2512
Anal. Chem. lg84, 56, 2512-2517
LITERATURE CITED (1) Gordon, J. E.; Selwyn, J. E.; Thorpe, R. L. J . Org. Chem. 1966, 31, 1925. (2) Pacholec, F.; Butler, H. T.; Poole, C. F. Anal. Chem. 1982, 5 4 , 1938. (3) Pacholec, F.; Poole, C. F. Chromatographia 1983, 17, 370. (4) Poole, C. F.; Butler, H. T.; Coddens, M. E.; Dhanesar, S. C.; Pachoiec, F. J . ChfOm8t0gf. 1984, 289, 299. (5) Onuska, F. L.; Comba, M. E.; Blstrlckl, T.; Wilkinson, R. J. J . Chromatogr. 1971, 142, 117.
(6) Peters, T. L.; Nestrlck, T. J.; Lamparski, L. L. Anal. Chem. 1982, 54, 2397. (7) Traitler, H.; Koiarovlc, L.; Sorlo, A. J . ChfOm8tOgr. 1983, 279, 69. (8) Traitier, H. J . Hlgh Resolut. Chromatogr. Chromatogr. Commun 1983, 6 , 60. (9) Schleke, J.; Pretorlus, V. J . Chrom8togr. 1977, 132, 217.
.
RECEIVED for review April 26,1984. Accepted June 21, 1984.
Pentafluorobenzenesulfonyl Chloride: A New Electrophoric Derivatizing Reagent with Application to Tyrosyl Peptide Determination by Gas Chromatography with Electron Capture Detection Abdellah Sentissi, Markus Joppich,’ Kathleen O’Connell? Albert Nazareth: and Roger W. Giese*
Department of Medicinal Chemistry in the College of Pharmacy and Allied Health Professions and Barnett Institute of Chemical Analysis and Materials Science, Northeastern University, Boston, Massachusetts 02115
Pentafluorobenzenesulfonyl chlorlde (PBSC) Is a new reagent for electrophore labellng of small tyrosyl peptldes, particularly onto thelr phenolic hydroxyl group, for analysls by gas chromatography wlth electron capture detection (GC-ECD). The products resist aqueous hydrolysis and have a response by GC-ECD close to that of Ilndane, B strong electrophore. Also examlned are the peak asymmetry and response characterlstlcs of these products as a functlon of the electrophore attachment site( s), N-methyl vs. N-plvaloyl nonpolar derlvatlzatlon, number of actlve hydrogens, and changes In the GC-ECD equlpment. Detectlon of 100 fg of the derlvatlzed dlpeptlde, N-plvaloyl-O-[(pentafluorophenyl)sulfonyl]glycyltyroslne ethyl ester, lowers the detectlon llmlt for peptide GC by loa.
Many types of chemical substances have been used as electrophoric derivatizing reagents, modifying analytes to improve their detection and other properties by gas chromatography with electron capture detection (GC-ECD) ( I ) . These reagents are also releveant to GC with detection by negative ion chemical ionization mass spectrometry (GCNICI-MS) (2). The best of these derivatizing reagents are available in a pure and stable form and form a stable adduct with the substance t o be analyzed including resistance to aqueous hydrolysis. This facilitates both sample cleanup after derivatization and the maintenance of standards. The electrophoric derivatizing reagent should also convert the analyte to a single product, and the excess reagent should be easily removed at the end of the reaction. Failure to do the latter can perturb the GC analysis (3)and shorten column lifetime (4). When further derivatization is required to overcome residual polar groups having unfavorable GC properties, the electrophore-labeled analyte should be stable to these conditions as well. Finally, the derivatized analyte should have adequate thermal stability, appropriate volatility, and high Present address: Ciba-Geigy,Basel, Switzerland. Present address: InstrumentationLaboratory, Lexington, MA. Present address: Carter-Wallace,Cranbury, NJ. 0003-2700/84/0356-2512$01.50/0
sensitivity for analysis by GC-ECD or GC-NICI-MS. Many of the available electrophoric derivatizing reagents have one or more serious shortcomings concerning these criteria. Since GC-ECD and GC-NICI-MS are important techniques, particularly now that bonded fused silica capillary columns are available along with advances in on-column injection, it is useful to improve the status of electrophoric derivatizing reagents. In this paper, we introduce pentafluorobenzenesulfonyl chloride as a new electrophoric derivatizing reagent. At least for the analysis of certain model amino acids and peptides by GC-ECD, with particular attention to a phenolic tyrosine moiety, this reagent does well in achieving the properties cited above. We also find that this reagent similarly is useful for labeling thymine or uracil nucleic acid bases (5). EXPERIMENTAL SECTION Chemicals and Reagents. Pentafluorobenzenesulfonyl chloride and methyl iodide were purchased from Aldrich Chemical Co. (Metuchen, NJ); phenylalanine, tyrosine, N-acetyltyrosine, glycylglycine, and trimethylacetic anhydride (which was distilled) were from Sigma (St. Louis, MO); glycylglycyltyrosinewas from Research Organics Inc. (Cleveland,OH); diisopropylethylamine and hexamethyldisilazane (HMDS) were from Pierce Chemical Co. (Rockford, IL); acetonitrile was from Burdick and Jackson Laboratories Inc. (Muskegon, MI);N-methylmorpholinewas from Eastman Kodak Co. (Rochester,NY) and distilled; ethyl acetate, anhydrous ether, ethanol, methanol, 1-butanol, chloroform,toluene, hexanes, and cyclohexane were obtained from J. T. Baker Chemicals (Phillipsburg, NJ); hydrogen chloride was obtained from Matheson (Gloucester, MA); Silica Gel 60, 230-400 mesh, was purchased from VWR Scientific (Boston, MA); and new fuchsin (tris(4-amino-3-methylphenyl)methane)was from Matheson, Coleman and Bell (Norwood, OH). High-Performance Liquid Chromatography (HPLC). The HPLC unit consisted of a Model llOA pump from Altex (Woburn, MA), a variable wavelength detector from LDC (Riviera Beach, FL), a Model HP7123 chart recorder from Hewlett-Packard (Pdo Alto, CA), and a Model CV6 VHPa-N-60 sampling valve with a 10-pL sample loop from Valco (Houston, TX). For HPLC, the water was deionized,distilled,filtered (0.6 pm poly(viny1chloride) membrane from Millipore, Medford, MA) and degassed under vacuum; the acetonitrile was filtered (0.5 pm fluoropore from Millipore); and the phosphate buffer was prepared from lOmM KH2P04by adjusting the pH to 2.1 with phosphoric acid, filtering, and degassing. The HPLC separations were accomplished on a 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY,
15 cm X 4.6 mm Supelco LC8 column (Supelco,Inc., Bellefonte, PA) using an isocratic system consisting of acetonitrile (60%)and phosphate buffer, pH 2.1 (40%) at a flow rate of 2 mL/min. Flash Column Chromatography. Silica Gel 60, 230-400 mesh, was used. The mobile phase consisted of 1/1 hexane/ether (v/v) or 2/1 ethyl acetate/hexane (v/v). The general technique is described elsewhere (6). Thin-Layer Chromatography (TLC). The separation was done on GHLF silica gel uniplates with fluorescence indicator, from Analtech Inc. (Newark,DE). The chromatographicsolvents were either 2/1 ethyl acetate/hexanes (v/v), 1/1 hexanes/ether (v/v), 9/1 chloroform/methanol (v/v), or 4/1/1 1-butanol/ water/acetic acid (v/v/v). TLC detection was done by fluorescence quenching,ninhydrin,Pauly reagent, new fuchsin, and ferric chloride. Staining with new fuchsin on a silica plate tends to produce purple spots against a pink background for organic compounds containing a perfluorinated group (7). We sprayed warm plates with a 10% new fuchsin aqueous solution and found that repeated heating and spraying enhanced the spots. Gas Chromatography. The instrument was a Model 3740 gas chromatograph from Varian Associates (Walnut Creek, CA) fitted, unless indicated otherwise, with a quartz direct vapor-injection insert (0.7-0.9 mm capillary bore, 11 cm length), a fused-silica capillary column (10 m X 0.25 mm i.d., DB5; J and W Scientific, Rancho Cordova, CA), a constant-current, pulse modulated, &Ni electron-capture detector, and a pressure regulator. A Varian Model 1095 on-column injector was also used as indicated, as was an experimental 350-rL ECD (8). The carrier and makeup gas was nitrogen (Matheson) with an actual flow rate in the column of 4 cms min-' at 160 OC and flow rates of 5 and 6 cm3min-' (measured at room temperature and uncorrected) at the detector base and detector insert base, respectively. The column head pressure was usually varied from 30 to 40 psi. The injector and detector temperatures were 320 OC; column temperatures ranging from 150 OC to 280 OC were employed. For low-flow silanizationof the conventional ECD, the column flow rate was 0.7 mL/min, and the detector makeup flow rate was approximately 1 mL/min, while 5 pL of 5% hexamethyldisilazane (HMDS) in toluene was injected slowly. After the HMDS-toluene peak eluted, the flows were reset to their normal values. Column and detector temperatures of 280 and 320 "C were used, respectively. High-temperature blue septa (Altech Associates, Deerfield, IL) and thermogreen LB septa (Supelco) were utilized. Direct injections into the gas chromatograph were made with silanized 10-rL syringes, type 1701 N (Hamilton Co., Reno, NY) fitted with a type 26 S needle. On-column injectionswere made with a syringe fitted with polyimide-sheathed vitreous silica needle (Scientific Glass Engineering, Austin, TX). All data were obtained in duplicate or triplicate. Synthesis. The structures of all produds were consistent with their infrared spectra and were confirmed by mass spectrometry. Unless noted otherwise, all products gave single peaks on TLC (using applicable detection techniques), HPLC (absorbance detection at 214 nm), and GC-ECD. L-PhenylalanineEthyl Ester. Ten millimoles of L-Phe (1.65 g) was dissolved in 60 mL of absolute ethanol. Dry hydrogen chloride (up to a final level of 220 mg of HCl/mL of ethanol) was passed into the solution, followed by heating with stirring at 60 OC for 1 h and then evaporation to dryness under reduced pressure. The residue was treated twice with toluene (20 mL) and rotary evaporated under vacuum to remove residual hydrogen chloride. The resultant powder was dried overnight under high vacuum. The same protocol was used to prepare the other amino acids and peptide ethyl esters, except that no heating was used for the esterificationof peptides to prevent acidic cleavage of the peptide bond. Complete conversion of starting acid to product ester was observed in each case based on both TLC and HPLC. N-[(Pentafluorophenyl)sulfonyl]phenylalanine Ethyl Ester. Five hundred milligrams (2.6 mmol) of phenylalanineethyl ester was dissolved in 5 mL of acetonitrile followed by the addition of 3 equiv (7.8 mmol, 870 pL) of N-methylmorpholine. Three equivalents of pentafluorobenzenesulfonylchloride (7.8 mmol,
VOL. 56, NO. 13, NOVEMBER 1984 2513
1.2 mL) was added at once and the solution was stirred for 2 h and evaporated. The oily residue was taken up in chloroform (10 mL) and washed three times with 0.01 N HCl, three times with 5% NaHCO, and three times with water, followed by drying over MgS04. The chloroform solution was reduced to a small volume by evaporation and applied to a flash chromatography column (25 X 1 cm) packed with Silicon Gel 60,230-400 mesh. Appropriate collected fractions based on TLC were placed overnight in the refrigerator. The crystals that appeared were recrystallized three times from cyclohexane: 68% yield; mp 86-87 OC. N,O-Bis[(pentafluorophenyl)sulfonyl]tyrosine Ethyl Ester. This compound was synthesized using the previous method, except that no 0.01 N HC1 was used in the cleanup step. The crystals were recrystallized three times from cyclohexane: 72% yield; mp 115-116 OC. N-Acetyl-0-[(pentafluorophenyl)sulfonyl]tyrosineEthyl Ester. The previous procedure was followed to derivatize N acetyltyrosine ethyl ester, yielding the product as an oil. N,O -Bis[ (pentafluorophenyl)sulfonyl]glycyltyrosine Ethyl Ester, The previous method was used to derivative glycyltyrosine ethyl ester. The product was recrystallized from cyclohexane/ether: 80% yield; mp 154-156 "C. GC-ECD showed two minor peaks (ca. 10% in area) before the major peak with direct injection. However, only one peak was seen on HPLC and GC-FID. N-[(Pentafluorophenyl)sulfonyl]glycylglycine Ethyl Ester. The same technique was used to derivatize glycylglycine ethyl ester. The product was recrystallized from cyclohexane/ ether: 78% yield; mp 155-156 OC. N-Methyl-N-[(pentafluorophenyl)sulfonyl]phenylalanine Ethyl Ester. Ten milligrams of N-[(pentafluorophenyl)sulfonyllphenylalanineethyl ester (0.024 mmol) was dissolved in 3 mL of acetonitrile by the addition of 10 equiv (40 pL, 0.24 m o l ) of diisopropylethylamine. Thirty equivalents of methyl iodide (43 pL, 0.72 mmol) was added and the solution was stirred for 2 h at room temperature, and then evaporated to dryness under reduced pressure. The residue was taken up in chloroform (2 mL), washed three times with water, and dried over MgS04. The product was obtained as an oil. N ,0-Bis[(pentafluoropheny1)sulfonyl]-N-methyItyrosine Ethyl Ester. The previous procedure was used to obtain a product that was recrystallized from cyclohexane/ether. N-Methyl-N-[(pentafluorophenyl)sulfonyl]glycylglycine Ethyl Ester. The same procedure was used to obtain this oily product. N-Pivaloyl-0-[(pentafluorophenyl)sulfonyl]tyrosine Ethyl Ester. Forty milligram of tyrosine ethyl ester (0.19 mmol) was dissolved in 3 mL of acetonitrile followed by the addition of 2 equiv (0.38 mmol, 30 pL) of triethylamine. Three equivalents of pivalic anhydride (0.57 mmol, 120 pL) was added at once, and the mixture was stirred for 2 h at room temperature and then evaporated under reduced pressure. The residue was dissolved in ethyl acetate and washed 3 times with 5% NaHCC&and 3 times with phosphate buffer pH 7.0. The organic layer was evaporated and the residue was taken up in 3 mL of acetonitrile. The Npivaloyltyrosine ethyl ester was then reacted with 35 pL of Nmethylmorpholine and 80 pL of pentafluorobenzenesulfonyl chloride. The solution was stirred for 2 h at room temperature and evaporated under reduced pressure giving an oily residue that was dissolved in ethyl acetate and washed 3 times with 0.01 N HC1 and 3 times with phosphate buffer, pH 7.0. The ethyl acetate solution was evaporated to a small volume and the product was purified on a flash column (25 X 1 cm) packed with Silica Gel 60, 230-400 mesh, giving an oil. N-Pivaloylglycyl-0-[ (pentafluorophenyl)sulfonyl]tyrosine Ethyl Ester. This compound was synthesized by using the previous protocol, yielding an oily product. N-Pivaloylglycylglycyl-0 -[(pentafluoropheny1)sulfonyl]tyrosine Ethyl Ester. This compound was prepared according to the previous protocol, yielding an oily product. RESULTS AND DISCUSSION Ultratrace chemical analysis of organic and biological substances by a strategy centrally involving electrophore-labeling along with nonpolar derivatization prior to separation/quantitation by gas chromatography has several at-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2514
Table I. GC-ECD Characteristics of PPS-Labeled, Nonpolar Derivatized Amino Acids and Peptides compd (ethyl ester)
derivativen no.
Phe
1 1 2
TYr
3 4 5 6
CH&O
(CH,)&CO
(N-)
(N-)
response
(F6CsS02)1,2
(N-)
(O-)
X
X X X
7
X X
8 GlyTyr
GlyGlyTyr
9 10 11
X
X X
6 GlyGly
CH3
(N,O-) ( N - )
X X X X
X
X X
X X X
X
X
(molar area re1 to
tR, min 4.1" 0.5d 4.5c 1.4d 1.5d 2.3d 2.5d 1.0e 3.lf 2.2f 1.28 3.6h 3.1h
A: 2.4 1.3 1.3 2.7 1.3 1.3 1.1 1.0
lindane) 1.2 2.0 1.2 1.9 1.2k 2.gk 3.2
6.4 (1.5)i 2.0 1.2 (1.0)iJ
0.4k
1.4 (1.O)j
0.3 0.9
2.2 (1,2)i
1.2 1.0
"The N substituent is always attached to the N terminal, a-amino nitrogen, based on reactivity considerations and analysis by mass spectrometry. bThe asymmetry value, A,, for the chromatographic peak is b/a where b and a are the back and front peak half widths, respectively,at 10% of the peak height. 'hThe column temperature is (c) 150 OC, (d) 180-230 OC at 40 OC/min with no hold after injection, (e) 200 OC, (f) 165 OC, (g) 190-260 "C as before, and (h) 190-290 "C as before. 'On-column injection is employed, as opposed to direct injection in all other cases. jA more inert, experimental ECD is used; see text. kThe same values were obtained within f10% when the analysis of these compounds was repeated 1.5 months later on the same equipment. tractive features: (1)bonded, fused silica capillary columns are now available offering improved performance in terms of analysis speed, column stability, solute recovery, and chromatographic efficiency (9);(2) elecrophores as labels can be chemically and physically stable, moderately small in molecular size, and variable in their properties by means of halogen substitution (1);and (3) both electron capture detection (ECD) and negative ion chemical ionization mass spectrometry (NICI-MS) are available as complementary detection techniques. The major negative features currently for this overall approach are the general problem of sample cleanup, including interferences and analyte recovery, the potential for incomplete reactions and other product heterogeneity from the labeling/derivatization steps, and the volatility/stability/ reproducibility limits of the modified analytes for ultratrace quantitation by GC-ECD and GC-NICI-MS. We have begun to develop this electrophore-labeling/nonpolar derivatization strategy for the ultratrace analysis of small peptides by GC. The analysis of such peptides could be of interest because of either their inherent biological properties or specific fragments derived from the enzymatic digestion of larger, biologically active polypeptides, Le., "fingerprint analysis". Toward this goal, we are particularly interested in electrophore-labeling the side chains of peptides, as opposed to their N-terminal amino or C-terminal carboxyl groups. This could enhance the selectivity of this labeling, due to the more distinctive nature of some of these side chains. We have begun with pentafluorobenzenesulfonyl chloride (PBSC) as a potential electrophoric reagent for labeling of a tyrosyl side chain. The choice of this reagent was prompted by the known ability of toluenesulfonyl chloride to attach firmly onto the side chain of this amino acid (10).For the subsequent nonpolar derivatization step, we chose to emphasize pivaloylation and methylation based on the successful use of these techniques by others (11) and our interest in nonpolar shielding derivatization. Glycyltyrosine and glycylglycyltyrosine were selected as the initial tyrosyl peptide analytes. We similarly labeled and derivatized the amino acids phenylalanine and tyrosine and the peptide glycylglycine as analogues of these tyrosyl peptides. Labeling and Aqueous Stability. Our first encouraging result was our ability to fully convert (based on TLC) tyrosine
ethyl ester to a single, doubly labeled (N,O)product by reaction with PBSC under mild conditions, involving treatment with 3 equiv each of PBSC and N-methylmorpholine in acetonitrile at room temperature for 2 h. Shorter reaction times were not examined. We were next pleased to find that our product was completely stable, as desired, when stored as an aqueous solution either in acidic buffer (0.1M sodium acetate, pH 4.0) or under nucleophilic basic conditions (tris(hydroxymethy1)aminomethane buffer, 0.2 M, pH 8.0) for 1 week at room temperature, based on analysis by HPLC (data not shown). Because this product also behaved well as a solute for GC-ECD (see below), we proceeded to label the other amino acids and peptides and also to conduct the nonpolar derivatization steps cited above. Our results are summarized in Table I, comprising chromatographic retention, asymmetry, and response. The relative standard deviation (SD/%) for the response data was at least better than *7% based on duplicate or triplicate injections, except for compound 2, where it was *E%. Retention Times. The retention times for the labeled/ derivatized peptides in Table I range from 1.2 to 3.6 min. These values are all low, a favorable result, particularly given the moderate column temperatures employed. Aside from other factors, this encourages an effort to analyze higher molecular weight solutes of this type by GC-ECD. It is also encouraging that nonpolar derivatization of an a-amino group with pivaloyl, as opposed to acetyl, evaluated for 0-[(pentafluorophenyl)sulfonyl]tyrosine ethyl ester ((pentafluorophenyl)sulfonyl, PPS), causes only a minor change in the retention time, 1.5 vs. 1.4 min, respectively. Apparently we are trading nonpolar for polar retention with the use of pivaloyl instead of acetyl, a useful strategy in GC analysis. Nonpolar shielding of polar groups has been cited before as a factor reducing the retention of solutes in GC, particularly on a polar column (12,13). For O-PPS-labeled glycyltyrosine ethyl ester, the pivaloyl group as an N substituent is seen to give a lower retention (1.2min) than a PPS group at the same site (3.6 min) under essentially the same column temperature conditions. Asymmetry, The significance of attaining a low value for peak asymmetry is to optimize peak detectability, and probably the long-term accuracy and precision of the analysis as well. For a chromatographic peak having a uniform composition, peak asymetry in GC ordinarily reflects strong sorption
ANALYTICAL CHEMISTRY, VOL. 56,
sites or dead volumes. Since we observe an asymmetry value of 1.0 for the highly derivatized solute, N-methyl-N,O-bis[(pentafluorophenyl)sulfonyl]tyrosineethyl ester at a column temperature of 200 OC as shown in Table I, we can rule out the latter event. As we shall subsequently demonstrate, we separately find (and also reduce) such sites in the injector, column, and detector, at least for the less derivatized solutes. Prior to defining the sources of the peak asymmetry in the GC-ECD, we may first note that the use of N-pivaloyl VS. N-acetyl substitution of our solutes minimizes their overall GC-ECD asymmetry. 0-PPS-tyrosine ethyl ester has an asymmetry of 1.3 when N substituted with pivaloyl, vs. an asymmetry of 2.7 when N substituted instead with acetyl and analyzed under the same GC-ECD conditions. We assume that the polar, N-terminal -CONH- group in this molecule is more shielded by an adjacent, bulky alkyl group of pivaloyl ([CH3I3C-) as opposed to the smaller alkyl group of acetyl (CH3-). The highest asymmetry value, 6.4, is observed for N[ (pentafluorophenyl)sulfonyl]glycylglycine ethyl ester. Two mechanisms are found to reduce this asymmetry significantly. The first is N-methylation of this compound at its N-terminal amide group, which reduces its asymmetry to 2.0 as shown in Table I. (This also establishes the compatibility of PPS labeling with subsequent methylation.) Secondly, the use of an on-column injector in place of the direct injector for the nonmethylated compound gives an asymmetry value of 1.5, demonstrating that the strong sorption sites causing the initial value of 6.4are mostly located in the direct injector. Similarly, the asymmetry for N-pivaloyl-0-[(pentafluorophenyl)sulfonyl]glycyltyrosineethyl ester is reduced from 2.2 to 1.2 by this latter technique. Our direct injector is a silanized, 110 mm by 0.7-0.9 mm i.d. quartz tube that connects directly to the column. Potential sources of the strong sorption sites in this direct injector are the inner surface of this tube, contamination (including septum particles and bleed) on this surface or at the top of the column, and the syringe needle. However, the main point here is the marked improvement in peak asymmetry with oncolumn as opposed to direct injection. The evidence for asymmetry-contributing sites in the column is the reduction in band asymmetry with a higher column temperature (14).This effect is seen most dramatically for a less derivatized solute such as N-[(pentafluorophenyl)sulfonyl]phenylalanine ethyl ester. The asymmetry value for this compound decreases from 1.8 to 1.1when the isothermal column temperature is increased from 150 to 180 OC, with no further improvement in peak asymmetry at a higher column temperature. Whether the residual asymmetry for this compound arises in the direct injector, column, or detector was not defined. We next investigated the contribution of a conventional Varian ECD to band asymmetry by replacing it with an experimental ECD fabricated by Varian to be more inert (8). In the latter ECD, an insert is present that should overcome exposure of the solute to ceramic and stainless steel surfaces encountered prior to the e3Nifoil region in the conventional ECD. With the experimental ECD, we obtain an asymmetry value of 1.0 for N-pivaloyl-0-[(pentafluorophenyl)sulfonyl]glycyltyrosine ethyl ester, as shown in Table I, using on-column injection. Our best asymmetry value under comparable conditions with a conventional Varian ECD after low-flow silanization is 1.2 for this compound. Thus, the direct injector, column, and conventional ECD all possess sorption sites potentially enhancing band asymmetry. Importantly, we are able to significantly overcome these sites with on-column injection, elevated column temperature, and a more inert ECD. This invites, along with the
NO. 13, NOVEMBER 1984 2515
low retention times cited before, even more polar, higher molecular weight peptides to be analyzed by this technique. Response. We turn now to our final topic of Table I, the GC-ECD response. The main point is the excellent response that is observed for all of our PPS substituted amino acid and peptide solutes, in the same general range (within a factor of 3) as that of lindane, a strong electrophore. It is important to appreciate that this response includes both the recovery of the solute throughout the GC-ECD, and the solute sensitivity in the ECD. Although we established at least a 10-fold linear range for the response of all of our compounds (except for N,0-bis[ (pentafluorophenyl)sulfonyl]glycyltyrosineethyl ester, to be discussed), and measured our responses within this range, this still does not define the role of solute recovery in the response. Thus, the response data in Table I must be interpreted with this uncertainty in mind. Let us first deal with the two compounds that gave anomalous results, at least under certain conditions. The first one was N,O-bis[(pentafluorophenyl)sulfonyl]glycyltyrosineethyl ester (compound lo), that consistently gave three GC peaks (a major peak, approximately 90%, and two earlier-eluting minor peaks, approximately 10% together) with direct injection. No change was seen in this pattern after repeated recrystallization of this modified dipeptide, and this “solute” also gave only a single peak by gradient HPLC and GC-FID. Changing to on-column injection by GC-ECD revealed at least four earlier eluting peaks that similarly comprised about 10% of the total pattern. Whether the compound is impure or unstable under the GC-ECD conditions was never established, but this behavior along with its relatively low response seems to be more consistent with the latter explanation, assuming that the earlier-eluting peaks are not detected by FID. The contrasting, good behavior for N-pivaloyl-0-[ (pentafluorophenyl)sulfonyl]glycyltyrosine ethyl ester suggests that any instability might involve the N-PPS substituent on this peptide. The second problem compound in terms of response, although only with direct injection, was N-pivaloyl-0-[ (pentafluorophenyl)sulfonyl]glycylglycyltyrosine ethyl ester (compound 111, a modified tripeptide. Successive, direct injections of this solute increased its peak area until a somewhat constant, maximal response was obtained. In contrast, the latter response was seen immediately and was constant with oncolumn injection, so this value is cited in Table I. We see that the overall range of response is 0.3-3.2 relative to a reference response for lindane of 1.0. Although this is a small range, it is worthy of some discussion because of the structural similarities of these solutes. To a first approximation, equal sensitivity (or, at most, two levels of sensitivity) might be expected for all of these compounds, because this sensitivity is based on the presence of the strong PPS electrophore in each molecule. Although some of the compounds have two PPS groups, these groups are not conjugated. Thus, no changes in sensitivity should derive from this aspect, given the equivalent responses reported by others for mono- and unconjugated didinitrophenyl-substitutedamino acids (15). Although one cannot predict the relative sensitivity of a phenolic 0-PPS vs. an N-PPS group, and this might give two levels of response, there is no apparent correlation of the response with this aspect of PPS attachment, For example, compounds 1,3, and 5 have N-PPS, 0-PPS, and N,O-PPS substitution, respectively, and yet have the same response of 1.2. Thus, it is interesting why a 10-fold range of response is observed. The response decreases with an increasing number of nonpivaloyl active hydrogens (nAH)defined as the number of NH sites except those also substituted on this N by a pivaloyl group. This latter group likely provides nonpolar shielding
2518
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
F
)”(
F
Flgure 2. Structure of N-plvaloyl-0-[(pentafluorophenyl)suifonyl]glycyltyroslne ethyl ester.
A
I
I
I
B
C
D
I
GC-ECD EQUIPMENT Figure 1. Variation in response as a function of different capillary GC-ECD equipment: the column (DB-5 for A, B, C; DB-1701 for D); injector (direct for A, B; on-column for C, D) and ECD (conventlonai for A, B, C experimental for D) were different for each of the four sets of GC-ECD equipment. The amounts of the solutes Injected ranged from 70 to 800 pg. The CV’s (SDIX) for the two to four InJections glving each point were 0 to 7 % , 8 to 15 % , and 16 to 22 % for 8 1, 15, and 4 % of the points. No compound consistently had a “poor” preclslon (SD/X > 7 % ), and most of the precision values above 7 % were due to a smaller value for X as opposed to a larger standard deviation for the data. Overall the best precision was obtained on equipment D, lnvolvlng the experlmental ECD, where the SD/X values were a11 in the 1-3% range except for two values (a%, 9 % ) having a low value for X. The designations for the compounds are deflned In Table I.
of any adjacent NH based on the prior retention and asymmetry data for our pivaloyl-substituted compounds. Thus, for compounds 2,4, and 6, having the highest response range of 2.0-3.2, nAH = 0; compounds 1, 3,5, 8, and 9 give an intermediate response of 1.1-1.2 and have nAH = 1; and compounds 7 and 10 have the lowest range of response, 0.3 to 0.4, with nAH = 2. The one exception is compound 11, having an intermediate response of 1.0 with nAH = 2, although 11 is also the largest of these molecules where additional shielding of N ” s might take place. Ordinarily one might interpret such data as reflecting differences in solute recovery, since the presence of active hydrogens is commonly known to degrade GC solute behavior. We attempted to prove this by substituting alternate as well as more inert components into our GC-ECD system in an attempt to raise or significantly vary the response, or to narrow its overall 10-fold range. Four different sets of GC-ECD equipment were employed, yielding the data shown in Figure 1. In this figure, GC-ECD system A, involving a direct injector, DB-5 column, and conventional Varian ECD was our initial system giving the response data shown in Table I. System B used a different injector, column, and detector, but all of the same type, and so did C along with an on-column injector. System D used a different on-column injector, a DB-1701 column, and an experimental ECD. All of these columns were approximately 10 m long. We see that the general level of response for our compounds remains fairly constant throughout all of this variation in equipment. Although there is some narrowing of the response range in going to the more inert system D, involving the on-column injector and experimental ECD, it is not fully convincing, and certainly the response does not collapse to one or two levels relating to 0-PPSvs. N-PPS substitution. (The low response for compound 11 in system D was later
shown to be a column effect, as discussed below.) Since the column, in spite of the several substitutions, was always nominally 10 m in length and exposed the solutes to a bonded, fused silica surface, it was still possible that the overall range of response developed here from differences in the recovery of our compounds in the column. However, we ruled out this component by observing no significant changes in response for representative compounds 3,6,and 7, when we varied the flow rate, temperature, and both for a given column, such that the retention times of these compounds increased at least 2-fold. Thus, changes in both the GC-ECD equipment and conditions neither change the general level or order of response for our compounds nor fully collapse this response to one or two levels. This raises the possibility that some of the overall difference in response may be due to a variation in sensitivity for our compounds. Since those having a lower number of active hydrogens also tend to have a larger fractional content of nonpolar groups, any variation in sensitivity might be related to the tendency of organic molecules to have a higher electron affinity when substituted, at least on polar sites, by larger alkyl groups (17). Along these lines, an increased GCECD response with alkyl replacement of an active hydrogen has been reported before, e.g., N-methyl-N-HFB-phenethylamine has twice the response of N-HFB-phenethylamine (16). Nevertheless, the relative contributions of sensitivity and recovery to the differences in response for both these and our solutes remain unclear. Superimposed on the general consistency in relative response for our compounds with changes in equipment are individual variations in response that, while subtle, are nevertheless reproducible (see footnote k in Table I, and also the caption to Figure 1 describing the precision for these data). Similar variations have been mentioned by others with a change in GC-ECD equipment (18). These variations are probably caused by differences in the surfaces to which the solutes are exposed in the several GC-ECD’s that were used. This was further investigated for the severe drop in response of compound 11 in going from equipment C to D, where the cause was found to be the DB-1701 column in system D. When the DB-5 column in C was installed in equipment D, the response for 11 remained as high as it was in C, whereas this response dropped severely when the DB-1701 column was installed in system C. For the other fluctuations in response, the sites are not defined, and neither is the nature (solute losses throughout the GC-ECD vs. the possibility of response enhancement in the ECD) of these variations. These aspecta, including the high specificity of these surface effects, are being studied in more depth with other solutes (19). Detection Limit. We chose N-pivaloyl-0-[(pentafluorophenyl)sulfonyl]glycyltyrosine,having the structure shown in Figure 2, for determination of a detection limit. The value was 100 fg at a signal-to-noise of about 3 as shown in Figure 3, representing approximately a lo3advance in the detection of a peptide by gas chromatography (20,21). This response for both peak height and area was linear at least up to 500
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER I984 280’ t 20’
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polar derivatization for the ultratrace analysis of peptides by GC has promise, including a potential application to even higher molecular weight and more polar peptides than investigated here. R e g i s t r y No. 1, 91860-42-3; 2,91860-43-4; 3, 91860-44-5; 4, 91860-45-6;5, 91860-46-7;6, 91860-47-8;7, 91860-48-9;8, 91860-49-0;9, 91860-50-3;10, 91860-51-4;11, 91860-52-5;Lphenylalanine, 63-91-2; L-phenylalanine ethyl ester, 3081-24-1; N-acetyltyrosine ethyl ester, 840-97-1; glycyltyrosineethyl ester, 19240-2&9;glycylglycine ethyl ester, 627-74-7; tyrosine ethyl ester, 949-67-7; pivalic anhydride, 1538-75-6; N-pivoyltyrosineethyl ester, 38453-21-3; pentafluorobenzenesulfonyl chloride, 832-53-1; glycyltyrosine, 658-79-7; glycylglycyltyrosine, 17343-07-6. LITERATURE CITED
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Flgure 3. Detectlon of 100 fg of N-plvaloyl-0-[(pentafluorophenyl) sulfonyl]glycyltyroslne ethyl ester by GC-ECD column, DB-5, 25 p m film thickness, 0.25 mm 1.d. X 10 m length; flow rate, 5.65 mL/mln of helium at 40 psl In the column (measured at room temperature and uncorrected), wRh nitrogen makeup in the ECD up to a total flow of 30 mL/mln measured slmllarly; ECD, 340 ‘C. The notations are (a) base llne disturbance due to the pressure jump (22); (b) and (d) contamination peaks observed with variable magnltude In the solvent blank injection, and (c) attenuation change of 128 X 1 to 1 X 1. Oncolumn injection and the more Inert experlmental ECD (see text) were used.
(1) Poole, C. F.; Zlatkis, A. J. Chromatogr. Libr. 1081, 20, 151-190. (2) Hunt, D. F.; Crow, F. W. Anal. Chem. 1978, 50, 1781-1784. (3) Gyllenhaal, 0.; Broteli, H.; Hartvlg, D. J. Chromatogr. 1078, 129, 295-302. (4) Cummins, L. M. I n “Recent Advances in Gas Chromatography”; Dom sky, I. I., Perry, J. A,, Eds.; Marcel Dekker: New York, 1971; pp 313-340. (5) Nazareth, A.; Jopplch, M.; O’Connell, K.; Sentlssi, A.; Glese, R. W. J. Chromatogr., In press. (6) Still, W. C.; Kahn, M.; Mltra, A. J. OIg. Chem. 1078, 43, 2923-2925. (7) Gagnon, J. E.; Mendel, A. Chem. Eng. News 1082, March 22, 36. (8) Wells, G. HRC CC,J. Hlgh Resolut. Chromatogr. Chromatogr. Commun. - ia83. - - - , 6. - , 651-654. - - ..
(12) (13) (14)
pg (data not shown). An on-column injector and the more inert, experimental ECD were used for these results.
(15) (16)
CONCLUSIONS We have introduced pentafluorobenzenesulfonyl chloride (PBSC) as a new electrophoric reagent, particularly for stable labeling of a phenolic hydroxy such as that occurring in a tyrosyl peptide. Both a dipeptide and tripeptide of this type are successfully analyzed by GC-ECD when these substances are converted to N”-pivaloyl-O-PPS ethyl ester derivatives, and inert GC equipment is used. Variations in response for these compounds raise interesting questions concerning the contributions of sensitivity, recovery, and highly selective surface effects that are pursued elsewhere (19). On the basis of our results, the strategy of electrophore-labeling and non-
(18)
(17)
(19)
(20) (21) (22)
Lipsky, S. R.; McMunay, W. J. J. Chromatogr. 1082, 239, 61-69. Stewart, J. M. I n “The Peptides”; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1981; Vol. 3, pp 170-201. Blau, K.; King, 0. S. “Handbook of Derivatives for Chromatography”; Heyden: London, 1978; pp 117, 201-233. Hattox, S. E.; McCloskey, J. A. Anal. Chem. 1974, 46, 1378-1383. Schomburg, G. J. Chromatogr. 1084, 14, 157-177. Grob, K. HRC CC,J . Hlgh Resolut. Chromatogr. Chromatogr. Commun . 1080, 3 , 585-586. Landowne, R. A,; Lipsky, S. R. Nature (London) 1083, 199, 141-143. Walie, T.; Ehrsson, H. Acta Pharm. Suec. 1970, 7 , 389-406. Janousek, B. K.; Brauman, J. I. I n “Gas Phase Ion Chemistry”; Bowers, M. T., ed.;Academic Press: New York, 1979; Vol. 2, pp 53-86. Moffat, A. C.; Horning, E. C.; Matin, S. B.; Rowland, M. J. Chrometogr. 1072, 66, 255-260. Nazareth, A.: O’Connell, K.; Sentissl, A.; Giese. R. W. J. Chromatogr.. in press. Dlzdaroglu, M.; Simlc, M. G. Anal. Biochem. 1980, 108, 269-273. Peralta, E.; Yang, H.-Y. T.; Hong, J.; Costa, E. J. Chromatogr. 1980, 190,43-51. Sentlssi, A.; Joppich, M.; Glese, R. W., manuscript In preparation.
RECEIVED for review April 2, 1984. Accepted July 6,1984. This work was supported by Oak Ridge Subcontract No. 19X4335C from the Reproductive Effects Assessment Group of the EPA, and NCI Grant CA35843. This is Publication No. 201 from the Barnett Institute.
