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Anal. Chem. 1985, 57, 2407-2409
mation of Q,. We employed here the K value determined from the positive peak rather than the negative one, because the latter gave somewhat scattered resulb owing to the uncertainty in measuring the broader peak area. The results are given in Table I and are in good agreement with those determined by the XAD column technique. Implicit in eq 1and 2 is the assumption that 1:l association occurs between the protein and the complex. This does not necessarily mean that only one guest molecule associates with a host molecule at a single site. It is desirable to appreciate the number of site on the protein available to accept the complex molecule; however, this was not demonstrated in the present work. Under the present experimental conditions, the ratio of Q,to Qp is as small as and hence a permissible assumption may be that the most active site@)on the protein would be concerned with association. If there are more than two active sites, whose affinity to the protein is approximately of the same order of magnitude, the association constant for each site can be estimated by dividing the observed K value by the number of sites. The K value obtained in this work is thus an overall association constant. Kinetic studies on electron-transfer reaction between plastocyanin and some metal complexes have assumed formation of association prior to electron transfer (IO). This was supported by NMR studies using redox inactive chromium complexes (11): a positively charged complex such as Crhen)^^+ associates preferentially at (or close to) Tyr 83 on plastocyanin. The overall charge on the protein is negative, PI=4.2 (121, and hence it is reasonable that acidic residues accept positively charged complexes. Compared to the association constants reported in the literature ( I O , I3), 167 M-l for Co(pheds3+and 176 M-l for C r ( ~ h e n )at ~ ~pH + 7.5 in 0.1 M NaCl solutions, the present result, 1.8 x lo3 M-l, for the divalent complex F e ( ~ h e n ) is ~ ~of+surprisingly large magnitude. It should be noted, however, that the constants derived from the kinetic studies only appreciate association to what extent the complexes contribute in the redox reaction with plastocyanin and that the kinetic studies have been made in 0.1 M NaCl solutions. The ionic strength which applies to the present studies is much lower, 0.01 M phosphate buffer solutions. The XAD column technique demonstrated here has three main advantages over the conventional gel chromatographic technique. Firstly, there is no interference due to coexisting hosts which can also associate with the same guest so long as the host of interest is followed by a suitable detector only sensitive to it. Secondly, Donnan exclusion which often causes serious problems in the gel filtration technique does not need
to be considered. Thirdly, if the guest molecules are highly hydrophobic, they can be effectively adsorbed by XAD resin beads in a small column. This allows a single run in quite a short time and also makes it possible to recover precious host sample free from contamination of the guest without any dilution. The most remarkable feature of the use of XAD resin for studying association between hydrophilic host and hydrophobic (or metal complexes with hydrophobic ligands) guest molecules is as follows: guest molecules being fixed on the resin surface, only one host molecule, even if it had several active sites on a single molecule, could associate with one fixed guest at a time. However, our comment regarding the possibility of such an association mechanism is largely speculative at the present stage. It is desired to get more data on the XAD column using various host-guest combinations and to compare these results with those on the gel column.
Registry No. Tris(1,lO-phenanthroline)iron(II),14708-99-7. LITERATURE CITED Hummel, J. P.; Dreyer, W. J. Biochim. Biophys. Acta 1982, 6 3 , 532. Sanemasa, I.; Mlzoguchi, T.; Deguchi, T. Bull. Chem. SOC. Jpn. 1984, 57, 1358. Korpela, T. K.; Himanen, J.-P. J . Chromatogr. 1984, 290, 351. Pletrzyk, D. J.; Chu C. H. Anal. Chem. 1977, 49, 860. Rotsch, T. D.; Pietrzyk, D. J. Anal. Chem. 1980, 52, 1323. Cantwell F. F.; Puon S. Anal. Chem. 1979, 51, 623. Plesnicar, M.;Bendall, D.S. Biochim. Biophys. Acta 1970, 216, 192. Madeja, K.; Koning, E. J. J . Inorg. Nucl. Chem. 1983, 25,377. Yoza, N. J . Chem. Educ. 1977, 54, 284. Segal, M. G.; Sykes, A. G. J . Chem. SOC.,Chem. Commun. 1977, 764. Cookson, D. J.; Hayes, M. T.; Wright, P. E. Biochim. Biophys. Acta 1980, 591, 162. Ramshaw, J. A. M.; Brown, R. H.; Scawen, M. D.; Boulder, D. Blochlm. Biophys . Acta 1973, 303, 269. Chapman, S. K.; Watson, A. D.; Sykes, A. G. J . Chem. SOC.,Dalton Trans. 1903, 2543.
Isao Sanemasa* Kei Toda Toshio Deguchi Department of Chemistry Faculty of Science Kumamoto University Kurokami 2-39-1, Kumamoto 860, Japan
A. Geoffrey Sykes Department of Inorganic Chemistry The University Newcastle upon Tyne NE1 7RU, England
RECEIVED for review April 4,1985. Accepted June 13,1985.
N-Ethylmaleimide as a New Electrophoric Derivatizing Reagent for the Gas Chromatography of Thiols Sir: The GC electron capture detector (ECD) is a specific detector with subpicogram sensitivity (2-4). Much of the sensitivity advantage is lost, however, because quite often the usual halogenated derivatizing agents also react with coextracted interfering components having a common reactive functional group. For example, the component isolated as a non-electron-capturing carboxylic acid is normally reacted to form an electrophoric ester (4-7). This may result in an
excessive chemical noise due to the background and a net loss in sensitivity and selectivity. Optimum stability, selectivity, and sensitivity for the blood level assay of captopril (compound I, Figure 1,Table I), a thiol angiotensin-converting inhibitor, has been achieved by converting I to the N-ethylsuccinimide derivative (11) by adding N-ethylmaleimide(NEM) to a blood sample immediately after collection. Compound I1 is then isolated and measured as its
0003-2700/85/0357-2407$01.50/00 1985 American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
n I
n
R~-s-cH~-FH-~-N-J-cooH
CH3 0
H5C2-N
5
(C6H8N02)
0
Flgure 1. Structures of captopril and related compounds (Table I).
Table I. Structures of Captopril and Related Compounds (Figure 1 ) O compound captopril (I) captopril-NES(11) 4-fluorocaptopril(111) 4-fluorocaptopril-NES (IV) S-benzoylcaptopril(V) 4-fluoro-S-benzoylcaptopril (VI)
S-acetylcaptopril(VII) SQ 26,333 (VIII) SQ 26,333-NES (IX) SQ 26,991 (X)
R1
RZ
H C&&Oz H C&8N02 C7H50 C7H50
H H F F H F H CBH5S C&S C6H5S
C2H30
H c~H8N02 C7H50
Key: C&I8NO2,N-ethylsuccinimide, NES; C7H50, benzoyl; C6H5S,phenylthio; C2H30,acetyl.
methyl ester by selected ion monitoring GC/MS (8-10). It was our objective to develop a GC-ECD method for the measurement of captopril-N-ethylsuccinimide(11). I t was a pleasant surprise to find, during a routine screening of electrophoric derivatives, that the methyl ester of I1 exhibited excellent electrophoric response, comparable to that of halogenated ester derivatives. As a thiol alkylating agent normally used to prevent the oxidation of the thiol group, NEM is ideally suited to forming N-ethylsuccinimides in whole blood (11-13). The reaction has been incorporated into a number of clinical assays for captopril including radiometric TLC (14), GC (15,16),GC/MS (8-10,17), and radioimmunoassay (18). Our finding that the N-ethylsuccinimide is an electrophoric group makes the thiol-NEM reaction even more useful. EXPERIMENTAL SECTION Reagents and Chemicals. Captopril (I) and analogues (Figure 1, Table I) were characterized pharmaceutical materials from E. R. Squibb & Sons (Princeton, NJ). A stock solution of captopril-N-ethylsuccinimide (11)was prepared by treating 50 mg of I with 250 mg of NEM in 10 mL of pH 7.0 buffer for 15 min, diluting to 100 mL with acetone, and filtering through a fine porosity sintered glass filter (8-10). Stock solutions of thiol containing analogues of I were similarly prepared by reacting 50 mg of each compound with 250 mg of NEM. Working diluted solutions were prepared as required by the appropriate dilution of the stock solutions with acetone. Methylation of I1 and related compounds was performed with methanolic hydrochloric acid as previously described (8). The hexafluoroisopropyl ester of I1 and related compounds was formed by heating with 100 pL of hexafluoroisopropyl alcohol (HFIP) and 10 pL of trifluoroacetic anhydride at 50 "C for 1 h and removing the reagents by evaporation under nitrogen. Pentafluorobenzyl ester of I1 and related compounds was formed by heating with 100 pL of acetonitrile, 0.2 pL of pentafluorobenzyl bromide (PFB), and 0.5 pL of triethylamine or diisopropylethylamine at 60 "C for 1 h, and removing the reagents by evaporation under nitrogen. The derivatized dried residues were reconstituted in toluene for chromatography. Gas Chromatography. A Hewlett-Packard 5840 gas chromatograph equipped with a constant current electron capture
Time
-
Figure 2. Chromatogram of methyl esters of compounds 11, I V , V, and V I at 1500, 750, 500, and 250 ng/mL, respectively (Table 11).
Table 11. Chromatographic Peaks of the Methyl Esters of Compounds 11, IV, V, and VI (Figure 2) peak 1 2
compound V
retention time 4.78 5.11
3
VI I1
4
If
5.87
5
IV IV
6.11
6
5.65
6.47
detector was used. The fused silica capillary column (10 m, 0.22 mm, i.d., 0.20 pm film thickness) was coated with bonded cyanopropylphenyl methylpolysiloxane phase (CP si1 19 CB, Chrompack). The carrier gas was helium with an inlet pressure of 137 kPa (20 psig) and the makeup gas for the electron capture detector was 5% methane in argon, at a flow rate of 30 mL/min. For methyl and HFIP esters of 11, the oven was operated isothermally at 180 "C for 1.0 min after injection, heated at a rate of 30 "C/min to 250 "C, and then held at the final temperature for 5 min. For the PFB ester of 11, the temperature was programmed from 230 "C to 290 "C, other conditions remaining the same. Injections were made by the splitless mode, with a split flow of 30 mL/min and a septum purge of 2.0 mL/min. The inlet purge was turned on 0.7 min after injection. The injector and detector temperatures were maintained at 280 "C and 310 "C, respectively.
RESULTS AND DISCUSSION A typical chromatogram for the methyl esters of 11, the 4-fluor0 analogue of I1 (IV), S-benzoylcaptopril (V), and 4fluoro-S-benzoylcaptopril(VI) is shown in Figure 2 (Table 11). Whereas V and VI gave a single peak, I1 and IV each gave two peaks. The reaction of the optically pure captopril diastereomer with NEM, in a nonstereospecific manner, leads to the formation of a diastereomeric pair. The resolving power of the capillary system employed was sufficient to resolve the two diastereomers, which was not achievable with packed column GC (8-10). The ECD response for the methyl ester of I1 and IV was excellent, and the sensitivity of IV, which has a fluorine atom, was better than that of I1 by only a factor of 2. On the other hand, the methyl ester of captopril (I) or of S-acetylcaptopril (VII) had no ECD response, which indicated that the Nethylsuccinimide (NES) group was responsible for the ECD
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Anal. Chem. 1985, 57,2409-2412
Table 111. GC-ECD Characteristics of the Methyl, Hexafluoroisopropyl (HFIP), and Pentafluorobenzyl (PFB) Esters of Captopril N-Ethylsuccinimide (11)and S-Benzoylcaptopril (V)
a
compound
no. of peaks
re1 responsea
methyl ester of I1 HFIP ester of I1 PFB ester of I1 methyl ester of V HFIP ester of V PFB ester of V
2 2 2 1 2 1
2 8 14 1 3 5
When the number of peaks is equal to two, the sum of the two used. The area count for 50 pg of injected methyl ester of V
was was
10000.
response. The methyl esters of the S-benzoylanalogues V and VI also gave excellent ECD response, with sensitivity equal to about half of that of I1 or IV. The 4-thiophenyl analogue of captopril (VIII) behaved like captopril in that the methyl ester of the NES derivative (IX) or S-benzoyl derivative (X) exhibited excellent ECD response whereas the methyl ester of the compound (VIII) had little ECD response. Table I11 summarizes the GC-ECD characteristics of the methyl esters of I1 and V vs. those of the hexafluoroisopropyl (HFIP) and pentafluorobenzyl (PFB) esters. There was no dramatic increase in the ECD response of I1 or V as a result of introducing the fluorinated esters. In addition, the chromatograms of the methyl esters were cleaner than those of the PFB or HFIP esters. A similar behavior was noticed with the methyl, HFIP and PFB esters of the 4-thiophenyl analogues, compounds IX and X. While disulfides and trisulfides are known to be electrophoric (19,201, reports of electrophoric sulfides are rare (21). As classified by Vessman (22), the methyl ester of I1 would fall into the category of conjugated electrophores, due to the NES group. We have verified that the thiobenzoyl derivative of captopril possesses good electrophoric response while the thiol (19,20) or thioacetyl (22,23) derivative has no response. While thioarylacyl derivatives could yield sufficient sensitivity to determine captopril in blood, this reaction is not exclusive to thiols and, hence, contributes to the background. Because the NES derivative is stable, it can be formed in body fluids and then isolated intact. The use of a selective reagent such as NEM, coupled with a specific extraction technique and high-resolution capillary column GC, greatly improves the chances of successfully using the electron capture detector for
the measurement of low levels of sulfhydryl compounds such as captopril in complex matrices such as blood, plasma, or urine. Registry NO.I, 62571-86-2; 111,72180-18-8;V, 75107-57-2; VII, 64838-55-7;VIII, 75176-37-3;X, 81872-10-8;NEM, 128-53-0.
LITERATURE CITED Peliizzarl, E. D. J. Chromatogr. 1974, 96, 323-361. Poole, C. F. HRC CC, J. High Resolut. Chromatogr. Chramatogr. Commun. 1982, 5 , 454-471. Conkill, J. A,; Joppich, M.; Kuttab, S. H.; Glese, R. W. Anal. Chem. 1982, 5 4 , 481-485. Zlatkls, A,; Poole, C. F. “Electron Capture. Theory and Practice in Chromatography”; Elsevier: Amsteldam, 1981. Blau, K., King, G. S., Eds. Handbook of Derivatives for Chroamtography”; Heyden: London, 1977. Knapp, D. R. “Handbook of Analytical Derivatization Reactions”; Wiiey: New York, 1979. Ziatkis, A.; Poole. C. F. Anal. Chem. 1980, 52, 1002A-1016A. Funke, P. T.; Ivashkiv, E.; Malley, M. F.; Cohen, A. I . Anal. Chem. 1980, 52, 1086-1089. Cohen, A. I.; Deviin, R. G.; Ivashklv, E.; Funke, P. T.; McCormick, T. J. Pharm. Sci. 1982, 71, 1251-1256. Ivashklv, E.; McKlnstry, D. N.; Cohen, A. I.J. Pharm. Scl. 1884, 73, 1113-1 117. Fontana, A.; Tonlolo, C. “The Chemistry of the Thiol Group”; Patai, S., Ed.; Wiley: New Yrok, 1974; pp 294-296. Pontanova. J. P.; Shrift, A. J. Chromatogr. 1977, 139, 391-394. Cohen, A. I.; Kripalani, K. J. US. Patent 4 179568, December 8, 1979. Migdalof, B. H.; Singhvi, S. M.; Kripalani, K. J. J. Liq. Chromatogr. 1980, 3, 857-865. Matsukl, Y.; Fukuhara, K.; Ito, T.; Ono, H.; Ohara, N.; Yui, T.; Nambara, T. J. Chromatogr. 1980, 168, 177-183. Bathala, M. S.; Weinstein, S. H.; Meeker, F. S., Jr.; Singhvi, S. M.; Migdalof, B. H. J. Pharm. Scl. 1984, 73, 340-344. Drummer, 0. H.; Jarrott, B.; Louis, W. J. J. Chramatogr. 1984, 305, 83-93. Tu, J.; Liu, E.; Nickoloff, E. L. Ther. Drug Manit. 1884, 6, 59-65. Oaks, D. M.; Hartmann, H.; Dlmlck, K. P. Anal. Chem. 1964, 36, 1560- 1565. Satouchl, M.; KoJlma, T. Anal. Left. 1972, 5, 931-942. Clozel, J. P.; Caille, G.; Taeymans, Y.; Theroux, P.; Biron, P.; Besner, J. G. J. Pharm. Scl. 1984, 73, 207-209. Vessman, J. J. Chromatogr. 1980, 184, 313-324. Gyilenhaal, 0.; Hawig, P. J. Chromatogr. 1980, 769, 351-357.
Mohammed Jemal* Allen I. Cohen Squibb Institute for Medical Research P.O. Box 191 New Brunswick, New Jersey 08903
RECEIVED for review April 19,1985. Accepted June 17,1985. Part of this work was presented at American Pharmaceutical Association Academy of Pharmaceutical Sciences National Meeting, Miami Beach, FL, Nov 13-17,1983, in a symposium entitled “Recent Advances in Capillary Gas Chromatography”.
AIDS FOR ANALYTICAL CHEMISTS Differential Density Method for Determination of Carbon Load on Chromatographic Packings Wei Cheng Beckman Instruments, Inc., 1716 Fourth Street, Berkeley, California 94710 Reverse-phasematerials are now the most popular packing materials in high-performance liquid chromatography (HPLC) and are widely used in routine analysis. Commercial silicabased packings are available with methyl-, n-propyl-, n-octyl-, and octadecyl-bonded functional groups.
Carbon load is generally considered as the most important characteristic of packings and its determination mainly relies on the conventional elemental analysis. Recently Hartwick et al. reported an alternative method based on the hydrolysis of packings and gas chromatography (1). Elemental analysis
0003-2700/85/0357-2409$01.50/00 1985 American Chemical Society
