Quantitative Metabolic Profiling of Urinary Organic Acids by Gas Chromatography-Mass Spectrometry: Comparison of Isolation Methods John A. Thompson and Sanford P. Markey’ 6.F. Stolinsky Research Laboratories, Department of Pediatrics, University of Colorado Medical Center, Denver, CO 80220
Several methods of isolation of organic acids from urine have been evaluated with regard to efficiency and reproducibility using quantitative gas chromatography and a GC-mass spectrometer-computer system to verify structures. Extraction by DEAE Sephadex anion exchange removed copious amounts of sulfate and phosphate with organic acids. A number of procedures designed to selectively eliminate these inorganic acids were investigated, resulting in the selection of preliminary barium precipitation. Aqueous solutions of standard organic acids of varying structural types were subjected to precipitation-anion exchange, continuous solvent, and manual solvent extractions. Recoveries by the first two methods were largely comparable, being higher than those of the last method. The best precision was obtained by anion exchange, and the efficiency of this method did not depend on acid concentration. Comparative extractions and quantitations of a urine specimen demonstrated that more organic acids and fewer interfering substances were isolated by anion exchange than by solvent extraction. These results and the fact that precision was much better by anion exchange indicate that it is the extraction method of choice for quantitation.
The concept of metabolic profiles was introduced by Horning and Horning ( 1 , 2), who coined the term to refer to qualitative and quantitative analyses of complex mixtures of physiological origin. Using gas or liquid chromatography ( 3 ) ,it is possible to produce characteristic patterns of various categories of metabolites such as urinary volatiles ( 4 ) ,steroids (5-7), polyols ( 8 ) ,and acids (9-11), as well as similar compound types from other physiological fluids (serum, amniotic fluid, cerebral spinal fluid, etc.). Such metabolic profiles can be considered a generalized use of the principles employed for many years in the determination of amino acid profiles from all types of biological (12) and extraterrestrial origins (13-15). Unlike the selective detection system employed for amino acid analyzers, other compound types require mass spectrometry to establish the structural nature of the many possible products present in biological extracts. Combination gas chromatography-mass spectrometry (GC-MS)-computer systems have been most successfully applied to these analyses (16). With the availability of elegant systems for studying the structures of compounds chromatographically separated, has come the need to produce extracts of physiological fluids which are worth studying in such detail. Urinary organic acids comprise a category reflective of many important metabolic routes in animals-amino acid, lipid, carbohydrate, biogenic amine, and bacterial end products are frequently low molecular weight acids. Various methods for isolating organic acids have been reported in the literature, Present address, Laboratory of Clinical Science, NIMH, NIH, Bethesda, MD 20014.
but there has been a paucity of data regarding comparison of these methods for qualitative and quantitative analyses. The simplest and fastest procedure is that of solvent extraction of a salt-saturated urine with ethyl acetate, diethyl ether, or pooled extracts of both (17).For some years, we have utilized this procedure for rapid analysis of patient populations likely to have a genetic disorder leading to an enzymatic block and the resulting accumulation of abnormally large amounts of acidic metabolites. Diseases such as methylmalonic aciduria, maple syrup urine disease, and glutaric aciduria have been readily detected by analyzing the methylated and/or silylated extracts. Most of these genetic disorders have a low incidence in the general population, with the result that greater than 99% of samples screened reveal interesting profiles which frequently change with the course of a patient’s condition. Consequently, it has been of considerable interest to refine metabolic profiling to reveal subtle, as well as gross, metabolic changes. In previous studies, we illustrated the variations which can be measured between individuals on the same diet ( 1 8 ) ,and the effects of ethanol upon the organic acid profile (19).The technique of solvent extraction was known to be unsatisfactory for many classes of organic acids, and the present study was initiated to compare techniques for reproducibly isolating organic acids from urine. Horning and Horning recommended that a DEAE-Sephadex anion exchange procedure ( 1 , 2 ) be employed when a “total acid” fraction was desired, and noted that solvent extraction strongly discriminates in favor of aromatic acids as opposed to polyhydroxy aliphatic compounds. In a series of recent papers, Chalmers and Watts have modified the Hornings’ procedure for qualitative and quantitative studies of urinary organic acids (20-23). Neutral and basic compounds are first washed through the column, and the acidic components are eluted with aqueous pyridinium acetate. The 0-ethyloximes of keto acids are formed prior to lyophilization of the sample under carefully controlled conditions. Finally, the sample is derivatized for GC-MS by trimethylsilylation of the dried residue. While the above methods have been well characterized in the literature, no qualitative and quantitative comparison of manual solvent extraction, continuous liquid-liquid extraction, and anion exchange techniques had appeared, and each of these techniques was known to have significant disadvantages. Solvent extraction suffers from lack of specificity due to the solubility of neutral compounds such as urea in both phases, as well as the solubility of water (and its contents) in the organic phase. The resulting extract will contain urea which chromatographically interferes with the quantitation of important low molecular weight acids, as well as other neutrals which may interfere in the interpretation of GC-MS data. Polar poly-hydroxy acids are difficult to detect in organic solvent extracts. Extracts prepared by anion exchange will contain copious amounts of inorganic acids, particularly phosphate and sulfate, which obscure c’iromatographic resolution in the range of 12-15 ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1313
Table I. Precision of Gas Chromatographic Quantitation-Repeated Injection of a Single Urine Extract ( N = 10) Peak'
Mean relative
Standard
peak areab
deviation
3 26 1.4 17 0.7 4, 5 27 0.9 6,7 10 29 2.0 12 107 8.7 13 13 0.5 14,15 18 0.5 54 1.7 21 23 26 0.9 24 14 0.5 27,28 17 0.7 a Refer to Figure 1A. Percent of internal standard n-tetracosane.
methylene units (MU), a region of considerable,interest in the study of inborn errors of metabolism. The availability of small computer data systems for online integration of GC data ( 2 4 ) has enabled the quantitation of multiple peaks in complex chromatograms. The following report is a comparison of methods of isolation of organic acids from urine using a GC-data system and GCMS-computer system to compare reproducibility and efficiency of these methods. Modifications of previously reported methods were contrived to permit isolation and derivitization of an extract which preserves maximum information with respect to organic acid content.
EXPERIMENTAL Standard solutions of organic acids were prepared by weighing an amount in the range 9.2-24.0 mg (see Tables I1 and 111, discussed below, for exact concentrations) of each free acid (commercially available) into a volumetric flask. The mixtures were dissolved in 10 ml of methanol and diluted to 100 ml with chloroform. Aliquots of 1.0 ml were carefully evaporated under a stream of nitrogen without heating, and 1.0 ml of water was added to dissolve the residue. In certain cases, aqueous solutions of inorganic ions were added to approximate thei; concentrations in normal urine (25).Amounts added to each 1.0 ml of a standard solution were: sodium chloride, 20 gl containing 5.8 mg; sodium sulfate, 10 gl containing 3.6 mg; and potassium dihydrogen phosphate, 20 gl containing 4.7 mg. The urine was a first-voided morning sample from a normal adult male which was stored by freezing without preservatives. The creatinine concentration was determined (26) to be 171.7 f 2.5 mg/100 ml. Column Methods. Columns of Amberlite XAD-2 and XAD-7 resins (Mallinckrodt Chemical Works) were prepared as previously described (27). A standard solution containing phosphate and the six organic acids (see Table I1 below) a t pH 1 was added to each column and washed with 6 ml of water. Nitrogen pressure applied to the top of each column forced out residue water, and each was then eluted with 20 ml of methanol. After evaporation of the solvent under a stream of nitrogen, 2 ml of benzene were added and evaporated to remove traces of water. Recoveries of the acids were determined by GC analyses as described below. Sephadex DEAE A-25 anion exchange resin (Pharmacia Fine Chemicals) was prepared by soaking 20 g in distilled water for a day, washing successsively with 0.5M HC1 (500 ml), water (until neutral), 0.5M NaOH (500 ml), and water (until neutral) on a sintered glass filter funnel and storing in distilled water until used. The resin was then packed in a 1- X 22-cm column to a height of 8 cm, and washed with 40 ml of 0.5M pyridinium acetate (28). The standard or urine sample to be extracted (1.0 ml) was treated with 3 ml of a 0.1M solution of Ba(0H)z in a centrifuge tube. The contents were immediately mixed and centrifuged for 15 sec. The SUpernatant was removed and treated with 200 pl of a hydroxylamine hydrochloride solution (15 mg) and heated a t 60' for 30 min. The solution was cooled, adjusted to pH 7-8 with 2N HC1, and added to the head of a prepared anion exchange column. The sample was allowed to drain into the resin, 30 ml of water were passed through, 1314
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
Mean relative
Standard
Peak
peak area
deviation
32,33 34 39 40 41 42,43 45 48 49 58 59
22 11 66 11 10 18 29 19 13 19 13
0.4 0.5 1.6 1.0 0.4 0.7 0.5 1.0 0.3 2.3 1.4
and the acids were eluted with 18 ml of 1.5M pyridinium acetate. This later effluent was collected and lyophylized a t pressures in the range 1-8 X lo-* Torr for the 4-5 hours necessary to attain dryness. The residue was dissolved in methanol, transferred to a centrifuge tube, and the solvent removed with a stream of nitrogen. Solvent Extractions. Manual solvent extractions of 1.0-ml aliquots of urine were preceded by the addition of 3 drops of 5N NaOH, 200 pl of the aqueous hydroxylamine hydrochloride solution, and heating a t 60' for 30 min (29). The samples were then acidified to pH 1 with 6 N HC1 and extracted with both ethyl acetate and diethyl ether as previously described (17). Continuous solvent extractions of standard and urine samples were performed after treatment with hydroxylamine hydrochloride and acidification as described above. The sample was then transferred to a micro (lighter-than-water) liquid-liquid extraction apparatus (Kontes Glass Co.), saturated with NaC1, and extracted for 15 hr with ethyl acetate. The solvent was evaporated under a stream of nitrogen. Benzene (2 ml) was added and similarly evaporated to remove traces of moisture. Trimethylsilylation. Samples were trimethylsilylated for GC analysis by adding 300 p1 of BSTFA (Supelco, Inc.) to the extract residues and heating a t 60' for 2 hr in a culture tube with a Teflon-lined screw cap. T o aid in the interpretation of GC-MS data, trimethylsilyl (TMS)-dg derivatives of two urine extracts were prepared by substituting deutero-Regisil (Regis Chemical Co.) for BSTFA. Gas Chromatographic Quantitation. Quantitation standards, stearic acid (197 pg) and n-tetracosane (194 pg) were each added to the extract residues as solutions in 0.50 ml of benzene, and the solvent was evaporated with a stream of nitrogen prior to TMS derivatization. Chromatography was performed on a HewlettPackard 7620A GC with a flame ionization detector (FID) and a lh in. X 6 ft glass column packed with 5% OV 22 on SO/lOO Supelcoport (Supelco, Inc.). GC conditions were: nitrogen flow, 17 ml/min, injector and detector temperatures, 270'; column oven operated isothermally a t 80' for 4 min and then programmed to 280' a t So/ min. Peak areas and retention times were determined with an online Digital Equipment Corporation PDP 8e computer equipped with a teletypewriter and utilizing a previously described program (241. Extraction efficiencies for the constituent acids of a standard solution were determined as follows. GC peak areas were measured relative to stearic acid and n-tetracosane after evaporating the methanol-chloroform solvent from 1.0-ml aliquots, adding the two GC standards, and forming TMS derivatives. The mean relative peak areas obtained from 10 such determinations were compared with those mean areas from a series of extractions of the identical mixture of acids from aqueous solution. Gas Chromatography-Mass Spectrometry. The GC column and conditions were similar to those described above, except that helium was used as the carrier gas. A Beckman GC-45 chromatograph was used with a ceramic frit GC-MS interface to an Associated Electrical Industries MS 1 2 mass spectrometer, and a Digital Equipment Corporation PDP 8/I computer (30, 3 1 ) . Mass spectra were recorded a t 70-eV ionizing voltage, 150-pA trap current, and a t an ion source temperature of 180'-200 "C. A complete spectrum was obtained every 10 sec.
I
Recoveries of Carbon-14 Acids. Prior to use, 50 pCi of each 14C-acid(citric, oxalic, and pyruvic from Amersham/Searle Corporation, and 2-ketoglutaric from New England Nuclear) were purified on a DEAE anion exchange column as described above. The lyophylized residues were dissolved in 10 ml of water and 20 pl added to each 1.0-mlaliquot of urine. When the corresponding unlabeled acid was added as “carrier”, it was deposited in the tube before the urine. After thorough mixing, 20 p1 were removed and counted in 10 ml of scintillation fluid (prepared from 32.2 grams of napthalene, 2.1 grams of PPO, 21 mg of NPO, 100 ml of ethanol, 160 ml of dioxane, and 160 ml of toluene) and the remaining solution was extracted by the DEAE anion exchange procedure. The lyophylized residue was redissolved in 1.0 ml of water and the radioactivity again determined in a 20-p1 aliquot.
I nil
ni I
I I Ill c
I’
RESULTS AND DISCUSSION Precision of the Quantitation Method. A single representative anion exchange extracted urine sample was chromatographed ten times and the areas of several of the peaks were computed relative to the internal standard ntetracosane, which has a retention time longer than the acids of interest. Areas of these peaks, whose identifying numbers correspond to those in Figure lA, are tabulated in Table I along with the standard deviations. Single, as well as partially overlapped peaks, were quantitated with good precision. The least reproducible were peaks 58 and 59 which had standard deviations of 11-12% of the peak area. These have relatively long retention times and column decomposition might cause some variation. The GC technique, including column conditions and computer determination of each peak area, were considered adequate to quantitate the constituents of a series of urine extracts. Anion Exchange Extraction. Removal of acids from urine by the use of DEAE Sephadex A-25 was discussed by Horning (28) who treated the lyophylization residue with diazomethane. The increased solubility of the resulting methyl esters in organic solvents, allowing them to be removed from an insoluble inorganic acid and polar conjugate residue and subsequently trimethylsilylated, may be responsible for the absence of sulfate and phosphate in the analyzed samples. The anion exchange method was later used by Chalmers and Watts who formed TMS-esterTMS-ether derivatives of the lyophylized extract and obtained sizable phosphate-TMS and sulfate-TMS peaks in their GC traces (22). In our hands, this ion exchange procedure evidently operated more efficiently to remove copious amounts of the inorganic acids from urine along with the organic acids of interest. Experiments using a standard column (see Experimental) demonstrated that when the effluent was collected in 1-ml aliquots, 96% of 14C-citrate eluted with 1.5M pyridinium acetate in fractions 5-10 along with 93% of 32P-phosphate, and that fraction 7 contained the maximum amount of each. Sulfate was similarly eluted in fractions 5-9 as determined by barium precipitation in the effluent. Because of the large concentration of these ions relative to the organic acids, their TMS derivatives obscured large portions of the chromatogram. In addition, repeated injections of these mixtures into the gas chromatograph greatly decreased column life, which may have been due to deposit of underivatized inorganic acids in the injector. A variety of standard trimethylsilylation conditions were tested in an attempt to derivatize the organic acids selectively (32). These, however, were unsuccessful and sulfate-TMS and phosphate-TMS GC peaks were not substantially reduced in size. Further work centered on removing these species from the sample prior to derivatization. The principle of operation of ion retardation resin (BioRad Laboratories, AG l l A 8 ) is that ions are retained in regions of high ionic strength on the resin as the remaining
1
48
1
0
5
10
15
20
25
MINUTES Figure 1. Comparative gas chromatograms of a urine specimen extracted by the ( A ) anion exchange, (B) manual solvent, and (C) continuous solvent methods. Each was oximated, trimethylsilylated, and analyzed on an OV 22 column isothermally at 80’ for 4 min, increasing to 280’ at 8’/min
constituents of a solution are eluted (33).Attempts to remove sulfate from a solution containing 14C-citric acid a t p H 1 were unsuccessful, as they eluted simultaneously. Molecular-sieve chromatography using Sephadex G-25 has been employed to separate ortho-, di-, and triphosphate ( 3 4 ) . We attempted a separation of 32P-phosphate and 14C-citrate on a column of G-25 resin. Citrate showed a slight tendency to be eluted ahead of phosphate, but the overlap was considerable and the method did not warrant further investigation. Because of the difference in molecular size between these two species, a much larger column and slower elution might have effected a separation. The time involved for such a procedure, however, would be unreasonable for analyses of routine clinical samples. In addition, separation of smaller organic acids might not be possible. Sephadex G-10 was even less effective, as phosphate and citrate were eluted simultaneously in the first few fractions. Organic acids extracted from fruit products have been isolated on an anion exchange column in the formate form, and subsequently eluted with 6N formic acid in acetone (35).We proceeded on the rationale that differences in solubility between organic and inorganic acids in the organic ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1315
Table 11. Percentage Recoveries of Standard Acids from Aqueous Solution with Amberlite XAD Resins ConcenAcid
2 -Hydroxyiso
-
tration,
Range of XAD-7
ug/ml
recoveriesa
92
51-78
XAD-2 re-
Continuous
coveries’
34
valeric Phenylacetic 118 45-81 38 Malic 97 7-24 0 Adipic 93 7 3 -a4 50 4 -Hydroxy 110 73-90 46 benzoic Citric 123 21-30 6 Results of three experiments. Quantitation was by gas chromatography (see Experimental). * Results of one representative experiment. eluant might be sufficient to remove the former while leaving major amounts of the latter acids adsorbed on the resin. A column of weak anion exchange resin (Bio-Rad Laboratories, AG3-X4A) was prepared in the formate form, and an aqueous solution containing sulfate and phosphate in addition to 2-hydroxyisovaleric, phenylacetic, malic, adipic, 4-hydroxybenzoic, and citric acids was analyzed. Inorganic acids appeared in much smaller amounts than were present in the DEAE extracted samples. On the other hand, while the aromatic acids were present in high yields, recoveries of 2-hydroxyisovaleric and adipic acids were only moderate and those of the more water soluble acids, malic and citric, were poor. In addition, several extraneous peaks appeared which were probably due to artifacts from the resin itself. These poor features of the method, as well as the potential for adverse effects of formic acid on labile acids, preclude its application to quantitative analyses of urinary acids. Sulfate should be separable from carboxylic acids by ion exchange because of the differences in pK, values. This, however, would require more careful experimental conditions and a longer time than is convenient for clinical analyses. Several carboxylic acids (e.g., oxalic and maleic) have lower pK,’s than phosphoric, rendering selective phosphate removal impossible by ion exchange. Some success has been achieved in the removal of organic acids from aqueous solution by the use of Amberlite XAD-7. This is a non-ionic acrylic ester resin which adsorbs lipophylic molecules from aqueous media. A solution of phosphate and organic acids was processed on a column of this resin. No phosphate could be detected in the extract by GC. Organic acid recoveries, as shown in Table 11, are highly variable with the more water soluble acids (malic and citric) isolated in low yield. This excessive variation precludes the method as a quantitative extraction technique. The experiment was repeated with Amberlite XAD-2, a non-ionic polystyrene resin. The recoveries were very low (Table II), and this extraction method was also sbandoned. Further experiments involved the removal of sulfate and phosphate from solution as insoluble salts prior to DEAE anion exchange. A perusal of solubility tables (36) revealed that barium salts of carboxylic acids are generally quite soluble in water, while barium phosphate and barium sulfate are highly insoluble. Barium precipitation has previously been employed to remove radioactive inorganic sulfate from aqueous solutions containing organic sulfate esters for quantitation purposes (37). A series of aliquots of a solution containing the acids listed in Table 111, as well as approximate physiologic concentrations of sulfate, phosphate, and NaCl, were treated with 0.1M barium hydroxide and 1316
Table 111. Percentage Recoveries of Standard Acid from Aqueous Solution by Anion Exchange and Solvent Extraction
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 . JULY 1975
solvent
Acid‘
2 -Hydroxyiso -
butyricb Glycolic‘ 2 -Hydroxyisovaleric Phenylacetic Malic Adipic 4 -Hydroxy benzoic Citric
Anion exchange,
extraction,
mean i S . D . d
mean i s . D . ~
78.4
* 7.5
69.0 87.5
+ 9.2 + 4.6
60.1
92.0 66.3 92.0 94.1
+ 7.6 + 3.7 + 3.5 + 3.6
85.0 + 11.9 75.1 i 8.6 82.6 i 3.9 4.3 85.0
17.4
* 2.7
44.3
... ... e
+ 8.6
* * 8.9
” Concentrations are equivalent to those
of Table 11. Concentration, 142 ug/ml. Concentration, 244 pg/ml. Mean f standard deviation of five extractions. Quantitation was by gas chromatography (see Experimental).e Not determined.
the supernatant was processed on a DEAE column (see Experimental). Average recoveries and their standard deviations, as determined by GC quantitation, appear in the table. Sulfate was not present, and only a low, quite tolerable, level of phosphate was detected by GC. The acids chosen represent a variety of structural types: mono-, di-, and tricarboxylic acids; hydroxy acids; and aromatic acids. Recovery data on the I4C-acids listed in Table V further extend the range structural types investigated to keto acids. Use of I4C-acids allows recoveries to be determined directly from urine rather than from a prepared solution in water. Recoveries of citric acid from both media agree, thereby demonstrating the significance of the data in Table 111. While recoveries are, in general, satisfactorily high, the low recovery of citric acid was expected. Other than barium tartrate (solubility, 0.3 mg/ml), barium citrate was the least soluble barium carboxylate (0.57 mg/ml) listed in our reference source (36).Anion exchange recoveries of citric acid were 85-90% from solutions not containing sulfate or phosphate and where barium hydroxide treatment was omitted. The good reproducibility of the precipitation-anion exchange method is evident from the standard deviations listed in Table 111, which represent a combination of variables due to the sample preparation procedure, as well as the GC quantitation method. Other than citric acid (where solubility is an additional factor), the highest variation occurs with the most volatile acids, 2-hydroxyisobutyric and glycolic, probably due to lyophylization losses. A comparison of recovery efficiencies of I4C-oxalic and 14C-2-ketoglutaric acids, with and without addition of unlabeled “carrier” acids (Table IV), demonstrates that the recoveries are essentially unaffected by concentration, as this represents a concentration range of approximately 1000. Additional evidence for such insensitivity is provided by GC recovery data for malic and 4-hydroxybenzoic acids tabulated in Table V. Each of these extractions represents a 100-fold difference in concentration, yet recoveries are essentially the same. Aliquots of distilled water were processed in an identical manner as the standard and urine samples. These procedural blanks showed no consistant interfering compounds by gas chromatography, only occasional small peaks, usually at long retention time.
Table IV. Percentage Recoveries of Carbon-14 Labeled Acids from Urine by Anion Exchange Concentration
Table V. Effect of Concentration on Recoveries of Standard Acids from Aqueous Solution by Anion Exchange Concentra-
of added Acid
unlabeled carrier Acid
Oxalic Oxalic Citric Pyruvic 2 -Ketoglutaric 2 -Ketoglutaric
acid, g g / m l a
0
167 0
120 0
162
Mean + S . D . b
32.0 f 1.0 35.9 i 0.7 18.3 * 1.5 72.0 i 0.7 69.7 i 1.5 61.7 i 2.5
An undetermined quantity of unlabeled endogenous acids were also naturally present in the urine. Mean f standard deviation of three extractions. Radioactivity was measured as described in the Experimental. a
Solvent Extraction. Extraction efficiencies for a series of standard acids were determined by the continuous liquid-liquid method (Table 111). Yields compare favorably with those obtained by anion exchange except that 2-hydroxyisovaleric is significantly lower and citric is about 2.5 times higher with continuous extraction. On the other hand, the standard deviations indicate that reproducibility is better by anion exchange. Difficulty of controlling solvent reflux rate may partially account for this variation. On several occasions, the results of a continuous extraction had to be disregarded because of operational problems (Le., solvent vapor leaking from the apparatus due to changes in condenser water pressure or through joints that had become dry by dissolution of the silicone grease). If such experiments are run without supervision, there is a much higher potential for problems than one encounters using manual extraction or anion exchange techniques. Procedural blanks gave essentially the same results as those obtained above for anion exchange extractions, except that small peaks a t long retention time were more numerous and consistent. Solvent extraction does not discriminate between acidic and non-acidic urinary constituents. The continuous method yielded large amounts of both phosphate and urea, which interfered with the GC analyses of organic acids. Smaller quantities of phosphate and urea were obtained on manual extraction, but the acids of interest were also less efficiently isolated. For example, while adipic and 4-hydroxybenzoic were obtained in approximately 80% yield, recoveries of 2-hydroxyisovaleric and malic acids were on the order of 45% and 14%, respectively. Comparison of Extraction Methods. One-ml aliquots of the same urine sample were extracted by DEAE anion exchange, manually with solvent, and continuously with solvent (methods A, B, and C, respectively). The extracts were separately dried, and equivalent amounts of standards (stearic acid and n-tetracosane) and BSTFA added to each. Equivalent GC injections were made and representative chromatograms appear in Figure 1. The peaks were quantitated and their mean areas and standard deviations, which are the result of five separate analyses, are listed in Table VI as percentages of the n-tetracosane peak. Unresolved peaks are represented by a single quantity as indicated by brackets. Peak numbers in the table correspond to those in the figure. Compounds extracted by each method were identified as completely as possible by GC-MS-computer techniques, making liberal use of a recently compiled library of mass spectral data (38) and other published spectra (39, 40). Identifications (positive and tentative) are listed also in Table VI. Where no identifications could be
Malic 4 -Hydroxy -
tion, ilglrnl
9.7 970 11.0
Mean GC peak area
s.D.'
54 * 5 61 i 2 120 i 4
benzoic 1100
124 f 4
Mean gas chromatographic peak area (as percent of the internal standard, n-tetracosane) f standard deviation of four determinations. The 100-fold difference in acid concentration was accompanied by a corresponding difference in n-tetracosane concentration. a
made, major ions are indicated. Many peaks could be recognized as inhomogeneous, and if significant differences in GC retention time could be distinguished between the components of a single GC peak (by analyzing successive MS scans), more than one number was assigned to that peak. Consideration of relative intensities of characteristic ions allowed an approximation of the contribution of each component to the peak, and this is indicated by pluses in the table. A quantitative comparison of methods A-C can be accomplished using several criteria, including yields of endogenous organic acids (accuracy), reproducibility of these yields (precision), and the content of interfering species which are not organic acids. A cursory examination of Figure 1 reveals some major characteristics. Manual extraction was obviously the least efficient, while chromatograms A and C are more or less comparable with regard to number and intensity of peaks. The latter method is somewhat better in the early region (3-9 min), probably due to losses of volatile acids during the lyophilization stage of method A. On the other hand, peak 8, a major peak in C does not contain organic acids. The region corresponding to peaks 11-13 of chromatogram C is also, a t first inspection, more impressive than that region of A; however, these peaks are mainly due to urea and phosphate. Procedural blanks demonstrated that more of the late peaks (after n-tetracosane) in C were due to artifacts than those in A . In addition, Table VI reveals that several more peaks (Le., 16, 22, 29, 34, 35, and 60) which do not represent organic acids, are present in extract C, but are absent in extract A. Several of these interfere with quantitation and identification of the acids of interest, and attempts were made to selectively remove the neutral compounds. Manual pre-extraction of basic urine with solvent failed to eliminate sufficient amounts of these species, while manual back-extraction of urine extracts with 0.1N HC1 (containing sodium chloride) removed these along with large quantities of the organic acids. Much of the information concerning relative efficiencies of anion exchange and solvent extraction, with regard to removal of organic acids from urine, is summarized in Tables VI1 and VIII. A total of 13 acids identified in extract A were not found in extracts B or C. Each of these acids contains three or more polar groups capable of hydrogen bonding with water and, with the exception of the aromatic acids, failure to extract them with solvent is not surprising. In addition, several more acids in this category were minor components of B or C while substantial amounts were present in A (Table VIII). On the contrary, solvent extracts contained only two acids (also minor components) which ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1317
~~~
~~~
Table VI. Composition and Relative Sizes of Peaks from Quantitative Gas Chromatographic and Gas Chromatographic-Mass Spectrometric Analyses of Urine Extracts
Peak
conlzibution'
Components
1 2 3
2-Hydroxyisobutyric
++++
Lactic Glycolic 3-Hydroxyisobutyric Glyoxylic Pyruvic 3-Hydroxyisovaleric Cresol Glycerol Artifacts 2,3-Dihydroxyisobutyric 2,3-Dihydroxybutyric (diastereomer A) Glyceric Urea Phosphate 2,3-Dihydroxybutyric ( d i a s t e r e o m e r B) Succinic 2,4-Dihydroxybutyric 3,4-Dihydroxybutyric
++++
4 5 6 7
8
9 10 11 12 13 14 15
++A
++++ ++++ ++ ++T
18
Erythritol o r Threitol Erythron0 o r T h r e o n o lactone M' 276
+++ --
'+ 1 .
+&JA'+&
'-++
25
26 27
28
29
1318
hexanoice) M' 364 (Dihydroxyhexanoic o r Hydroxy pentanedioic) 3 -Methyladipic
261(13)219(12) 217(14) 189(16)147(30) 103(23) M+ 364 (Dihydroxyhexanoic o r Hydroxy pentanedioic ) M' 378 (Dihydroxyheptanoic o r Hydroxyhexanedioic ) Deoxypentonic M* 3 64 (Dihydroxy hexanoic o r Hydroxypentanedioic) Hydroxybenzoic Pentitol
10 * 4 12 f 5 12 * 3
1.3 i 4
++++ ++++
15 i 5
+++
16 i 7
1
1
++++
++
25*3 6i4
0
2*1
1
i 0
+-+
+'+
++-
98 i 10 15 i 3
2*1
1
+
0
1
++++
+ +'+ +'+ 1-
0
-.
I
_1
...
0
OSf
++
++++
'ic
13 + 8
3il
1 I
J
I
+++' ++ ++ +
+++
10 i 1
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
+
+++
1
+++
++
+++-e
2i1 9i2
++ ++ +
28 i 12 17 i 6 15 + 4
1
612
19*2
1
l-
54*13 2*1
0 0
211
J-+
t+
++
++
+' 4*2
0
1
OSf
++' +'+
++t
++
... ...
+A+
0
7i3
0 0
0
++
+A++
0
19*3
+'
I
++
49 i 9
0
0
T+
-+
... *.. 10i2
A+'
0
1 p
0 0
it+
i
2i1
29 i 5 17 * 2
+++
++++
+
I
++
0
.
12 * 5
++++ ++++ 0
+ T
26i2
6*2 28 f 18 37 i 9
++++ ++++
...
+++
55*5
++A+
0
++7*3
Peak area
++
++
...
contribution
11 * 7
+++
++++
1
++++
342 (2)319 (1)262 ( 7 )247 (25) 0 M' 364 (5,6-Dihydroxy-
++++ ++++ ++
i++
333 (1)259(4)215(5)116(22) 0
24
2*1 10 i 3 26 i 3
+
20 21
Adipic
Peak area
_1
M' 364 (2,4-Dihydroxyhexanoice)
22 23
contribution
1
0
19
E r y t h r o n i c o r Threonic 3-Methylglutaconic Artifact 171(100) E r v t h r o n i c o r Threonic
I-
J-
256(6)244 (18)241 (12) 231 (12)191(87)147 (60) 16 17
c
7
Peak aread
++it
+++
Component
Component
Component
*
NO.a
Continuous extraction
Manual extraction
Anion exchange
0 0
1*1
+ +++
1 t
28*6
713
Table VI (Continued) Anion exchange
Peak
Component Componentsb
30 31
32 33 34 35
36 37
contribution'
Hydroxymethylfuroic M' 302 M* 364 (Dihydroxyhexanoic o r Hydroxy pentanedioic)
+ ++
289 (10)258(10)230(11) 156 (87) 2-Ketoglutaric
0
Hydroxybenzoic 3 -Hydroxyphenylacetic 4 -Hydroxyphenylacetic Pentonice FunanOsee FunanOsee Suberic 0-Glycerophosphate
M' 552, 345 (100)
38 39 40 41 42 43 44 45 46
47 48 49 50 51 52 53 54 55 56 57 58 59 60
+++ +++ + ++++
Manual extraction
1 !l I: 1 Component
Peak aread
0 0
+++ + +++
13 + 2
... 9*3 22 i 2
+++ 0
1850
Homovanillic Glucuronic (oxime), peak A Glucuronic (oxime), peak B Dihydroxymethoxy phenylacetic Glucuronic, peak A Glucuronic, peak B
++ ++++
30
+++ ++++
16 ~t2 65 + 5 13 * 3 9*2
++++ ++++ +++
M+ 398 Palmitic M+ 479 Hippuric Indole a c e t i c S t e a r i c (Internal Standard) Uric n - T e t r a c o s a n e (Internal Standard) 3 -Hydroxyhippuric 5-Hydroxyindole acetic 4 -Hydroxyhippuric Glucuronide conjugatee Glucuronide conjugatee Phthalate a r t i f a c t
+++
+++
0
7 + 1
17 + 3
++++
...
0
++++ + + ++ ++
78i35 22 f 3
++++ +++
33 * 2
0
22 f 4
++ ++++ +++ 0
++ +++ 0
++++ + + ++ ++
0
0
0
0
ii:
i-:
] # i > 1 5
0
3a*37
++++
0
+ o
lo* 1 4+2 38 * 3
... OSf
62 i 8
29-6
... lil
4*1
++
...
0
++++ +++ +
2*1 8*1
...
0
...
++++ +++
34 54 14 + 3 OSf
+
OSf
...
++++ ++++ ++++
...
+++ ++++ ++++ ++++
92 + 8
0
++++
... ...
++++
... ... ...
0
...
0
12 f 4
0 0
... ... 11 * 3
211
++++ ++++ +++
...
9f1
OSf
+
14 + 2
0 0 0 ++++ 0 0
... ...
++++ ++++ ++++
+++
lo* 1 19 2
0
0
+++
++-F
++ ++++ ++++
0
11 2 19 * 2 16 i 2
Peak area
87 + 9
3*1 7
+
contribution
2 * 4 1 ]
+++
24 i 2
++ ++++ ++++ + + ++ ++ ++
Peak area
+
15+2
Aconitic a -Glycerophosphate Citric Azelaic Isocitric Dihydroxybenzoic Hydroxymethoxybenzoic 1,5 Hexonolactonee 1,5-Hexonolactonee Dihydroxyphenylpro pionic Me thoxymandelic Homogentisic
+++
Component
conhibution
+
+++
++
Continuous extraction
4f1
... 14 * 3
0
3*3
... 2*1 ... ...
0
6f2 18 + 7 15 i 1
...
++ 0 0
...
0
0
++ +++ ++++
16 f 7 4*2
5*4
Refer to Figure 1. Positive and tentative identifications by gas chromatography-mass spectrometry (see Experimental). Where no identifications could be made, prominent ions (and their relative intensities) are given. Molecular ions are designated by M A . Estimated by mass spectrometric ion intensities: 0 = component does not contribute to the peak; = trace contribution; ++ = partial contribution; + + + = principal contribution; + + + = peak is essentially pure. Mean f standard deviation of five analyses. Areas are percentages of the internal standard n-tetracosane (194 pg/ml of urine). e Tentative identification. f Peak off-scale.
+
+
ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1319
Table VII. Organic Acids Isolated from Urine Exclusively by Anion Exchange o r by Solvent Extraction Method
Acid
Anion exchange
2.3 -Dihydroxyisobutyric
Solvent Extrac tion
Organic Acids Grouped According to Quantitation Precision Peak”
2,3 -Dihydroxybutyric (diastereomer A) Dihydroxyhexanoic or Hydroxy pentanedioic Deoxypentonic Dihydroxyhexanoic or Hydroxypentanedioic Pentonic (Y -Glycerophosphate p-Glycerophosphate Homovanillic Uric 3 -Hydroxyhippuric Glucuronide conjugate s Homogentisic
Total S o . of peaksb
9
Ran e of relative rtandar! deviations, %
10
0-10 11-20 21-30 Over 30
25 28 28 34 37 38 44 54 56 58,59 42
Refer to Figure 1.
Table VIII. Organic Acids Isolated from Urine with Much Higher Efficiency by Anion Exchange or by Solvent Extraction Anion exchange
Solvent extraction
Acid
Peak
2,4 -Dihydroxv butyric 3 , 4 -Dihydroxybutyric Erythronic Threonic 5,6 -Dihydroxy hexanoic Glucuronic
Acid
Peak
14
2 -Hydroxybutyric
1
15
Lactic
2
21 23 24
3 -Methyladipic Aconitic Citric
25 37 39
4548
a
a
a
Dihydroxybenzoic Dihydroxymethoxyphenylacetic
40 46
Refer io Figure 1.
were not found in extract A. Of the acids found in A, but more efficiently extracted with solvent, 2-hydroxybutyric and lactic may have been partially lost during lyophylization because of the relatively high volatility of these compounds. Citrate was shown to be partially lost. during the barium precipitation phase of method A. Quite possibly the yield of aconitic acid was also decreased in this way because of its structural similarity. Yields of the two aromatic acids in Table VIII, and homogentisic in Table VII, were higher by solvent extraction, although homovanillic and 3-hydroxyhippuric (Table VII) were exclusively removed by anion exchange. These results, together with the well documented efficiency of solvent extraction of aromatic acids (41) and the nearly equal recoveries of phenylacetic and 41320
‘
Anion exchange
,Manual extraction
9 10 5 6
2 6 0 8
Continuous extraction
4 4 3 7
a The standard deviation of the mean area of each peak was computed as a percent of that peak area (data in Table VI). Only “onscale” peaks predominantly composed of organic acids. and wlth areas which are at least 4% of the internal standard, are included.
49
Palmitic a
Table IX. G a s Chromatographic P e a k s of Urinary
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
hydroxybenzoic acids by methods A and C from aqueous solutions (Table 111),indicate that neither method is clearly advantageous with regard to aromatic acids. A peak-by-peak comparison of the quantitative precision obtained by the three extraction procedures is complicated, as most peaks differ in composition (Table VI). The data in Table IX represent an attempt to evaluate reproducibilities. In each chromatogram, only those peaks predominantly composed of compounds identified as organic acids, and, therefore, suitable for quantitation, are considered, not including hippuric and citric (since the former is off-scale in all and the latter is off-scale in one chromatogram). Only those of a size greater than 3% of the internal standard were included. The table reveals that a total of 30 peaks in the anion exchange extract could be quantitated, while only 16 and 18 peaks could be quantitated in the solvent extracts. The superior reproducibility of method A is obvious from the fact that 19 peaks were quantitated with standard deviations which are 20% or less of the mean peak area, while only 8 peaks were measured with similar precision in each of the solvent extractions. Half of the peaks in method B and nearly half in method C had standard deviations greater than 30% of the mean. CONCLUSIONS Anion exchange has been shown to be generally more effective for isolating organic acids from urine than solvent extraction. This is especially true for polyhydroxy acids, several of which could not be detected in solvent extracts by GC-MS. Substances other than organic acids are minimal constituents of anion exchange extracts, but interfere substantially with the GC quantitation and GC-MS identification of organic acids isolated by solvent methods. Additionally, these acids were isolated in more reproducible quantities by anion exchange. Many laboratories will undoubtedly continue to use manual solvent extractions because of the short sample preparation time required. While this is a satisfactory method for detecting gross metabolic errors, it is inadequate for quantitating a series of acids to search for possible subtle metabolic changes. Continuous solvent extraction has neither the information advantages of anion exchange nor the speed of manual extraction. The complicated nature of gas chromatograms of urinary organic acid extracts suggests that quantitation would be facilitated by more efficient peak resolution. The use of open tubular glass capillary columns ( 4 2 ) might allow the separate quantitation of several acids which were measured in combination with another acid (or acids) as a single peak.
ACKNOWLEDGMENT The authors thank Barbara G' Urban' and Stephen P. Levine for their professional assistance a t
''
various stages of this project.
LITERATURE CITED E. C. Horning and M. G. Horning. Clin. Chem., 17, 802 (1971). E. C. Horning and M. G. Horning. J. Chromatogr. Sci,, 9, 129 (1971). J. E. Mrochek. W. C. Butts, W. T. Rainey, Jr., and C. A. Burtis, Clin. Chem., 17, 72 (1971). R . Teranishi, T. R. Mon, A. B. Robinson, P. Cary, and L. Pauling, Anal. Chem., 44, 18 (1972). M. G. Horning, A. Hung, R . M. Hill, and E. C. Horning. Clln. Chim. Acta, 34, 261 (1971). C. H. L. Shackelton, J.-A. Gustafsson, and J. Sjovall, Steroids, 17, 265 (1971). R. Reinmendal and J. B. Sjovall, Anal. Chem., 45, 1083 (1973). H. Haga. T. imanari. A. Tamura, and A. Momose. Chem. Pharm. Bull., 20, 1805 (1972). S. P. Markey, W. G. Urban, A. J. Keyser. and S. I. Goodman, Adv. Mass Spectrom., 6, 187 (1973). 0. A. Mamer. J. C. Crawhill. and S. S. Tjoa, Clin. Chim. Acta, 32, 171184 (1971). E. Jellum. 0. Stokke. and L. Eldjarn, Clln. Chem., 18, 800 (1972). "Handbook of Biochemistry", H. A. Sober Ed., Chemical Rubber Co., Cleveland, OH, 1968, Section B. P. B. Hamilton and B. Nacy, Space Life Sci., 3, 432 (1972). S. W. Fox, K. Harada, and P. E. Hare, Space Life Sci., 3, 425 (1972). C. W. Gehrke, R . W. Zumwalt, K. Kuo, J. J. Rash, W. A. Aue, D.L. Staliing, K. A. Kvervolden, and C. Ponnamperuma, Space Life Sci.. 3, 439 (1972). C. E. Costello. H. S. Hertz, T. Sakai, and K. Biemann, Clin. Chem., 20, 255 (1974). K. B. Hammond and S. I. Goodman, Clin. Chem., 16,212 (1970). T. A. Witten, S. P. Levine. J. 0. King, and S. P. Markey, Clin. Chem., 19, 586 (1973). T. A. Witten. S. P. Levine. M. T. Killian, P. J. R . Boyle, and S. P. Markey, Clin. Chem., 19, 963 (1973). R. A. Chalmers and R. W. E. Watts, Analyst (London), 97, 951 (1972). R . A. Chaimers and R. W. E. Watts, Analyst (London), 97, 224 (1972).
(22) R . A. Chalmers and R. W. E. Watts, Analyst(London), 97, 958 (1972). (23) A. M. Lawson. F. L. Mitchell, R . A. Chalmers, P. Purkiss. and R. W. E. Watts, Adv. Mass Spectrom., 6, 235 (1973). (24) S. P. Levine, J. L. Naylor, and J. P. Pearce, Anal. Chem., 45, 1560 [ 1973). (25) A. White, P. Handler, and E. Smith, "Principles of Biochemistry", McGraw-Hill Book Co., New York, NY, 1973, p 942. (26) D.O'Brien, F. A. Ibbott, and D. 0. Rodgerson. "Laboratory Manual of Pediatric Microbiochemical Techniques", Harper and Row, New York. NY. 1968, pp 114-116. (27) J. A. Thompson and J. L. Holtzman. J. Pharmacol. Exp. Ther., 186, 640 (1973). (28) M. G. Horning in "Biomedical Applications of Gas Chromatography". H. A. Szymanski Ed.. Vol. 2, Plenum Press, New York. NY, 1968, p 56. (29) G. Lancaster. P. Lamm, C. R . Scriver, S. S. Tjoa, and 0. A. Mamer, Clin. Chim. Acta, 48, 279 (1973). (30) J. R. Planner and S. P. Markey, Org. Mass Spectrom., 5, 463 (1971). (31) S. P. Markey. Anal. Chem., 42, 306 (1970). (32) "Handbook of Silylation". Pierce Chemical Co. Handbook GPA-30, Rockford, IL, 1972. (33) Bio-Rad Laboratories Tech. Bull. 1005, Richmond, CA, 1973. (34) S. Ohashi, N. Yoza, and Y. Veno. J. Chromatogr., 24, 300 (1966). (35) D. W. Baker, J. Assoc. Off. Anal. Chem., 56, 1257 (1973). (36) "Solubilities of Inorganic and Metal Organic Compounds", W. F. Linke, Ed., Vols. I and 11, American Chemical Society, Washington, DC, 1958. (37) B. Wengle. Acta Chem. Scand.. 18, 65 (1964). (38) "Mass Spectra of Compounds of Biological interest", S. P. Markey, W. G. Urban, and S. P. Levine Ed., National Technical information Service, U S . Department of Commerce, Springfield, VA. 1974. (39) G. Peterson, Tetrahedron. 3413 (1970). (40) G. Peterson, Org. Mass Spectrom., 6, 565 (1972). (41) C. M. Williams and C. C. Sweeley in "Biomedical Applications of G a s Chromatography". H. A. Szymanski Ed., Plenum Press, New York, NY, 1964. (42) A. L. German, C. D. Pfaffenberger, J-P Thenot, M. G. Horning, and E. C. Horning, Anal. Chem., 45, 930 (1973).
RECEIVEDfor review November 25, 1974. Accepted March 6, 1975. This work was supported by NIH Grants HD04870 and HD-04024 in addition to MCH Project Grant 252.
Gas Chromatographic Determination of Apomorphine in Urine and Feces Robert
V. Smith
Drug Dynamics Institute, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712
Andrew W. Stocklinski Division of Medicinal Chemistry and Natural Products, College of Pharmacy, The University of lowa, lowa City, IA 52242
A method for the determination of apomorphine in rat urine has been devised based on liquid-liquid extraction with ethyl acetate, derivatization with N,O-bis(trimethylsily1) acetamide and gas chromatography on an OV-17 column. The developed method can be incorporated into a scheme that permits selective determination of apomorphine and its isomeric 0-methyl metabolites. Attempts to devise similar procedures for the determination of these compounds in rat feces were only partially successful. Urinary elimination of apomorphine in Sprague-Dawley rats, following intraperitoneal injection of this drug, was determined.
The mammalian metabolism of aporphine alkaloids is being systematically investigated in these laboratories ( I 3 ) . As part of these studies, a procedure for measuring apomorphine (1) in rat urine and feces in the presence of its potential metabolites, apocodeine (2), isoapocodeine (3),
and norapomorphine (4) was desired. We have previously demonstrated that 2 and 3 can be separated from 1 by extraction with 1%isomyl alcohol in n-heptane (1%INH) a t p H 8.6. 2 and 3 were subsequently quantiated as such by gas chromatography (GC) ( 4 ) . A method for analyzing 1 and 4 in urine, based on thin-layer chromatographic fluorescence quenching ( 5 ) has also been developed though it is somewhat tedious to perform since it requires over twentyfour hours to complete. An earlier method used to estimate I in biological fluids requires mercuric chloride oxidation to the ortho-quinone, 5 , and subsequent colorimetric measurement (6). Although this method provides sufficient sensitivity to detect 1 a t sub-microgram levels, it is not entirely suitable since i t cannot distinguish 1 from a number of its potential metabolites. That is, Cava et al. ( 7 ) have recently shown that compounds such as 2 , 3 , and 4 can be converted to 5 (or its homolog) upon treatment with mercuric chloride. Other colANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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