Critical Experimental Parameters in Gas Chromatographic-Mass Spectrometric Analysis of Oligopeptide Hydrolysates at the Picomole Level Willi Frick, Ding Chang, and Karl Folkers Institute for Biomedical Research, University of Texas at Austin, Austin, Texas 78712
G. Doyle Daves, Jr." Department of Chemistry, Oregon Graduate Center, Bea verton, Oregon
97005
A method for gas chromatographlc-mass spectrometric (GC-MS) analysis of amino acids In hydrolysates of low plcomole quantities of oligopeptides is described. The method utilizes trifluoroacetyl n-butyl ester derlvatives and involves selected ion monitorlng of the gas chromatographic effluent. Analysis of amino acids released by hydrolysls of picomole quantities of ollgopeptides requires strlngent precautlons to prevent fortuitous introduction of amlno acid or peptide containing contaminants into the sample. I n the present study, such contamination was minimized by use of vacuum line and microtechniques Involving solution and reaction mlxture volumes of - 1 1L. It was also important to protect the gas chromatographic column by means of a solvent/reagent vent valve from reactive reagents present In the amlno acid derlvatizatlon reactlon mixture.
and solvent transfer methods which are based on the work of Burzynski (6,7). This report details the techniques we have devised for these exacting microanalyses and describes their use for analyses of representative oligopeptides.
Hypothalamic hormones occur in very low concentrations and as a consequence, large numbers (50000-500000) of hypothalami must be pooled and processed to obtain quantities of hormones adequate for structural elucidation. The availability of nanogram quantities of biologically active fractions which appeared homogenous based on chromatographic criteria ( I , 2) led us to seek a method for amino acid analysis which is suitable for use when the total peptide sample available is 1-100 pmol (0.5-100 ng). To this end, we have developed techniques based on gas chromatographymass spectrometry (GC-MS) which permit hydrolysis, derivatization, and amino acid analysis of 25-50 pmol of a decapeptide. We have found that successful accomplishment of amino acid analysis of low picomole quantities of oligopeptide hydrolysates by GC-MS requires that two critical problems, not encountered when nanomole quantities of peptide are available, be overcome. These two problems-(a) avoidance of the fortuitous introduction of amino acids (or protein) into the sample during processing and (b) maintenance of the required high performance characteristics of a gas chromatographic column despite the necessity of injecting highly reactive reagents (e.g., trifluoroacetic acid, trifluoroacetic anhydride) used for or formed during amino acid derivatization-were recognized earlier by Gehrke and associates (3-5) during amino acid analyses of lunar samples. The technique we have used to avoid gas chromatographic column degradation is essentially that of Zumwalt et al. (4) and involves use of a bypass valve and a precolumn to remove solvent and reagents prior to adsorption onto the chromatographic column. To avoid fortuitous contamination of our samples with extraneous amino acids we have developed specialized microtechniques including vacuum-line reagent
EXPERIMENTAL Materials and Instrumentation. Methanol was heated under reflux over Mg-turnings and a trace of iodine for 6 h and then distilled. n-Butanol was glass-distilled from magnesium sulfate; dichloromethane was distilled from P205. Acetyl chloride, trifluoroacetic anhydride (TFAA) and 6.09 M hydrochloric acid (constant boiling HCl) were repeatedly distilled before use. Hydrolysis and esterification reactions were carried out in Teflon-sealed vials as described (8) or were run in capillary tubes (length, 126 mm; i.d., 0.28 mm). Trifluoroacylation was accomplished in 1-mm (i.d.) capillaries. AU capillaries were annealed at 600 "C for at least 3 h before use to remove organic residues. Gas chromatography-mass spectrometry (GC-MS)experiments were carried out using a DuPont 21-491B mass spectrometer interfaced with a Varian 2700 gas chromatograph. This system is equipped with a four-channel DuPont MSID accessory for selected ion monitoring (SIM) analyses. Outputs from the gas chromatograph-flame ionization detector and the selected ion monitors were recorded on a Gould Brush 260 six-channel recorder. The 1.8-m glass gas chromatography column (i.d., 2 mm) was equipped with an all glass solvent/reagent vent system (Figure 1, right) functionally equivalent t o the more elaborate device described by Gehrke ( 4 ) and was packed with 3% OV-210 on Gas-Chrom Q 80/100 mesh. The pre-column between injection port and cold spot was packed with 1.5% OV 101 on Gas-Chrom Q 100/120 mesh (packingmaterials obtained from Applied Science Laboratories). Peptide Hydrolysis. A capillary (id., 0.28 mm; o.d., 1.4 mm) was sealed at one end and a small bulb (-2.5-mm 0.d.) was blown 8 mm from the sealed end (capillary in Figure 1, left). After annealing the capillary, 0.5-1pL of an aqueous peptide solution containing 25-50 pmol of peptide was transferred into it with a micropipet. The bulb in the capillary prevented the solution from creeping back along the pipet and the inner wall of the capillary. The sample was moved to the sealed end of the capillary by centrifugation. The capillary was then attached to a vacuum line as shown (Figure 1,left) with the solvent/reagent reservoir empty and the water was evaporated under cooling of the capillary tip in ice water. Then 0.75 WLof 6 N hydrochloric acid was placed in the capillary using a micropipet. After centrifugation of the reagent to the sealed end and repeated flushing with nitrogen, the open end of the capillary was sealed. Repeated inversion of the capillary followed by centrifugation assured thorough mixing of the peptide with the aqueous hydrochloric acid. The hydrolysis was then accomplished by immersion of the capillary in an oil bath (110 "C) for 18 h. After cooling and centrifugation of the reaction mixture to the opposite end, the end of the capillary possessing the bulb was broken off and the aqueous solution was evaporated using the vacuum line as described before. To remove traces of water from the hydrolysate, the capillary was left on the vacuum line at 40 "C overnight. ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
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Flgure 1. Left. Vacuum line manifold used for solventlreagent transfer to capillary reaction vessel. I t is used also for evaporation of solvents from capillaries when the solventlreagent reservoir is empty. Middle. Schematic diagram showing procedure for coupling small (0.28-mm i.d.) capillary containing dry amino acid-n-butyl ester mixture to large (1-mm i.d.) capillary containing acylation reagents. Right. Schematic design of simplified ( 4 ) all-glass solventheagent vent system for gas chromatography
'
Esterification of Amino Acids in Peptide Hydrolysates. Using the vacuum line apparatus (Figure 1, left), 0.8 p L of methanol was condensed into the capillary. To accomplish this transfer, it was necessary to heat the methanol in the reservoir to near its boiling point while the tip of the capillary was cooled with liquid nitrogen. The capillary was then removed from the vacuum line, the open capillary end was covered, and the methanol was centrifuged to the sealed end. Then, in similar fashion, 0.2 WLof acetyl chloride was condensed into the capillary. After centrifugation, the capillary was sealed and the esterification reaction mixture was mixed thoroughly by repeated centrifugation. Esterification was accomplished by heating the capillary at 50 "C for 30 min. After cooling to room temperature and centrifugation, one end of the capillary was broken off and the solvent was evaporated as described. Transesterification of the amino acid methylesters to n-butyl esters was done similarly by using n-butanol instead of methanol and a reaction temperature of 100 "C. Following completion of the reaction, solvent was removed as described and the capillary was left on the vacuum line overnight at 40 "C to ensure complete removal of n-butanol. Acylation of the Amino Acid n-Butylesters. For the acylation reaction, a larger capillary was used so that the solution containing the derivatives could be removed by syringe for injection into the gas chromatograph for GC-MS analysis. This capillary (id., 1 mm) contained a bulb (o.d., 3.5 mm) about 6 mm from the sealed end. After annealing and flushing the capillary with nitrogen, 2.5 pL of dichloromethane and 2.5 FL of TFAA were placed into it with a micropipet. The capillary containing the mixture of amino acid n-butyl esters was then joined to it with a piece of shrinkable Teflon tubing (Figure 1, middle). To achieve effective mixing, the dichloromethane/TFAA solution was centrifuged into the end of the capillary containing the esterified sample, sonicated for 1 min, inverted, and centrifuged again. This procedure was repeated ten times. Then, with the solution in the larger capillary, it was sealed and placed in an oil bath at 150 "C for 5 min. The capillary was opened under cooling just before injection into the gas chromatograph. GC-MS Analysis of the Trifluoroacetyl-n-butylesters. Analyses were accomplished using the instrumentation described modified to include the solvent/reagent vent system shown (Figure 1,right). Prior to analysis, the gas chromatographic column was equilibrated using a mixture containing 10 nmol of each selected amino acid derivative prepared as described (8). Before sample injection, the oven of the gas chromatograph was stabilized at 50 "C and the "cold spot" was cooled with a stream of air. Then 1242
ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
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Figure 2. Three-channel selected ion monitoring (SIM) program for analysis of 17 trifluoroacetyl amino acid-n-butyl esters. The column emergence temperatures of the various amino acid derivatives, the points at which changes are made in the ions monitored, and the assignment of ions to SIM programs (I or 11) and to ion monitoring channels (A, B, or C) are indicated. In program I, a fourth ion channel is monitoring a perfluorokerosene reference ion at m l e 119; in program 11, the reference ion monitored is m l e 193. See Table Ifor identification of the specific ions selected for monitoring
the solvent/reagent vent valve was opened and the sample (0.51.5 p L ) was injected. After 45 s, the solvent/reagent vent valve was closed, the air stream at the cold spot was stopped, and the gas chromatograph heating program (8"/min) was started. At 100 "C oven-temperature, the mass spectrometer ion current was started and the sample was analyzed according to the program shown schematically in Figure 2 using three different ion monitoring channels A, B, and C (a fourth channel was used for a fluorocarbon reference ion). Two sample injections (and two ion monitoring programs, I and 11, Figure 2) were necessary to analyze for all selected amino acids (Figure 2, Table I).
RESULTS AND DISCUSSION We elected to use GC-MS techniques for amino acid analysis of the picomole quantities of hypothalamic oligopeptide hormones available to us because of the high sensitivity and specificity inherent in mass spectrometric detection. Surprisingly, little experience with GC-MS analysis of amino acids has been reported in the literature; the available reports,
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R IRF: Figure 3. Selected ion recording for a GC-MS analysis resulting from injection (-20%) of the derivative mixture prepared using 50 pmol of the decaoeDtide listed in Table 11. IV)retention times of the derivativesof amino acids of the peptide (omitting Trp destroyed by hydrolysis);(V)retention times for other amino acid derivatives included in the analysis program E'" k t -
although not addressing all of the limiting constraints of our analytical problem, were highly encouraging. Abramson et al. (9) used trimethylsilyl derivatives and demonstrated that, in favorable instances, detection of femtomole quantities of amino acid derivatives is feasible by GC-MS selected ion monitoring (SIM) techniques. Summons and co-workers (10) used a GC-MS computer system for the analysis of 12 amino acids in biological fluids. In contrast, gas chromatographic methods for analysis of amino acids have been well studied ( I 1 ) and analytical procedures, developed principally by Gehrke and colleagues (8),are convenient for applications in which nanomolar quantities of amino acids or peptides are available. I t has been recognized (4-6), and we confirmed in initial phases of the current study, that protocols for nanomole level analyses are inadequate for use when only picomole samples are available. As a result, we undertook a systematic study to determine which experimental parameters are critical and to make necessary procedural modifications. For our study, we used the conventional procedure of peptide hydrolysis-heating with 6 N hydrochloric acid a t 110 "C for 18 h (12). This procedure, although it has well known limitations (8), is convenient, gives reproducible results and, unlike more sophisticated methods which spare sensitive amino acids (13), utilizes only volatile, readily purified reagents. Similarly, we elected to use standard amino acid derivatization techniques involving preparation of n-butyl (14) or isoamyl (15, 16) esters followed by acylation using heptafluorobutyric (15-1 7) or trifluoroacetic (14,18)anhydrides. The more conveniently prepared trimethylsilyl derivatives were not selected for our use owing to experimental difficulties experienced by several investigators (11, 18, 19). In Table I are experimental data concerning the GC-MS behavior of 18 trifluoroacetyl amino acid n-butyl esters. This derivative was employed in our fully developed procedure after initial work using heptafluorobutyryl isoamyl ester and trifluoroacetyl isoamyl ester derivatives. It is noteworthy that, in contrast to the experience of a number of other workers
Table I. Experimental Parameters for Gas Chromatography-Mass Spectrometry Selected Ion Monitoring (SIM) Method for Analysis of Trifluoroacetyl Amino Acid n-Butyl Esters Ion Relative Relative ion SIM retention monitored Amino acid time' ( m / e ) b intensity' programd Ala 0.46 140 2.5 I-A Gly 0.49 126 0.9 I-B 0.51 168 1.6 Val I-c Thr 0.53 153 1.6 I-A Leu/Ile 0.55 182 0.9 I-c Ser I-B 0.57 139 1.6 Pro 166 3.4 0.65 I-A Met 0.69 22Te 0.2 11-A Phe 0.4 0.72 204= 11-B 184e, 240 0.6 0.74 I-c, 11-c ASP His 302e 0.03 0.79 11-A Glu 180e, 198 1.00 0.83 I-B, 11-B 260e 0.9 0.84 11-c TYr 180 1.4 0.92 I-c LYS 1.00 166e 0.3 I-A Arg 226e Trpf cysg
240e
Typical values obtained using column and experimental parameters as described in Experimental. Except as noted, ions are those described by Summons et al. (1 0). Amino acid derivatives determined in SIM Program I are normalized to Glu m / e 180; SIM Program I1 values are normalized to Glu m l e 198. For Asp, only the SIM I result is given. Values recorded are averages of 3-11 determinations involving both amino acid mixtures and peptide hydrolysates and injections of 1-100 M. Coefficients of variation are in the range 5-25%. 'See Figure 2 for SIM program details. e Met, C,H,,NO,F,; Phe, CI,HI,O,; Asp, C,H,NO,F,; His, C,H,N,O,F,; Glu, Not inC,H,NO,F,; Tyr, C,,H,O,F,; Arg, C,H,NOF,. cluded in SIM Program because Trp is destroyed during hydrolysis of peptides (8). Not included in SIM Program because of high column temperature (> 250 " C ) required. a
(10,11,15,20),we found histidine to be observed reliably using this method, although the monitored ion intensity was much ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
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