MS for Determination of the

Anal. Chem. 1997, 69, 5146-5151

A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide: Combination of Marfey’s Method with Mass Spectrometry and Its Practical Application Kiyonaga Fujii,† Yoshitomo Ikai,‡ Hisao Oka,‡ Makoto Suzuki,† and Ken-ichi Harada*,†

Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan, and Aichi Prefectural Institute of Public Health, Tsuji-machi, Kita, Nagoya 462, Japan

The “advanced Marfey’s method” proposed in our preceding paper has been developed to nonempirically determine the absolute configuration of constituent amino acids in a peptide using liquid chromatography/mass spectrometry (LC/MS). For the establishment of this method, we had to resolve the following three problems: (1) elucidation of the limitation of Marfey’s method, which is chosen as the chromatography technique, and its separation mechanism, because this proposed method relies on the elution order of an amino acid derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA) to determine its absolute configuration; (2) how to effectively combine Marfey’s method with mass spectrometry in order to detect and identify a desired amino acid without a standard sample; and (3) how to obtain the corresponding enantiomer from either the L- or D-amino acid in a peptide sample. In a preceding paper, we investigated problem 1 and finally described the rational application guideline for Marfey’s method to elucidate the elution order of a desired amino acid according to the proposed separation mechanism. In this paper, we further investigated the two remaining problems. Because the sensitivity of the amino acids derivatized with the original derivatizing reagent, L-FDAA, was poor for LC/ MS analysis using any interfaces due to their possible thermal instability and low hydrophobicity, we chose electrospray ionization and frit-fast atom bombardment (Frit-FAB) as the interface and developed 1-fluoro-2,4dinitrophenyl-5-L-leucinamide (L-FDLA) instead of LFDAA as a new derivatizing reagent in order to combine Marfey’s method with mass spectrometry. Furthermore, we introduced a racemization procedure using 1-fluoro2,4-dinitrophenyl-5-DL-leucinamide (DL-FDLA), the “DLFDLA derivatization”, instead of the conventional chemical racemization for obtaining the corresponding enantiomer from either the L- or D-amino acid. Thus, we have established a nonempirical method using LC/MS, the “advanced Marfey’s method”. The method was suc-

cessfully applied to the characterization of constituent amino acids in microcystin LR produced by cyanobacteria.

5146 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

S0003-2700(97)00289-8 CCC: $14.00

The purpose of this study is to establish a nonempirical method using liquid chromatography/mass spectrometry (LC/MS) for determination of the absolute configuration of constituent amino acids in peptides, which can determine the absolute configuration without a standard sample and can simultaneously analyze any amino acids in peptides using a simple technique. Our proposed method consists of a chromatography technique for the separation of the amino acids into each enantiomer, the detection of an amino acid by mass spectrometry, and a procedure for obtaining the corresponding enantiomer from either the L- or D-amino acid. This method has been designated as the “advanced Marfey’s method”,1 because we chose Marfey’s method2 as the chromatography technique, which is based on the principle that the D- and L-amino acids can be separated into each enantiomer by HPLC after the derivatization with the chiral reagent, 1-fluoro-2,4-dinitrophenyl5-L-alaninamide (L-FDAA). In our proposed method, the peaks of any amino acids in peptides are identified without a standard sample using mass spectrometry, and the absolute configuration of a desired amino acid is deduced from its retention times of the L-FDAA derivative of the original amino acid and its corresponding enantiomer. Because this proposed method relies on the elution order of an amino acid derivatived with L-FDAA to determine its absolute configuration, it is very important to clarify why an enantiomeric mixture is separated into each enantiomer after the derivatization. In a preceding report,1 Marfey’s method was applied to commercially available amino acids to confirm its applicability, and the separation behavior was examined in detail as the first step. In the subsequent step, its separation mechanism was elucidated based on the experimental results. Finally, we described the limitations of the method and the rationale of the proposed separation mechanism.1 Thus, the elution order of a * Corresponding author: (tel) 81-52-832-1781, ext. 334; (Fax) 81-52-834-8780; (e-mail) [email protected]. † Meijo University. ‡ Aichi Prefectural Institute of Public Health. (1) Fujii, K.; Ikai, Y.; Mayumi. T.; Oka, H.; Suzuki, M.; Harada, K.-I. Anal. Chem. 1997, 69, 3346-3352. (2) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596. © 1997 American Chemical Society

desired amino acid can be elucidated from the average retention time of L- and D-amino acid derivatives according to the proposed separation mechanism for Marfey’s method. For the establishment of the advanced Marfey’s method, we have to further resolve two remaining problems; how to combine Marfey’s method with mass spectrometry and how to obtain the corresponding enantiomer from either the L- or D-amino acid. In the present study, several chiral reagents were synthesized for the combination of electrospray ionization (ESI) or frit-fast atom bombardment (Frit-FAB) mass spectrometry with Marfey’s method, and a practical procedure was proposed for obtaining the corresponding enantiomer from either the L- or D-amino acid in a peptide sample. Finally, this method was applied to the characterization of constituent amino acids in a naturally occurring peptide. EXPERIMENTAL SECTION Chemicals and Materials. Derivatizing reagents, 1-fluoro2,4-dinitrophenyl-5-L-alaninamide (L-FDAA) and its analogs, 1-fluoro2,4-dinitrophenyl-5-L-valinamide (L-FDVA), -5-L-phenylalaninamide (L-FDPA), -5-L-isoleucinamide (L-FDIA), -5-L-leucinamide (L-FDLA), and -5-D-leucinamide (D-FDLA, mp 171-173 °C; FABMS m/z 315 [M + H]+), were synthesized from each amino acid amide hydrochloride (Kokusan Chemicals, Tokyo, Japan) and 1,5difluoro-2,4-dinitrobenzene (Nacalai Tesque, Kyoto, Japan) according to the procedure of Marfey.1,2 Amino acids were purchased from Sigma (St. Louis, MO), Wako (Osaka, Japan), Tokyo Kasei Kogyo (Tokyo, Japan), or Nacalai Tesque. Derivatized amino acids with each derivatizing reagent were prepared in the same manner as L-FDAA.1,2 Bacitracin A was isolated and purified from the bacitracin complex which was purchased from P-L Biochemicals (Milwaukee, WI), and microcystin LR was obtained from surface bloom samples in Lake Suwa, Japan, as previously described.3,4 Reagent-grade acetonitrile from Nacalai Tesque was distilled and filtered for LC, and water was purified using a purification system (Barnstead, IA). All other chemicals and solvents were of analytical grade. Sample Preparation. A 100 µg sample of bacitracin A was hydrolyzed at 110 °C for 24 h with 200 µL of 6 N HCl. This solution was evaporated to dryness, and the residue was dissolved in 50 µL of water. To this amino acid solution was added 20 µL of 1 M sodium bicarbonate and then 100 µL of 1% L-FDLA in acetone. The solution was vortexed and incubated at 37 °C for 60 min. After the reaction was quenched by the addition of 20 µL of 1 N HCl, the reaction mixture was diluted with 810 µL of methanol and 6 µL of this solution was analyzed by ESI LC/MS. A 400 µg sample of microcystin LR was hydrolyzed at 110 °C for 16 h with 200 µL of 6 N HCl. This solution was divided into two portions, and each portion was derivatized with L- or D-FDLA. After dilution with 810 µL of acetonitrile, 4 µL of L-FDLA derivative and an equal mixture of the L- and D-FDLA derivatives were analyzed by ESI LC/MS. HPLC Conditions. HPLC was performed using a Tosoh (Tokyo, Japan) dual-pump delivery system. Separations were carried out on a TSK gel ODS-80Ts (150 × 4.6 mm i.d., Tosoh) column maintained at 40 °C. Acetonitrile-0.01 M trifluoroacetic acid (TFA) was used as the mobile phase under a linear gradient (3) Ikai, Y.; Oka, H.; Hyakawa, J.; Matsumoto, M; Saito, M.; Harada, K.-I.; Mayumi, T.; Suzuki, M. J. Antibiotics 1995, 48, 233-242. (4) Watanabe, M. F.; Harada, K.-I.; Matsuura, K.; Oishi, S.; Watanabe, Y.; Suzuki, M. Toxic. Assess. 1989, 4, 487-497.

elution mode (acetonitrile, 25-65%, 45 min). The flow rate was 1 mL/min with UV detection at 340 and 250-500 nm by photodiode array detection. Frit-FAB Conditions. The separation of the DL-amino acid derivatized with L-FDAA and L-FDLA was performed on a TSK gel ODS-80Ts (150 × 4.6 mm i.d., Tosoh) column maintained at 40 °C using a LC-100P (Yokogawa Electric, Tokyo, Japan). Acetonitrile-water containing 0.01 M TFA and 1% m-nitrobenzyl alcohol was used as the mobile phase under the linear gradient elution mode (acetonitrile, 30-50%, 25 min) at a flow rate of 0.5 mL/min. The mass spectrometer and the data system used were a JMS-AX505W (Jeol, Tokyo, Japan) and a JMS-DA5000 (Jeol), respectively. The temperature of the ion source was maintained at 60 °C, and a neutral xenon beam was used as the primary beam for the ionization of the sample by FAB. The LC/MS data were obtained by scanning from m/z 100 to 1000 at a cycle time of 5.5 s. The HPLC and the mass spectrometer were interfaced by a laboratory-made flow splitter and Frit-FAB probe (Jeol). The effluent from the HPLC was split at a ratio of 1:125, and the smaller portion of the effluent was introduced into the mass spectrometer at a flow rate of 4 µL/min. ESI LC/MS Conditions. The separation of the L- and DLFDLA derivatives of hydrolyzed bacitracin A and microcystin LR was performed on a Develosil ODS-HG-5 (150 × 2.0 mm i.d., Nomura Chemical, Seto, Japan) column maintained at 40 °C using a HP1050 (Hewlett-Packard, Novi, MI). Acetonitrile-water containing 0.01 M TFA was used as the mobile phase under a linear gradient elution mode (acetonitrile, 30-65%, 40 min for bacitracin A, 30-80%, 50 min for microcystin LR) at a flow rate of 0.2 mL/ min. The mass spectrometer used was a Finnigan TSQ700 and 7000 (Finnigan-Mat, San Jose, CA). All mass spectra were acquired using Q1 as the scanning quadrupole. In the case of bacitracin A, the ESI voltage was 4.5 kV with the auxiliary and sheath gas nitrogen pressure set at 5 units and 70 psi, respectively, and the capillary was heated to 200 °C. A mass range of m/z 250-800 was covered with a scan time of 1 s, and data were collected in the negative ion mode using an electron multiplier voltage of 1400 V. In the case of microcystin LR, the ESI voltage was 4.5 kV with the auxiliary and sheath gas nitrogen pressure set at 5 units and 60 psi, respectively, and the capillary was heated to 220 °C. A mass range of m/z 300-1100 was covered with a scan time of 1.5 s, and data were collected in the negative ion mode using an electron multiplier voltage of 1200 V. RESULTS AND DISCUSSION Combination of Marfey’s Method and Mass Spectrometry. In Marfey’s method, the derivatized amino acids with L-FDAA are separated by the usual reversed-phase HPLC and are detected at UV 340 nm.2 In our proposed method,1 the UV detection has to be replaced by a mass spectrometer. In other words, the amino acid derivatives are analyzed by LC/MS. In order to smoothly change from the conventional HPLC conditions to appropriate LC conditions for the LC/MS analysis, we have to overcome two problems: injection volume of the HPLC eluent into the mass spectrometer should be considerably reduced, because the ESI and Frit-FAB were finally used as an interface, and nonvolatile buffers in the mobile phase should be replaced by volatile buffers such as ammonium acetate or TFA. In this study, the first problem was solved by the use of a semimicrocolumn (150 × 2.1 mm i.d.), because the method using this column gave enough separation between the L- and D-amino acid derivatives. In the Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

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Table 1. Comparison of Synthesized Derivatizing Reagents RTa (min) L-FDAA L-FDVA L-FDPA L-FDIA L-FDLA D-FDLA

17.4 28.5 37.3 35.5 36.4 36.4

[M + H]+ (m/z) 273 301 349 315 315 315

Table 2. Analysis of L-FDLA Derivatives of Amino Acids by HPLC

[M + H]+/m/z 307b 0.8 11.2 1.5 7.0 32.1 32.1

a The retention times (RT) of each derivatizing reagent were analyzed under similar conditions (see ref 1). b The ratios of the ion abundance of [M + H]+ of each derivatizing reagent and that of [2M + H]+ (m/z 307) of m-nitrobenzyl alcohol as the matrix under the FAB conditions.

case of Frit-FAB LC/MS, however, the solvent flow from the HPLC should be further reduced to 5 µL/min using an appropriate splitter. Another problem was also solved by using volatile TFA or ammonium acetate buffer (pH 3) as the mobile phase, which showed almost the same separation behavior as that using the conventional phosphate buffer solvent system. Goodlett et al. and Krishnamurthy have reported Marfey’s method using LC/MS with ESI and thermospray (TSP) as the interface, respectively.5,6 We also carried out fundamental approaches for the LC/MS analysis of amino acid derivatized with L-FDAA as the derivatizing reagent using four types of interfaces, ESI, atmospheric pressure chemical ionization (APCI), TSP, and Frit-FAB. Polar amino acid derivatives could not be detected using TSP and APCI interfaces. Although any amino acid derivatives tested could be detected using ESI and Frit-FAB interfaces, their sensitivities were extremely poor. These results may be due to the thermal instability and low hydrophobicity of the L-FDAA derivatives of the amino acids and indicated strongly the need to prepare a new derivatizing reagent with higher hydrophobicity. Additionally, ESI and Frit-FAB were selected as the interface, because thermally induced effects can be limited in these interfaces. It is well-known that a compound with more hydrophobicity property gives generally higher ionization efficiency in FABMS.7 We considered that the low hydrophobicity of L-FDAA was responsible for the previously mentioned results. In order to obtain a higher hydrophobicity of the amino acid derivative without the loss of the UV spectral characteristic of the original L-FDAA derivatives and with the common proposed separation mechanism, four derivatizing reagents, L-FDVA, L-FDPA, L-FDIA and L-FDLA, were tried. Table 1 shows the retention times, the protonated molecules ([M + H]+), and the ratios of the ion abundance of [M + H]+ for each derivatizing reagent and that of [2M + H]+ (m/z 307) of m-nitrobenzyl alcohol as the matrix under the FAB conditions. These results showed that the retention times of any of the derivatizing reagents tested become longer than that of L-FDAA and the ratios also become larger compared to L-FDAA. In particular, the ion abundance of [M + H]+ (m/z 315) of L-FDLA was the strongest at ∼30 times that of L-FDAA, suggesting that L-FDLA is the most suitable for the derivatization (5) Goodlett, D. R.; Abuaf, P. A.; Savage, P. A.; Kowalski, K. A.; Mukherjii, T. K.; Tolan, J. W.; Corkum, N.; Goldstein, G.; Crowther, J. B. J. Chromatogr., A 1995, 707, 233-244. (6) Krishnamurthy, T. J. Am. Soc. Mass. Spectrom. 1994, 5, 724-730. (7) Naylor, S.; Findeis, A. F.; Gibson, B. W.; Williams, D. H. J. Am. Chem. Soc. 1986, 108, 6359-6363.

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amino acid alanine valine leucine isoleucine allo-isoleucine methionine phenylalanine tyrosine (mono-R) tyrosine (di) proline serine homoserine threonine allo-threonine glutamine asparagine glutamic acid aspartic acid β-hydroxyaspartic acide β-hydroxyaspartic acidf lysine (mono-R) lysine (di) ornitine (mono-R) ornitine (di) histidine (mono-R) histidine (di) arginine citruline

elution order L

fD fD LfD LfD LfD LfD LfD LfD LfD LfD LfD LfD LfD LfD LfD d LfD LfD d DfL DfL LfD DfL DfL DfL LfD DfL DfL L

tRLa (min)

tRDb (min)

∆tc (min)

19.1 22.9 26.5 26.1 26.1 22.6 27.0 20.7 39.7 19.1 14.9 14.9 14.5 15.4 14.7 13.3 16.8 15.3 11.1 14.0 12.6 38.1 11.8 36.1 11.2 25.8 13.3 15.2

23.5 30.8 35.2 34.8 34.8 29.1 33.3 23.2 43.5 22.2 15.7 16.4 19.8 17.4 14.9 13.3 18.1 16.3 11.1 13.2 10.4 40.4 9.0 34.8 8.5 27.4 11.1 13.8

4.4 7.9 8.7 8.7 8.7 6.5 6.3 2.5 3.8 3.1 0.8 1.5 5.3 2.0 0.2 0.0 1.3 1.0 0.0 -0.8 -2.2 2.3 -2.8 -1.3 -2.7 1.6 -2.2 -1.4

a The retention times of the L-amino acid derivatives. The retention times of D-amino acid derivatized with D-FDLA were identical within (0.01 min. b The retention times of the D-amino acid derivatives. The retention times of L-amino acids derivatized with D-FDLA were identical within (0.01 min. c ∆t ) tRD - tRL. d The diastereomers were not resolved. e β-threo-Hydroxyaspartic acid. f β-erythro-Hydroxyaspartic acid.

of amino acids in our procedure. Therefore, we selected L-FDLA as the new derivatizing reagent instead of L-FDAA, and its applicability was evaluated by HPLC analysis. As shown in Table 2, L-FDLA gave the same or better separation behavior than L-FDAA and was expected to be an efficient derivatizing reagent for the LC/MS analysis.1 In order to confirm the applicability of the L-FDLA, the derivatized amino acids with L-FDLA were analyzed using LC/ MS with Frit-FAB or ESI as the interface. For comparison of the ionization between the L-FDAA derivatives and L-FDLA derivatives of the amino acids under similar conditions, the mixtures of equal amounts of both derivatives were applied to the LC/MS analysis. Figure 1 shows the positive ion mass chromatograms of DL-alanine derivatized with L-FDAA and L-FDLA monitored at their [M + H]+ and their whole mass spectra using Frit-FAB as the interface. The sensitivity of the L-FDLA derivatives was increased ∼3 times higher than that of the L-FDAA derivatives in the mass chromatograms. This tendency was common to any usual amino acids. In the case of ESI LC/MS, the sensitivity of the L-FDLA derivatives was also increased from 2 to 4 times higher than that of the L-FDAA derivatives in both the positive and negative modes. Throughout these experiments, the applicability of Marfey’s method using LC/MS was improved using L-FDLA instead of L-FDAA as the derivatizing reagent, and it was confirmed that L-FDLA was useful not only for HPLC analysis but also for LC/ MS analysis. Therefore, it is concluded that the present combination method comprised of Marfey’s method and mass spectrometry can be reliably applied to our proposed method, because

Figure 1. Mass chromatograms of the L-FDAA derivatives ([M + H]+, m/z 342) and the L-FDLA ([M + H]+, m/z 384) of alanine using Frit-FAB LC/MS.

detected peaks can be identified not only by their retention times but also by the mass spectral information. The detection limits were estimated as about 10 and 5 pmol using Frit-FAB and ESIMS, respectively. In order to confirm the applicability of this method, it was applied to bacitracin A, an antibiotic peptide containing 12 amino acid residues with L- and D-amino acids. The resulting hydrolysate8 (6 N HCl, 110 °C, 24 h) of bacitracin A was analyzed by LC/MS using ESI as the interface after derivatization with L-FDLA. Figure 2 shows the reconstructed ion chromatogram (RIC, m/z 368-800) and the mass chromatograms monitored at m/z values for the deprotonated molecules ([M - H]-) of the L-FDLA derivatives of each constituent amino acid from bacitracin A together with the HPLC chromatogram at UV 340 nm. While it is laborious to simultaneously assign all peaks by the usual UV detection, the present method can readily and specifically find these peaks by mass chromatography. DL-FDLA Derivatization. Usually, naturally occurring peptides are composed of amino acids with either a D or L configuration and each constituent amino acid gives only one peak on the chromatogram, whose absolute configuration cannot be determined using any other method. Therefore, a racemization procedure of amino acids is essential to obtain both enantiomers from either the L- or D-amino acid in a peptide for our method, and we initially used the following procedure: a hydrolysate is heated with acetic anhydride and triethylamine to racemize each amino acid through an oxazolone intermediate, and the resulting racemized N-acetylamino acids are subsequently hydrolyzed again. Although this procedure is simple and applicable to small amounts of amino acids from a peptide, it is time consuming and it is difficult to complete the racemization. In addition, the procedure has an additional drawback that it does not give an enantiomeric mixture, but a diastereometric mixture in the case of amino acids with two asymmetric carbons such as threonine and isoleucine. (8) The N-terminal dihydrothiazol moiety in bacitracin A consists of cysteine and isoleucine. On hydrolysis, cysteine decomposes and isoleuine is epimerized to give a mixture of D-allo- and L-isoleucine: Koningsberg, W.; Hill, R. J.; Craig, L. C. J. Org. Chem. 1961, 26, 3867-3870.

Figure 2. HPLC chromatogram and reconstructed ion and mass chromatograms (under negative ion mode) of L-FDLA derivatives of the hydrolyzed bacitracin A.

We considered that the above chemical racemization process is not necessary if 1-fluoro-2,4-dinitrophenyl-5-D-leucinamide (DFDLA) is available as an additional derivatization reagent. As shown in Figure 3, the relationship between the L-FDLA derivatives of the D-amino acids (D-L type) and those of the L-amino acids (L-L type) is diastereomeric, usually giving different retention times on a chromatogram. On the other hand, the relationships between the L-D and D-L types and between the D-D and L-L types are enantiomeric and each pair will show the same retention times. Namely, the D-FDLA derivative of a desired amino acid and the L-FDLA derivative of its enantiomer of the desired amino acid have the same retention time. Accordingly, it is expected that an equal mixture of D- and L-FDLA can be used as the derivatizing reagent instead of only L-FDLA to obtain the same effect as the previously mentioned racemization. In order to confirm this consideration, D-FDLA was prepared in the same manner as L-FDLA and commercially available amino acids were derivatized with D- and L-FDLA. The derivatives were separated Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

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Figure 3.

DL-FDLA

system.

by HPLC, and the retention times of each amino acid derivative were compared. These experiments clearly show that two enantiometric pairs, the D-D and L-L type and D-L and L-D type derivatives, have the same retention time. These results indicate that a derivatization with DL-FDLA is applicable to the racemization step in the advanced Marfey’s method in place of the chemical procedure. This procedure is designated as the “DL-FDLA derivatization”. When this method was applied to amino acids with two asymmetric centers, the resulting two peaks correspond to the L-FDLA derivatives of the original amino acid and its enantiometric isomer. Additionally, a combination of a chemical racemization with the DL-FDLA derivatization can form four probable isomers on the HPLC chromatogram in the case of amino acids with two asymmetric centers. Thus, we have established the advanced Marfey’s method including LC/MS and DL-FDLA derivatization. Application of the Advanced Marfey’s Method. We have studied the structural determination of naturally occurring peptides and isolated various peptides from cyanobacteria and applied the advanced Marfey’s method with DL-FDLA derivatization to the characterization of the constituent amino acids in microcystin LR produced by cyanobacteria. Microcystin LR is a toxic cyclic peptide and is composed of seven amino acid residues, of which β-methylaspartic acid (β-MeAsp) has two asymmetric centers in its molecule9-11 (Figure 4). The hydrolysate12 of microcystin LR was divided into two portions, and each portion was derivatized with L- or D-FDLA. The L-FDLA derivatives alone and the equal mixture of the L- and D-FDLA derivatives were analyzed using ESI LC/MS in the negative ion mode after UV spectra of their peaks were checked by HPLC with photodiode array detection. Figure 5a shows the mass chromatograms monitored at m/z values of the [M - H]- for L-FDLA derivatives of glutamic acid, β-MeAsp, (9) Botes, D. P.; Vilien, C. C.; Kruger, H.; Wessels, P. L.; Williams, D. H. Toxicon 1982, 20, 1037-1042. (10) Botes, D. P.; Wessels, P. L.; Kruger, H.; Runneger, M. T. C.; Santikarn, S.; Barna, J. C. J.; Williams, D. H. J. Chem. Soc., Perkin Trans. 1 1985, 27472748. (11) Carmichael, W. W.; Beasly, V. R.; Bunner, D. L.; Eloff, J. N.; Falconer, I. R.; Gorham, P.; Harada, K.-I.; Krishnamurthy, T.; Yu, M.-J.; Moore, R. E.; Rinehart, K. L.; Runneger, M. T. C.; Skulberg, O. M.; Watanabe, M. F. Toxicon 1988, 26, 971-973. (12) N-Methylamine was derived from Mdha moiety on hydrolysis and could be detected at m/z 438 of the [M + TFA - H]- as shown in Figure 5a,b: Sivonen, K.; Carmichael, W. W.; Namikoshi, M.; Rinehart, K. L.; Dahlem, A. M.; Niemela¨, S. L. Appl. Environ. Microbiol. 1990, 56, 2650-2657.

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Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

Figure 4. Structure of microcystin LR from cyanobacteria.

alanine, and leucine and the mass chromatogram monitored at m/z values of the [M + TFA - H]- for L-FDLA derivatives of arginine. The desired peaks of the five amino acids can be definitely detected in the chromatogram monitored at the respective m/z values. This method has an additional advantage that it is possible to find any reaction products other than normal amino acids. In this case, we could sensitively detect two methanolloss products derivatized with L-FDLA from 3-amino-9-methoxy10-phenyl-2,6,8-trimethyl-deca-4,6-dienoic acid (Adda) at m/z 706 of the [M + TFA - H]-.13 In the mass chromatogram of the resulting derivatized hydrolysate with DL-FDLA, the corresponding enantiomers newly appear (Figure 5b) and we can conclude that arginine and leucine have L-configurations and that glutamic acid, alanine, and β-MeAsp have D-configurations in microcystin LR on the basis of their elution orders. Because the relative configuration of β-MeAsp from microcystin LR had already been elucidated to be erythro, it has the D-erythro configuration.10 In addition, comparison of the retention times of peaks on the mass chromatograms monitored at m/z 706 showed that the L-FDLA derivatives are eluted before the D-FDLA derivatives, estimating that the asymmetric β-carbon at C-3 in Adda possesses the S-configuration according to our separation mechanism.1 These results are also consistent with those obtained from chemical derivatization.14 (13) Namikoshi, M.; Rinehart, K. L.; Dahlem, A. M.; Beasly, V. R.; Carmichael, W. W. Tetrahedron Lett. 1989, 30, 4349-4352. (14) Rinehart, K. L.; Harada, K.-I.; Namikoshi, M.; Chen, C.; Harvis, C. A.; Munroe, M. H. G.; Blunt, J. W.; Mulligan, P. E.; Beasly, V. R.; Dahlem, A. M.; Carmichael, W. W. J. Am. Chem. Soc. 1988, 110, 8557-8558.

Figure 5. Mass chromatograms of the L-FDLA derivatives (a) and the LC/MS under negative ion mode.

In addition, our proposed method using the conventional racemization was successfully applied to the characterization of the constituent amino acids in anabaenopeptins,15 which contain homotyrosine and N-methylalanine as unusual amino acids.16 Furthermore, we also applied this method with DL-FDLA derivatization to the determination of the absolute configuration of 3-amino-6-hydroxy-2-piperidone, one of the constituent amino acids in aeruginopeptin 228-A.17,18 CONCLUSIONS For the establishment of the advanced Marfey’s method, we had to resolve two remaining problems. One was how to effectively combine Marfey’s method with mass spectrometry. Because the sensitivity of the amino acids derivatized with L-FDAA was poor for LC/MS analysis using any interfaces due to their possible thermal instability and low hydrophobicity, we chose ESI and Frit-FAB as the interface and developed L-FDLA instead of L-FDAA as the derivatizing reagent. Consequently, the applicability of Marfey’s method including LC/MS was improved by the (15) Harada, K.-I.; Fujii, K.; Shimada, T.; Suzuki, M.; Sano, H.; Adachi, K.; Carmichael, W. W. Tetrahedron Lett. 1995, 36, 1511-1514. (16) Harada, K.-I.; Fujii, K.; Mayumi, T.; Hibino, Y.; Suzuki, M.; Ikai, Y.; Oka, H. Tetrahedron Lett. 1995, 36, 1515-1518. (17) Harada, K.-I.; Mayumi, T.; Shimada, T.; Suzuki, M.; Kondo, F.; Watanabe, M. F. Tetrahedron Lett. 1993, 34, 6091-6094. (18) Harada, K.-I.; Fujii, K.; Hayashi, K.; Suzuki, M.; Ikai, Y.; Oka, H. Tetrahedron Lett. 1995, 36, 1515-1518.

DL-FDLA

derivatives (b) of the hydrolyzed microcystin LR using ESI

use of L-FDLA. Another problem was how to obtain the corresponding enantiomer from either the L- or D-amino acid. We introduced the “DL-FDLA derivatization” for this step instead of the conventional chemical racemization. The derivatization with an equal mixture of D- and L-FDLA can surely give the desired enantiomer from its L or D-amino acid on the HPLC chromatogram. Thus, we have established a nonempirical method using LC/MS, the “advanced Marfey’s method”, proposed in our preceding paper,1 which includes HPLC with a rational guideline, a sensitive derivatizing reagent, L-FDLA, and a racemization procedure using DL-FDLA. The wide utility of this method has been shown in several peptides and is now being applied to other naturally occurring peptides. ACKNOWLEDGMENT The authors thank Drs. Hideo Takashina, Kenji Matsuura, and Tatsuo Ikei of Santen Pharmaceutical Co. for providing the ESI LC/MS spectra.

Received for review March 17, 1997. Accepted September 30, 1997.X AC970289B X

Abstract published in Advance ACS Abstracts, November 15, 1997.

Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

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