1372
Anal. Chem. 1988, 58, 1372-1375
ACKNOWLEDGMENT We gratefully acknowledge the work of Bob Cunico, Laura Correia, and Tim Wehr for data supplied on the analysis of PTH amino acid and peptide samples. LITERATURE CITED (1) Yang, F. J. HRC CC, J . Hlgh Resolui. Chromatogr. Chromatogr. Commun. 1983, 6 , 348. (2) Schwartz, H. E.;Berry, V. V. LC Mag., Liq. Chromatogr. HPLC Mag. 1985, 3 , 110. (3) Schwartz, H. E.; Karger, 6. L.; Kucera, P. Anal. Chem. 1883, 55.
1752. (4) Snyder, L. R.; Saunders, D. L. J. Chromatogr. Sci. 1969, 7 , 195. (5) Katz, E.; Scott, R. P. W. J . Chromatogr. 1982, 253, 159. (6) Bakaiyar, S:R.; McIiwrick, R.; Roggendorf, E. J. Chromatogr. 1977, 142, 353.
(7) Quarry, M. A.; Grob, R. L.; Snyder, L. R. J . Chromatogr. 1984, 285, 1. (8) Wehr, C. T.; Cunico, R. L. Am. Biotechnol. Lab. 1983, Dec. (9) Cunico, R. L.; Simpson, R.; Correia, L.; Wehr, C. T. J. Chromatogr. 1984, 336, 105. (IO) Wehr, C. T.; Correia, L. “High Sensltivity Peptide Mapping Using Microbore Reversed Phase Chromatography”. Presented at I V Internationai Symposium of PeptMes, Proteins, and Polynucleotides, Monte Carlo, Nov 1983.
RECEIVED for review March 18,1985. Resubmitted January 13,1986. Accepted January 13,1986. This paper was presented in part at the Eastern Analytical Symposium,New York, 1983, and in part at the National American Chemical Society Meeting, Chicago, IL, Sept 1985.
Fluorometric Determination of 5-Aminolevulinic Acid after Derivatization with o-Phthaldialdehyde and Separation by Reversed-Phase High-Performance Liquid Chromatography Hans-Ulrich Meisch* and Bernhard Wannemacher Fachbereich 15.2, Biochemie, der Universitat des Saarlandes, 0-6600 Saarbrucken, FRG
The well-known reaction of o-phthaidlaidehyde with amino compounds in the presence of P-mercaptoethanoi leads to a fluorescent product that Is often used for a sensitive assay of cy-amino acids. This method Is now extended to the quantitative determination of 5-aminoievuiinic acid (ALA) by optimlzlng the analytical conditions (derivatizatlon at pH 10.0 for 3 h at 97 “C) with the advantage of eiiminating interfering fluorescent compounds of other amino acids. The ALA derivative Is detected fluorometricaily after separation by reversed-phase high-performance liquid chromatography. lhus, ALA could be measured either in aqueous solution or in cell-free extracts of the green alga Chlorella vulgaris, down to the low picomole range (detection limit 0.8 pmol).
As it was first described by Roth ( I ) , o-phthalaldehyde (OPA) reacts with amino acids in alkaline solution in the presence of 2-mercaptoethanol (ME) by giving rise to strongly fluorescent adducts (excitation,,A = 340 nm, emission A, = 455 nm), which permits the fluorometric assay of amino acids down to the picomole range. OPA and ME condense with primary amines including a-amino acids (2)to form highly fluorescent, thio-substituted isoindoles ( 3 , 4 ) . This reaction has found broad application for amino acid analysis using chromatographic techniques, whereby both pre and postcolumn derivatization have been successfully performed (5,6). The OPA reagent, however, cannot be used for the determination of secondary amines like proline or hydroxyproline (1, 7),and the reaction with cysteine and cystine yields a product with low fluorescence (1,8). A low sensitivity of the system is also reported for amines and amino acids lacking an a-hydrogen atom ( 2 ) as well as for amino acids having their amino group in other than the a position (9). The determination of 5-aminolevulinic acid (ALA),which belongs to the latter group, is of particular interest because
* Correspondence to Hans-Ulrich Meisch, Fachrichtung Biochemie,
Universitat des Saarlandes, D-6600 Saarbrucken, FRG.
0003-2700/86/0358-1372$01.50/0
of its clinical relevance in body fluids where it is found as an important metabolite during tetrapyrrole biosynthesis. ALA is the first committed step in this pathway, and it is unique for tetrapyrrole formation in plants, bacteria, and animal tissue (10). Normally, ALA concentrations in human blood plasma are very low, but in some diseases like hereditary porphyrias, hereditary tyrcsinaemia, and during lead poisoning, the plasma levels of ALA become markedly elevated (11). In green plants and photosynthetic bacteria, ALA synthesis is the rate-limiting step for chlorophyll biosynthesis,its formation being controlled by light (12). Therefore, quantitative ALA determination is not only of clinical interest but is often used as a tool for investigations on ALA and chlorophyll biosynthesis and their regulation (13). The classic method of ALA determination has been established by Mauzerall and Granick (14): ALA is allowed to react with a 1,3-diketone(ethyl acetoacetate or acetylacetone) to form a pyrrole that is then transformed in the presence of 4-(N-dimethylamino)benzaldehyde(Ehrlich’s reagent) into a red dye, which can be used for quantitation by its absorption at 550 nm. The main disadvantage of this method is its lack of specificity, since Ehr1ich’s.reagent is known to react with other compounds like pyrroles, amino ketones, or indole derivatives (15). Furthermore, the sensitivity of the color reaction is limited to the nanomole range (14). Recently, a new method of ALA determination has been developed, depending on the condensation of ALA with 2-amino-3-hydroxynaphthalene in alkaline medium, followed by extractions, HPLC separation, and fluorometric detection of the ALA derivative (16). Compared to the color reaction with Ehrlich‘s reagent, a 10Ox higher sensitivity had been achieved with the HPLC method, thus allowing ALA determination down to the picomole range (16). This method, however, requires an almost complete separation of the ALA derivative from a large excess of 2-amino-3-naphtholprior to HPLC separation, since the reagent itself has fluorescent properties too. The present work describes the reaction of ALA with OPA and ME in alkaline solution, yielding a highly fluorescent adduct, which can be successfully applied for a simple but sensitive and specific method of ALA determination in biological samples. 0 1986 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7,JUNE 1986
EXPERIMENTAL SECTION Chromatography. The ALA derivative was analyzed in a high-performance liquid chromatograph, equipped with a Waters 6000 A pump (Waters ASSOC.,Milford, MA), a Rhecdyne injection valve, a Perkin-Elmer 650/LC fluorescence detector set at 366 nm (excitation) and 432 nm (emission), and a Fuci 1100 multirange recorder or a Hewlett-Packard 3380 A integrator. A reversed-phase system was used consisting of a Merck HIBAR RP-18 column (25 cm X 4.6 mm, Lichrosorb C-18, particle size 5 pm), which was eluted at 25 "C with a degassed mobile phase of methanol/O.l M potassium phosphate buffer (pH 6.8) 47:53 (v/v) at a flow rate of 1.2 mL/min. Derivatization of ALA. The reagent for ALA derivatization was prepared according to Cronin et al. (9)and modified as follows: 50 mg of OPA (Merck) was dissolved in 5 mL of ethanol, to which 200 pL of ME was added. This solution wm then brought to a final volume of 100 mL with 0.5 M potassium borate buffer (pH 10.0). After deaeration by bubbling with nitrogen, 7.5 mL of a 30% Brij-35 solution (Merck) was added. This mixture was kept dark under a nitrogen atmosphere and allowed to age at least 24 h prior to use. The reagent strength was maintained by adding 20 pL of ME every 2 days. Under these conditions, the OPA reagent could be used for at least 1 week. For ALA determination, 20 pL of sample was allowed to react with 100 pL of OPA reagent in 1-mL Eppendorf reaction vials at 97 "C for 3 h. Five microliters of this solution was then directly injected into the HPLC system. Derivatization of ALA in algal extract was performed as above by adding synthetic ALA (ALA-HC1,Merck) to cell-free preparations of Chlorella vulgaris (Collection of Algae, Gottingen). For this purpose C. vulgaris was autotraphically grown for 3 days in a mineral nutrient medium according to Meisch and Bielig (27) then harvested by centrifugation and homogenized in a Na2P207/HC1buffer (0.1 M, pH 7.8) that contained 300 mM glycerol in a Buhler Zellmuhle (Buhler, Tubingen). Centrifugation a t 15OOOg yielded a supernatant that was used as a cell-free extract for ALA determinations. RESULTS AND DISCUSSION Conditions for ALA Derivatization. In a first series of experiments, ALA was allowed to react with the OPA reagent using conditions that are commonly used for the determination of a-amino acids, i.e., a reaction time of 1-5 min a t room temperature (18-20), but no fluorescent product could be detected. The reaction temperature was then raised and the reaction time was extended. At temperatures above 70 "C, a fluorescence occurred whose intensity was optimized by variation of pH, temperature, and reaction time (Figure 1). As shown in Figure la, maximal fluorescence intensity is observed at pH 10.0. Optimal fluorescence yield at this pH was obtained when the reaction temperature was raised up to 97 "C, while a t higher temperatures the fluorescence intensity decreased again (Figure lb). Variation of the reaction time then showed that the fluorescence reached its maximum after 180 min at 97 "C and pH 10.0 (Figure IC). Cronin et al. (9) reported that with an increase in the temperature of the reaction with OPA/ME, the fluorescence yield for amino acids having a fully substituted carbon atom in the a position with respect to the amino group can be substantially improved, whereas amino acids having no hydrogens in the a position gave fluorescenceyields that declined sharply at reaction temperatures above 25 "C. We found that with ALA, although belonging to the latter group, the fluorescence yield increased markedly when the temperature was raised. On the other hand, Cronin et al. (9) used a short reaction time, while in our case, maximal fluorescence was observed after 3 h of condensation. Additionally the high
1373
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Figure 1. Optimization of the reaction of ALA with OPA/ME: dependence of (a) pH, (b) temperature at pH 10.0,(c) derivatization time M; at pH 10.0and 97 "C. Conditions are as follows: ALA, 5 X OPA, 3 X M; ME, 2.5 X lo-' M in 0.5 M potassium borate buffer.
reaction temperature of 97 "C and the extended condensation time have the great advantage of eliminating nearly all other interfering fluorescent products of other amino acids present in biological samples. Spectral Properties of the ALA Derivative. The interaction of OPA, ME, and ALA is demonstrated by their absorption spectra (Figure 2). In 0.5 M potassium borate buffer of pH 10.0, OPA (3 X M) itself exhibits a maximum at 285 nm, and the presence of ALA (5 X M) had no influence. In the presence of OPA and ME (2.5 X M), however, this absorption band diminished and a new peak emerges at 243 nm. When OPA, ME, and ALA are present altogether, the absorption at 243 nm was strongly reduced, while new absorption maximum at 366 nm (strong) and 413 nm (weak) arose. Figure 2 shows that the main absorption of the OPA/ME/ALA complex at 366 nm is clearly distinct from all other absorptions of either of the components. Excitation of the ALA derivative at 366 nm gives rise to a fluorescence emission with a maximum at 432 nm (Figure 3). Stability of the ALA Derivative and Its Quantitative Determination. The stability of the OPA/ME/ALA adduct was examined under several conditions as a function of time
1374
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7,JUNE 1986
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450
Figure 2. Absorption spectra of OPA and Its derivatives with ME and ALA. The spectra were measured in 0.5 M potassium borate buffer, pH 10.0, containing 3 X 10" M OPA, 2.5 X lo-* M ME, and 5 X M ALA. The spectrum of the OPA/ME/ALA derivatlve was measured after 5-fold dllution with buffer: (- -) OPA, (- - -) OPAIME, (- -)
OPAIALA, (-)
OPAIMEIALA.
standard additions of ALA. Conditlons are as follows: excitation at 366 nm and emission at 432 nm; for further conditions see the Experimental Section.
-
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Fluorescence spectrum of the derivative of ALA wlth OPAlME after excitation at 366 nm. Figure 3.
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Figure 4. Stability of the ALA derivative with OPAIME. (A-A) without protection from air and light, ( 0 4 )derivatives stored dark derivatives ) stored dark in air. under nitrogen, (H
during 56 h (Figure 4). From Figure 4 it can be seen that storage in bright light and in the presence of oxygen leads to a rapid loss of fluorescence intensity, which means that after 24 h only about 50% of the initial intensity is still present. Storage under nitrogen in the dark extends the stability of the ALA derivative to about 36 h, followed by a small decrease during the next 20 h, while storage in the dark without protection from air guarantees a stability sufficient for ALA analysis within the first 12 h. Under these conditions, ALA
15
Figure 6. Separation of the derivatlve of ALA with OPA/ME by reversed-phase HPLC. HPLC condltlons are 10 pmol of ALA, eluent potassium phosphate buffer (0.1 M, pH 6.8)/methanol53:47,flow rate 1.2mL/min. Derivatization of ALA in a cell-free extract of C. vulgaris gives the following peak Identiflcation: (1, 2)derivatives of ALA with ME, (3)ALA/OPA/ME, and (4)not identified.
could be quantitatively determined, the fluorescence intensity of ita OPA/ME derivative being linear between 1and 50 pmol of ALA (Figure 5). The precision of the ALA determination in water was checked by 10-fold analysis at the 5- and 50-pmol level: with 5 pmol of ALA a relative standard deviation of 8.7% was obtained, while with 50 pmol of ALA 3.8% could be calculated. Serial dilution of aqueous ALA standard solutions, followed by derivatization with OPA/ME, and fluorescence analysis lead to a detection limit of about 0.8 pmol of ALA (signalto-noise value 101). The retention time of the ALA derivative during HPLC analysis was 5.1 f 0.05 min (Figure 6). The method of ALA analysis was also checked with algal extracts. For this purpose, a cell-free homogenate of Chlorella vulgaris was used. Standard additions of ALA to these extracts yielded a recovery of 90-97% (at 50 or 5 pmol, respectively) compared to ALA analysis in water (Figure 5). The corresponding relative standard deviations as determined by 10 independent measurements were found to be 9.0% (5 pmol) and 4.2% (50 pmol). With the method demonstrated in the present work OPA now serves as a highly sensitive and specific reagent for the determination of ALA. With respect to the classic colorimetric ALA determination by reaction with Ehrlich's reagent (14) and to the fluorometric method after derivatization with 2-amino-3-naphthol and subsequent separation by HPLC (16), the new procedure of ALA analysis presented
Anal. Chem. 1986, 58, 1375-1379
here has the great advantage to be more selective without the necessity to separate the fluorescent byproducts from the ALA derivative, combined with the highest sensitivity ever reported for the determination of ALA (detection limits: Ehrlich's reagent, 4 nmol (14); 2-amino-3-naphtho1, 40 pmol (16); OPA/ME, 0.8 pmol). The OPA reagent has the outstanding property of being nonfluorescent until it reacts with the amines (21). A disadvantage may be the still potentially reactive excess of OPA remaining in solution. As indicated by the additional peak at 242 nm in the UV spectrum of the reaction product (Figure 2), the excess OPA reacts with 2-mercaptoethanol to form a 1:l hemimercaptal (22). Nevertheless it is unnecessary to remove this product prior to injection into the HPLC system. In his first publication, Roth ( I ) considered the possibility that the same fluorescent species is produced regardless of the nature of the primary amine. The reaction product has not been isolated up to now, but its possible structure has been extensively discussed by Simons and Johnson (3,22),who identified the structure of the fluorescent adduct as an 1-alkylthio-2-dkyl-substituted-isoindole. In the case of the reaction of OPA/ME with ALA, no information about the structure of the fluorogen is available. In view of the drastically different reaction conditions used in this work (high temperature, long time) and the presence of a reactive carbonyl group, it seems unlikely that a simple alkyl-substituted isoindole has been formed with ALA, OPA, and ME. Further discussion of the product structure should therefore wait on its isolation or characterization. On the other hand, temperatures above 80 OC gave rise to two additional peaks in the elution profile (peaks 1 and 2 in Figure 6). Since isoindoles are quite reactive, these peaks may result from derivatives formed from OPA and ME at elevated temperatures. As Figure 4 indicates, the reaction product of ALA with OPA/ME is very sensitive toward oxygen and light. The adduct has been shown to be stable at room temperature in borate buffer of pH 10.0 for up to 1.5 h, but it decomposed to about 50% within 24 h. Investigations on the structure of the decomposition products suggested that the fluorescent adduct had undergone a spontaneous, albeit slow, intramolecular sulfur-to-oxygen rearrangement (3). This decompo-
1375
sition can easily be prevented by storing the reaction product under nitrogen in the dark. As a fluorometric reagent, OPA is particularly suited for automated use (20) and can be applied for the determination of picomolar amounts of amino compounds (8) like amino acids, primary amines, peptides (8), and recently even as a fluorescence probe of aldolase active site (23). With the new method presented here, the OPA reagent might also be useful as an additional tool for elucidating the biosynthetic pathway to ALA and the chlorophylls in green plants. Registry No. ALA, 106-60-5; OPA, 643-79-8; ME, 60-24-2.
LITERATURE CITED (1) Roth, M. Anal. Chem. 1971, 43, 880-882. (2) Cronin, J. R.; Hare, P. E. Anal. Biochem. 1977, 81, 151-156. (3) Simons, S. S.,Jr.; Johnson, D. F. J. Am. Chem. SOC. 1976, 9 8 , 7098-7099. (4) Slmons, S. S., Jr.; Johnson, D. F. Anal. Biochem. 1977, 8 2 , 250-254. (5) Larsen, B. R.; West, F. 0. J. Chromatogr. Sci. 1981, 19, 259-265. (6) Roth, M. J. Clin. Chem. Clin. Biochen,. 1976, 14, 361-364. (7) Benson, J. R.; Hare. P. E. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 619-622. (8) Lee, L. S.;Drescher, D. G. Int. J . Biochem. 1978, 9 , 457-467. (9) Cronin, J. R.; Pizarello, S.;Gandy, W. Anal. Biochem. 1979, 9 3 , 174-179. 10) Granlck, S.; Sassa, S. I n "Metabollc Pathways"; Vogel, H. J., Ed.; Academic Press: New York, 1971; Vol. 5, pp 77-141. 11) Gorchein, A. Biochem. J. 1984, 219, 883-889. 12) Granick, S.;Beale, S. I . I n "Advances in Enzymology"; Meister, A,, Ed.; Wlley: New York, 1978; Vol. 46, pp 33-204. (13) Castelfranco, P. A.; Beale, S. I.Ann. Rev. Plantfhysiol. 1983, 34, 241-278. (14) Mauzerall, D.; Granick, S . J. Biochem. 1956, 219, 435-446. (15) Dalgliesh, C. E. Biochem. J. 1952, 5 2 , 3-14. (16) Melsch, H.-U.; Reinle, W.; Wolf, U. Anal. Blochem. 1985, 149, 29-34. (17) Meisch, H.-U.; Bielig, H.J. Arch. Microbiol. 1975, 105, 77-82. (18) Lindroth, P.; Mopper, K. Anal. Chem. 1979, 5 1 , 1667-1674. (19) Roth, M.; Hampal, A. J. Chromatogr. 1973, 8 3 , 353-356. (20) Bohlen, P.; Schroeder, R. Anal. Biocbem. 1982, 126, 144-152. (21) Chen, R. F.; Scott, C.; Trepman, E. Biochlm. Biophys. Acta 1979, 576, 440-455. (22) Simons, S. S., Jr.; Johnson, D. F. J. Org. Chem. 1978, 43, 2886-2891. (23) Palczewski, K.; Hargrave, P. A.; Kochman, M. Eur. J. Biochem. 1983, 127, 429-435.
RECEIVED for review November 8, 1985. Accepted January 13, 1986.
High-Performance Liquid Chromatographic Determination of Electrically Neutral Carbohydrates with Conductivity Detection Tetsuo Okada* and Tooru Kuwamoto Department of Chemistry, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606, Japan Hlgh-performance liquid chromatographic determination of electrically neutral carbohydrates was carrled out with conductometrlc detectlon. Although these compounds are not detected directly by a conductlvlty detector because of thelr low dissociation, most of thelr borate complexes are strong enough acids to be detected with a conductlvlty detector. Therefore, use of a boric acid solutlon as an eluent permltted these compounds to be determined wlth conductometric detection. Moreover, this method can be applied to simultaneous determination of carbohydrates with organic aclds. However, only a ilmlted number of organic acids can be measured together wlth carbohydrates because of the llmltatlon of using a low-acidity eluent. The detectlon limits for carbohydrates typically ranged In 1 X lo-' M levels, and this method can be used for determlning carbohydrates In food samples.
High-performance liquid chromatographic analysis of carbohydrates has been investigated by many workers from the viewpoints of both the separation and detection (1-16). Carbohydrates can be separated with silica gel (1-4), an amino-bonded silica gel (5-9),a cation-exchange resin (7,10-13), and an anion-exchangeresin (14,15). Every stationary phase has particular advantages. For example, silica-basedstationary phases are superior in the separation of the structural analogues of carbohydrates, and a cation-exchange resin permits the use water as a mobile phase for separating many watersoluble carbohydrates. There remain, thus, few problems concerning the separation. On the contrary, there are many problems in the detection as follows: refractive index detection (2, 7-11, 16), which is used generally for detection of carbohydrates, is less sensitive; most of the postcolumn reaction (I,12,14,15)and precolumn
0003-2700/88/0358-1375$01.50/00 1986 American Chemical Society
