Use of specific bacteria for the determination of mutagenic and

Use of specific bacteria for the determination of mutagenic and carcinogenic ... between genotoxic potency, mutagenic potency, and carcinogenic potenc...
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Anal. Chern. 1987, 59, 504-508

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(89) Olson, B. Anal. Chlm. Acta 1982, 136, 113-118. (70) Kok, G. L.; Holler, T. P.; Lopez, M. B.: Nachtrieb, H. A.; Yuan, M. Environ. Sci. Technol. 1978, 12, 1072. (71) Hwang, H.; Dasgupta, P. K. Anal. Chim. Acta 1985, 170, 347. (72) Hinze, W. L.: Igarashi, s.,submitted for publication in Anal. Lett.

RECEIVED for review May 13, 1986. Accepted September 16, 1986. This research was supported by the National Science

Foundation (CHE-8215508) and, in part, by Wake Forest University through a Research and Publication Fund Grant. We thank a referee for helpful comments. This work was presented at the Symposium on Processes in Organized Assemblies, Santiago, Chile, December 9, 1985 [Abstr. No. 31 and at the 191st National Meeting of the American Chemical Society, New York, NY, April 14,1986 [Abstr. No. ANAL 161.

Use of Specific Bacteria for the Determination of Mutagenic and Carcinogenic Compounds Stanislav MiertuEi,*' Jozef Svorc,' Ernest Sturdjk: and Helena Vojtekovii' Department of Analytical Chemistry and Department of Biochemical Technology, Slovak Polytechnical University, 812 37 Bratislava, Czechoslovakia

PosslMmles of the use of €scher/ch/a CON K-12 in the determination of mutagenic compounds have been tested. Analylkai parameters, e.g., the range of linearity of the analytical curve, the detection limit and the serrsitivlty, the accuracy, and the precision of analysls, have been evaluated for a series of nitrofurans. The detection ilmit Is In the range of lO-'-lO5 mol L-'. Anaiyses of real samples (determination of 5-nltro-2-furyiacrylicackl in wine and nitrovin in chicken meat) have been completed without any preseparatlon.

The mutagenic and carcinogenic hazards of many chemical compounds are some of the most topical problems in the protection of the living environment. The number of such compounds is still increasing. They are spread in different areas of the living environment-in the atmosphere, water, and soil or as contaminants in food. Many of them are present in industrial processes (1, 2). Analysis of these samples by classical analytical procedures is often complicated due to the complexity of the samples as well as the low specificity and sensitivity of the methods. That is why more sophisticated methods are urgently needed. As regards the specificity of analytical methods, biological systems belong to the most specific ones. This possibility also exists in the case of sensitive microorganisms with a specific response to the mutagenic compounds. Certain bacteria, e.g., Salmonella typhimurium (3)or Escherichia coli (4-7)) have been frequently used for the detection of mutagenicity. However, there have only been a few attempts to use that systems in the analytical determination of mutagenic compounds. Karube et al. (8) have constructed a Salmonella microbial electrode and correlated the current decrease with mutagen concentration. The goal of the present work is to study the possibilities of the use of the response of biological systems, namely, bacteria Escherichia coli K-12, to the mutagenicity of chemical compounds for analytical purposes. Recently, Quillardet et al. (4-6, 9) have specifically manipulated bacteria strain E. coli K-12 to produce SOS response after DNA damage by mutagenic compounds (the so-called SOS chromotest) (Figure 1). The SOS response is manifested by increased production 'Department of Analytical Chemistry. Department of Biochemical Technology.

of enzyme /3-galactosidase. For details of genetic manipulation, see original papers of Quillardet et al. (4,6).The amount of produced enzyme correIates with the amount of mutagenic compound. The level of enzyme alkaline phosphatase is also determined. This enables detection of the proteosynthesis inhibition, which can occur at higher concentrations of mutagenic compounds (4). The direct assay consists of incubating the tester strain with increasing concentrations of the agent to be tested. After a time for protein synthesis, @-galactosidase and alkaline phosphatase are determined. The SOS chromotest has been exclusively used until now for tests of mutagenic potency and sensitivity to various compounds, namely, benzofurans, naphthofurans, nitrosamines, fungal toxins, and antibiotics (4, 6). In spite of the fact that there is quantitative relation between SOS response and amount of mutagenic compound, there has been no attempt until now to use this specific bacteria for analytical purposes, i.e., for determination of the amount of mutagenic compound. That is why we focus our attention in this paper to the study of analytical application of the SOS chromotest. We have studied in detail the following analytical parameters: (1) the linearity range of dependence between the concentration of mutagen and response of bacteria; (2) detection limits and sensitivity for different compounds; (3) accuracy and precision of the analysis. The above-mentioned tasks have been solved for 10 nitrofurans. This type of compounds was chosen for the following reasons: (i) the mutagenic potency of these compounds has not been tested by SOS chromotest; (ii) they are considered by many authors as a representative direct mutagens (10); (iii) they are still widely used in clinical and veterinary medicine and in the food industry (10, 11). We have also examined the possibilities of determination of mutagenic compounds in real complex samples without any preseparation. We have determined 5-nitro-2-furylacrylicacid (NFAA) directly in wine. This compound was used as a wine stabilizer (12). In the second example, nitrovin (used as a growth stimulator (11))is determined directly in chicken meat.

EXPERIMENTAL SECTION Chemicals. The formulas of the studied nitrofurans are shown in Table I. 5-Nitro-2-furylacrylicacid (NFAA) (1)was obtained from Slovakofarma Hlohovec (Czechoslovakia). Alkyl estersand amides of NFAA (2-9) were synthesized according to Sturdik et al. (13, 14). Nitrovin (9) was obtained from Chemapol Praha (Czechoslovakia),and nitrofurantoin (10) was purchased from Sigma (St. Louis, MO). Other chemicals of analytical grade

0003-2700/87/0359-0504$01.50/0 C 1987 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987

505

0-galactosidase IONPG)

'f I 12

compound

E

COLI

9

6 I alkaline phosphatase ( PNPP I

Figure 1. Main steps of the procedure for the SOS chromotest. and p -nltrophenyl phosphate (PNPP) are the substrates for determlnation of the enzymes @galactosidaseand alkaline phosphatase, respectlvely. (See text for details.) (0 -Nitrophenyl)-@-D-galactopyranoslde (ONPG)

(s),

Table I. Survey of 5-Nitrofurans Studied: Sensitivity Detection Limit (nL), and Concentration Detection Limit (CL)

no.

structure"

5 6 7 8

RCH=CHCOOH RCH=CHCOOCH3 RCH=CHCOOCzH5 RCH=CHCOOC3H7 RCH=CHCOOCH(CHJZ RCH=CHCOOCdHg RCH=CHCONHZ RCH=CHCONHCH3

9

RCH=CHCCH=CHR

1

2 3 4

S,

nL,

lO%L,

nmol-'

nmol

mol L-'

4.8 14.4

0.42

0.69

0.14

0.23

9.9 6.7 1.1

0.20 0.29

5.8 12.8 38.7

0.34 0.08 0.15 0.05

0.33 0.48 2.90 0.57 0.13 0.25

9.9

0.20

It

24.4

1.74

0.09

NNHCNHNH,*HCI ( n it r o v i n )

10

RCH=NNCO,

lCO/"

0.33

(nitroturantoin)

R = 5-nitro-2-furyl. (dimethyl sufoxide (Me2SO),components of buffers) and enzyme substrates (0-nitrophenyl)-P-D-galactopyranoside (ONPG) and p-nitrophenyl phosphate (PNPP)were provided by Lachema Brno (Czechoslovakia). Bacteria Strain. Strain Escherichia coli K-12, recombinant PQ 37, was granted by Maurice Hofnung, Institut Pasteur Paris. Medium and Buffers. Bacteria were cultured in LB medium (10 g of Bacto tryptone, 5 g of Bacto yeast extract, 10 g of NaCl/liter) supplemented with ampicillin at final concentration, 20 pg/mL. Z buffer was prepared according to Miller (15),Le., 16.1 g of Na2HP04.7H20,5.5 g of NaH2P04-H20,0.75 g of KC1, 0.25 g of MgS04.7H20,and 2.7 mL of @-mercaptoethanolper liter adjusted to pH 7.0. T buffer was 1 mol L-' tris(hdyroxymethy1)aminoethane (Tris) adjusted to pH 8.8 with HCl ( 4 ) . Procedures. SOS Chromotest. The test consists of colorimetric assay of enzymatic activities after incubation of the tester strain in the presence of various amounts of test compounds. A detailed procedure was published by Hofnung and Quillardet (4, 6,9).Briefly, an exponential-phase culture grown to Am = 0.2 in LB medium with ampicillin at 37 "C was diluted 1:lO into fresh medium. Fractions (0.6 mL) were distributed into glass test tubes containing 10-60 pL of the diluted compound to be tested. After 2 h of incubation at 37 "C with shaking, @-galactosidaseand alkaline phosphatase activities were assayed. Enzyme Assays. Alkaline Phosphatase (4-6). T buffer (0.9 mL) was added to 0.1 mL of cell culture. Cell membranes were disrupted by adding 2 drops of 0.1% sodium dodecyl sulfate solution and 2 drops of chloroform and mixing vigorously. Tubes were equilibrated at 28 "C. The reaction was started by addition of 0.2 mL of PNPP (4 mg/mL in T buffer) and stopped by addition of 0.34 mL of 2 mol L-' HCl. After 5 min, 0.34 mL of 2 mol L-' Tris was added to restore the color, which was measured spectrophotometrically at 420 nm.

3

0

0.5

10

15

20

n [ nmo~] Flgure 2. Dependence of Induction factor I, vs. amount n (in nmol) of selected nitrofurans: W, NFAA; e, methyl ester; 0,ethyl ester: A, n-propyl ester; A, isopropyl ester; 0, butyl ester NFAA.

@-Galactosidase(15). The protocol was the same as for alkaline phosphatase except that Z buffer replaces T buffer, ONPG replaces PNPP, and the reaction was stopped with 0.65 mL of 1 mol L-l Na2C03. The enzyme activities are expressed in the units defined by Miller (15). The ratio R for the activities of @-galactosidaseand alkaline phosphatase is calculated as

R = A@42,tP/AP420t@

(1)

where ARm, to, AP420,and tP represent the absorbance at 420 nm and the reaction time from the @-galactosidaseand alkaline phosphatase assays (6). Because the time of the enzymatic reactions is the same (mostly 1 h), the ratio is R = AR420/Ap42p. To compare results obtained in different experiments, it IS convenient to normalize the ratio R, obtained in the presence of the mutagenic compound by dividing it by its value Ro obtained at the absence of the mutagenic compound. The induction factor If is thus If = R,/Ro

(2)

The dependence of the induction factor Ifvs. amount of mutagenic compound (n) is linear for a certain interval. SOS-inducing potency (SOSIP) is calculated as a slope from the linear range of If = f(n)and represents the change of the induction factor If per nanomole of the compound tested ( 4 ) . Preparation of Real Samples. Wine Sample. NFAA solution in Me2S0 mol L-l) was diluted 10 times with distilled water and then different amounts were added to the wine (white wine "RaEianskf vfber", VZB Bratislava) to obtain appropriate concentrations. The control contained a wine sample diluted in the same way but without NFAA. mol Chicken Liuer Sample. Nitrovin solution in Me2S0 L-l) was diluted directly to appropriate concentrations in the chicken liver homogenate prepared by the homogenization of 10 g of liver in 5 mL of distilled water by a Potter homogenizer. The control contained only the sample of liver homogenate diluted in the same way. Statistics. For a statistical evaluation of the analytical parameters of the method (accuracy, precision, detection limit, sensitivities, etc.) six parallel measurements were performed at each concentration of mutagen. Data evaluation was carried out according to the recommendations of this journal and used statistical procedures are individually described and cited in Results and Discussion.

RESULTS AND DISCUSSION Linearity Range and Choice of Model Calibration Curve. Utilizing the SOS chromotest in quantitative analysis, we ascertained the linear range of the induction factor If dependence w. the amount (n) of mutagen. Examples of these dependences for several nitrofurans are shown in Figure 2. The survey of studied compounds is given in Table I. Nitrofurans were applied in the range 0.1-2 nmol. This de-

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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987

Flgure 3. Dependence of induction factor 1, on added volume of measured sample at constant amount of mutagen ( n = 0.2 nmol of NFAA

amide).

pendence is linear within the range 0.1-1 nmol for compounds 2 and 4. For compounds 9 and 10 linearity is within the range 0.1-0.5 nmol. For other nitrofurans the dependence If = f ( n ) is linear in the whole interval of applied amounts, i.e., 0.1-2 nmol. The linear range of dependence If = f ( n )can be described by the straight line equation If = a S n (3)

0

0,2

0,L

0.6

p[nmoi] Figure 4. Dependence of percentage accuracy (relative error of measurement, 6) vs. true content ( p ) of sample NFAA.

$4 4 -

+

However, if mutagen is not present in the system ( n = 0) the value of If is equal to 1, according to the definition of the induction factor, and the value a should be always equal to 1. We have proved by the statistical F test (16) that the y axis intercept value was not different from 1 for all compounds. Detection Limit and Sensitivity of the Method. For detection limit values of mutagen amount (nL), we can use eq 3, namely

3-

5

0

10

15

20

'3

~[nmol-

Figure 5. Dependence of percentage accuracy (6) vs. sensitivity of

method S (in nmol-I).

Accuracy and Precision of the Results of Analysis. where S is the slope of linear dependence of eq 3, i.e., senis the limit value of the signal (induction factor), sitivity, which should be k times higher than the background signal, If(,,).Background signal is measured from activity of enzyme @-galactosidaseand alkaline phosphatase in the absence of mutagenic compound; however, according to eq 2 the value of is always equal to 1. To distinguish unambigufrom background, various values of k are recomously If(lim) mended but most often k = 3 (17). It follows from eq 4 that the detection limit is inversely dependent on the slope of the line of eq 3, Le., sensitivity S. Detection limits expressed in units of substance amount can be converted to limit concentrations of mutagen in the sample. However, it is necessary to know the maximal volume of the sample that can be added to the bacteria without a decrease of the signal. Thus we studied the dependence of induction factor vs. sample volume for equal amounts of the mutagen. This dependence for amide of 5-nitro-2-furylacrylic acid (NFAA amide) a t the amount of 0.2 nmol is shown in Figure 3. It follows from this dependence that the maximal sample volume added to the bacteria is about 60 pL. This VL volume was used for the evaluation of the concentration detection limit cL as follows: (5) The sensitivity of the method is expressed by the equation S = A I f / h ( 4 ) ;thus the sensitivity represents the slope of the linear part of dependenceIf = f ( n )(eq 3). Detection limits n~ and cL and sensitivites of the method for representative samples of the selected groups of mutagens are given in the Table 1.

The accuracy of the results was evaluated by using percentage accuracy, 6. Percentage accuracy is defined as follows: 6 = 1OO(ii - p ) / p , where ii is the mean of individual measurements (found contents) and p is true content of mutagen. The values of the true content of mutagens have been represented by the value of the amount (weight) of mutagen added to the bacteria. Figure 4 shows the dependence of percentage accuracy 6 vs. true NFAA amide content, p. It follows from the given dependence that the value is minimal within the range 0.16-0.18 nmol; Le., the method is the most accurate in this region. If the determined content of the compound approaches the detection limit or it reaches values under the detection limit, the relative error of measurement represented by 6 increases sharply. Further the dependence of percentage accuracy 6 of measurement vs. sensitivity S of the method for different compounds was constructed. An example of this dependence for 0.2 nmol content of compounds with different sensitivity (NFAA, ethyl and methyl amide, and NFAA amide, respectively) is shown in Figure 5. It follows from the given dependence that the relative error of measurement (percentage accuracy 6) decreases with the increasing sensitivity. The precision of the results of the analysis has been evaluated by calculating the relative standard deviation S, = lOO(s/ii) (in %), where k

s = [

(Izi

- ii)Z/(k - 1)]1/2

i = l

where r i is the mean of the set of individual found contents (ni)and k is the number of measurements. The values of S, (in percent) are plotted in Figure 6 at difference amounts of determined NFAA. The minimum of this dependence is in the region of 0.2-0.4 nmol of NFAA where the relative

ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987

0.2

0.6

507

1.0 n [nmoi]

Figure 8. Dependence of induction factor I , vs. amount of NFAA n (In nmol) in the absence (- - -) and in the presence (-) of wine.

L Oh

I

I

0.8

12

I

n [nmol]

Flgure 8. Dependence of percentage precision (s,)vs. amount of NFAA n (in nmol).

0.1

0.3

0.5 n [nmd]

Figure 7. Dependence of Induction factor If vs. amount of nttrovin n (in nmol) in the absence (-) and in the presence (- - -) of liver

homogenate.

standard deviation reaches values up to 4%. For higher concentrations the relative standard deviation is in the range of 8-12%. These are quite acceptable values regarding the complexity of the method. Analysis of Real Samples. In this part of the work we verified the possibility of determining two substances-NFAA and nitrovin-in real samples. NFAA was used as a wine stabilizer (12). However, after mutagenic properties were found in NFAA, the use of this substance as a stabilizer was forbidden in several countries. Nitrovin is still used as a growth stimulator for livestock (11). The procedures for the preparation of real samples are described in the Experimental Section. The control contained only the wine sample or the sample of liver homogenate without NFAA or nitrovin, respectively. In both cases the measurement was carried out for comparison purposes also in the absence of chicken liver homogenate and wine, respectively. Four parallel determinations in all cases were done. The dependence of induction factor If vs. nitrovin and NFAA substance amounts (n)was measured. Values of slopes of If = f(n)were calculated. The coincidence of arithmetical means of slopes (sensitivities was tested by the statistical F test (16)for determinations in the presence and absence of wine and liver homogenate, respectively. With the F test it was found that in the case of nitrovin the slopes (8)of dependences If = f(n)were not congruent (Figure 7). In the presence of liver homogenate the slope is equal to 36.1 nmol-'; in its absence is equal to 38.1 nmol-l. The liver homogenate causes little inhibition of nitrovin mutagenic effect probably due to components present in the liver that could

s)

s

s

react with nitrovin. A similar test has been carried out for the blank experiment (Le., without nitrovin). The ratios of Ro (see eq 2) were the same in the presence as well as in the absence of liver homogenate; that means neither mutagenic activity nor inhibition of proteosynthesis by matrix (liver homogenate) was observed. In the case of NFAA the slopes of dependences, It = f ( n ) , were congruent in both the presence and absence of wine (5 = 4.6 nmol-') (Figure 8). Also the values of Ro ratios were congruent; the wine sample showed no increased mutagenic activity either, and the analysis can be carried out without preseparation. In both cases detection limits were calculated (see part Detection Limit and Sensitivity of the Method). The values of 0.3 mg of nitrovin/l kg of liver and 1mg of NFAA/1 L of wine have been obtained. These values correspond to those conditions of the experiment when a real sample (liver homogenate, wine) is directly added to the bacteria. In some cases the sample must be diluted by distilled water before being added to the bacteria because protein synthesis can be inhibited by some sorts of wine at high concentrations and the relation of If = f ( n )is not accurate (pure liver homogenate could cause problems with solution turbidity, etc.). If, however, the corresponding real sample is diluted, the detection limit is increased equivalently. Thus the optimal wine dilution was achieved for some sorts of wine. It was found that 2-fold wine dilution for all types tested is sufficient (50% wine); thus the detection limit is 2 mg of NFAA/L of wine. As a dose of 10-20 mg of NFAA was added per 1 L of wine for stabilization purposes (12),the NFAA content can be determined also with greater than optimal dilution directly without separation. In a similar way, the optimal liver homogenate dilution can be determined. Careful homogenization of liver has enabled us to approach the theoretical detection limit, Le., 0.3 mg of nitrovin/l kg of liver. Summarizing obtained results, we can state that the detection limits for the given method are in the range 9 X lo-' to 8 X mol L-' and the sensitivities lie between 0.4 and 40 nmol-'. We can compare these analytical parameters with those obtained by classical analytical method. For example, the detection limit of colorimetric determination of nitrofurans (18) is - lo4 mol L-l, which is a comparable value with those obtained by our approach. The possibility of determination of mutagenic compounds directly in real samples of foods was found. NFAA in wine and nitrovin in chicken liver were chosen to serve as model mixtures. The detection limitis are sufficiently low in both cases (2 mg/dm3 for NFAA and 0.3 mg/kg for nitrovin) to determine these mutagens in real samples, e.g., in imported wines or in products of the podtry industry as well as in other foods directly, without preseparation. Such determination is fully correct when the sample contains a single mutagen

508

Anal. Chem. 1987. 59. 508-513

without any interferring substances. When the sample contains several mutagens, the determination of the individual constituents is more questionable. A preseparation is then recommended.

ACKNOWLEDGMENT We are grateful to Maurice Hofnung,Institut Pasteur, Paris, for providing the bacterial strain and to JBn Garaj, Slovak Technical University, Bratislava, for stimulating support of this work. Registry No. 1, 6281-23-8; 2, 1874-24-4; 3, 1874-12-0; 4, 90147-18-5;5,90147-21-0;6,90147-19-6;7,710-25-8 8,14308-65-7; 9, 804-36-4; 10, 67-20-9.

LITERATURE CITED Fishbein, L. Potenfkl Indusfrlal Carcinogens and Mutagens ; Amsterdam, Oxford, New York, 1979; Vol. 4. Qtoenen. P. J.; de Cook-Bethbedef. M. W.; Bouwman, J.; Dhont, J. H. "N-Nitroso Compounds: Analysis, Fwmatkn and Occurrence; Walker, E. A., *Me, L., Castegnaro, M., M r a h y i , M., Eds.; Lyon, 1980. Maron. D. M.; Ames, B. N. Mutat. Res. 1983, 173, 173-215. Quhrdet, Ph.; Huisman, 0.; D'Ari, R.; Hofnung, M. R o c . Natl. Acad. SCi. U . S . A . W 8 2 , 5971-5975.

Quillardet, ph.; Hofnung, M. Mutaf. Res. 1985, 147, 65-78. Qulllardet, Ph.; de Beicombe, Ch.; Hofnung, M. Mutat. Res. 1985, 147, 79-95. Hayes, S.; Gordon, A.; Sadowski, I.; Hayes, C. Mutat. Res. 1984, 130, 97-106. Karube, I.; Nakahara, T.; Matsunaga, T.; Shuichi, S. Anal. Chem. 1982, 54, 1725-1727. Hofnung, M., special publication, Institute Pasteur, Paris, 1982. McCaila, D. R. Environ. Mutagen. 1983, 5 , 745-765. Atkinson, R . L. Nutr. Rep. Inf. 1971, 3. 363-367. FarkaZ, J. C/ym.-Ab~fr.~1978, 84, 119900. KellovB, G.; Sturdlk, E.; Stibrinyi, L.; Drobnica, i.;Augustin, J. Folia Mlcrqbbl. (Pfague) 1984, 29, 23-34. EbringSturdik, E.; Rosenberg, M.; Stibrinyi, L.; BaPi, 5.; Chreiio, 0.; er, L.; Iiavskg, D.; VBgh, D. Chem.-Biol. Interact. 1985, 53, 145- 153. Miller, J. H. Experiments in Molecular Genetics; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1972. Eckschlager, K.; StQinek, V. Information Theory as Applied to Chemical Analysis ; Wley-Interscience: New York, 1979. Kaiser, H.; Menzies, A. C. The Llmif of Defection of a Complete Analytical Procedure; Adam Hilger: London, 1968. Borgatzi, A. R.; Tarozzi. F.; Grisetig, G.; Brusco, A. Nuova Vet. 1970, 46. 6-21.

RECEIVED for review April 14,1986. Accepted September 23, 1986.

Characterization of Beech Milled Wood Lignin by Pyrolysis-Gas Chromatography-Photoionization Mass Spectrometry Wim Genuit' and Jaap J. Boon* Mass Spectrometry of Macromolecular Systems Unit, FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, T h e Netherlands

Oskar Faix Bundesforschungsanstalt fur Forst- und Holzwirtschaft, Institut fur Holzchemie und chemische Technologie des Hokes, Leuschnerstrasse 91, 2050 Hamburg 80, West Germany

A Cwle-pobrt pyrdysls-gas chromatography-photolonlzath maw spectrometry Instrument has been used for characterIzaHon of beech mllled wood Ilgnln. The llght source for photolonlretlon Is a rare gas resonance lamp. The main pyrolyds products have been Identffled and are Wed Into a propoeed thennal degradation scheme for the beech Hgnln. I t Is shown that the responses for these products depend strongly on lonizlng energy. The cumulatlve photoionization mass spectra at two photon energie0, 11.8 eV (Ar I)and 10.6 eV (Kr I)y are compared to a 15-eV electron impact pyrolysis mass spectrun. The Ar I photdonlratlon and 1 5 4 electron Impact IOnizeUon mass spectra both show a hbgh abundance of fragment Ions, whereas In the Kr I mass spectrum predominantly molecular Ion peaks are observed.

Curie-point pyrolysis is a useful general method for thermal depolymerization of macromolecular materials, producing characteristic chemical units amenable to gas chromatographic or mass spectrometric analysis (I,2). A large variety of materials has been characterized by automated Curie-point pyrolysis-mass spectrometry (Py-MS) (2),and many groups of biomaterial8 have been differentiated by subjecting their 'Present address: Shell Research Laboratory, Badhuisweg 3,1031 CM Amsterdam. T h e Netherlands.

Py-MS fingerprints to advanced numerical data analysis methods (2-6). However, mass spectrometric results on the composition of pyrolysates depend strongly on the way in which the pyrolysate is transferred from the pyrolysis zone to the ion source and on the ionization conditions (6-9). The transfer problem can be avoided by applying in-source pyrolysis, but this in turn leads to rapid source contamination and sets and undesirable restriction to the heating rate, to prevent high-pressurepulses in the vacuum system. In the instrument described here a chromatographic inlet is used, which provides well-defined transfer conditions. Furthermore, this method enables the identification of individual pyrolysis products (Py-GC-MS mode), as well as the production, by spectrum accumulation, of a useful mass spectral fingerprint (Py-MS mode). Py-MS studies (2) generally apply low-energy (10-16 eV) electron impact ionization (EI) to enhance the characteristics of the mass spectral fingerprints by reducing fragmentation compared to 70-eV EI. At these low energies, however, small variations in electron energy lead to large differences in mass spectra, which is one of the main factors involved in poor interlaboratory reproducibility in Py-MS (8). Photoionization (PI) by means of a vacuum-UV light source is an attractive alternative to low-energy EI, because of the well-defined, highly constant, and reproducible ionizing energy. Although the ion yield of PI is much lower than that of 70-eV EI, it can be expected to be equal to or larger than that of E1 at energies near threshold, because photoionization cross sections have

0003-2700/87/0359-0508$01.50/00 1987 American Chemical Society