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Determination of δ-aminolevulinic acid in blood plasma and urine by gas-liquid chromatography
BIOCHEMICAL
MEDICINE
17, 31-44
(1977)
Determination of SAminolevulinic Acid in Blood Plasma and Urine by Gas-Liquid Chromatography JOSEPH MACGEE, SANDY M. B. RODA, SAMI V. ELIAS, ARTHUR LINGTON, MARVIN W. TABOR, AND PAUL B. HAMMOND Basic Science Laboratory, Veterans Adminisrration Hospital, 3200 Vine Street, Cincinnati, Ohio 45220 and the Departments of Environmental Health, Biological Chemistry. and Medicine, College of Medicine, University of Cincinnnti. Cincinnati. Ohio 45267 Received
May
28. 1976
&Aminolevulinic acid (ALA) is the first intermediate in the pathway of heme biosynthesis from glycine. Some changes in heme synthetic pathways, such as occur in acute intermittent porphyria and lead poisoning, are accompanied by elevation of ALA in tissues, plasma, and urine. Most analytical methods for ALA in biological materials are variations of the method of Mauzerall and Granick (l), which depends on the formation of a colored complex between a pyrrole derivative of ALA and p-dimethylaminobenzaldehyde (DMAB). The original method had two significant limitations. Sensitivity was limited to the microgram range (the limit of detection was 0.5 pg of ALA) and the method was nonspecific. DMAB produces colored complexes with pyrroles formed by Knorr condensation of many aminoketones other than ALA, notably aminoacetone (AA), which occur in biological fluids. Modifications of the method of Mauzerall and Granick have been described which separate AA from ALA prior to coupling with DMAB (2, 3). These separation procedures are based on selective elution from resin columns either of the aminoketones or of pyrrole derivatives formed during the analytical procedure. A gas-liquid chromatography (GLC) procedure for aminoketones involving condensation with ethyl acetoacetate and preparation of trimethylsilyl derivatives of the resultant pyrroles has been described recently, but the procedure was not sufficiently sensitive for the measurement of normal concentrations of ALA in serum samples (4). The present investigation was undertaken to develop an analytical method for determination of ALA in nanogram quantities, with specific application to blood plasma. Since the method represents a departure from previous approaches, its application to the determination of ALA in urine was characterized also.
MATERIALS
AND METHODS
Reagents ALA.HCI: &Aminolevuhnic acid hydrochloride (5amino-4-oxopentanoic acid hydrochloride) was purchased from Sigma. Internal standard: 6-Amino-5oxohexanoic acid hydrochloride was synthesized by substituting 4-carbomethoxybutyryl chloride for 3-carbomethoxypropionyl chloride in the preparation of ALA. HCI described by Aranowa rt (11. (5). Details of the preparation and characterization of this analog of ALA are given below. Gibson et crl. (6) prepared this same compound by a different synthetic route. Internal standard solution: An aqueous solution of 1 pg of internal standard/ml was used. It was stored in the freezer. TMPI: Trimethylphenylammonium iodide (Eastman Organic Chemicals) was recrystallized from water. TMPH: A 1 M solution of trimethylphenylammonium hydroxide was prepared by mixing 1.05 g of TMPI with 1 g of silver oxide (Fisher Purified) and 4.0 ml of water in a test tube on a Vortex mixer. After several minutes with occasional mixing, the resultant silver iodide and excess silver oxide were removed by centrifugation. It was usually necessary to recentrifuge the supernatant 1 M TMPH. The clear reagent was stored in a capped test tube at room temperature in the dark. Acetylacetone: The Fisher Reagent product was redistilled through a IO-cm vigreux column at atmospheric pressure. When a 60-ml charge was distilled, the first 5 ml was discarded and the next 30 ml was collected. When smaller amounts of acetylacetone are redistilled, care must be taken to avoid any late cuts generated from charred reagent in the still pot. When stored in the refrigerator in the dark, the reagent can be kept for at least 10 weeks. MIBK: Methyl isobutyl ketone (Fisher Certified ACS). Hexane: Hexanes (Fisher Certified ACS). Ethyl ether: The anhydrous product from Fisher or Mallinckrodt. AG-1 x 8 column: BioRad AG-1 x 8, chloride form, 200 to 400 mesh, was stored in the refrigerator. Columns were prepared in the following manner: A slurry of the resin in water was transferred to a 25 cm x 10 mm chromatograph tube with a wet glass-wool plug in the bottom. The height of the settled resin bed was 2 cm. Columns can be prepared several days in advance if they are stored submerged in a beaker of water in the refrigerator. The columns were rinsed with 30 ml of water at room temperature just prior to use. TCA (40%): Forty grams of trichloroacetic acid (Mallinckrodt Analytical Reagent) was dissolved in water to a total volume of 100 ml. The solution was stored in the refrigerator. Preparation
of Itlternal
Standard
The 6-amino-5-oxohexanoic acid was prepared by a modification of the two-step synthesis of ALA. HCI described by Aranowa et al. (5). The synthesis of the internal standard is shown in Fig. I.
GLC DETERMINATION SYNTHESIS HIPPURIC
OF INTERNAL
33
OF ALA STANDARD
ACID 0
a--+
+
N Ii
“woc”” O
0 GLUTARVL CHLORIDE METHYL ESTER
OH IHCI.
t-
6,
N~~~zL%~;;~~~
HCI. BENZOIC
Ii20
0
3H20
ACID
-I+ +
0 co*.
CH30H
“Cl.“,Nq~oH 0
0
b-AMINO-5-OXOHEXANOIC
FIG.
1.
The synthesis of 6-amino-Soxohexanoic
ACID
HCI
acid hydrochloride.
Gfutaryf chloride methyl ester. The starting material, glutaryl chloride methyl ester, was prepared by a standard thionyl chloride procedure (7). Twenty-five grams (170 mmoles) of glutaric acid monomethyl ester (Aldrich) was mixed with 37 ml (515 mmoles) of thionyl chloride (Aldrich) in a lOO-ml round-bottom flask fitted with a pressure-equalizing dropping funnel and a reflux condenser. The condenser was equipped with a drying tube connected to a sodium hydroxide trap for acid gases. The thionyl chloride was added at a rate to ensure constant, but not vigorous, efflux of acid gases while stirring the reaction mixture at room temperature with a magnetic stirrer. After complete addition of the thionyl chloride, the mixture was heated under reflux for 2 hr. The excess thionyl chloride was then removed in vacua by heating the mixture in the same flask with a water bath at 50°C. The yield of the dark reddish-brown product was 28.5 g. 2-Phenyl-4-(4-carbomethoxybutyryl)-oxaz.olinone-5.
The crude glutaryl chloride methyl ester (28.5 g) was slowly added from a pressureequalizing dropping funnel to a mechanically stirred solution of 15.2 g (85 mmoles) of hippuric acid (Aldrich) in 82.5 ml of 4-picoline (Aldrich) in a 500-ml round-bottom flask fitted with a thermometer. The temperature of the reaction was maintained between -5” and 0°C with an ice-salt bath during the addition of the acid chloride ester and for an additional 4 hr. At this time, a mixture of 60 ml of 12 N hydrochloric acid in 350 g of crushed ice was added. The ice-salt bath was removed, and the mixture was stirred until all the ice in the flask had melted. The maroon-colored crystals of the substituted oxazolinone were collected on a sintered glass funnel and washed on the funnel with 10 ml of ice-cold distilled water. The air-dried crude product (28.5 g: mp, 115-120°C) was transferred to a
125-m] Erlenmeyer flask and stirred briefly in an ice bath with 50 ml of ethyl acetate. After collection on a sintered glass funnel, the product was washed on the funnel with an additional 10 ml of ice-cold ethyl acetate. Fifteen grams of 2-phenyl-4-(4-carbomethoxybutyryl)-oxazolinone-5 was obtained (mp 135-138°C). 6-Amitlo-5-oxohrxottoic crcid hydrochloride. The 15 g of substituted oxazolinone was heated under reflux for 8 hr with 250 ml of 6 N hydrochloric acid. After cooling to room temperature, the crystalline benzoic acid was removed by filtration. Seven grams of powdered activated charcoal was added to the filtrate, and the mixture was boiled for 15 min while stirring. The hot mixture was filtered, and the hydrochloric acid and water were removed in I’UCUO from the filtrate at 50°C to yield a semicrystalline mass of the product. This product was dissolved in 7.3 ml of 12 N hydrochloric acid with brief heating, and after cooling in an ice bath, 73 ml of acetone was added slowly with stirring. After an additional 30 min of stirring, the product was collected by filtration and washed with 7 ml of acetone. The yield of crystalline pressed and air-dried product was 2.99 g (16.5 mmole, 32% yield based on the oxazolinone, 17% yield based on hippuric acid). The light-tan crystals exhibited a structure change at 178-180°C and melted at 210-21X. Recrystallization of 300 mg of the product from hot aqueous isopropyl alcohol yielded 176 mg of crystals (single mp, 128-131°C). which were subjected to analysis and were used as the internal standard for GLC of ALA. Nuclear magnetic resonance analysis of the final product yielded results consistent with the proposed structure. Combined GLC-mass spectrometric analysis performed on the methylated pyrrole arising in the course of the GLC analytical procedure also supports the proposed structure, yielding a fragmentation pattern completely analogous with that exhibited by the dimethylated pyrrole of ALA. Apparatus
A Hewlett-Packard Model 402B dual column, dual flame-ionization detector gas chromatograph with a 1-mV recorder was used. The glass columns, 6 ft x 0.25 in. (4 mm i.d.), with graphite ferrules, were packed with 5% OV-17 (Applied Science) on GasChrom Q, loo-120 mesh (Applied Science). The injector temperature was 290°C the column temperature was 220°C and the nitrogen carrier flow rate was 70 ml/min. Procedures Plasma
Three milliliters of the sample was mixed in a centrifuge tube with 0.30 ml of the internal standard solution for a few seconds on a Vortex mixer. One-half milliliter of 40% TCA was added and the sample was mixed for
GLC
DETERMINATION
OF ALA
35
0.5 min on the Vortex mixer. After centriguation for 5 min, the sample was remixed briefly and recentrifuged for an additionai 5 min. The supernatant fluid was transferred with a disposable Pasteur pipette to a 125 x 16-mm screw-capped test tube with a Teflon-lined cap (Kimble 45066A), 5 ml of ethyl ether was added, and the tube was shaken vigorously by hand for 1 min. The upper ether layer was discarded and the aqueous phase was reextracted with an additional 5 ml of ethyl ether. After discarding the second ether wash, 1 ml of 1 M NaH,PO, and 50 ~1 of acetylacetone were added. After 1.5 min of mixing on the Vortex mixer, the sample was heated loosely stoppered for 10 min in a boiling water bath. The sample was cooled and extracted with 5 ml of hexane by shaking vigorously by hand for 1 min. After 1 min of centrifugation, the clear upper hexane phase was discarded. If an emulsion occurred at this point, the shaking and centrifugation steps were repeated. After discarding the hexane phase, 5 ml of MIBK was added. The tube was shaken vigorously by hand for 1 min and then centrifuged for 1 min. With a disposable Pasteur pipette, nearly all of the upper MIBK extract was transferred to a Concentritube (Laboratory Research Co., P.O. Box 36509, Los Angeles, California 90036). Quantitative transfer of the MIBK phase is neither possible nor necessary, but none of the aqueous phase or any emulsion layer should be transferred. While mixing the extract on the Vortex mixer, 5 ~1 of TMPH was introduced and the mixing was continued for 1.5 min. The tube was stoppered and centrifuged for 2 min. A lO+l syringe was prewetted with water and used to draw up all of the small lower TMPH extract and then to inject the sample slowly (15 to 20 set) into the GLC unit. No interference is introduced if some of the MIBK layer is drawn into the syringe and injected into the chromatograph. Urine
Urine samples were collected in bottles containing 0.5 g of benzoic acid and stored in the freezer until analyzed. The sample (0.10 ml of urine), 0.30 ml of the internal standard solution, and 2 ml of 1 M NaH,PO, were mixed in a test tube. The mixture was passed through a washed AG-1 x 8 column, and two l-ml water rinses of the test tube were used to wash the column. The column effluent and the rinses were collected in the same type of screw-capped tube as was used for plasma analysis. After addition of 50 ~1 of acetylacetone, the remainder of the urine procedure was identical with the plasma procedure, starting with the 1.5-min mixing and heating steps. Standard Curve and Quantitation
The standard curve was prepared by adding ALA. HCl to outdated blood bank plasma over the range of 0 to 1 pg of ALA/ml of plasma (6
points) and subjecting these samples to the plasma analytical procedure. Peak heights were measured from straight baselines drawn for the ALA and internal standard (IS) peaks, in which the minimum tangent points before and after each of the two peaks were connected. The baselines were drawn as shown in Fig. 3. Plotting the peak height ratio (ALA/IS) 1’s ALA added (calculated as the nonhydrochloride) resulted in a straight line with a small positive y-intercept. Extrapolation of this line to the x--intercept indicated that the apparent residual ALA in the outdated plasma was 7 rig/ml. The slope (peak height ratio/nanograms of ALA per milliliter) was calculated from this line and was used for the calculation of the ALA concentration in plasma and urine samples as follows. For 3-ml plasma samples, the peak height ratio (ALA/IS) was divided by the standard curve slope to yield results expressed as nanograms of ALA per milliliter of plasma. For urine samples, the peak height ratio was divided by a second slope value in order to correct for the fact that 0.1 ml of urine is used with the same amount of internal standard employed with 3 ml of plasma and to yield results expressed as micrograms of ALA per milliliter of urine. This second slope value was obtained by multiplying the plasma standard curve slope value by 0.03. Figure 3 illustrates typical gas-liquid chromatographic elution patterns obtained when a fresh blood plasma specimen was analyzed by the plasma procedure with and without the addition of ALA and internal standard, each added at 100 r&ml. Peak height measurements, rather than peak area measurements obtained with an electronic digital integrator, were used for quantitation. The advantage of this procedure derives from the fact that small, incompletely
NORMAL
URINE + IS
MINUTES
2. Gas-liquid chromatogram showing peaks for 6-aminolevulinic acid (ALAI from normal human urine and the 300-ng addition of the internal standard (IS). The ALA content of this specimen was measured to be 2.81 Fgiml. FIG.
GLC DETERMINATION
OF ALA
37
FIG. 3. Gas-liquid chromatograms of human blood plasma with and without added Gaminolevulinic acid (ALA) and internal standard (IS). The top pattern, plasma + ALA + IS, resulted from adding 300 ng each of ALA and IS to 3 ml of fresh human blood plasma. The middle pattern, plasma + IS. was obtained from 3 ml of the same plasma on addition of 300 ng if IS only. The bottom pattern, plasma blank, had no additions. The ALA content of this specimen was measured to be 8.4 rig/ml.
resolved peaks near the two peaks of interest were frequently incorporated in the areas of the desired peaks. Their contributions were minimized when peak height measurements were used. Peak area measurements, in which the areas are calculated by multiplying the peak heights by their widths at half-height, can be used, but they offer no advantage over peak heights alone and require additional operations. The ideal internal standard for gas chromatography should be added to the measured sample before any other operation in the analysis is performed. It should be a very close analogue of the compound to be measured, so that it will then behave exactly the same as the compound of interest in its physical and chemical properties and, thus, will partition between the various solvent-solvent and sorbent-solvent systems and react with any derivatizing reagents in a manner identical to the compound to be measured. The only step in the procedure where it should behave differently is in the gas chromatographic column. Here it should be completely separated from the material to be measured, but it should elute close enough to avoid the necessity for changing conditions to allow a reasonable analysis time. A considerable advantage is offered by this type of internal standard in that it automatically corrects for losses of the compound of interest and it makes it unnecessary to transfer the various phases quantitatively. Any loss of a given phase results in proportional losses of compound and internal standard. The internal standard synthesized for this procedure, 6-amino-Soxo-
hexanoic acid, differs from ALA in having a single additional methylene group between the carboxyl and carbonyl groups of ALA, making the side chain butyric acid instead of propionic acid. Its functional groups, the carboxyl and the Lu-aminoketone structures, are identical to those of ALA; so all the reactions that ALA will undergo will be equally achieved with the internal standard. The extra methylene group merely causes the methylated derivative of the internal standard pyrrole to elute slightly later than the same derivative of ALA (See Figs. 2 and 3). A synthesis of 6-amino-5oxohexanoic acid was reported in 195.5 by Gibson ef al. (6). We have prepared this compound by a shorter, more recent route, i.e., a minor modification of the synthesis of ALA. HCl described by Aranowa et ~1. (5). The synthesis of ALA. HCl was accomplished by Aranowa et al. by condensing succinyl chloride methyl ester with hippuric acid to yield 2-phenyl-4-(3-carbomethoxypropionyl)-oxazolinone-5, which on acid hydrolysis yielded the hydrochloride of ALA in good yield. Our modification involves the use of glutaryl chloride methyl ester in place of succinyl chloride methyl ester to yield 2-phenyl4-(4-carbomethoxybutyryl)-oxazolinone-5, which in turn generates the hydrochloride of the desired analogue of ALA in good yield on acid hydrolysis. Some precautions are necessary in the course of the synthesis. Adequate sodium hydroxide must be used to trap all the acid gases generated in preparation of the glutaryl chloride methyl ester. The reaction temperature must be maintained below 0°C while preparing the substituted oxazolinone to prevent formation of glytaryl-(4-picoliniuml methyl ester chloride. A temperature at 50°C or lower should be maintained during removal of the excess thionyl chloride in \YICIIO after the preparation of the glutaryl chloride methyl ester and during removal of the excess hydrochloric acid and water in the preparation of the final product. Excess heat at either of these two steps could lead to polymerization or decomposition of the desired compounds. Finally, the final product should be stored in a well-stoppered container, in the dark, at a temperature below 0°C. The internal standard partitions exactly the same as ALA in the extraction steps used in the procedures described in this report. Three aliquots of a mixture of the two compounds were treated in different ways to establish this fact. The first sample, the control, was processed by the usual plasma procedure. The other two aliquots were also processed by the same procedure, except that the second sample was washed four times with ethyl ether instead of the usual two times and the final TMPH extract of the third sample was reprocessed through the reaction with acetylacetone, washed with hexane. extracted into MIBK. and reextracted from the MIBK with TMPH. Had there been a difference in
GLC
DETERMINATION
OF ALA
39
partitioning of the ALA and internal standard between ether and the aqueous phase, the first and second samples would have yielded different peak height ratios. Had there been a difference in partitioning of the two pyrroles in either the hexane wash or the MIBK or TMPH extractions, the first and third samples would have exhibited different peak height ratios. All three samples yielded identical peak height ratios on GLC analysis. Six samples each of the same specimen of urine were analyzed by both the calorimetric procedure of Marver et al., a well-accepted procedure that involves the use of two ion exchange columns, pyrrole formation, and reaction with DMAB (3), and our GLC method for ALA in urine. The calorimetric procedure yielded values 16% higher than the GLC procedure (2.19 + 0.06 vs 1.84 ? 0.04 pg of ALA/ml, mean +- standard deviation). The same ALA solution was used to calibrate both methods. While the method of Marver et al. ranks among the best calorimetric procedures, we strongly suspect that it measures non-ALA materials. When a sample of ALA was carried though the analytical procedure and chromatographed on an OV- 17 column in an LKB-9000 combined gas chromatograph-mass spectrometer with a System 150 (Systems Industries) computer, the ALA derivative yielded a mass spectrometric fragmentation pattern consistent with two methylations of the ALA pyrrole-one methylation on the carboxyl group and the other on the pyrrole ring nitrogen. The base peak at m/e 223 and fragments of m/e 181 (P(parent ion)-42, loss of ketene from acetyl), m/e 180 (P-43, loss of acetyl), and m/e 150 (P-73, loss of carbomethoxymethylene from the carboxyl methyl ester end) were found. The approach used in the development of the method described here was based on that developed in this laboratory for the gas chromatographic analysis of a number of important acidic compounds (8-11). This approach involves extracting the acidic and neutral compounds into a suitable organic solvent and back-extracting only the acidic compounds into a very small volume of an aqueous quaternary ammonium hydroxide. Injection of the resultant quaternary ammonium salts into the hot vaporizer of the gas chromatograph results in thermal decomposition of the salts to yield alkylated derivatives of the acidic compounds and a tertiary amine (12). Selection of a suitable chromatographic stationary phase and chromatographic conditions achieves separation of the reaction products. Such an approach has the added advantages of simplicity, speed, and the elimination of any tedious, time-consuming, and sometimes destructive evaporation steps. The fact that ALA occurs in normal plasma samples at concentrations about three orders of magnitude lower than the materials analyzed in most of our past experiences has made it necessary to develop a slightly more
40
MAC‘GEE
Ii7 ill
complex procedure to eliminate potential interfering substances from the plasma and reagents. As a result, instead of requiring about 6 min to prepare a sample ready for injection into the chromatograph (8, 9), the present procedures require about 30 min to reach the same point. Being an amino acid, ALA is not readily partitioned into organic solvents from aqueous phases. This allowed us to remove the vast majority of interfering materials along with the trichloroacetic acid from the trichloroacetic acid supernatant fluid by two simple ether washes. By converting the ALA and the internal standard to their respective pyrroles after the ether wash, we were then able to extract them as their pyrroles from an aqueous solution into methyl isobutyl ketone. Back-extraction into a very small volume of aqueous trimethylphenylammonium hydroxide yielded a sample ready for injection and flash methylation in the 290°C vaporizer of the gas chromatograph. Aminoacetone (AA) does not interfere with the measurement of ALA in the procedures described in this report. While this aminoketone forms a pyrrole with acetylacetone, and causes some of the spectrophotometric methods to yield falsely high ALA values, addition of 1 pg of AA/ml of plasma did not change the ALA analysis of the plasma by our procedure. Since the AA pyrrole does not contain the carboxyl group present in either the ALA or internal standard pyrroles, it is probably not extracted from the MIBK by the alkaline TMPH reagent. An experiment in which [14C]ALA in plasma was carried through the entire procedure showed that 70% of the ALA was found in the final alkaline extract. Since quantitative separation of the phases is not accomplished in this procedure, some variation in the overall recovery can be expected from analysis to analysis, but the nature and method of use of the internal standard make any recovery corrections unnecessary. Three plasma samples with different concentrations of ALA were each analyzed six times by the procedure described in this report. The following values (mean & standard deviation) were obtained: 6.33 + 0.90,43.70 t 2.39, and 73.50 + 2.30 ng of ALA/ml. Four aliquots of the same urine sample were analyzed on two separate days (eight analyses) with the following result: 2.46 & 0.10 pg of ALA/ml. Three commercially available toxicology control samples, two sera and one urine, were analyzed by the procedures described in this report, except that 10 times the usual amount of urine was analyzed. No internal standard was added in order to determine if any of the commonly used (and abused) drugs interferes with our procedures. Table 1 lists the drugs and their concentrations in the three control samples. The only potentially interfering peaks in the elution patterns were seen in Serum B. Performance of the analysis on samples of methaqualone and methprylon did not result in any interfering peaks. We conclude from this
GLC DETERMINATION
41
OF ALA
study that none of the drugs tested at these concentrations (Table 1) will interfere with the procedures described in this report, but the source of the interfering peaks in Serum B remains unknown. As can be seen in Fig. 3, there are late peaks in the GLC elution pattern that could interfere with subsequent analyses. The retention times of these peaks were highly reproducible relative to the ALA and internal standard peaks, so it was possible to place each of the peaks to be measured in a clear window by injecting the next sample at a time that was twice the retention time of the ALA peak. This rule holds even for the slight differences in retention times that can occur because of differences in column temperature, carrier flow rate, or the age of the column. A TABLE I DRUG CONTENT OF TOXICOLOGY CONTROL SAMPLES Drug concentration
DWiT Alcohol Amobarbital Amphetamine sulfate Chlorodiazepam hydrochloride Codeine Diazepam Diphenylhydantoin Ethchlorvynol Glutethimide Meperidine Meprobamate Methamphetamine Methaqualone Methyprylon Morphine Pentobarbital Phenobarbital Procainamide hydrochloride Propoxyphene hydrochloride Quinidine sulfate Secobarbital Sodium bromide Sodium salicylate Sulfanylamide
Serum A” 1000 20 2 20
&$rnl)
Serum B*
Urine’
20
5 5 3
3 20 20 10
20
20
20
20
20 20 3 20 20 5 2 6 20 500 250 50
a Hyland Toxicology Serum Control (unassayed). b Lederle Diagnostics Serum Toxicology Control-Drugs ’ Lederle Diagnostics Urine Toxicology Control-Drugs
20
5
5
A. I for analytical use only.
slight difference in retention time of this type can be seen on comparing Figs. 2 and 3. We have used the same OV-17 column for this and other projects for over 18 months. After several months of use, a slight broadening of the peaks was noted. This loss of efficiency was corrected by replacing the first 10 cm of packing of the inlet end of the column with fresh packing material. Before replacing the packing, we removed any injection residues with TMH-24 (tetramethylammonium hydroxide, 24% in methanol, Southwestern Analytical Chemicals, Austin, Texas) on a cotton-tipped applicator stick. The residual TMH-24 was removed with water and then with methanol on clean cotton-tipped applicator sticks and the inlet was allowed to dry in air before it was replaced. After about 1 hr at operating conditions, the column was as good as new. This rejuvenation has been performed twice with equal success, and we anticipate a long and useful life for this column. A sample of outdated blood bank plasma containing 5 ng of ALA/ml was incubated with a crude ALA dehydrase preparation in order to remove the ALA. After a brief incubation with the enzyme, the ALA concentration fell below 1 rig/ml. This convinced us that there were no significant non-ALA peaks cochromatographing with the ALA derivative. The crude enzyme preparation was a dialyzed 25,OOOg supernatant fraction obtained from rat liver. The preparation of the homogenate and the conditions for dialysis and incubation followed the procedures described by Wilson et al. (13). A similar experiment performed with rabbit plasma, however, left a significant peak on the chromatogram. This peak had a retention time about 30 set shorter than the ALA derivative and would be assayed as 56 ng of ALA/ml. Reexamination of the apparent ALA peak on the chromatogram generated from rabbit plasma before treatment with the enzyme showed that the peak was significantly broader than the ALA peaks obtained from human plasma specimens. Examination of the ALA peaks on chromatograms obtained from blood plasma specimens of rat, cat, dog, sheep, and goat did not demonstrate broadening of the ALA peaks, and the ALA peaks had exactly the same retention times as are found with human plasma. Of the seven mammalian species tested to date, only rabbit plasma exhibits broadening of the apparent ALA peak due to the unknown compound. The instability of ALA in alkaline solution is well documented (141, but the pyrroles resulting from condensation of ALA and the internal standard with acetylacetone are very stable in alkaline solutions. This has been demonstrated in two ways. When solutions of ALA pyrrole and internal standard pyrrole in 1 M TMPH were stored at room temperature and analyzed by GLC periodically over a several-week storage period,
GLC DETERMINATION
OF ALA
43
the peak height ratio remained constant and the peaks retained their original heights within injection volume precision. The second and most important test is the stability of the final extract. When the TMPH extract under the MIBK phase was stored at room temperature for over 1 week, GLC analyses revealed no loss of either the ALA or internal standard derivatives. This observation allows for the simultaneous preparation of multiple samples and GLC analysis when it is most convenient. In this way, three samples can be analyzed each hour with a single gas chromatographic column. Also, in the event of any minor instrumental difficulties, valuable samples are not lost while the difficulties are being rectified. SUMMARY
Procedures have been developed for the quantitative determination of normal and elevated concentrations of &aminolevulinic acid (ALA) in 3 ml of blood plasma or 0.1 ml of urine by gas-liquid chromatography (GLC) with a flame-ionization detector. Selective partitions, pyrrole formation with acetylacetone, and flash methylation in the vaporizer of the GLC unit allow for a convenient and rapid procedure for the analysis. An analogue of ALA, 6-amino-5-oxohexanoic acid, was prepared and used as an internal standard. A single analysis of plasma required about 30 min for preparation and about 20 min for GLC. Multiple samples can be prepared simultaneously and stored for at least 1 week before GLC analysisallowing for the analysis of up to 24 samples/day on a single GLC unit. ACKNOWLEDGMENTS The authors thank Dr. Daniel A. Garteiz for the combined gas chromatography-mass spectrometric analyses. These studies were supported by Veterans Administration funds (Grant MRIS 5421-02) and by a grant from the International Lead-Zinc Research Organization (LH-229).
REFERENCES I. Mauzerall, D., and Granick. S.. J. Bid/. Chem. 219, 435 (1956). 2. Urata, G., and Granick, S., J. Biol. Chem. 238, 811 (1963). 3. Marver, H. S., Tschudy, D. P.. Perlroth, M. G.. Collins, A.. and Hunter, G., Anal. Biochem. 14, 53 (1966). 4. Gibbs, B. F., Itiaba, K., and Crawhall, J. C., Biochem. Med. 11, 165 (1974). 5. Aranowa, N. I. S., Machowa, N. W., Sawjalow, S. I., Wolkenstein, J. B., and Kunizkaja, G. M., Federal Republic of Germany Patent No. 2 208 800, 1973. 6. Gibson, K. D., Neuberger, A., and Scott, J. J., Biochem. J. 61, 618 (1955). 7. Fieser, L. F., and Fieser. M., “Reagents for Organic Synthesis,” Vol. 1. Wiley, New York, 1967. 8. MacGee, J., Anal. Chem. 42, 421 (1970). 9. MacGee, J., C/in. Chem. 17, 587 (1971). 10. MacGee,
J., and Allen,
K. G., J. Chromatog.
100, 35 (1974). Environ. Micro&o/.
11. Tabor. M. W., MacGee. J., and Holland, J.. Appl.
31, 25 (1976).
44
MACGEE
15-7 Al
12. MacGee, J.. in “Methods of Analysis of Anti-Epileptic Drugs. Proceedings of the Workshop on the Determination of Anti-Epileptic Drugs in Body Fluids. Noordweijkerhout, the Netherlands, 13-14 April 1972” (J. W. A. Meijer. H. Meinardi. G. Gardner-Thorpe, and E. Van Der Kleijn, Eds.). p. I I I. Excerpta MedicaiAmerican Elsevier. Amsterdam, 1973. 13. Wilson, E. L., Burger. P. E.. and Dowdle, E. B., Eur. .I. Biocllrm. 29. 563 t 1972). 14. Haeger-Aronsen, B.. J. Lob. C/in. /nr,e.rt. 12, Suppl. 47. 5 (1960).
MEDICINE
17, 31-44
(1977)
Determination of SAminolevulinic Acid in Blood Plasma and Urine by Gas-Liquid Chromatography JOSEPH MACGEE, SANDY M. B. RODA, SAMI V. ELIAS, ARTHUR LINGTON, MARVIN W. TABOR, AND PAUL B. HAMMOND Basic Science Laboratory, Veterans Adminisrration Hospital, 3200 Vine Street, Cincinnati, Ohio 45220 and the Departments of Environmental Health, Biological Chemistry. and Medicine, College of Medicine, University of Cincinnnti. Cincinnati. Ohio 45267 Received
May
28. 1976
&Aminolevulinic acid (ALA) is the first intermediate in the pathway of heme biosynthesis from glycine. Some changes in heme synthetic pathways, such as occur in acute intermittent porphyria and lead poisoning, are accompanied by elevation of ALA in tissues, plasma, and urine. Most analytical methods for ALA in biological materials are variations of the method of Mauzerall and Granick (l), which depends on the formation of a colored complex between a pyrrole derivative of ALA and p-dimethylaminobenzaldehyde (DMAB). The original method had two significant limitations. Sensitivity was limited to the microgram range (the limit of detection was 0.5 pg of ALA) and the method was nonspecific. DMAB produces colored complexes with pyrroles formed by Knorr condensation of many aminoketones other than ALA, notably aminoacetone (AA), which occur in biological fluids. Modifications of the method of Mauzerall and Granick have been described which separate AA from ALA prior to coupling with DMAB (2, 3). These separation procedures are based on selective elution from resin columns either of the aminoketones or of pyrrole derivatives formed during the analytical procedure. A gas-liquid chromatography (GLC) procedure for aminoketones involving condensation with ethyl acetoacetate and preparation of trimethylsilyl derivatives of the resultant pyrroles has been described recently, but the procedure was not sufficiently sensitive for the measurement of normal concentrations of ALA in serum samples (4). The present investigation was undertaken to develop an analytical method for determination of ALA in nanogram quantities, with specific application to blood plasma. Since the method represents a departure from previous approaches, its application to the determination of ALA in urine was characterized also.
MATERIALS
AND METHODS
Reagents ALA.HCI: &Aminolevuhnic acid hydrochloride (5amino-4-oxopentanoic acid hydrochloride) was purchased from Sigma. Internal standard: 6-Amino-5oxohexanoic acid hydrochloride was synthesized by substituting 4-carbomethoxybutyryl chloride for 3-carbomethoxypropionyl chloride in the preparation of ALA. HCI described by Aranowa rt (11. (5). Details of the preparation and characterization of this analog of ALA are given below. Gibson et crl. (6) prepared this same compound by a different synthetic route. Internal standard solution: An aqueous solution of 1 pg of internal standard/ml was used. It was stored in the freezer. TMPI: Trimethylphenylammonium iodide (Eastman Organic Chemicals) was recrystallized from water. TMPH: A 1 M solution of trimethylphenylammonium hydroxide was prepared by mixing 1.05 g of TMPI with 1 g of silver oxide (Fisher Purified) and 4.0 ml of water in a test tube on a Vortex mixer. After several minutes with occasional mixing, the resultant silver iodide and excess silver oxide were removed by centrifugation. It was usually necessary to recentrifuge the supernatant 1 M TMPH. The clear reagent was stored in a capped test tube at room temperature in the dark. Acetylacetone: The Fisher Reagent product was redistilled through a IO-cm vigreux column at atmospheric pressure. When a 60-ml charge was distilled, the first 5 ml was discarded and the next 30 ml was collected. When smaller amounts of acetylacetone are redistilled, care must be taken to avoid any late cuts generated from charred reagent in the still pot. When stored in the refrigerator in the dark, the reagent can be kept for at least 10 weeks. MIBK: Methyl isobutyl ketone (Fisher Certified ACS). Hexane: Hexanes (Fisher Certified ACS). Ethyl ether: The anhydrous product from Fisher or Mallinckrodt. AG-1 x 8 column: BioRad AG-1 x 8, chloride form, 200 to 400 mesh, was stored in the refrigerator. Columns were prepared in the following manner: A slurry of the resin in water was transferred to a 25 cm x 10 mm chromatograph tube with a wet glass-wool plug in the bottom. The height of the settled resin bed was 2 cm. Columns can be prepared several days in advance if they are stored submerged in a beaker of water in the refrigerator. The columns were rinsed with 30 ml of water at room temperature just prior to use. TCA (40%): Forty grams of trichloroacetic acid (Mallinckrodt Analytical Reagent) was dissolved in water to a total volume of 100 ml. The solution was stored in the refrigerator. Preparation
of Itlternal
Standard
The 6-amino-5-oxohexanoic acid was prepared by a modification of the two-step synthesis of ALA. HCI described by Aranowa et al. (5). The synthesis of the internal standard is shown in Fig. I.
GLC DETERMINATION SYNTHESIS HIPPURIC
OF INTERNAL
33
OF ALA STANDARD
ACID 0
a--+
+
N Ii
“woc”” O
0 GLUTARVL CHLORIDE METHYL ESTER
OH IHCI.
t-
6,
N~~~zL%~;;~~~
HCI. BENZOIC
Ii20
0
3H20
ACID
-I+ +
0 co*.
CH30H
“Cl.“,Nq~oH 0
0
b-AMINO-5-OXOHEXANOIC
FIG.
1.
The synthesis of 6-amino-Soxohexanoic
ACID
HCI
acid hydrochloride.
Gfutaryf chloride methyl ester. The starting material, glutaryl chloride methyl ester, was prepared by a standard thionyl chloride procedure (7). Twenty-five grams (170 mmoles) of glutaric acid monomethyl ester (Aldrich) was mixed with 37 ml (515 mmoles) of thionyl chloride (Aldrich) in a lOO-ml round-bottom flask fitted with a pressure-equalizing dropping funnel and a reflux condenser. The condenser was equipped with a drying tube connected to a sodium hydroxide trap for acid gases. The thionyl chloride was added at a rate to ensure constant, but not vigorous, efflux of acid gases while stirring the reaction mixture at room temperature with a magnetic stirrer. After complete addition of the thionyl chloride, the mixture was heated under reflux for 2 hr. The excess thionyl chloride was then removed in vacua by heating the mixture in the same flask with a water bath at 50°C. The yield of the dark reddish-brown product was 28.5 g. 2-Phenyl-4-(4-carbomethoxybutyryl)-oxaz.olinone-5.
The crude glutaryl chloride methyl ester (28.5 g) was slowly added from a pressureequalizing dropping funnel to a mechanically stirred solution of 15.2 g (85 mmoles) of hippuric acid (Aldrich) in 82.5 ml of 4-picoline (Aldrich) in a 500-ml round-bottom flask fitted with a thermometer. The temperature of the reaction was maintained between -5” and 0°C with an ice-salt bath during the addition of the acid chloride ester and for an additional 4 hr. At this time, a mixture of 60 ml of 12 N hydrochloric acid in 350 g of crushed ice was added. The ice-salt bath was removed, and the mixture was stirred until all the ice in the flask had melted. The maroon-colored crystals of the substituted oxazolinone were collected on a sintered glass funnel and washed on the funnel with 10 ml of ice-cold distilled water. The air-dried crude product (28.5 g: mp, 115-120°C) was transferred to a
125-m] Erlenmeyer flask and stirred briefly in an ice bath with 50 ml of ethyl acetate. After collection on a sintered glass funnel, the product was washed on the funnel with an additional 10 ml of ice-cold ethyl acetate. Fifteen grams of 2-phenyl-4-(4-carbomethoxybutyryl)-oxazolinone-5 was obtained (mp 135-138°C). 6-Amitlo-5-oxohrxottoic crcid hydrochloride. The 15 g of substituted oxazolinone was heated under reflux for 8 hr with 250 ml of 6 N hydrochloric acid. After cooling to room temperature, the crystalline benzoic acid was removed by filtration. Seven grams of powdered activated charcoal was added to the filtrate, and the mixture was boiled for 15 min while stirring. The hot mixture was filtered, and the hydrochloric acid and water were removed in I’UCUO from the filtrate at 50°C to yield a semicrystalline mass of the product. This product was dissolved in 7.3 ml of 12 N hydrochloric acid with brief heating, and after cooling in an ice bath, 73 ml of acetone was added slowly with stirring. After an additional 30 min of stirring, the product was collected by filtration and washed with 7 ml of acetone. The yield of crystalline pressed and air-dried product was 2.99 g (16.5 mmole, 32% yield based on the oxazolinone, 17% yield based on hippuric acid). The light-tan crystals exhibited a structure change at 178-180°C and melted at 210-21X. Recrystallization of 300 mg of the product from hot aqueous isopropyl alcohol yielded 176 mg of crystals (single mp, 128-131°C). which were subjected to analysis and were used as the internal standard for GLC of ALA. Nuclear magnetic resonance analysis of the final product yielded results consistent with the proposed structure. Combined GLC-mass spectrometric analysis performed on the methylated pyrrole arising in the course of the GLC analytical procedure also supports the proposed structure, yielding a fragmentation pattern completely analogous with that exhibited by the dimethylated pyrrole of ALA. Apparatus
A Hewlett-Packard Model 402B dual column, dual flame-ionization detector gas chromatograph with a 1-mV recorder was used. The glass columns, 6 ft x 0.25 in. (4 mm i.d.), with graphite ferrules, were packed with 5% OV-17 (Applied Science) on GasChrom Q, loo-120 mesh (Applied Science). The injector temperature was 290°C the column temperature was 220°C and the nitrogen carrier flow rate was 70 ml/min. Procedures Plasma
Three milliliters of the sample was mixed in a centrifuge tube with 0.30 ml of the internal standard solution for a few seconds on a Vortex mixer. One-half milliliter of 40% TCA was added and the sample was mixed for
GLC
DETERMINATION
OF ALA
35
0.5 min on the Vortex mixer. After centriguation for 5 min, the sample was remixed briefly and recentrifuged for an additionai 5 min. The supernatant fluid was transferred with a disposable Pasteur pipette to a 125 x 16-mm screw-capped test tube with a Teflon-lined cap (Kimble 45066A), 5 ml of ethyl ether was added, and the tube was shaken vigorously by hand for 1 min. The upper ether layer was discarded and the aqueous phase was reextracted with an additional 5 ml of ethyl ether. After discarding the second ether wash, 1 ml of 1 M NaH,PO, and 50 ~1 of acetylacetone were added. After 1.5 min of mixing on the Vortex mixer, the sample was heated loosely stoppered for 10 min in a boiling water bath. The sample was cooled and extracted with 5 ml of hexane by shaking vigorously by hand for 1 min. After 1 min of centrifugation, the clear upper hexane phase was discarded. If an emulsion occurred at this point, the shaking and centrifugation steps were repeated. After discarding the hexane phase, 5 ml of MIBK was added. The tube was shaken vigorously by hand for 1 min and then centrifuged for 1 min. With a disposable Pasteur pipette, nearly all of the upper MIBK extract was transferred to a Concentritube (Laboratory Research Co., P.O. Box 36509, Los Angeles, California 90036). Quantitative transfer of the MIBK phase is neither possible nor necessary, but none of the aqueous phase or any emulsion layer should be transferred. While mixing the extract on the Vortex mixer, 5 ~1 of TMPH was introduced and the mixing was continued for 1.5 min. The tube was stoppered and centrifuged for 2 min. A lO+l syringe was prewetted with water and used to draw up all of the small lower TMPH extract and then to inject the sample slowly (15 to 20 set) into the GLC unit. No interference is introduced if some of the MIBK layer is drawn into the syringe and injected into the chromatograph. Urine
Urine samples were collected in bottles containing 0.5 g of benzoic acid and stored in the freezer until analyzed. The sample (0.10 ml of urine), 0.30 ml of the internal standard solution, and 2 ml of 1 M NaH,PO, were mixed in a test tube. The mixture was passed through a washed AG-1 x 8 column, and two l-ml water rinses of the test tube were used to wash the column. The column effluent and the rinses were collected in the same type of screw-capped tube as was used for plasma analysis. After addition of 50 ~1 of acetylacetone, the remainder of the urine procedure was identical with the plasma procedure, starting with the 1.5-min mixing and heating steps. Standard Curve and Quantitation
The standard curve was prepared by adding ALA. HCl to outdated blood bank plasma over the range of 0 to 1 pg of ALA/ml of plasma (6
points) and subjecting these samples to the plasma analytical procedure. Peak heights were measured from straight baselines drawn for the ALA and internal standard (IS) peaks, in which the minimum tangent points before and after each of the two peaks were connected. The baselines were drawn as shown in Fig. 3. Plotting the peak height ratio (ALA/IS) 1’s ALA added (calculated as the nonhydrochloride) resulted in a straight line with a small positive y-intercept. Extrapolation of this line to the x--intercept indicated that the apparent residual ALA in the outdated plasma was 7 rig/ml. The slope (peak height ratio/nanograms of ALA per milliliter) was calculated from this line and was used for the calculation of the ALA concentration in plasma and urine samples as follows. For 3-ml plasma samples, the peak height ratio (ALA/IS) was divided by the standard curve slope to yield results expressed as nanograms of ALA per milliliter of plasma. For urine samples, the peak height ratio was divided by a second slope value in order to correct for the fact that 0.1 ml of urine is used with the same amount of internal standard employed with 3 ml of plasma and to yield results expressed as micrograms of ALA per milliliter of urine. This second slope value was obtained by multiplying the plasma standard curve slope value by 0.03. Figure 3 illustrates typical gas-liquid chromatographic elution patterns obtained when a fresh blood plasma specimen was analyzed by the plasma procedure with and without the addition of ALA and internal standard, each added at 100 r&ml. Peak height measurements, rather than peak area measurements obtained with an electronic digital integrator, were used for quantitation. The advantage of this procedure derives from the fact that small, incompletely
NORMAL
URINE + IS
MINUTES
2. Gas-liquid chromatogram showing peaks for 6-aminolevulinic acid (ALAI from normal human urine and the 300-ng addition of the internal standard (IS). The ALA content of this specimen was measured to be 2.81 Fgiml. FIG.
GLC DETERMINATION
OF ALA
37
FIG. 3. Gas-liquid chromatograms of human blood plasma with and without added Gaminolevulinic acid (ALA) and internal standard (IS). The top pattern, plasma + ALA + IS, resulted from adding 300 ng each of ALA and IS to 3 ml of fresh human blood plasma. The middle pattern, plasma + IS. was obtained from 3 ml of the same plasma on addition of 300 ng if IS only. The bottom pattern, plasma blank, had no additions. The ALA content of this specimen was measured to be 8.4 rig/ml.
resolved peaks near the two peaks of interest were frequently incorporated in the areas of the desired peaks. Their contributions were minimized when peak height measurements were used. Peak area measurements, in which the areas are calculated by multiplying the peak heights by their widths at half-height, can be used, but they offer no advantage over peak heights alone and require additional operations. The ideal internal standard for gas chromatography should be added to the measured sample before any other operation in the analysis is performed. It should be a very close analogue of the compound to be measured, so that it will then behave exactly the same as the compound of interest in its physical and chemical properties and, thus, will partition between the various solvent-solvent and sorbent-solvent systems and react with any derivatizing reagents in a manner identical to the compound to be measured. The only step in the procedure where it should behave differently is in the gas chromatographic column. Here it should be completely separated from the material to be measured, but it should elute close enough to avoid the necessity for changing conditions to allow a reasonable analysis time. A considerable advantage is offered by this type of internal standard in that it automatically corrects for losses of the compound of interest and it makes it unnecessary to transfer the various phases quantitatively. Any loss of a given phase results in proportional losses of compound and internal standard. The internal standard synthesized for this procedure, 6-amino-Soxo-
hexanoic acid, differs from ALA in having a single additional methylene group between the carboxyl and carbonyl groups of ALA, making the side chain butyric acid instead of propionic acid. Its functional groups, the carboxyl and the Lu-aminoketone structures, are identical to those of ALA; so all the reactions that ALA will undergo will be equally achieved with the internal standard. The extra methylene group merely causes the methylated derivative of the internal standard pyrrole to elute slightly later than the same derivative of ALA (See Figs. 2 and 3). A synthesis of 6-amino-5oxohexanoic acid was reported in 195.5 by Gibson ef al. (6). We have prepared this compound by a shorter, more recent route, i.e., a minor modification of the synthesis of ALA. HCl described by Aranowa et ~1. (5). The synthesis of ALA. HCl was accomplished by Aranowa et al. by condensing succinyl chloride methyl ester with hippuric acid to yield 2-phenyl-4-(3-carbomethoxypropionyl)-oxazolinone-5, which on acid hydrolysis yielded the hydrochloride of ALA in good yield. Our modification involves the use of glutaryl chloride methyl ester in place of succinyl chloride methyl ester to yield 2-phenyl4-(4-carbomethoxybutyryl)-oxazolinone-5, which in turn generates the hydrochloride of the desired analogue of ALA in good yield on acid hydrolysis. Some precautions are necessary in the course of the synthesis. Adequate sodium hydroxide must be used to trap all the acid gases generated in preparation of the glutaryl chloride methyl ester. The reaction temperature must be maintained below 0°C while preparing the substituted oxazolinone to prevent formation of glytaryl-(4-picoliniuml methyl ester chloride. A temperature at 50°C or lower should be maintained during removal of the excess thionyl chloride in \YICIIO after the preparation of the glutaryl chloride methyl ester and during removal of the excess hydrochloric acid and water in the preparation of the final product. Excess heat at either of these two steps could lead to polymerization or decomposition of the desired compounds. Finally, the final product should be stored in a well-stoppered container, in the dark, at a temperature below 0°C. The internal standard partitions exactly the same as ALA in the extraction steps used in the procedures described in this report. Three aliquots of a mixture of the two compounds were treated in different ways to establish this fact. The first sample, the control, was processed by the usual plasma procedure. The other two aliquots were also processed by the same procedure, except that the second sample was washed four times with ethyl ether instead of the usual two times and the final TMPH extract of the third sample was reprocessed through the reaction with acetylacetone, washed with hexane. extracted into MIBK. and reextracted from the MIBK with TMPH. Had there been a difference in
GLC
DETERMINATION
OF ALA
39
partitioning of the ALA and internal standard between ether and the aqueous phase, the first and second samples would have yielded different peak height ratios. Had there been a difference in partitioning of the two pyrroles in either the hexane wash or the MIBK or TMPH extractions, the first and third samples would have exhibited different peak height ratios. All three samples yielded identical peak height ratios on GLC analysis. Six samples each of the same specimen of urine were analyzed by both the calorimetric procedure of Marver et al., a well-accepted procedure that involves the use of two ion exchange columns, pyrrole formation, and reaction with DMAB (3), and our GLC method for ALA in urine. The calorimetric procedure yielded values 16% higher than the GLC procedure (2.19 + 0.06 vs 1.84 ? 0.04 pg of ALA/ml, mean +- standard deviation). The same ALA solution was used to calibrate both methods. While the method of Marver et al. ranks among the best calorimetric procedures, we strongly suspect that it measures non-ALA materials. When a sample of ALA was carried though the analytical procedure and chromatographed on an OV- 17 column in an LKB-9000 combined gas chromatograph-mass spectrometer with a System 150 (Systems Industries) computer, the ALA derivative yielded a mass spectrometric fragmentation pattern consistent with two methylations of the ALA pyrrole-one methylation on the carboxyl group and the other on the pyrrole ring nitrogen. The base peak at m/e 223 and fragments of m/e 181 (P(parent ion)-42, loss of ketene from acetyl), m/e 180 (P-43, loss of acetyl), and m/e 150 (P-73, loss of carbomethoxymethylene from the carboxyl methyl ester end) were found. The approach used in the development of the method described here was based on that developed in this laboratory for the gas chromatographic analysis of a number of important acidic compounds (8-11). This approach involves extracting the acidic and neutral compounds into a suitable organic solvent and back-extracting only the acidic compounds into a very small volume of an aqueous quaternary ammonium hydroxide. Injection of the resultant quaternary ammonium salts into the hot vaporizer of the gas chromatograph results in thermal decomposition of the salts to yield alkylated derivatives of the acidic compounds and a tertiary amine (12). Selection of a suitable chromatographic stationary phase and chromatographic conditions achieves separation of the reaction products. Such an approach has the added advantages of simplicity, speed, and the elimination of any tedious, time-consuming, and sometimes destructive evaporation steps. The fact that ALA occurs in normal plasma samples at concentrations about three orders of magnitude lower than the materials analyzed in most of our past experiences has made it necessary to develop a slightly more
40
MAC‘GEE
Ii7 ill
complex procedure to eliminate potential interfering substances from the plasma and reagents. As a result, instead of requiring about 6 min to prepare a sample ready for injection into the chromatograph (8, 9), the present procedures require about 30 min to reach the same point. Being an amino acid, ALA is not readily partitioned into organic solvents from aqueous phases. This allowed us to remove the vast majority of interfering materials along with the trichloroacetic acid from the trichloroacetic acid supernatant fluid by two simple ether washes. By converting the ALA and the internal standard to their respective pyrroles after the ether wash, we were then able to extract them as their pyrroles from an aqueous solution into methyl isobutyl ketone. Back-extraction into a very small volume of aqueous trimethylphenylammonium hydroxide yielded a sample ready for injection and flash methylation in the 290°C vaporizer of the gas chromatograph. Aminoacetone (AA) does not interfere with the measurement of ALA in the procedures described in this report. While this aminoketone forms a pyrrole with acetylacetone, and causes some of the spectrophotometric methods to yield falsely high ALA values, addition of 1 pg of AA/ml of plasma did not change the ALA analysis of the plasma by our procedure. Since the AA pyrrole does not contain the carboxyl group present in either the ALA or internal standard pyrroles, it is probably not extracted from the MIBK by the alkaline TMPH reagent. An experiment in which [14C]ALA in plasma was carried through the entire procedure showed that 70% of the ALA was found in the final alkaline extract. Since quantitative separation of the phases is not accomplished in this procedure, some variation in the overall recovery can be expected from analysis to analysis, but the nature and method of use of the internal standard make any recovery corrections unnecessary. Three plasma samples with different concentrations of ALA were each analyzed six times by the procedure described in this report. The following values (mean & standard deviation) were obtained: 6.33 + 0.90,43.70 t 2.39, and 73.50 + 2.30 ng of ALA/ml. Four aliquots of the same urine sample were analyzed on two separate days (eight analyses) with the following result: 2.46 & 0.10 pg of ALA/ml. Three commercially available toxicology control samples, two sera and one urine, were analyzed by the procedures described in this report, except that 10 times the usual amount of urine was analyzed. No internal standard was added in order to determine if any of the commonly used (and abused) drugs interferes with our procedures. Table 1 lists the drugs and their concentrations in the three control samples. The only potentially interfering peaks in the elution patterns were seen in Serum B. Performance of the analysis on samples of methaqualone and methprylon did not result in any interfering peaks. We conclude from this
GLC DETERMINATION
41
OF ALA
study that none of the drugs tested at these concentrations (Table 1) will interfere with the procedures described in this report, but the source of the interfering peaks in Serum B remains unknown. As can be seen in Fig. 3, there are late peaks in the GLC elution pattern that could interfere with subsequent analyses. The retention times of these peaks were highly reproducible relative to the ALA and internal standard peaks, so it was possible to place each of the peaks to be measured in a clear window by injecting the next sample at a time that was twice the retention time of the ALA peak. This rule holds even for the slight differences in retention times that can occur because of differences in column temperature, carrier flow rate, or the age of the column. A TABLE I DRUG CONTENT OF TOXICOLOGY CONTROL SAMPLES Drug concentration
DWiT Alcohol Amobarbital Amphetamine sulfate Chlorodiazepam hydrochloride Codeine Diazepam Diphenylhydantoin Ethchlorvynol Glutethimide Meperidine Meprobamate Methamphetamine Methaqualone Methyprylon Morphine Pentobarbital Phenobarbital Procainamide hydrochloride Propoxyphene hydrochloride Quinidine sulfate Secobarbital Sodium bromide Sodium salicylate Sulfanylamide
Serum A” 1000 20 2 20
&$rnl)
Serum B*
Urine’
20
5 5 3
3 20 20 10
20
20
20
20
20 20 3 20 20 5 2 6 20 500 250 50
a Hyland Toxicology Serum Control (unassayed). b Lederle Diagnostics Serum Toxicology Control-Drugs ’ Lederle Diagnostics Urine Toxicology Control-Drugs
20
5
5
A. I for analytical use only.
slight difference in retention time of this type can be seen on comparing Figs. 2 and 3. We have used the same OV-17 column for this and other projects for over 18 months. After several months of use, a slight broadening of the peaks was noted. This loss of efficiency was corrected by replacing the first 10 cm of packing of the inlet end of the column with fresh packing material. Before replacing the packing, we removed any injection residues with TMH-24 (tetramethylammonium hydroxide, 24% in methanol, Southwestern Analytical Chemicals, Austin, Texas) on a cotton-tipped applicator stick. The residual TMH-24 was removed with water and then with methanol on clean cotton-tipped applicator sticks and the inlet was allowed to dry in air before it was replaced. After about 1 hr at operating conditions, the column was as good as new. This rejuvenation has been performed twice with equal success, and we anticipate a long and useful life for this column. A sample of outdated blood bank plasma containing 5 ng of ALA/ml was incubated with a crude ALA dehydrase preparation in order to remove the ALA. After a brief incubation with the enzyme, the ALA concentration fell below 1 rig/ml. This convinced us that there were no significant non-ALA peaks cochromatographing with the ALA derivative. The crude enzyme preparation was a dialyzed 25,OOOg supernatant fraction obtained from rat liver. The preparation of the homogenate and the conditions for dialysis and incubation followed the procedures described by Wilson et al. (13). A similar experiment performed with rabbit plasma, however, left a significant peak on the chromatogram. This peak had a retention time about 30 set shorter than the ALA derivative and would be assayed as 56 ng of ALA/ml. Reexamination of the apparent ALA peak on the chromatogram generated from rabbit plasma before treatment with the enzyme showed that the peak was significantly broader than the ALA peaks obtained from human plasma specimens. Examination of the ALA peaks on chromatograms obtained from blood plasma specimens of rat, cat, dog, sheep, and goat did not demonstrate broadening of the ALA peaks, and the ALA peaks had exactly the same retention times as are found with human plasma. Of the seven mammalian species tested to date, only rabbit plasma exhibits broadening of the apparent ALA peak due to the unknown compound. The instability of ALA in alkaline solution is well documented (141, but the pyrroles resulting from condensation of ALA and the internal standard with acetylacetone are very stable in alkaline solutions. This has been demonstrated in two ways. When solutions of ALA pyrrole and internal standard pyrrole in 1 M TMPH were stored at room temperature and analyzed by GLC periodically over a several-week storage period,
GLC DETERMINATION
OF ALA
43
the peak height ratio remained constant and the peaks retained their original heights within injection volume precision. The second and most important test is the stability of the final extract. When the TMPH extract under the MIBK phase was stored at room temperature for over 1 week, GLC analyses revealed no loss of either the ALA or internal standard derivatives. This observation allows for the simultaneous preparation of multiple samples and GLC analysis when it is most convenient. In this way, three samples can be analyzed each hour with a single gas chromatographic column. Also, in the event of any minor instrumental difficulties, valuable samples are not lost while the difficulties are being rectified. SUMMARY
Procedures have been developed for the quantitative determination of normal and elevated concentrations of &aminolevulinic acid (ALA) in 3 ml of blood plasma or 0.1 ml of urine by gas-liquid chromatography (GLC) with a flame-ionization detector. Selective partitions, pyrrole formation with acetylacetone, and flash methylation in the vaporizer of the GLC unit allow for a convenient and rapid procedure for the analysis. An analogue of ALA, 6-amino-5-oxohexanoic acid, was prepared and used as an internal standard. A single analysis of plasma required about 30 min for preparation and about 20 min for GLC. Multiple samples can be prepared simultaneously and stored for at least 1 week before GLC analysisallowing for the analysis of up to 24 samples/day on a single GLC unit. ACKNOWLEDGMENTS The authors thank Dr. Daniel A. Garteiz for the combined gas chromatography-mass spectrometric analyses. These studies were supported by Veterans Administration funds (Grant MRIS 5421-02) and by a grant from the International Lead-Zinc Research Organization (LH-229).
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J., and Allen,
K. G., J. Chromatog.
100, 35 (1974). Environ. Micro&o/.
11. Tabor. M. W., MacGee. J., and Holland, J.. Appl.
31, 25 (1976).
44
MACGEE
15-7 Al
12. MacGee, J.. in “Methods of Analysis of Anti-Epileptic Drugs. Proceedings of the Workshop on the Determination of Anti-Epileptic Drugs in Body Fluids. Noordweijkerhout, the Netherlands, 13-14 April 1972” (J. W. A. Meijer. H. Meinardi. G. Gardner-Thorpe, and E. Van Der Kleijn, Eds.). p. I I I. Excerpta MedicaiAmerican Elsevier. Amsterdam, 1973. 13. Wilson, E. L., Burger. P. E.. and Dowdle, E. B., Eur. .I. Biocllrm. 29. 563 t 1972). 14. Haeger-Aronsen, B.. J. Lob. C/in. /nr,e.rt. 12, Suppl. 47. 5 (1960).