2308

Anal. Chem. 1991, 63, 2306-2313

Determination of Site-Specific Carbon Isotope Ratios at Natural Abundance by Carbon- 13 Nuclear Magnetic Resonance Spectroscopy Valerie Caer, Michel Trierweiler, Gerard J. Martin, a n d Maryvonne L.Martin* Laboratoire de RMN et Rbactiuite‘ Chimique, URA-CNRS 472, Universit6 de Nantes, 2 rue de la Houssinisre, F-44072 Nantes Cedex 03, France

SIte-speMc natural Wope fractknation of hydmgen studled by deuterium NMR (SNIF-NMR) spectroscopy Is a powerful source of information on hydrogen pathways occurring in bkyM”InnatuaiC0ndltknr. Thepotentlalofthecarbon counterpart of this method has been investlgated and compared. Wee typlcai mokcuiar spedes, ethad, acetk acid, a d vanilh, have been conrklered. laking into account the requirements of quantitative lac NMR, appropriate experimental procedures have been defined and the repeatability and reproducibility of the Isotope ratio determinations have been checked in different conditions. It is shown that the carbon version of the SNIF-NMR method is capable of detecthgrmdl~inthecarbon-13cont~oftheelhyl fragment of ethanols from different botanical or synthetic origins. These results are in agreement with mass spectrometry determinations of the overall carbon isotope ratios. Deviations with respect to a statistical distribution of lachave been detected in the case of acetic acid and vanillin. However, since the method is very sensitive to several kinds of systematk error, only a rdathre dgntfkance can be attached at preunt to the internal parameters directly accessible. Isotope dilution experiments have also been carried out in order to chock the condstency of the results. I n the present state of exporlmental accuracy, the lacNMR method is of more limited potential than *H SNIF-NMR spectroscopy. However it may provide complementary information. Moreover it is particularly effkient for detecting and quantifying addleratknr that aim to mknk the overall carbon-13 content of a natural compound by adding a selectivity enriched specks to a kss expenrlve substrate from a different origin.

INTRODUCTION Isotope ratio mass spectrometry (IRMS) is very efficient for determining isotope contents at natural abundance (1-3). However since the compound must be combusted in order to provide C 0 2 and H20,which are subsequently used for measuring the 13C/12Cand 2H/1H ratios, only overall parameters of the considered molecule are obtained. The direct access to site-specific isotope ratios of hydrogen at natural abundance by NMR spectroscopy (SNIF-NMR) ( 4 , 5 ) has provided original approaches to the study of biosyntheses in natural conditions and has been the source of new analytical applications in food, drug, and forensic sciences (6-8). In principle, site-specific isotope contents can also be obtained by IRMS but appropriate chemical transformations into smaller molecules are required in order to isolate the considered molecular fragment. Deviations with respect to a random distribution of carbon-13 have been indirectly detected in this way for several kinds of molecular species (914). In the case of fatty acid synthesis for example it could be shown that isotope fractionation occurring in the formation

of acetylCoA by pyruvate dehydrogenase results in a relative 13C depletion in the carbonyl group (12,13). Such behavior is also supported by analyses of carbon NMR spectra of mixtures of various isotopically labeled amino acid species extracted from algae grown on 13C02(15). On the basis of the performances reached by the 2H NMR method, direct determination of the site-specific isotope ratios of carbon at natural abundance by 13CNMR spectroscopy is a priori very attractive, since it avoids the need for tedious chemical degradations possibly accompanied by undesirable fractionation effects. However, in spite of the higher natural abundance of 13C with respect to deuterium, the access to natural carbon isotope ratios with a suitable degree of accuracy remains a challenging problem. This situation results from both the restricted range of variation of the carbon isotope ratios and the relatively limited precision and accuracy of quantitative ‘3c NMR spectroscopy. Thus,although very low ‘3c contents have been measured in sedimentary hydrocarbons derived from methanotrophic bacteria (16),the common range of the relative overall isotope ratios is only about 50% in the 6 scale (17) (eq 10). In contrast, a range of more than 400% is occupied by the hydrogen isotope parameter defined similarly (18). Moreover, whereas quantitative deuterium NMR measurements can take advantage of the narrow range of the chemical shift frequencies, of the relatively short 2H quadrupolar relaxation times, and of the absence of discriminating nuclear Overhauser effects, quantitative 13CNMR spectroscopy suffers from several theoretical and technical limitations (19). Consequently, although the detection of some variability in the 13C contents of several molecular species has been mentioned (20), insufficient short-term repeatability and long-term reproducibility have prevented the development of this spectroscopy for the determination of carbon isotope ratios. It must be emphasized that a difference of 5% in the relative isotope contents (eq 10) of two carbon sites is associated with a difference in the signal intensities of the corresponding 13C NMR signals of only 0.5%. If it is recalled for example that the range of the overall isotope ratios of sugars from different origins is not higher than 20% (21-23), the requirement for very precise and accurate quantitative determinations becomes obvious. Dedicated protocols must therefore be developed. The purpose of this work was to define methodologies adapted to the determination of sitespecific carbon isotope ratios and to estimate the analytical potzntial of the method in the study of different kinds of organic molecules. In order to check the consistency of the results the method has been applied to typical examples well documented in the field of IRMS: ethanols, acetic acids, and vanillins from different botanical or synthetic origins. EXPERIMENTAL SECTION Materials. The biogenic alcohols were obtained by fermentation of sugars from different origins by Saccharomyces cereoisiae, in standardized conditions (6).The ethanol samples were

0003-2700/Q1/0383-2308$02.50/0 0 1991 Amerlcen Chemical Soclety

ANALYTICAL CHEMISTRY, VOL. 63, NO. 20, OCTOBER 15, 1991

extracted from the fermentation medium by distillation in strictly controlled conditions in order to minimize the kinetic isotope The acetic effects due to liquid-vapor isotope fractionation (24). acids are from a commercial origin or were extracted from alcohol, grape, or rice vinegars according to well-defined procedures (25). All vanillin samples are from a commercial origin except one which was extracted from beans of Vanilla planifolia. The selectively labeled samples used in the isotopic dilution experiments are from a commercial origin. NMR Experiments. The NMR experiments were performed at 62.8 and 100.6 MHz by using Bruker WM 250 and AM 400 spectrometers, respectively, with a probe accepting 10 mm 0.d. tubes. Since the volumes of samples available were not limited, quantities on the order of 1 g were mixed with about 0.5 g of the working standard, 0.03 g of the relaxation reagent, and 1mL of the deuterated solvent (C&, CDCl,) in order to lock the field to the frequency of the spectrometer. However, the problem of sensitivity is much less severe for than for 2H NMR spectroscopy and good signal-to-noise ratios are easily obtained with 0.1 g of product. Gated decoupling techniques were applied in order to obtain quantitative results (19). The pulse intervals, D, were selected (D > 92'' mas) on the basis of the longitudinal measured beforehand by the inversion rerelasation times, TI, covery Fourier transform (IRFT) method (Tables 1-111). Experimental conditions appropriate to each molecular species have been defined by checking the influence of systematic variations of the spectral parameters on the precision and accuracy of the isotopic ratios. In the case of vanillin investigated at 100.6 MHz, improved results were obtained by dividing the spectrum into three domains observed separately (Table In). We have also compared the results obtained in the absence and in the presence of the paramagnetic complex Cr(acac1,. This reagent strongly reduces the values of the relasation times and ensures a significant increase in the signal to noise ratio accessible at constant experimental time (Tables 1-111). Variations in the decoupling performance are a source of instability in signal shapes and in the accuracy of the area measurements. In the present state of our equipment the broad-band noise decoupling technique leads to a lower dispersion of the results than the Waltz decoupling sequence (26)provided that the offset frequency and the irra-

Table I. NMR Parameters of Ethanol, I'CH,CH,OH (l),and CHa1'CH20H(2), Determined at Two Spectrometer Frequencies u,, v,('%)

param

= 62.8 MHz

Hz/ref 1180 -1294 v('H), Hz/ref 207 -416 8 Ti('%), 'S 10.7 v('%),

Ti('Q, sd

1.4 3.2 350

;;g;pcc

= 100.6 MHz

v,('%)

ref

2

1

ref

2

1

O(b) O(b)

00 1891 -2071 -669 0" 333 7.6O 10.6 8.3 16Bb 1.5 1.2" 0.8 0.9 1.3b 3.6 3.6" 3.6 3.6 4.00 350 250 450 450 350

OThe sample contains 300 g L-' of tetramethylurea. bThe sample contains 170 g L-'of cyclohexane. Investigated in the absence of the relaxing agent C~(acac)~. dInvestigated in the presence of the relaxing agent Cr(acad3 ( ~ 1 g3 L-'). eThe half-height line width, AuIl2, and the signal to noise ratio, S I N , are determined from spectra which result from 72 (62.8MHz) and 32 (100.6MHz) accumulations and involve an exponential multiplication of the free induction decay associated with a line broadening of 2 Hz. Table 11. NMR Parameters of Acetic Acid Determined at 100.6 MHza

co

CH3

2307

C6H12

-674 0 Hz/CgHIZ 15040 v('H), Hz[C&iz 280 0 10.5 19 Ti("C), B 35 Ti('%), 8' 0.9 0.9 1.5 A u l p Hzbd 3.0 3.4 3.0 OCyclohexane has been added (180 g L-') aa a reference. bThe sample is investigated in the absence of the relaxation reagent. The sample is investigated in the presence of the relaxation reagent Cr(aca& dThe half-height line-width, Avllz, and the signal to noise ratio ( S I N > 130) are determined from spectra which result from 256 accumulations and involve an exponential multiplication of the free induction decay associated with a broadening of 2 Hz. v('%),

Table 111. NMR Parameters of Vanillin

A

molecular site

1

v(13C)Hz/ref u('H). Hz/ref T ~ ( ' ~ Csa) , Avi Hz lJ(k-H), Hz

9622 1750 0.4 1.6 173.5

21

2 7198

3 6909

0.5 1.6

5757

0.6 1.6

0.5 1.3

part 1 molecular site B

v(13C),Hz/ref v('H), Hz/ref T1('3C), sc T1('3C), sd

1

6

7

8

reP

5558 1157 0.4 2.1 167

4841 1058 0.3 1.6 159.7

4528 1157 0.4 2.1 160.5

1109 274 0.5 1.6 144.4

0 0 0.7 1.6 134.6

part 2 re@

re@

2

3

2889 0 0 -1095 -1592 630 0 0 0.5 4.6 4.6 12.5 19.4 0.3 0.3 0.3 0.3 0.4 3.0 3.8 4.7 3.7 3.7 >250 >250 >110 >110 >110 HZ 'J('%-H), HZ 173.5 221 221

S"P

5

4

4 -3450

5

6

7

-3645 -4881 -5316 -295 -463 -295 12.6 1.2 1.3 1.3 0.4 0.3 0.2 0.3 3.7 4.9 4.6 5.0 >110 >110 >110 >110 159.7 160.5 167

part 3 re@ 8 0 -1087 0 -65 5.5 2.2 0.5 0.3 2.2 4.6 >180 >180 144.5 144.4

"In experiments A a sample of vanillin dissolved in acetonitrile (4.2 mol L-') and containing 1.1 mol L-' of the reference compound experiments B a sample tetramethylurea carefully weighted is investigated at 62.8 MHz. The signal-to-noiseratio is higher than 130. of vanillin dissolved in CDC13(2.8mol L-') and containing 4.5 mol L-' of formic acid (parts 1 and 2) and 3.0 mol L-' of dioxane (6% = -23.1) (part 3) is studied at 100.6 MHz by performing three separate observations of selected spectral ranges. 'The samples are investigated in the absence of the relaxation reagent (Cr(acac)&. dThe samples are investigated in the presence of the relaxation reagent (Cr(acac)&. 'The half-height line width, AulI2, and the signal to noise ratio, S I N , are determined from spectra which result from 256 accumulations and involve an experimental multiplication of the free induction decay associated with a line-broadening of 2 Hz.

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ANALYTICAL CHEMISTRY, VOL. 63, NO. 20, OCTOBER 15, 1991

Table IV. Overall Carbon Isotope Ratios of Ethanol Samplesa Determined by Isotope Ratio Mass Spectrometry origin c3

sample

species

6, 4bO

A, 7'0

1 2

beet root beet root grape grape mean

-26.9 -26.5 -26.6 -26.5 -26.6 -26.5 -13.7 -14.4 -11.6

1.0817 1.0821 1.0820 1.0821 1.0820 1.0821 1.0962 1.0954 1.0985 1.0990

3 4

lit. 5 6

7 8

fossil

9

maize maize cane cane mean lit. oil lit.

-11.1 -12.7

-11.0 -28.7 -30.0

1.0973

1.0991 1.0797 1.0783

a The samples are derived from plants with C3 or C, photosynthetic metabolisms or from oil (fossil). The results are expressed in the relative 6 scale (eq 10) or as fractional abundances (eq 1). Average values over literature data (14, 30, 31) are also given.

diation power have been carefully selected. Typical values of the experimental parameters are the following: ethanol (at 62.8 MHz) acquisition time (At) 1.64 s, spectral width (SW) 5OOO Hz, memory size (SI) 16K, pulse width (for a pulse angle of 90°) (PW) 20 ps, delay time (D) 20 s, temperature (T)300 K, zero filling (Z) 16K, number of transients (NS) 72, number of experiments per sample (NE) 9; ethanol (at 100.6 MHz) At = 1.36 s, SW = 6024 Hz, SI = 32 K, PW = 20 ps, D = 20 s, T = 308 K, NS = 32, NE = 9; vanillin (at 62.8 MHz) At = 0.54 s, SW = 15151 Hz, SI = 16K, PW = 20 ps, D = 10 s, T = 300 K, NS = 320, NE = 8; vanillin (at 100.6 MHz) SI = 32K, PW = 21.8 M, D = 5 s, 5" = 308 K, NS = 128, NE = 8; part 1 A t = 3.4 s, SW = 4800; part 2 At = 2.6 s, SW = 6410 Hz;part 3 At = 5.1 s, SW = 3205 Hz. Usually, the acquisition of data requires 2-4 h of spectrometer time, depending on the nature of the chemical species studied. Referencing methods are required to compare the isotope ratios of samples from different botanical or synthetic origins. The isotope contents associated with the different molecular sites of the unknown are compared with that of a standard previously calibrated, as described in eqs 1-9. In the case of ethanol, tetramethylurea (TMU) is a convenient standard from both the observation and decoupling points of view since its 13C and 'H methyl chemical shifts are intermediate between those of the methyl and methylene positions of the ethyl moiety (Table I). TMU has already been adopted as a reference for calibrating the *H/lH isotope ratios (27) and a collaborative study coordinated by the Community Bureau of Reference (BCR) in Brussels was organized in order to measure the deuterium contents in the methyl and methylene sites of ethanols from different origins with Ethanol-TMU mixtures calibrated respect to that of TMU (28). for the measurement of hydrogen isotope ratios are now provided by the BCR. The 13C/1*Cratio, expressed in the 6 scale (see eq 10) was determined independently by mass spectrometry for the same pool of TMU as that used for deuterium (Table V). However the situation is less favorable in the case of carbon since the measured value corresponds to a mean over the two different carbon positions of TMU. In practice the area of the 13C NMR signals of ethanol are referred to that of the methyl signal of ThKJ which should be known specifically. As a first approximation we have retained the overall '%/'% value (6 = -25%) as a reference. This may be the source of a systematic deviation in all the subsequent determinations of the 613C values of the unknowns but does not influence the analysis of the results. When large spectral ranges have to be investigated, better results are obtained by implementing a multireferencing method. Thus formic acid and dioxane are shown to be suitable for quantitative determinations performed with the three domains procedure adopted in the study of vanillin (Table 111). Each of these two substances exhibits a single 13CNMR line and is characterized by a specific 613C value accessible by isotope ratio mass spectrometry. Precisely known amounts of the two standards are added to the sample and the area of the 19CNMR signals of the different sites of vanillin are

compared to that of the nearest standards in the spectrum (Table 111). Treatment of the NMR Signalsand Analysis of the Data. Usually it is convenient to submit the free induction decay to an exponential multiplication associated with a line broadening of 2 Hz. Careful adjustment of the phases is of prime importance. Several integration procedures were checked and the truncation effects were estimated in the conditions of our experiments which compare NMR lines with similar intensities. The numerical integration procedures implemented in the spectrometer software give satisfactory results when the peaks to be compared have nearly identical half-height line widths (Tables 1-111) and are therefore defined within similarly truncated domains. Curvefitting procedures available in the NMR-1 program (New Methods Research Inc. Syracuse, NY)are more efficient in particular when lines relatively close to each other (Tables 1-111) must be compared. Isotope Ratio Mass Spectrometry (IRMS) Experiments. The overall carbon isotope ratios of the samples and of the standards have been measured using a Finnigan Delta E mass spectrometer equipped with a Carlo Erba microanalyzer. DEFINITIONS AND SYMBOLS The carbon isotope content can be expressed either as the absolute ratio of the numbers of 13C and 12C isotopes, R = 13C/12C,or as the fractional abundance A (eq 1). If we

A=

13C

1zc + 13c

=

R R +1

(1)

consider two samples Q and Q of the same chemical species (acetic acid for example) but containing different amounta of carbon-13, the fractional abundance, AM,of a mixture, M, consisting of molar fractions x and (1- x ) of Q and Q obeys eq 2. At natural abundance the carbon-13 isotopes are mainly A M = xAQ + (1 - x ) A q (2) distributed in monolabeled isotopomers. In the case of acetic acid for example the two main lines in the proton-decoupled 13C NMR spectrum are assigned to the 13CH312COOH,and WHJVOOH isotopomers (Table 11). The small satellite lines resulting from the bilabeled molecules are not easily detected in the conventional spectrum. A fractional abundance associated with a given isotopomer i in a compound Q, Ais, may be defined as the ratio of the number of 13C atoms in site i to the total number of 12Cand 13C atoms corresponding to the same position

(3) The site-specific fractional abundance Ais can be expressed as a function of the overall fractional abundance AQ of carbon-13 in compound Q by eqs 4 and 5. =

fi EAQ

(4)

In these equations, fi is the effective molar fraction of isotopomer i defined with respect to the whole population of the monolabeled 13C isotopomers, Fi is the corresponding statistical molar fraction that would result from a random distribution of 13C over the molecule (Fi = 0.5 for both sites of acetic acid), and n is the number of isotopomers directly observed in the 13C NMR spectrum. fi can be derived from the area, Si, of the NMR signals measured in the proton decoupled spectrum. n

fi = Si& ill

ANALYTICAL CHEMISTRY, VOL. 63, NO. 20, OCTOBER 15, 1991

2309

Table V. Natural Abundnncar" of the Methylene and Methyl Monolabeled Isotopomers of Ethanol Sampler 1-9 from Different Origins (Table IV)

exp. protocol A

1 2

Ca

3 4 X

S

A, % 6, 960 5 6 7

c4

8 X

S

A, % 6, 960

fossil

9

S

B

A, % 6, 960 1 2 3

c3

4 X

S

A, % 6, % 5 6 7

c4

8 X

8

A, % 6, 960

fossil

external comparison reduced isotopic abundances

internal comparison molar fractions

orimn

9

S

A, % 6, 960

0.5018 0.5015 0.5012 0.5016 0.5015 0.0022 1.0852 -23.7 0.4991 0.4997 0.4982 0.4999 0.4992 0.0025 1.09a55 -14.3 0.4998 0.0019 1.0795 -28.9 0.5017 0.5015 0.5008 0.5016 0.5014 0.0015 1.0850 -23.8 0.4988 0.4999 0.4980 0.5019 0.4996 0.0012 1.0964 -13.3 0.5006 0.0010 1.0810 -27.5

0.4983 0.4985 0.4988 0.4984 0.4985 0.0020 1.0787 -29.6 0.5009 0.5003 0.5018 0.5001 0.5008 0.0031 1.0990 -11.1 0.5002 0.0021 1.0800 -28.4 0.4982 0.4985 0.4987 0.4982 0.4959 0.0017 1.0748 -33.1 0.5011 0.5003 0.5019 0.4980 0.5003 0.0015 1.0979 -12.2 0.4992 0.0012 1.0780 -30.2

CH2

CH3

C2H6

0.9912 0.9996 0.9890 1.oooO 0.9950 0.004 1.0784 -30.1 1.0039 1.0074 1.0077 0.9963 1.004 0.005 1.0881 -21.0 0.9882 0.0035 1.0707 -36.8 1.0052 1.0069 1.0029 1.0085 1.006 0.003 1.0932 -16.4 1.0158 1.0149 1.0156 1.0189 1.016 0.002 1.1015 -8.8 0.9962 0.002 1.0795 -28.9

0.9847 0.9920 0.9858 0.9936 0.9890 0.004 1.0718 -36.1 1.0080 1.0120 1.0136 0.9938 1.007 0.006 1.0913 -18.1 0.9898 0.004 1.0729 -34.9 0.9961 0.9992 0.9926 0.9987 0.997 0.003 1.0802 -28.3 1.0135 1.0118 1.0121 1.0225 1.015 0.003 1.100 -10.2 0.9968 0.002 1.0800 -28.4

0.9880 0.9956 0.9874 0.9968 0.9920 0.006 1.0751 -32.9 1.oO60 1.0097 1.0106 0.9952 1.0055 0.007 1.0897 -19.6 0.9890 0.007 1.0718 -35.9 1.OOO6 1.0031 0.9978 1.0036 1.001 0.005 1.0851 -23.8 1.0147 1.0133 1.0138 1.0207 1.0155 0.005 1.101 -9.3 0.9965 0.004 1.080 -28.6

"Each value is the mean over two replications in the case of biogenic ethanols and over four replications in the case of synthetic ethanol 9. Each replication is itself an average over nine spectra. x is the mean value corresponding to the C3 or C4 family and s is the standard deviation. The spectra determined at 62.8 MHz were analyzed by numerical integration in case A and by curve-fitting in case B. It has been assumed as a first approximation that the isotopic abundance in the methyl position of tetramethylurea (TMU) used as a reference is equal to the overall isotope content of TMU measured by IRMS (6 = -25960,A = 1.084%). and Fi is obtained from eq 7, where Pi is the number of equivalent carbon positions at site i. Fi = Pi/CPi = P i / P (7) i

In order to compare the isotope contents in different molecular sites i and j, it is convenient to define relative parameters rilj which respresent the ratios of the fractional abundances in the considered sites and are directly accessible from the I3CNMR spectrum

NMR spectrum (eq 6) and A ~ Q is computed by means of eq 4 using a value of the overall fractional abundance AQ mea-

sured by IRMS. In the internal referencing method, A , is calculated from eq 9 (81,where m and M are masses and molecular weights, S ~ Q and S w s are the signal intensities of sites i and WS, and t is the purity (w/w) of the sample.

Aig =

mWS SiQ AWS MQ -PWS

MWS

(9)

sWS

mQ

The results will also be expressed in the 6 scale usually adopted by mass spectroscopists (1 7) The site-specific isotope contents, A ~ Qcan , be obtained either by a combination of NMR spectroscopy and mass spectrometry measurements or by NMR experiments only using a working standard, WS, with a known '3c content, Aws. In the first method, the effective molar fractions, f i t are directly derived from the signal intensities, Si, of the quantitative 13C

613C (%o)

=

(;= -

- 1)1000

(10)

where PDB denotes the international reference characterized by an isotope ratio RpDB = 0.0112312 and a fractional abundance ApDe = 0.111233 (29).The 6 parameter is related

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ANALYTICAL CHEMISTRY, VOL. 83, NO. 20, OCTOBER 15, 1991

to the fractional abundance A by

Table VI. Comparison of the Discriminating Potential of the Mass Spectrometry and NMR Methods” a (fwil/C4)

a (CdC4)

CH2 CHS

In order to compare the isotope content of site i in samples Q and Q from different origins, it is convenient to define the ratio “QIQ:

CH2 CHS

CzHs

IRMS lit.

this work num integ (A)

RESULTS AND DISCUSSION Nine samples of ethanol, seven samples of acetic acid, and eight samples of vanillins from different botanical or synthetic origins have been investigated in order to appraise the potential of the method to detect possible variabilities in the isotope contents of specific molecular sites and to discriminate between different origins of the precursors. These three types of molecules are illustrative of different spectral behaviors that require appropriate experimental conditions. Relatively large differences in the overall 13C contents of synthetic or biosynthetic compounds derived from plants with C3, C4, or CAM photosynthetic metabolisms have been determined by mass spectrometry (2). With the exception of synthetic products, the natural dispersion of the isotopic parameters associated with a given origin is relatively small with respect to the NMR precision. Typical average values of the overall isotopic abundances calculated from literature data (14,30,31)and from our own IRMS measurements may therefore be retained as reference parameters for checking the consistency of the NMR determinations. Isotopic Analysis of Ethanol. Four ethanol samples were obtained by fermentation of sugars extracted from maize and cane, which are plants with a C4 photosynthetic metabolism, and four samples resulted from the fermentation of beet and grape sugars,which are produced by a C3photosynthetic cycle. One commercial sample has a synthetic origin from fossil oil. The overall isotope ratios measured by mass spectrometry are given in Table IV. The two-line spectrum of ethanol seems relatively favorable from both the observation and decoupling points of view since the ranges of the proton and carbon resonances are relatively narrow. The low volatility of tetramethylurea makes it a suitable candidate for quantitative referencing. Moreover its methyl signal is adequately situated between the methylene and methyl signals of ethanol (Table I). Gated decoupling techniques were applied at two spectrometer frequencies in the presence of the relaxation reagent Cr(acacI3,as described in the Experimental Section. The area measurements were performed independently by numerical integration and by curve-fitting. The molar fractions of the methyl and methylene isotopomers determined from the direct comparison of the corresponding signal area (eq 6) are given in Table V. Reduced isotopic abundances were also defined by referring the specific isotopic abundances, Aiq, to that of the methyl signal of tetramethylurea, AWS,considered as unity. These reduced parameters were calculated from the signal area of the methyl and methylene signals of ethanol or their sum and from the area SWSof the methyl signal of TMU, according to eq 9. Standard deviations were computed over the nine series of experiments performed for every sample, as described in the Experimental Section. In a first series of signal analyses, denoted as case A, the spectra obtained at 62.8 MHz are analyzed by the numerical integration method (Table V). The average standard deviation over the replications is of the order of 0.003 for the molar fractions and 0.007 for the reduced isotopic abundances. Expressed in the 6 scale, the confidence

CIH, 0.9845 0.9861

0.9811 0.9840

NMR

internal comp 0.990 0.982 0.986 0.985 0.983 0.984 external comp 0.991 0.982 0.987 0.984 0.983 0.983 curve-fitting (B) internal comp 0.990 0.979 0.985 0.986 0.982 0.984 external comp 0.992 0.982 0.987 0.980 0.982 0.981 The relative overall and site-specific isotope contents, characterized by the parameter a and defined in eq 12,are compared for samples pertaining to the Cs and C, families or to the fossil and C4 families. The values have been calculated either by internal comparison (eq 4 and Table V) or by external comparison (eq 9 and Table VI. intervals at 95% is about *8%. When the spectra are analyzed by a Lorentzian curve-fitting procedure (results denoted B in Table V) the standard deviation on the isotopic abundances is lowered to 0.003 and the confidence interval at 95% is then equal to *2% in the 6 scale. An analysis of the variance has been performed on the two series of experiments in order to detect possible differences in the isotope contents of the two ethyl sites. The analysis corroborates a slight ‘3c enrichment in the methylene site as compared to the methyl site in the case of C3 samples whereas nearly no distinction is detected in the C4 family (Table V). However, the occurrence of small systematic deviations related to decoupling and offset effects cannot be excluded. Therefore in the present state of the technique, it is only concluded that if differences exist in the carbon isotopic ratios of the two positions in the ethyl moiety of the investigated ethanols, they are lower than 1.5%. Whereas powerful analytical criteria are provided by the site-specific deuterium fingerprints, the relative internal 13C contents are not easily exploitable for characterizing the biogenic or synthetic origin of ethanol either because they do not exhibit sufficient differences or because the experimental accuracy remains insufficient. In this respect, more fruitful results are obtained by comparing the isotopic ratios associated with a given isotopomer, among samples from different origins. This external comparison is less subject to discriminating effects, resulting in particular from differences in the offset frequencies and in the shapes of the NMR signals. An analysis of the variance shows that samples pertaining to the C3 and C4families on one hand and to the C4 and fossil families on the other hand are unambiguously distinguished in the two series of experiments. The average ratios a (eq 12) of the isotopic contents in the two sets of partners are compared in Table VI for the individual methyl and methylene sites and for the overall isotope contents determined by NMR spectroscopy and IRMS. The results are quite consistent not only with each other but also with literature data (Table IV). The NMR method is therefore capable of characterizing the relative depletion of C3 and fossil ethanols with respect to C4 ethanols. The reliability of the NMR method is further corroborated by an isotope dilution technique. Four different mixtures containing a precisely known amount of a substrate ethanol (613C = -26.6%) and of a tracer enriched at 99% on the methylene site have been prepared. The mass ratios of these mixtures are situated between 30 and 350. Experimental values of the isotopic abundances were determined by using a single site reference, cyclohexane, the isotope ratio of which

ANALYTICAL CHEMISTRY, VOL. 63, NO. 20, OCTOBER 15, 1991

was determined by IRMS (6'C = -29.0%). A very good linear relationship is obtained between the experimental and theoretical values A* 0.0001 0.960(0.02)A0, (R = 0.999) (13)

Table VII. '42 Abundances of Vanillin Samples from Different Origins"

+

Isotope Parameters of Acetic Acids. Acetic acids from different biogenic origins have been extracted from vinegars. Since the experimental conditions were maintained constant, differenciating isotope fractionation effects possibly introduced by the extraction procedure (W)may be reasonably neglected. Experimental conditions were selected with a view to circumvent the technical problems due to the large differences in the '9c and 'H chemical shifts and in the relaxation times of the carboxyl and methyl groups (Table 11). The hydroxyl hydrogen was eliminated by chemical exchange with DzO. In these conditions, the decoupling frequency can be situated in the low-frequency region of the spectrum. An improvement in the digital resolution was ensured by a procedure involving two offset values of the carrier frequency. In order to conveniently excite the CO and CH3 carbons, one frequency is positioned 150 Hz from the CO resonance on the high-frequency side and the other one 150 Hz from the CH3 signal on the low-frequency side. A 1000-Hz sweep width associated with a memory size of 16 or 32 K can be used while the pulses are alternatively applied at the two different frequencies. The internal ratio rlI2, defined in eq 8, was measured in seven samples from a different origin. Since the occurrence of systematic errors cannot be excluded, only relative parameters (~t!/?)Q/q which refer the (r1/2)Qvalue of a sample from a given origin, Q, to the (rl/z)q parameter of a reference sample, Q , from a synthetic origin, are given below: origin Q beet

grape apple rice

(al,g)Q/e1.020 1.021

synthesis synthesis

1.012 1.022 1.006

0.981

These results, which are associated with a standard deviation of about 0.005 (five experiments), indicate that the biogenic samples are characterized by higher ratios of the '9 contents in the carboxyl site with respect to the methyl site than samples from a chemical origin. Isotope dilution experiment have also been performed in order to check the consistency of the NMR determinations. A sample of commercial synthetic acetic acid, characterized by an overall isotope ratio 613C = -39% obtained by IRMS, was mixed in various proportions with acetic acid selectively labeled at the methyl site (9970). A value 613C = 30L of the methyl group of the unlabeled sample was retained on the basis of literature data (9-11). Cyclohexane (613C = -29%0) was used as a reference. A very good linear relationship is computed from the couples of theoretical and experimental isotopic abundances associated with the four dilution experiments Ath = -0.0003 + 1.030(0.002)A0,, ( R = 1.00) (14) Several attempts to determine site-specific 13Ccontents of acetic acids by mass spectrometry have been performed by resorting to appropriate chemical transformations. Thus 13C depletions in the methyl site with respect to the carboxyl site ranging between 3 and 18% have been measured in biogenic acetic acids (10, 11, 32,33). Acetate intervening in lipid synthesis exhibits the opposite behavior (131, and either depletions or enrichments of the carboxyl site with respect to the methyl site have been determined in various synthetic acids (9, 11). Our results are in satisfactory qualitative agreement with these observations. Moreover by using external referencing in the NMR method the 13C contents of individual sites or their sums can be compared among samples from different origins. It is corroborated in this way that acetic acid extracted from vinegar produced from beet sugar ethanol

2311

site-specific isotope

overal

ratios

isotope

origin

param

CHO

Ar

OCH,

content

vanilla

R, %

1.074 1.061 1.062 1.051 1.067 1.056

1.113 1.101 1.102 1.090 1.102 1.090

1.061 1.049 1.066 1.055 1.026 1.016

1.101 1.089 1.093 1.081 1.089 1.077

A, %

lignin

guaiacol

R, % A, % R, % A, %

" The overall isotope contents have been measured by mass spectrometry (IRMS), and the site-specific isotope ratios have been determined by NMR spectroscopy at 100.6 MHz by using a multireferencing technique. The Ar value is the mean over the site-specific parameters measured for the six aromatic positions. The standard deviations over the nine series of experiments performed for every sample are on the order of 0.003-0.007 for all fractional abundances.

-

exhibits a significant 13C enrichment with respect to a commercial sample with synthetic origin: & ! ~ ~ / n ~ * e t i c 1.02. Isotopic Parameters of Vanillin. Four samples of vanillin derived from lignin, three samples of synthetic vanillin from guaiacol, and one sample of natural vanillin extracted from V.planifolia Andrews have been investigated. The multisite system of vanillin combines all the previous sources of difficulties. Due to the large chemical shift range of the vanillin protons, we are faced in particular with the problem of the homogeneity of decoupling. The reliability of the results must be considered in terms of repeatability and reproducibility. In the present context, both terms refer to the closeness of agreement between successive results obtained with the same sample, apparatus, and protocol but within a short or a long period of time, respectively. According to these definitions, the repeatability variance 9, over p series of NE spectra recorded successively is equal to the mean variance, ( C S : ) / p , of the p experiments and contains only short-term random deviations. The reproducibility, L, should be understood as the variance of the means yi of different series of experiments, corrected for the short-term deviations St, i.e.

where yi is the general mean over the whole set of experiments. Then, as far as 13C NMR spectroscopy is concerned, the reproducibility is expected to express some of the systematic deviations due to the long-term instability of the system (decoupler, receiver, ...). The mean precision of 1.5%,which is reached with the optimum decoupling power (4.9 kHz at 62.8 MHz), is of the same order of magnitude as the differences in the measured isotope contents. Improved performances are obtained when the decoupler offset is swept by steps of 100 Hz, every scan of a block containing 32 FID. The reproductibility parameters, L, then reach values close to those of the repeatability parameters,, : S 4-8960. In these conditions the lignin and guaiacol families can be distinguished. Thus variance analyses performed on the different series of data indicate that site 1 (CHO) and site 8 (OCH3) are discriminating indicators. The ratios of the 13C contents in sites 1 and 8 are smaller for a lignin origin than for synthetic samples from guaiacol: di 1Rin/wiacol = 0.96 (eq 12). However, since systematic biases may result from the large range of observation offsets, a multireferencing technique has been implemented. Formic acid and dioxane are suitable references, and three domains of the vanillin spectrum can be separately observed (Table I11 and Experimental Section). The isotopic ratios,

2312

ANALYTICAL CHEMISTRY, VOL.

63,NO. 20, OCTOBER 15, 1991

Table VIII. Site-Specific Carbon Isotope Ratio Determined in Isotopic Dilution Experiments' site-specific isotope ratio

Param exp 1

R, % A, %

AT, R, % A, %

exp 2

substrate

AT, % R, %

1

A, %

R, %

2

A, %

1

2

3

4

5

6

7

8

2.080 2.038 20.3 1.674 1.646 20.8

2.884 2.803 34.8 2.130 2.086 34.8

2.916 2.833 35.0 2.164 2.118 35.2

1.117 1.105 1.15 1.127 1.114 1.48

1.133 1.120 1.47 1.144 1.131 2.09

1.098 1.086 0.84 1.122 1.110 1.45

1.077 1.066 0.95 1.096 1.084 1.45

1.613 1.587 11.4 1.404 1.385 12.1

1.063 1.052 1.060 1.049

1.083 1.071 1.079 1.067

1.104 1.092 1.101 1.089

1.113 1.101 1.116 1.104

1.114 1.102 1.112 1.100

1.107 1.105 1.106 1.094

1.085 1.073 1.084 1.072

1.063 1.052 1.065 1.054

-

"Labeled vanillin has been added at two concentrations (0.0304 w/w in experiment 1 and 0.0544 w/w in experiment 2) to a vanillin substrate derived from lignin (6 -28.2%). Eight series of spectra have been performed in every experiment and the results of two replications are given separately for the substrate. The isotope parameters measured in experiments 1 and 2 have been used to compute the fractional abundances of the labeled tracer. A,. %. Table IX. Carbon Isotopic Abundance8 of Two Commercial Samples 1 and 2 Supposed To Be of a Natural Origin"

ratios collected in Table IX that the methoxy site exhibits an abnormally high isotopic abundance.

site

CONCLUSION The 13C NMR method can be used for directly detecting small differences in the natural-abundance carbon isotope ratios at specific molecular positions provided that appropriate experimental procedures are defined. Thus deviations with respect to a statistical distribution of carbon-13 have been directly detected in acetic acid and vanillin samples. By resorting to external referencing, differences in the isotopomeric contents of samples from different origins can be unambiguously characterized. However, in the present state of experimental accuracy the method is not easily exploitable on an absolute quantitative basis. Although improvements may still be achieved, the carbon version of the SNIF-NMR method seems less powerful than its 2H counterpart. Nevertheless, the 13C NMR method already provides useful complementary information with respect to 13C IRMS and *H SNIF-NMR spectroscopy. In particular it is of unique value for detecting false natural compounds simulated by addition of seletively labeled species. Finally, it should be emphasized that, although they are relatively small, the deviations with respect to a statistical repartition of the 13Cisotope may affect quantitative analyses of mixtures performed by '3c NMR. This phenomenon should not be ignored in very accurate analytical determinations.

sample 1

R, % A, %

2

R, % A, %

CHO

Ar

OCH3

vanillin

1.085 1.073 1.077 1.066

1.101 1.086 1.099 1.087

1.126 1.113 1.131 1.117

1.100 1.088 1.101 1.089

"The overall isotope ratios are in agreement with a natural status (Table VII) but an abnormally high isotope content of the methoxy site (+2% in 1 and +6.5% in 2) is detected.

R, and the fractional abundances, A (eq 1and 91,determined for the three categories of samples are given in Table VII. Both the aldehyde and methoxy sites exhibit a lower 13C content than the aromatic positions, and the methoxy site is particularly depleted in the synthetic sample. Although the intramolecular variability seems to be exaggerated by systematic biases, the NMR method is able to characterize the three kinds of samples on a relative basis. The observed behaviors are in reasonable agreement with some partial determinations performed by mass spectrometry on degradation products of vanillin (34-36). The consistency of the NMR method is further checked by isotope dilution experiments involving mixtures of a vanillin substrate from lignin with a commercial labeled sample. Two concentrations of the tracer were investigated and eight spectra were obtained for every mixture and pure substrate (Table VIII). The isotopic fractional abundances, AT, of the tracer computed from the results of the two dilution experiments are in reasonable agreement. (It can be noted that this commercial tracer is conveniently adapted to mimic the isotopic content of natural vanillin by addition to ex-lignin vanillin!) From a related point of view, we have checked the efficiency of the NMR method to detect possible adulteration of vanilla extract by 13C-labeledvanillin. Thus in order to circumvent the IRMS method, natural ex-beans vanillin, which has an overall 13C content of about -20%, can be easily simulated by mixing an appropriate quantity of vanillin selectively enriched on the OCH3 or CHO group or on both of them with hemisynthetic ex-lignin vanillin ( P C 27%). For example a quantity of 0.6-0.7 g/kg of a sample labeled on the methoxy position gives the desired result. In this respect we investigated two commercial samples for which a natural status was excluded on the basis of 2H SNIF-NMR, although the overall 13C contents (-20.8 and -20.5%) were in the natural range. It is obvious from the values of the site-specific carbon isotope

-

ACKNOWLEDGMENT We thank the scientific group "Aromes et Bioconversions" (CNRS-SANOFI) for helpful discussions. Registry No. 13CH3CH20H, 14770-41-3; CH3l9CHZ0H, 14742-23-5; CH3CH20H,64-17-5; acetic acid, 64-19-7; vanillin, 121-33-5; carbon-13, 14762-74-4; carbon, 7440-44-0. LITERATURE CITED (1) Santrock, J.; Studiey, S. A.; Hayes, J. M. Anal. Chem. 1985, 57. 1444-1448. (2) O'Leary, M. ti. R?ytoche&try 1981,20. 553-588; BlOSclence 1988, 38, 328-338. (3) Schmidt. ti. L.; Forstel, ti.; Heinringer, K. Stable Isotopes; Analytlcal Chemistry Symposia Serles; Eisevler: Amsterdam 1982; Vol. 11. (4) Martin, 0. J.; Martin, M. L. C . R. A a d . Scl., Sw. 2 1081, 293, 3133; TetrahedronLett. 1881 22, 352-3528. (5) Martin, 0. J.; Martln, M. L.; Mabon, F.; Michon, M. J. Anal. C b m . 1882, 54, 2380-2382. (8) Martin, 0. J.; Zhang, B. L.; Naulet, N.; Martin, M. L. J . Am. Chem. SOC. 1986, 108, 5118-5122. ( 7 ) Martin, 0. J.; Guiilou. C.; Martln, M. L.; Cabanis, M. T.; Tep, Y.; Aerny, J. J . A r k . FoodChem. 1988, 36, 318-322. (8) Martin, G. J.; Martin, M. L. In Modern methods of p&nt ana&&; Llnkens, H. F., Jackson, J. F., Eds.; Sprlnger-Verlag: Berlln, 1988: Vd. 8, p 258. (9) Yankwich, P. E.; Promislow, A. L. J . Am. Chem. SOC. 1853, 75, 488 1-4882.

Anal. Chem. 1991, 63, 2313-2323 (10) Mol",W. G.; Rlnaldi, (3. G. L.; Heyes, J. M.; Schoiler. D. A. Bkmed. MeSS Sp&tWl. 1974, 1 , 172-174. (11) Schmmld, E. R.; Grundmenn, H.; Fogy, I.; Papesch, W.; Rank, D. Bkmed. Mess Spect". 1981, 8 , 496499. (12) Darlro, M. J.; Epstdn, S. Scknce 1977, 197, 261-263. (13) M o n a , K. D.; Hayes, J. M. J . Bld. Chem. 1980, 225, 1143511441; Geochhn. Cosmochlm. Acta 1982. 46. 139-149. (14) Rossmann, A.; SchmM, H. L. 2.Lebensm.-lhm.--Forsd,. 1909, 188, 434-438. (15) Bengsch, E.; Grivet, J. P.; Schulten, H. R. 2.NahKforsch. 1981, 368, 1289- 1296. (16) Freeman, K. H.; Hayes, J. M.; Trendl, J. M.; Albrecht, P. Nature 1990, 343. 254-256. (17) Craig, H. Oeochlm. C o s m h l m . Acta 1959, 53-92; Science 1961, 133, 1833-1834. (18) Hagemann, R.; Nief, G.; Roth, E. Tellus 1970, 2 2 , 712-715. (19) Martin, M. L.; Delpuech, J. J.; Martin, (3. J. FracllcelNM Spechoscopy; Wiley-Heyden: London-Phliadeiphia-Rhelne, 1980 Chapter 9. (20) Bengsch. E. French Patent FR 2530026, July 9, 1982. (21) Smlth, B. N. hlehnwlsssnscheften 1976, 62, 390-390. (22) Mcout. J.; Fontes, J. C. Ann. Falslf. Experf. Chlm. 1974, 211-215. (23) bunbar, J. Fresenlus' 2.Anal. Chem. 1982. 311, 578-580. (24) WSm, 1.; MUkt, N.; Martin, M. L.; Martin, 0. J. J . f h y s . Chem. 1990, 94. 8303-8309. (25) Shnon, H.: Rauschenbach, P.; Frey, A. z. Lebnsm.-untef.FwsCh. 1988, 136, 279-284.

2919

(26) Shaka, A. J.; Keeler, J.; Frenklel, T.; Freeman, R. J . Magn. Res. 1983, 52, 335-338. (27) Guiilou, C.; Trlerweiler, M.; Martin, G. J. Mgn. Reson. Chem. 1988, 2 6 , 491-496. (28) Martln, 0.J.; Naulet, N. Fresenlus' 2. Anal. Chem. 1988. 332, 648-651. (29) Craig, H. Geochlm. Cos".Acta 1957, 12, 133-149. (30) Rauschenbach,P.; Simon, H.; Stichler, W.; Moser, H. Z.Mturforsch. 1979, 34C, 1-4. (31) Misselhorn, K.; Bruckner, H.; MussigZuflka, M.; Grafahrend, W. Branntwelnwktscheft 1983, 162- 170. (32) Rlneldi, 0.; Melnscheln, W. G.; Hayes, J. M. Blomed. Mass. spscbwn. 1974. 1 , 412-414. (33) Krueger, D. A.; Krueger, H. W. Biomed. Mass Spectrom. 1984, 1 1 , 472-474. (34) Hoffman, P. G.;Salb, M. J . Agric. Fwd Chem. 1979, 2 7 , 352-355. (35) Brlcout, J.; Kozlet, J.; Derbesy. M.; Beccat, B. Ann. Fa&. Exp. Chlm. 1981, 74, 691-696. (36) Martin, G. E.; Alfonso. F. C.; Flgert, D. M.; Burggraff, J. J . Assoc. Off. Anal. Chem. 1981, 64, 1149-1153.

RECEIVED for review March 7, 1991. Accepted July 16,1991. This work was supported in part by a grant from Sanofi Bioindustries and the CNRS.

Nonlinear Multivariate Calibration Using Principal Components Regression and Artificial Neural Networks Paul J. Gemperline,* James R. Long,l and Vasilis G. Gregoriou2 Department of Chemistry, East Carolina University, Greenuille, North Carolina 27857

ThkmMlecrlptdescdbwmethodsfor detecting and modeling nonlinear regions of spectral response In multivariate, multicomponent spectroscopic assays. Slmuiated data and exparimenta1 UV/vklble data were used to 8tudy the capablilty of multivariate Ilnear models to approximate nonlinear response. Tho sources of real and apparent noniinearlty dmulated Included nonilnear Instrument response functions (e.g. dray light), concentrationdependent wavelength shifts, and concentratlon dependent absorption bandwidth changes. A weighting algorithm was devised to reduce the Influence of nonlnear spectral reglons in principal component regresdon (PCR) caiibratlons, thereby improvlng the performance of muitivariate linear calibration models. Secondorder caiibrat h methods using quadratic principal component scores and nonilnear calibration methods using artlficlal neural networks were compared to unwelghted and welghted i h a r callbration methods. Orthogonal transformation of the input variables was used to dgnlficantly improve neural network trainlng speed and reduce callbration error. Some conditions where wc-r and nonitnear calibrationtechniques outperform Ilnear callbration techniques have been ldentlfled and are dercribod.

INTRODUCTION For the past three years, our laboratory has been working in collaboration with scientists at Burroughs Wellcome Co.

* Corresponding author.

Current address: Air Force and Engineering Services Center, Tyndall AFB, FL 32403-6001. Current address: Department of Chemistry, Duke University, Durham, NC 27706. 0003-2700/91/0363-2313$02.50/0

to develop UV/visible spectroscopic assays for routine determination of the active ingredients and preservatives in p h c e u t i c a l products. Our goal has been to develop simple, accurate (f0.2%),and reliable assays using digitally stored calibration models from UV/visible diode array spectra. Simple check standards are used to verify the accuracy of the stored calibration models so that lengthy recalibration procedures can be avoided. We quickly encountered problems with some of the assays, which we attributed to nonlinear spectral response (1). The symptoms included a lack of short-term and long-term reproducibility (large bias between days, ca. &l.O%) and curvature in the residuals for the estimated concentrations. Such errors are unacceptable in a heavily regulated industry like the pharmaceutical industry. In order to understand this problem more thoroughly, we undertook a study to examine the effeds of nonlinear spectral response on PCR calibration models. During the course of the study we identified some circumstances where PCR and PLS satisfactorily model nonlinear response by inclusion of extra factors or latent variables in the calibration model. We have also identified some circumstances where extra factors do not give a satisfactory model of the nonlinear response. To handle the latter circumstances, we report a weighting method for reducing the effects of nonlinear spectral regions on PCR calibration models. We also report the capability of PCR models with quadratic terms and artificial neural networks (ANN) to model nonlinearity.

BACKGROUND Principal component regression (PCR) and partial least squares (PLS)are two multivariate full-spectrum calibration techniques that have received considerable attention in the chemometrics literature (2-6). Many researchers have observed circumstances where inclusion of extra principal components or latent variables in the calibration model can model @ 1991 Amerlcan Chemical Sockly