Anal. Chem. 1995, 67,4000-4003
Effect of Hydrogen Rearrangement on the Determination of the Enrichment of [15N]Leucine by GC/MS Annabelle m a y , Bien Dang-Vu, Jean ChrSstophe Moreau, and Franqois Guyon*
Laboratoire de Chimie Analytique, Facult& de Pharmacie. Universith Rem Descartes, 4 avenue de I'Obselvatoire, 75270 Paris cedex 06, France
In the determination of the enrichmentof [15Nlleucineby GUMS, the measured ratio of 15/14N-labeledleucine may be affected by H rearrangement. This effect was investigated using 11 esters of I5N-labeled and nonlabeled N-(heptafhorobutyry1)leucine. The H rearrangement is dependent on the nature of the alcohol used for the esterification. The labeling ratio increases with the length of the alkyl chain of the ester and with the number of the H atoms at the #&siteand, to a lesser extent, at the y-site on this chain. For the measurement of the enrichment of [15Nlleucine,better standard curves were obtainedwhen ion fragments not affected by H rearrangement were used. Gas chromatography/mass spectrometry (GC/MS) is a selective and rapid method for the determination of the abundance of [lSNlleucineand other amino acids in isotopic tracer In these experiments, mixtures of 1s/14N-labeledleucine are transformed into volatile esters of N-(heptafluorobutyry1)leucine by a two-stage derivatization before GUMS analysis.' Selected ions containing an atom of nitrogen are monitored and quantified. The ions from lSN-labeledleucine can be discriminated from the corresponding ones coming from nonlabeled leucine by a shift of the m/z values by +1 mass unit as a result of the replacement of an atom of I4N by an atom of 15N. The relative area counts of the associated ions are then used to calculate the [lSNlleucine abundance. However, a shift by +1 mass unit, which is supposed to characterize [15N]leucine,may also occur when an ion containing an atom of I4N captures an atom of hydrogen by rearrangement. When the mass spectrometer has low resolution, it cannot discriminate between an ion containing an atom of IsN and an isobaric ion containing an atom of I4N and a captured H atom. This may affect the determination of the abundance of [lsN]leucine. As H rearrangements are frequently encountered with esters of carboxylic acids5and are dependent on the nature of the alcohol used for the formation of the ester, we prepared the esters of N- (heptafiuorobutyry1)leucine using 11 different alcohols and investigated their influence on the determination of the abundance of [lSN]leucinein different mixtures of labeled and nonlabeled leucine. (1) Coulter, J. R; Hann, C. S. J. Chromatogr. 1968,36,42-49. (2) Adams, R. F. J. Chromatogr. 1974,95,189-212. (3) Rhodes, D.; Myers, A C.; Jamieson, G. Plant Physiol. 1981,68,11971205. (4) Golan-Goldhirsh, A.; Hogg, A. M.; Wolfe, F. H. J Agn'c. Food Chem. 1982, 30,320-323. (5) McLafferty, F.W. Anal. Chem. 1959,31, 82-86.
4000 Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
EXPERIMENTAL SECTION Materials and Reagents. The GC/MS system was a Fisons Instruments Model MD 800 with an 8000 series gas chromatograph. The capillary column used was a DB-5 (30 m x 0.25 mm i.d.) J&W Scientific instrument from Interchim (Paris, France). L-Leucine, acetyl chloride, 2-butanol, and 2-methyl-2-propanolwere obtained from Labosi (Paris, France); [lSNlleucinewas from S i a (St. Quentin Fallavier, France); heptafluorobutyric anhydride (HFB) was from Fluka (St. Quentin Fallavier, France); ethanol and 2-propanol were from Carlo Erba (Rueil Malmaison, France); 2,2-dimethyl-l-propanolwas from Aldrich (St. Quentin Fallavier, France); 1-butanolwas from Merck (Paris, France); and methanol, 1-propanol,2-butanol,n-pentanol,n-hexanol, and ethyl acetate were from Prolabo (Paris, France). Procedures. Leucine was derivatized according to the procedure of MacKenzie and T e n a s c h ~ kwith ~ , ~ minor modifications: mixtures of labeled leucine and nonlabeled leucine at different concentrations (10 pg/mL) in aqueous solution were evaporated under a flow of dry nitrogen. To the residues was added 0.5 mL of a freshly prepared solution of alcohol-HC1 (1 vol of acetyl chloride mixed with 5 vol of ice-cold alcohol at 4 "C). The sealed vials (closed with a Tefloncoated cap) were vigorously shaken and then heated to 110 "C for 30 min. When they were cooled, excess alcohol-HC1 was removed under a flow of dry nitrogen. HFB (50 pL) was added. The vials were sealed and heated anew at 60 "C for 30 min. The cooled samples were evaporated under dry nitrogen, and the ethyl acetate (500 pL) was added. The sealed vials were vigorously shaken to ensure complete dissolution. Next, 1 pL of the solution of derivatized leucine mixture was injected, using a CTC MOOS autosampler injector. GUMS analysis was performed under the following conditions: inlet temperature, 250 "C; detector temperature, 280 "C; oven temperature, 6 min at 130 "C, then 15 "C/min to 210 "C (run time, 12 min). RESULTS AND DISCUSSION Results. Under the adopted GC conditions, the retention times of all the esters were less than 10 min. The mass spectra of 1-propyl N-HFB and 2-propyl N-HFB esters of leucine are represented in Figure 1. They show the same major ion fragments at m/z 240, 241, 282, and 283, which appear also in the mass spectra of other amino acid derivatives. These ions contain an atom of nitrogen and may have the structures presented in Figure 2. A possible mechanism for the formation of ion fragment at (6) Mackenzie, S. L.; Tenaschuk, D. J. Chromatogr. 1974,97,19-24. (7) Mackenzie, S. L.; Tenaschuk, D. J. Chromatogr. 1979,171,195-208.
0003-2700/95/0367-4000$9.00/0 0 1995 American Chemical Society
240.00
I
283.00
m/Z Figure 1. Mass spectra of two N-HFB esters of nonlabeled leucine: (a) 1-propyl [14N](heptafluorobutyryl)leucineand (b) 2-propyl [14N](heptafluorobutyryl)leucine.
area % ratio (R;)versus the molar % enrichment (E),with i = 241 or 283. Ri is calculated using eq 1, where Ai is the peak area of the ion fragment at m/z 241 or 283, and Ai-1 is the peak area of the mlr 202
0
0
mlz 240
(Aj +AiAj-1 )
Ri=
x
100
ion fragment at m/z 240 or 282. E is known from the composition of the samples and is calculated using eq 2, where ['5NNLeulis the
Figure 2. Possible structures of two major ion fragments of esters of N-(heptafluorobutyry1)leucine containing an atom of nitrogen.
m/z 282 has been described previously.* The formation of an ion fragment at m/z 240 may imply a McLafferty rearrangement: For the determination of 15N abundance, one can use either fragment 240 or fragment 282. For [15Nlleucine,the masses of these fragments were shifted by 1 mass unit. The ratio of the concentration of [15N]leucine to that of [14N]leucinemay be assumed to be proportional to the ratio of the peak area of the ion fragment at m/z 283 (or m/z 241) to that of the ion fragment at m/z 282 (or 240). Standard curves were drawn by plotting the (8) Silverstein, R. M.; Bassler, G. C.; Momll, T. C. Spectrometn'c Ident$cafion oforganic Compounds; John Wiley & Sons, Inc.: 1991;p 38. (9) Budzikiewicz, H.; Djerassi, C.; Williams, D. H. Mass Specfromety of Organic Compounds; Holden-Day, Inc.: San Francisco, CA, 1967; p 158.
concentration of labeled leucine and ['4NNLeulis the concentration of nonlabeled leucine. Table 2 shows the intercepts, slopes, and correlation coefficients of the standard curves for esters of N-(heptafluorobutyry1)leucine obtained using either the two ion framents at m/z 240 and 241 or the two ion fragments at m/z 282 and 283. According to the structures of ions at m/z 240 and 282 (Figure 2) and because of the natural abundances of 15N, I3C, and 2H, ions at m/z 241 and m/z 283 will also be present in the spectra of nonlabeled leucine derivates. For nonlabeled leucine, the relative intensities of these ions can be predicted and are expected to be independent of the nature of the alcohol used for N-(heptafluoAnalytical Chemistry, Vol. 67, No. 21, November 1, 1995
4001
different from the ones observed with ions at m/z 240 and 241, each ester gave a different intercept, which may be as low as 11.2 and as high as 27.2. Discussion. The intercept represents the peak area % ratio found with nonlabeled leucine. Theoretically, it should be independent of the nature of the alcohol used for the esterification of N-(heptafluorobutyry1)leucine and must be close to the predicted value calculated on the basis of the natural abundances of the higher isotopes of C, H, and N. This is the case when the two ions at m/z 240 and 241 are used: the measured values of the intercept are practically the same for the two alcohols (Rz41= 6.7 f 0.3%) and are close to the predicted value of R241 = 6.96%. However, when the two ions at m/z 282 and 283 are used, the ~ very different from the predicted value (R283 intercepts ( R zare = 9.33%). They depend on the nature of the alcohol used for the derivatization of leucine. Thus, it is not possible to assume that the ion fragment at m/z 283 is exclusively an isotopic ion of ion fragment at m/z 282, generated by replacement of one atom of I2C, 'H, or I4N by one atom of the corresponding higher isotope. It must also have other origins which depend on the nature of the derivates. As the mass of an ion fragment is shifted by 1unit when it captures a H atom by rearrangement, the dependence of R283 on the nature of the derivatives may be an artifact due to H rearrangement. This rearrangement is frequently encountered with esters of carboxylic acids5 and is dependent on the nature of the alcohol used for the formation of the esters. We suggest a probable mechanism for the formation of ion at m/z 283 through a two-steps H rearrangement (Figure 3). As H rearrangement is not observed for methyl esters of of the carboxylic acids, we have measured the area % ratio (&3) ion at m/z 283 and 282 using the methyl ester of nonlabeled N-(heptafluorobutyry1)leucine. We found effectively for R283 the value of 9.65%,which is close to the theoretical value of 9.33%. The experimental value of R283 for other esters of N-(heptafluorobutyry1)leucine are presented in Table 3, along with the number of H atoms and their location on the a b 1 chain of the esters. It can be noted from Table 3 that R283 increases, as expected, with the number of hydrogens in the p-site. This is very likely due to a sixcenter cycle McLafferty rearrangement, which is favored by an increase in the number of hydrogens at the p-site. Variation of R283 versus the number of hydrogens at the p-site is of second order (Figure 4). For the normal chain esters, which contain the same number of hydrogens at the p-site,R2a increases also as a second-order equation with the length of the chain as measured by the number of carbon atoms on the chain (Figure 5). That may be explained by an inductive effect of the alkyl chain, which may influence differently the H rearrangement. It is to be noted that in 2,2-dimethyl-l-propanol,there is no H atom at the p-site; the rearrangement must come through a sevencenter cycle
I mlz = 283 Figure 3. Suggested mechanism for the formation of the ion fragment at m/z 283 by H rearrangement.
Table 1, Influence of the Nature of the Alcohol Used for the Esterlflcatlonof Nonlabekd Leuclne on RI ( I = 241 or 283)
alcohol used for derivatization 1-propanol, CH3(CH2)20H (predicted value)" 2-propanol, (CH&CHOH (predicted value)=
&a3
(%)
12.8
R241
(%)
(9.33)
6.4 (6.96)
(9.33)
(6.96)
26.9
6.4
nValue calculated on the basis of the natural abundance of the higher isotopes of C, H, and N.
robutyry1)leucineesterification. We found that this is true when the two ion fragments at m/z 240 and 241 were used, but not when the two ion fragments at m/z 282 and 283 were used. Table 1 illustrates this difference. The area % ratios Rj,in the case of m/z 241, are the same regardless of whether esterification is carried out with 1-propanolor 2-propanol,whereas in the case of m/z 283, the Ri values are different (Table 1). A similar observation may be made in the case of the standard curves. When the ions at m/z 240 and 241 are used, all the data fit the same curve: R241= 6.7 0.69E. When the standard curves were drawn on the basis of the peak area of ion fragments at m/z 282 and 283, each alcohol led to a different curve: R283 = 12.58 0.66E for the 1-propyl N-HFB ester of leucine and R283 = 27.22 0.57E for the 2-propyl N-HFB ester of leucine. We present in Table 2 the slopes, the intercepts, and the correlation coefficients of the standard curves for the ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, and 2,2-dimethyl-l-propylesters. All the standard curves obtained with the two ions at m/z 240 and 241 have high correlation coefficients (Z > 0.745) and not very different slopes (0.61-0.76) and intercepts (6.6-7.2). On the contrary, when the two ions at m/z 282 and 283 are used, the correlation coefficients are lower (0.92 > Z > 0.41). Although the slopes are not very
+
+
+
Table 2. Dependence of the Intercepts, the Slopes, and the Correlation Coefficients of the Standard Curves on the Nature of the Ester of W(Heptafluorobutyryl)leuclne and on the Choice of the Ions (at m/z 240 and 241 or at mlz 282 and 283)Used To Measure Rj
ethanol
1-propanol
2-propanol
1-butanol
2-butan01
m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z 282, 283 240,241 282,283 240,241 282,283 240,241 282,283 240,241 282,283 240, 241
intercept slope corrcoeff, 12
11.17 0.46 0.41
7.20
12.58
0.69
0.66
0.78
0.82
6.80
0.62 0.75
27.22 0.57 0.45
4002 Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
6.63 0.77 0.90
15.56
0.57 0.79
6.65 0.76 0.92
17.18 0.69 0.72
6.87 0.66 0.86
2,2-dimethyl-l-propanol m/z
m/z
282,283
240,241
26.63
6.73
0.47 0.74
0.70 0.87
Table 3. Variation of Rm3 (YO) with the Number and the Position of Hydrogens In the Ester Chain
no. of hydrogens /3-site y-site
alcohol used for esterification
0 3 6 9 2 1 0
methanol ethanol 2-propanol 2-methyl-2-propanol 1-propanol 2-b~tan0l 2,2-dimethyl-l-propanol
R(%) 30
R283
(%) (exptl)"
9.65 10.80 26.85 54.51 12.76 17.36 26.37
0 0 0 0 3 6 9
0 The value for R28 calculated on the basis of the natural abundance of the higher isotopes of C, H, and N is 9.33%,regardless of the nature of the alcohol used for esterification.
R(%)
T
20
-
15
-
y = 0,2x2 + 1,6x + 8,4
5t 0
0
T
1
2
3
4
5
6
Number of C atoms Figure 5. Variation of &83 with the number of carbon atoms in the normal chain of nonlabeled N-(heptafluorobutyry1)leucineesters.
a six-center cycle is more important than that through a sevencenter cycle. The values of R283 for 2-methyl-2-propanol (9 H in the #?-siteand 0 H in the y-site, R ~ S = 54.51%)and for 2,2-dimethyl1-propanol (0 H in the ,%site and 9 H in the y-site, R283 = 26.37%) proved this (Table 3). CONCLUSION
0
2
4
6
8
I 10
Number of H in C, Figure 4. Variation of leucine esters.
R283
with the number of p-site H of nonlabeled
and not through a six-center cycle. Although the number of hydrogens at the p-site decreases from ethanol to 2,2-dimethyl1-propanol, R283 increases, as does the number of hydrogens at the y-site. When H atoms are present at both the #?-siteand in the y-site, the two rearrangements contribute to the increase of R283. Contribution of the H atoms in the y-site is less important than that of H atoms in the p-site. McLafferty rearrangement through
Our results show that H rearrangement may affect the determination of [15Nlleucine abundance in isotopic dilution experiments. This rearrangement depends quantitatively on the nature of the alcohol used for the esterification of leucine. Better results and better standard curves were obtained when they were based on the use of the two ion fragments at m/z 240 and 241 instead of the more frequently used ion fragments at m/z 282 and 283, which may be affected by H rearrangement. Further investigations are needed to ascertain the formation of the ion fragment at m/z 283, for instance, through the use of 2H-labeled alcohol for the esterification of leucine. Received for review May 8, 1995. Accepted July 26,
1995.B AC950434Q @Abstractpublished in Advance ACS Abstracts, September 1, 1995.
Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
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