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Determination of Stable Isotopic Enrichment in Individual Plasma Amino Acids by Chemical Ionization Mass Spectrometry D. E. Matthews," E. Ben-Galim, and D. M. Bier Departments of Medicine and Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 631 10
Using chemical ionization-selected ion monitoring-gas chromatography-mass spectrometry (CI-SIM-GCMS) of the N-acetyl, n-propyl ester amino acid derivatives, isotopic enrichment can be measured in individual plasma amino acids regardless of choice of label (I5N, I3C, "0, *H) or position in the molecule. Results using "N-labeled amino acids show that CI-SIM-GCMS can detect 0.08 at. % excess isotope in plasma amino acids from less than 100 FL of plasma,
Since Schoenheimer and Rittenberg first reported the measurement of nitrogen-15 enrichment in amino acids after feeding '%-ammonia to rats ( I ) . nitrogen-15 labeled compounds have played an important role in investigating amino acid metabolic pathways. Yet, quantitative information concerning in vivo amino acid transamination rates is still not available. Although the sum of 15Nenrichments of all amino acids in a plasma sample can be measured easily, there is no convenient technique for measuring I5N enrichment (or 13C, l8O, or 'H enrichment for that matter) in each individual amino acid in a physiological sample. The only study to report 'jNenrichments in each of the amino acids in man was the study by Giordano et al. ( 2 ) who administered "N-urea to normal and uremic subjects and measured the incorporation of 'jN into the amino acids of serum albumin. For each hydrolyzed serum albumin sample, these workers separated the amino acids using fraction collecting liquid chromatography, reacted each amino acid separately to N2, and then, measured each Nzsample for '% enrichment using a dual-inlet dual-collector isotope ratio mass spectrometer. For a single sample, the entire process requires more than 50 hours effort. An alternative to liquid chromatography for the separation of the amino acids is gas chromatography. When combined gas chromatography-mass spectrometry (GCMS) is used, the amino acids can be separated and their isotopic enrichments measured in one step. While good in theory, this idea has not been widely applied in practice. Summons et al. ( 3 ) ,and Shulman and Abramson ( 4 ) have used GCMS to quantitate amino acids by adding deuterated amino acids as internal standards, requiring the measurement of the ratio of natural (unenriched) amino acid to deuterated amino acid. These workers have been able to obtain a precision of slightly better than 1070. The variance is due to several factors-principally, the mass spectral noise associated with monitoring amino acid fragment ions. For most amino acids, the molecular ion is very weak, and the more intense fragment ions are less than half the mass of the molecular ion. In this low-mass fragment-ion range, either fiormal contaminants of physiological samples, other amino acids, or GC column bleed can produce peaks a t the same nominal mass. For a particular amino acid, the analysis problems can be recognized, conditions optimized, and an isotopic enrichment determined with a precision of 1-2% (51, but optimum conditions cannot be generally maintained when multiple amino acids are measured. 0003-2700/79/0351-0080$01 .OO/O
If the mass spectra of the amino acids could be simplified, the stringent requirements for optimum conditions could be relaxed, allowing "N to be measured in multiple amino acids with the same precision obtained when conditions are optimized for measuring only one. Two solutions to this problem have been devised. The first has been reported recently by Matthews and Hayes (6). These authors placed a combustion oven in between the gas chromatograph and mass spectrometer to convert each amino acid emerging from the gas chromatograph to Nz, COz, and HzO before entering the mass spectrometer. In this way, the technique, termed "isotoperatio-monitoring gas chromatography-mass spectrometry" (IRM-GCMS), avoids the complex electron impact fragmentation pattern of the amino acids by measuring I3C enrichments from COS,and "N enrichments from N2 for all the amino acids. The second method of avoiding the complex amino acid mass spectra is to use a "gentler" form of ionization, for example, chemical ionization using methane as the reactant gas. The N-acetyl, n-propyl ester amino acid derivatives form several ion molecule reaction products when methane chemical ionization is used: an intense molecular ion with a proton attached, [M + HI+; other cluster ions, [M + C2H5]+and [M + C3H5]+;and a few smaller fragments (7). Figure 1 compares the electron impact (EI) and the methane chemical ionization (CI) mass spectra of N-acetyl, n-propyl leucine. In contrast to the strong chemical ionization [M + H]+ ion for N-acetyl, n-propyl leucine, no molecular ion can be seen in the electron impact mass spectrum-consistent with published spectra of other leucine derivatives (8,9). Not only are the mass spectra of leucine and other amino acids simplified by chemical ionization, but also are the spectra of the background and other minor contaminants present in physiological samples. Furthermore, the lower intensity cluster ions [M + CzH5]+ and [M + C3Hj]+,can be monitored in place of the [M + H]+ ion for concentrated amino acids in a sample, thus retaining the advantages of monitoring the molecular ion while, at the same time, attenuating concentration differences between amino acids. This scheme allows stable isotopic enrichment of multiple plasma amino acids to be measured in a single gas chromatographic run even though greater than an order of magnitude concentration difference between amino acids is encountered. This paper describes the use of chemical ionization-selected ion monitoring-GCMS (CI-SIM-GCMS) to measure stable isotopic enrichment in amino acids, and compares this technique with the IRM-GCXIS method.
EXPERIMENTAL Instrumentation. The chemical ionization-GCMS system used is a Finnigan 3300 dual EI/CI quadrupole mass spectrometer under computer-control. The computer system and software have been previously described (10). The interface between the gas chromatograph and the mass spectrometer is direct. Included in the transfer line is a short tee t o a valve and vacuum pump. The valve is opened at the start of the GC run t o divert the majority of the solvent peak a w a y from the mass spectrometer. C 1978 American Chemical Society
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Figure 1. Mass spectra of N-acetyl, n-propyl leucine. Electron impact was used to obtain t h e u p p e r spectrum while methane chemical ionization produced the lower spectrum
computer-controlled Finnigan 3200 mass spectrometer (10). An ionizing electron energy of 70 V was always used. Gas Chromatography. Amino acids were derivatized to the ;"\I-acetyl,n-propyl esters according to the method of Adams (13). The gas chromatographic separation of the amino acid derivatives was effected using a 2 mm x 1 m glass column packed with a mixed phase (0.3% Carbowax 20M, 0.370 Silar 5CP, and 0.05% Lexan) on Chromosorb W AM: 120-140 mesh (J. Graff Associates, Santa Clara, Calif.). After sample injection, the column temperature was held at 120 "C for 2 min before programming a t a rate of 8 "C/min. At 13 min, the rate was increased to 20 OC/min until 250 "C was reached. The injector and GCMS transfer line were kept a t 250 "C. The trimethylsilyl amino acid derivatives were prepared by the procedure of Gehrke and Leimer (14) and chromatographed by temperature programming a 2 mm X 2 m glass column packed with 1070 OV-11 as previously described (14). The lysine Ndimethylaminomethylene methyl ester derivative was prepared by the method of Thenot and Horning 115) and chromatographed at 200 "C on a 2 mm X 2 m glass column packed with 3% SE-30 on Chromosorb W HP. Samples. ~-['~N]leucine (96.4% "N)was obtained from Merck, Sharpe and Dohme Canada Limited (Quebec, Canada). ~ - [ l "CC]leucine (92.7% I3C) and L-[cu-"N]lysine (97.0% 'jN)were obtained from KOR Isotopes (Cambridge, Mass.). Leucine of natural abundance was obtained from Sigma Chemical Co., (St. Louis. Mo.). Standard solutions of isotopic enrichment leucine for l3C and 'jN in the range of 0-5 at. 70excess were prepared from the above materials. A 20-kg dog was fasted for 24 hours. A polyethylene catheter was placed into a forelimb vein for constant infusion of L["N]leucine at a rate of 0.35 mmol/h for 9 h. Another catheter was inserted retrograde into the abdominal aorta by way of the femoral artery for sampling. Before the start of the infusion and 9 h thereafter, 3-mL blood specimens were drawn from the arterial catheter and the plasma was separated immediately by centrifugation a t 4 "C. The plasma was then stored a t -60 "C until preparation for GCMS by the method of Adams (13).
Methane is used both as the chemical ionization reactant gas and as the GC carrier gas. The ion source pressure (ca. 1 Torr) is set by adjusting the carrier gas flow rate (ca. 10 mL/min). The analyzer is differentially pumped to better than 10 Torr. A special ion current measurement circuit (11) is used to amplify the signal from the electron multiplier. The circuit has a tested dynamic range of 256000 and a linearity of 0.1% ( 1 1 ) . T o measure stable isotopic enrichments in the amino acids, selected ion monitoring was used instead of scanning mass spectrometry. Two masses were chosen for each amino acid; usually these were the molecular ion [M + HIt and one mass unit higher [M + H + 11' for measurement of the natural abundance species and the monoisotopically labeled species, respectively. Multiple-labeled compounds were not used in this report. The concentrations of the different amino acids can vary widely within a sample. For example, in plasma the concentration of alanine is an order of magnitude higher than the concentration of phenylalanine. To avoid taxing the dynamic range of the ion current amplifier, the less intense [ M + C,H,]+ peak was chosen for alanine (and other very intense amino acids) instead of the [M + H]+ peak. The presence of the [M + C2H5]+cluster ion allows the most concentrated amino acids in a sample to be attenuated without perturbing the ion current electronics or without losing any isotopic information. If two ions are chosen for each amino acid and 15 amino acids are measured, 30 ions would have to be monitored. The advantage of selected ion monitoring is the maximization of the time spent measuring the ions of interest-an advantage which is lost when 30 ions are monitored. Thus, the amino acids were placed into four groups (by GC retention time) and no more than a maximum of 5 amino acids or 10 ions were measured at any one time. As the GC program progresses and the last amino acid of a group elutes, the computer program automatically switches over to the next group of 4 amino acids. The EI-SIM-GCMS data for lysine in Table I1 were obtained using a PDP-12 computer-controlled LKB-9000 mass spectrometer (12). Other E1 mass spectral data were obtained using a similar
RESULTS AND D I S C U S S I O N Comparison of 15N a n d 13C measurements. Since CISIM-GCMS uses the [ M + HIf ion for isotopic measurement, a label (or labels) can be placed anywhere in a n amino acid and its isotopic enrichment determined. Under E1 conditions, this statement cannot be made. For example, isotopic enrichment in the carboxyl position of derivatized 13C-labeled amino acids is especially difficult to measure by E1 since fragmentation predominantly cleaves off the carboxyl group as part of a neutral species. Only the trimethylsilyl amino acid derivatives produce a n ion thal; contains the carboxyl group for all t h e amino acids. This ion, m / e = 218, is generated by cleaving off t h e amino acid functional group. However, the intensity of the 218 ion is weak and highly variable from one amino acid to the next (9). In contrast, the [ M + H]+ion is t h e base peak in the methane CI spectra of t h e LV-acetyl,n-propyl amino acid esters, and isotopic enrichment is easily measured. Data for CI-SIM-GCMS measurement of standard samples of known isotopic composition for ["'Nlleucine and [ l-13C]leucine are given in Table I. A regression line of t h e form: R, = a + bR, where R, is t h e observed isotope ratio and Re is the expected mole ratio, was calculated separately for t h e 15N and 13C data. T h e regression line values for t h e slope, intercept, corresponding standard deviations, and coefficient of determination are also listed in Table I. As expected, no significant difference between t h e [ '"N]leucine and [ 1-13C]leucine d a t a was found. Comparison of E1 a n d C I Measurements. When the same amino acid sample is measured by E1 and CI, the same values of isotopic enrichments should be obtained. Likewise, different amino acid derivatives should also produce the same isotopic measurements. While there are obviously poor choices of amino acid derivatives, several good alternatives exist. T h e
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, J A N U A R Y 1979 Leu
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TIME, MIN Figure 2. CI-SIM-GCMS ion chromatogram for the plasma amino acids. The chromatogram was obtained for the free amino acids extracted from a dog plasma sample and derivatized as the N-acetyl, n-propyl esters. Either the [M -I-HI+ or [M C2H5]+ion trace for each amino acid was used (see text)
+
Table I. Calibration Data for [l-’3C]leucineand [ 15N]leucineby CI-SIM-GCMSa expected observed mole ratio* isotope ratioC isotopic x 100R, x 100R, label __ 0.00 12.59 1-13c 0.425 13.05 1-13c 0.857 13.50 1-I 3c 1.713 14.31 1-’3c 4.271 17.16 ”N 0.434 13.09 lSN 0.868 13.48 I5N 1.290 13.97 I 5N 1.714 14.45 sN 2.580 15.42 5N 3.496 16.42 15N 5.144 18.02 R , = a -Ib R , a + sa b * St, r2 l-13C 12.57 c 0.04 1.067 c 0.017 0,999 lSN 12.60 t 0.03 1.067 i 0.013 0.999 The leucine was chromatographed as the W-acetyl, ngropyl ester, and the ions m/e = 216, 217 were monitored. Calculated from isotopic dilution of standard solutions. Measured from the intensity ratio of 217/216. Each point was determined in duplicate data in Table I1 compare 15N measurement for four amino acids by CI-SIM-GCMS and EI-SIM-GCMS. The CISIM-GCMS measurements used the N-acetyl, n-propyl ester amino acid derivatives; the EI-SIM-GCMS measurements used either the trimethylsilyl or the dimethylaminomethylene methyl ester amino acid derivatives. For a stringent test, the samples measured were not “clean” amino acid standard solutions, but were plasma amino acid samples taken from two different metabolism studies. In one study ~-[~’N]leucine was infused into dogs (samples 1-7 in Table 11);in the other study L- [ ~ u - ~ ~ N ] l y s was i n e infused into adult volunteers (samples 8 and 9 in Table 11). Each measurement in Table I1 was performed in duplicate or triplicate; the precision of
Table 11. Comparison of CI and E1 Methods for 15N Measurement in Plasma Amino Acidsa at. % excess 15N sample no. amino acid CI E1 1 leucine 4.29 4.23 4.93 4.91 2 leucine 0.82 0.87 3 isoleucine 0.89 0.88 4 isoleucine 0.63 0.61 5 alanine 0.64 0.61 alanine 6 7 alanine 0.84 0.89 8 lysine 0.71 0.74 9 lysine 0.83 0.81 a Each measurement was made in duplicate or triplicate. The CI measurements were made using N-acetyl, n-propyl ester amino acid derivatives. The E1 measurements were made using trimethylsilyl-leucine, isoleucine, and alanine and dimethylaminomethylene methyl lysine. Samples 1 - 7 were derived from dog plasma samples; s a m ples 8 and 9 were obtained from human plasma samples. measurement was better than &0.1 at. 7 c excess (precision will be discussed in detail later). The CI and E1 results compare very closely; the maximum difference in any measurement being 0.06 at. 70excess I5N. This is especially remarkable since comparisons for all amino acids agree even though different derivative, chromatographic, and mass spectrometric conditions were used. These results demonstrate that with proper care (use of standard curves, proper selection of chromatographic and mass spectrometric conditions, etc.), experimental results obtained either by CI-SIM-GCMS or EI-SIM-GCMS are virtually identical, as they theoretically should be. T h e authors are unaware of other literature studies that document similar quantitative comparative data for GCMS measurement of isotopic enrichment by various derivative and ionization techniques. M e a s u r e m e n t of lSN i n M u l t i p l e Amino Acids. T h e strength of the CI-SIM-GCMS method lies not in measurement of stable isotopic enrichment in a particular amino acid alone, but in measurement of enrichment in multiple
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Table 111. * SNIncorporation into Dog Plasma Amino Acids during Continuous Infusion of [ 5N]leucine amino acid Ala Val
m le isotope ratio isotope ratio monitoreda a t 0 h, X 100b a t 9 h, x 100b (201, 202) 11.236 t 0.047 11.872 t 0.062 201, 202 11.354 r 0.064 12.054 t 0.107 GlY (188, 1 8 9 ) 10.242 i 0.058 10.344 t 0.066 Ile 216, 217 12.462 i 0.063 13.464 t 0.106 Leu 216, 217 12.450 t 0.091 18.110 d Pro 200, 201 11.402 t 0.095 11.506 t 0.050 Thr 246, 247 12.460 2 0.133 12.612 i 0.053 S er 232, 233 11.370 i- 0.055 11.636 t 0.062 ASP 260, 261 13.658 i 0.093 13.748 i- 0.074 Met 234, 235 12.338 t 0.072 12.350 * 0.037 Phe 250, 251 15.672 i- 0.044 15.760 t 0.060 Glu (302, 303) 16.684 i 0.123 17.030 i 0.068 'IkY 308, 309 18.072 i 0.270 18.072 I 0.115 Orn 259, 260 13.914 i- 0.096 14.234 i 0.066 LYS (301, 302) 17.224 i 0.098 17.144 t 0.134 a m l e values in parentheses are the [M + C,H,]+ ion pair rather than the [M iHI' ion pair. n = 5. Ratio difference 5 95% confidence limits (one-tailed f-value, 8 degrees of freedom).
amino acids during a single GC run. This is illustrated by the multiple ion chromatogram for the various plasma free amino acids shown in Figure 2. Although two ions (one mass unit apart) for each amino acid are monitored, Figure 2 is simplified, showing only the lower mass ion of each pair. Adams (13)described N-acetyl, n-propyl ester formation for 21 plasma amino acids (included in that number are glutamine and asparagine which lose their amide nitrogens during derivatization and are co-chromatographed with glutamate and aspartate, respectively), although great care must be taken to obtain good derivative formation for more than 17. In our hands, the latter number is easily derivatized and shown as resolved peaks in Figure 2. Measurement of isotopic enrichment in multiple amino acids for in vivo metabolism studies requires the CI-SIMGCMS method (i) detect a t least 0.1 at. % excess isotope (corresponding to a 0.001 difference in the nominal 0.1-0.2 [ M + H + 1 ] / [ M + HI ratio), (ii) measure a wide range of ion intensities that occur from the widely varying concentrations between amino acids in biological systems, and (iii) monitor a pair of ions for each amino acid (15 different amino acids require 30 different ions). These difficulties have been resolved or simplified in the following manner: (i) an ion current amplifier after the electron multiplier with a wide dynamic range of 2.5 X lo5 is used ( 1 1 ) . (ii) The signals of the most concentrated amino acids are attenuated by monitoring either the [M + C2H5]+or [M + C,H5]+cluster ions rather than the [M + HI+ ions. These cluster ions contain the same isotopic information present in the [M + H]+ ion but are 30-5070 as intense. In Figure 2 the [ M + C2H5]+cluster ion was used to attenuate the alanine, glycine, proline, glutamate, and lysine signals. For alanine, the [M C2H5]+ion and the valine [M + H]+ ion are a t the same nominal mass, and only two ions instead of four need be monitored. Likewise, isoleucine and leucine are of equal mass and only one pair of ions is required for their measurement. In Figure 2, the total number of ions monitored has been reduced from 30 to 26; yet, the advantage of selected ion monitoring over repetitive scanning is maximization of the time spent measuring the ions of interest. The chromatographic resolution between certain amino acids is large enough to group the amino acids into four clusters of five amino acids or less separated by 0.5 min or more retention time. Therefore, (iii) a maximum of only 8-10 ions instead of 26-30 need be monitored a t any one time. The computer shifts attention from one group of ions to the next automatically, the time interval between groups being sufficient to allow for run-to-run variation in retention time. No attempts have been made in this work to press the sensitivity of the technique. A good indication of its sensi-
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ratio difference 9 h - 0 h, X 100' 0.636 t 0.065 0.700 i 0.104 0.102 t 0.0'73 l . 0 0 2 t 0.102 5.660 i 0.162 0.104 t 0.089 0.152 i- 0.119 0.266 t 0.069 0.090 i- 0.099 0.012 2 0.067 0.088 t 0.061 0.346 i 0.117 0.000 ?; 0.244 0.320 i- 0.097 -0.084 t 0.138 Mean -t standard deviation; For n = 2 rather than n = 5.
tivity, however, can be obtained from the amount of serum required to generate Figure 2 and the precision of measurement obtained (vide infra). For the conditions of this experiment, amino acids from 0.5 mL of serum were extracted and derivatized. Subsequently, only 4 % of the total derivatized sample (corresponding to a modest 20 pL of plasma) was injected into the mass spectrometer. Replicate analysis can easily he performed with less than 100 pL of plasma-an acceptable sample size even for neonatal studies. T o demonstrate the precision that can he obtained using CI-SIM-GCMS, the "N incorporated into the free plasma amino acids were measured after I.-[ 15N]leucine had been continuously infused into a dog. Two plasma samples were analyzed: one drawn before the infusion was started and one drawn 9 h after the infusion was begun. Table 111 lists each amino acid measured, the pair of ions monitored for each, the observed isotope ratios a t 0 and 9 h , and the difference between the 0- and 9-h ratios. T o give a true presentation of the isotope ratio measurement precision, the standard deviations of the isotope ratios and not standard errors of the ratios are shown-for the latter, the listed values would be divided by x '5. The average standard deviation of a single ratio measurement of the Table I11 data is 0.00080. When the uncertainty in the isotope ratio of the unenriched amino acid is small, Le., the ratio has been repeatedly determined (as is normally the case), the uncertainty in the at. % excess calculation from measuring the enriched isotope ratio once is approximately equal to the enriched isotope ratio uncertainty or, on the average, 0.08 at. c70 excess. T h e average precision, computed from averaging the relative standard deviation of each amino acid isotope ratio, is 0.60%. The last column in Table I11 lists the difference in isotope ratios between the 9- and 0-h samples and their 95% confidence limits. This column shows ihe extent of I5N incorporation into the various plasma amino acids. T h e average 95% confidence limits for the ratio differences presented in Table I11 is 0.0010 or about 0.10 at. % excess. The information desired from this study was: which amino acids have become significantly enriched with "N? This question can be answered using a one-tailed t-test. Even when the stringent p < 0.005 level of significance is applied, the amino acids alanine, valine, isoleucine, leucine, serine, glutamate, and ornithine show 15N incorporation. Lysine, an amino acid known not to incorporate I5N under the conditions of this experiment ( I @ , shows no enrichment. Comparison of CI-SIM-GCMS and IRM-GCMS. T h e only other technique reported for isotopic measurement in multiple amino acids is the method of isotope-ratio-monitoring gas chromatography-mass spectrometry (IRM-GCMS) (6).
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T h e technique differs from conventional GCMS in that a combustion oven is placed between the gas chromatograph and the mass spectrometer. Amino acid I3C and I5N enrichments are determined from the COz and N2 produced as each amino acid elutes from the gas chromatograph and is combusted. T h e IRM-GCMS technique is inherently more sensitive to 13Cand 15N Lhan the CI-SIM-GCMS method since the IRM-GCMS measures a small difference in isotopic enrichment on top of a small natural abundance background (13C02/12C02 = 0.010 or 15N'4N/14N2= 0.0074) while CISIM-GCMS measures A small difference in isotopic enrichment on top of a large tiatural abundance background from multielement multiatoiri ions (for the amino acids the [M H + l ] / [ M + HI ratio r
