmalonic TMS 2 Valine TMS z Ketoleucine TMS 2 Octanoic acid TMS Keto valine TMS 2 Ethanolamine TMS 3 Leucine TMS 2 Ethylmalonic TMS 2 Phosphate TMS 3 Proline TMS 2 lsoleucine TMS z Maleic TMS 2 Glycine TMS 3 Ketoisoleucine TMS 2 Succinic acid TMS 2 Ketoisoleucine TMS 2 Fumaric acid TMS 2 Pipecolic acid TMS 2 Serine TMS 3 Threonine TMS 3 Glutaric TMS 2 Propionylglycine TMS 2 fl-Aminoisobutyric TMS 3

1.000 0.194 0.200 0.207 0.219 0.237 0.256 0.269 0.278 0.312 0.382 0.348 0.392 0.395 0.449 0.463 0.459 0.479 0.490 0.500 0.502 0.522 0.528 0.535 0.544 0.549 0.552 0.564 0.618 0.645 0.664 0.716 0.731 0.771 0.833

1.000 0.152 0.701 0.095 0.049 1.089 1.058 0.016 1.992 0.431 1.152 0.420 0.026 1.354 0.045 0.665 0.100 1.883 1.530 0.031 0.320 1.964 1.456 0.171 1.058 0.111 0.167 0.111 0.520 3.769 1.345 0.460 0.206 1.252 1.152

118:1000 191:162 173:652 205:60 217:162 116:1000 102:1000 190:35 116:I000 191:594 102:1000 102:1000 247:66 144:1000 259:94 210:540 245:114 174:1000 158:1000 261:21 314:152 142:1000 158:830 245:67 174:1000 259:96 247:81 259:96 245:1000 156:1000 204:1000 218:446 261:133 158:815 174:1000

129

A U T O M A T E D S C R E E N I N G OF U R I N E SAMPLES TABL E I (continued) Row

Target compound a

RRT

RF

No. 37 38 39 40 41 42 43 44 45 46 47 48 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

Mass:intensity (versus 1000) of quantification ion

Homoserine TMS 3 Niacinamide TMS Niacinamide TMS 2 Butyrylglycine TMS z Malic acid TMS 3 Adipic acid TMS z Hydroxyproline TMS 2 Methionine TMS z Isovaleryglycine TMS 2 Aspartic acid TMS 3 y-ABA TMS 2 Hydroxyproline TMS a Cysteine TMS 4 Creatinine TMS 3 Cysteine TMS 3 Tiglyglycine TMS 2 2-Oxoglutarate Phenylalanine TMS 2 Ornithine TMS 3 Glutamic acid TMS 3 Suberylglycine TMS 3 Hexanoylglycine TMS a Homocysteine TMS 3 Ribose TMS 4 Asparagine TMS 3 Ribose TMS 4 Suberic acid TMS 2 fl-Met Crotonylglycine TMS z Phenylpyruvic acid TMS Lysine TMS 3 Quinolinic acid TMS 2 Xylose TMS 4 Xylitol TMS 5 Orotic acid TMS a Homovanillic TMS z Glutamine TMS 3 Xylose TMS 4 3-Methylhistidine TMS 3 Hippuric acid T M S / Hippuric acid TMS Ornithine TMS 4 Citric acid TMS 4 Fructose TMS 4 M H P G TMS a Mannose TMS 5

0.834 0.837 0.852 0.893 0.915 0.923 0.937 0.940 0.942 0.966 0.967 0.971 0.991 1.000 1.012 1.017 1.105 1.110 1.118 1.121 1.155 1.160 1.176 1.187 1.195 1.204 1.216 1.220 1.225 1.251 1.254 1.267 1.278 1.300 1.312 1.331 1.341 1.352 1.355 1.387 1.397 1.400 1.401 1.406 1.408

1.541 0.461 0.461 0.997 0.778 1.208 2.233 0.992 0.424 1.170 2.944 2.233 0.540 1.000 0.540 0.625 0.076 0.922 3.377 1.060 4.477 0.651 0.947 5.825 4.766 5.825 0.204 0.369 0.429 1.268 0.209 3.691 0.522 0.721 1.272 0.408 3.691 0.541 0.477 0.477 3.377 2.158 0.419 6.717 4.335

218:1000 179:1000 151:433 172:565 233:185 111:788 156:1000 176:1000 288:266 232:700 174:1000 230:1000 220:1000 115:901 220:1000 286:537 347:135 218:906 142:1000 246:957 188:1000 200:433 234:902 217:1000 231:727 217:1000 187:334 170:1000 293:264 84:974 296:198 204:1000 307:286 254:1000 209:948 156:1000 204:1000 96:1000 105:1000 206:837 142:1000 273:1000 437:143 297:1000 204:1000

(Continued on p. 130)

130

J. D. S H O E M A K E R , W. H. E L L I O T T

TABL E I

(continued)

Row No.

Target compound"

RRT

RF

83

Homogentisic acid TMS

1.411

0.783

384:489

84 85 86 87

Fructose TMS 4 Galactose TMS 5 Normetanephrine TMS 3 Formiminoglutamic TMS 4

1.414 1.427 1.437 1.439

0.419 4.335 1.957 0.997

437:161 217:1000 297:1000 241:864

Hydroxy lysine TMS 4 Sebacic acid TMS 2 Vanillylmandelic TMS 3 Galactose TMS 5 Galactonic acid TMS s Glucose TMS 5 Metanephrine TMS 3 Histidine TMS 3 Lysine TMS 4 4-Pyridoxic acid TMS Tyrosine TMS 3 Glucitol TMS 3 Ascorbic acid TMS 4 Phenylpropionylglycine TMS z Pantothenic acid TMS 4 Glucuronic acid TMS 6 Glucose TMS 5 Gluconic acid TMS 6 Galactonic acid TMS 6 Glucaric acid TMS 6 Glucuronic acid TMS 6 N-Acetyl glucosamine TMS 5 Kynurenine TMS 3 lnositol TMS 6 Uric acid TMS 4 N-Acetylglucosamine TMS s N-Acetylglucosamine TMS s Kynurenine TMS 4 Tryptophan TMS 3 5-HIAA TMS 3 Cystathionine TMS 4 Xanthurenic acid TMS 3 Cystine TMS 4 Pseudouridine TMS 5 Lactose TMS 8 Sucrose TMS 8 Maltose TMS 8 Maltose TMS 8 Lactose TMS 8

1.452 1.460 1.463 1.468 1.492 1.494 1.503 1.507 1.519 0.816 0.817 0.830 0.830 0.842 0.845 0.846 0.862 0.868 0.869 0.876 0.881 0.897 0.906 0.909 0.918 0.918 0.921 0.940 0.951 0.954 0.954 0.964 0.974 1.000 1.091 1.092 1.096 1.119 1.119

0.656 0.579 5.221 4.335 1.674 5.570 5.239 1.856 1.268 0.811 2.966 2.723 0.725 0.160 0.325 0.415 5.570 0.819 0.921 3.337 0.415 3.884 0.975 2.335 1.744 3.884 3.884 0.510 4.303 3.807 1.256 2.184 0.458 3.981 2.690 0.234 2.409 2.409 1.431

232:384 331:366 297:1000 204:1000 217:876 204:1000 116:1000 154:719 174:1000 309:674 218:1000 319:894 332:427 351:403 291:482 292:152 204:1000 333:465 292:436 333:1000 292:156 245:1000 120:1000 318:330 441:760 173:1000 245:1000 307:523 202:1000 290:1000 128:687 406:1000 146:846 217:999 204:796 437:105 204:1000 204:1000 191:532

88 89 90 91 92 93 94 95 96 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 115 I 17 118 119 120 121 122 123 125 126 127 128 129 130 131

Mass:intensity (versus 1000) of quantification ion

ABA = aminobutyric acid; H I A A = hydroxyindoleacetic acid; M H P G = 3-methoxy-4-hydroxyphenyl glycol.

AUTOMATED SCREENING OF URINE SAMPLES

131

gram are provided in Table I. A single-ion chromatogram of a window of ten to fifteen scans on either side of a given relative retention time was searched and the scans of maximum intensity were matched against eight-peak mass spectra stored in a custom library file. The area of the peak yielding the best match was used to calculate the concentration of the compound relative to the internal standard, d3-creatinine, using the relative response factor (RF) listed in Table I. These response factors were calculated from five-point standard curves previously run for each compound. The five concentrations were generally between 25 and 1000 nmol per sample. Standards were run in groups of ten to twenty at a time. A custom program then calculated the concentrations in terms of the endogenous creatinine (target compounds 49 and 51), added together multiple derivative peaks of some compounds and printed the results.

Verification and statistical methods The urea nitrogen and creatinine concentrations of twenty samples were measured by a clinical autoanalyzer, either the Beckman 'Synchron' CX-3 or the Baxter Paramax. Both instruments measured urea by urine conductance. The creatinine procedure utilized the dual-wavelength absorbance ratio of the chromophore produced by picric acid. The first 21 clinical samples received from out-patients over six months of age which were found to be within previously established normal ranges for excretion of the target compounds were statistically analyzed using the Lotus 123 spreadsheet software. Means and standard deviations were calculated from these 21 subjects and appear in Table II. T A B L E II M E A N S A N D S T A N D A R D D E V I A T I O N S F O R 103 C O M P O U N D S IN U R I N E Data were from 21 outpatients, over six m o n t h s of age, none of whom had values outside established clinical norms. Patients older than 0.5 years: mean creatinine excretion 3.04/zmol/ml of urine. Compound

Organic Acids Lactic acid Pyruvic acid Glycolic acid Oxalic acid Hexanoic acid Octanoic acid Succinic acid

Concentration Compound (#mol/mmol of creatinine)

Concentration (#mol/mmol of creatinine)

Mean

S.D.

Mean

S.D.

50.73 9.79 38.10 3.26 0.91 0.39 12.60

53.54 9.34 27.35 9.04 1.05 0.37 12.13

0.09 4.93 0.85 3.67 1.08 4.75 0.06

0.10 3.37 3.17 2.12 2.95 5.38 0.06

Neurotransmitters ~-ABA Homovanillic acid Normetanephrine Vanillylmandelic acid Metanephrine HIAA MHPG

(Continued on p. 132)

J. D. S H O E M A K E R , W. H. E L L I O T T

132 T A B L E II (continued) Compound

Glutaric acid Fumaric acid Maleic acid Adipic acid Suberic acid Sebacic acid Ketoisoleucine Ketovaline Ketoleucine fl-Hydroxybutyric acid Methylmalonic acid Ethylmalonic acid Homogentisic acid Phenylpyruvic acid Malic acid Pipecolic acid Hexanoylglycine Phen Prop glycine Propionylglycine Citric acid 2-Oxoglutarate Hippuric acid Uric acid Phosphate

Concentration (#mol/mmol of creatinine) Mean

S.D.

1.08 0.95 0.00 2.87 4.34 0.60 0.05 0.16 0.00 2.60 0.92 0.14 0.04 0.25 0.46 0.03 0.41 0.00 0.00 226.32 43.46 871.36 118.09 1364.27

1.24 0.93 0.02 2.50 5.99 0.78 0.16 0.48 0.00 3.50 1.97 0.27 0.17 0.43 0.79 0.07 1.72 0.00 0.00 98.12 53.46 1024.84 114.57 915.27

0.90 2.99 I 1.16 0.10 0.56 0.67 1.05 0.30 42.76 63.47 63.65

0.71 5.25 8.88 0.17 0.91 1.34 1.22 0.51 22.84 32.76 142.96

70.38 11.65 262.58 143.14 2.96 6.08 6.14 6.35 26.69

107.54 15.42 881.72 399.78 1.85 6.07 5.12 4.59 23.74

Nutritionals Formiminoglutamic acid 4-Pyridoxic acid Pantothenic acid Xanthurenic acid Kynurenine Quinolinic acid Orotic acid Niacinamide 3-Methylhistidine Pseudouridine Ascorbic acid

Compound

Concentration (/lmol/mmol of creatinine) Mean

Ethanolamine

71.32

S.D. 33.29

Amino acids and glycine conjugates Butyrylglycine Suberylglycine Isovalerylglycine Tiglyglycine fl-Methyl Crotglycine Serine Glycine Sarcosine Alanine fl-Alanine Proline Hydroxyproline Hydroxylysine Asparagine Ornithine Tryptophan Leucine Valine Isoleucine Lysine Histidine Homoserine Threonine Methionine Cysteine Homocystine Cystine Cystathionine Aspartic acid fl-Aminoisobutyric acid Glutamine Phenylalanine Tyrosine Glutamic acid

0.24 0.29 0.37 0.50 0.48 74.67 513.54 1.35 113.35 0.23 4.14 88.17 0.35 0.96 2.19 13.52 7.58 12.30 3.75 31.45 194.90 0.29 36.20 1.21 30.32 0.07 10.05 0.35 1.36 82.35 118.96 23.59 27.35 6.87

0.36 0.24 1.19 0.93 0.64 37.74 360.61 1.21 69.81 0.73 2.85 49.59 0.62 0.65 3.21 7.37 4.10 7.69 2.75 52.32 144.31 0.37 25.38 1.20 24.58 0.10 28.34 0.65 1.06 136.96 89.94 l 6.02 13.98 3.51

33.82 43.25 3.91 9.74 6.11 35.23 l 8.79 16.29

20.95 28.14 5.53 10.36 6.25 29.19 29.28 26.02

Carbohydrates Fructose Mannose Galactose Glucose N-Acetylglucosamine Lactose Maltose Ribose Xylose

Glucuronic acid Galactonic acid Glucaric acid Glucitol Inositol Gluconic acid Sucrose Xylitol

A U T O M A T E D S C R E E N I N G OF U R I N E SAMPLES

133

RESULTS A N D DISCUSSION

The stepwise reduction in urea concentration achieved by this method is illustrated in fig. 1A-C. The presence of urea did not preclude acquisition of data from the rest of the chromatogram, but large amounts of the silylating agent were consumed by urea and other constituents may not have been completely derivatized in its presence. A typical TIC chromatogram is shown in Fig. 2A. Three of the 85 clinical samples retained significant amounts of urea after the treatment described. Two of these samples were found to have been acidified below pH 1, apparently in preparation for amino acid analysis by liquid chromatography. Neutralization of these two samples allowed removal of urea. The third sample was presumed to contain heavy metal ions known to inhibit urease [9] and was therefore pretreated by bubbling hydrogen sulfide gas through it for a few seconds until a slight precipitate appeared, then filtering and proceeding as above. This permitted removal of urea. The nature of the inhibitor is not known, but unusual concentrations of lead, iron or calcium were not found in the sample by atomic absorption spectrophotometry. 731

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134

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AUTOMATED SCREENING OF URINE SAMPLES

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acid; 641, 2-pentenedioic acid; 734, hydroxyproline; 753, threitol; 767, tetrose; 799, creatinine; 814 and 842, tetronic acids; 970, deoxypentonic acid; 1055, ribitol; 1112, 3-methylhistidine; 1134, hippuric acid; 1157, citric acid; 1245, histidine; 1254, lysine; 1293, ascorbic acid; 1341, glucose; 1351, galactonic acid; 1381, glucuronic acid; 1432, uric acid; 1499, tryptophan; 1583, pseudouridine; 1730, sucrose. The peak numbers in B correspond to the row numbers in Table I.

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J.D. SHOEMAKER, W. H. ELLIOTT

The success or failure of urea removal can be estimated from the area of a single-ion chromatogram for mass 189 in the region of scans 200-400, as this is a major ion in the mass spectrum of the most common TMS derivative of urea, urea di-TMS. When urea remains in very large amounts, the tri-TMS derivative, containing masses 245,261 and 276 all at about 20% of base-peak intensity, may also be observed at earlier retention times (Fig. 1A, peak 160). Urease itself was found to add few contaminants to the samples, even if left in the mixture during dehydration and derivatization by omission of the acetone precipitation and filtration steps. Citric acid was the only significant contaminant and was found to add 11.8/tmol citrate per mmol creatinine when creatinine was present at 10 #mol/ml. The range of urinary urea concentration in twenty samples from children analyzed by an automated clinical analyzer was 32-596 mg urea nitrogen per dl (11-210 mmol/1). Samples from adults may contain higher concentrations of urea and require dilution prior to urease treatment. Fig. 3 shows the mass spectra for native and trideuterated creatinine. Creatine is quantitatively converted to creatinine during the procedure described above, as long as alkaline conditions are maintained throughout the aqueous steps. This is in contrast to a previous report [10] stating that with mineral acids and bases, acidity favored creatinine and alkalinity creatine formation. A creatine tri-TMS derivative elutes near citric acid tetra-TMS, but is not detectable when the present procedure is used. The agreement between the two methods of urinary creatinine measurement (isotope dilution in the method above and the automated clinical analyzer method based on picric acid) depended on the concentration. In twenty samples analyzed by both methods, the correlation coefficient was 0.923 (P

Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease.

Eighty-five clinical urine samples and nineteen urine samples previously found by other laboratories to suggest genetic metabolic defects were prepare...
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