JOURNAL OF MASS SPECTROMETRY, VOL. 31, 1163-1168 (1996)

Gas Chromatography/Mass Spectrometry of 13 Resin Acids as their PFB Esters

F. Dethlefs', K. 0. Gerbardtz, H.-J. Stan' Institute of Food Chemistry, Technical University of Berlin, Gwtav-Meyer-Allee 25,13355 Berlin, Germany Experiment Station Chemical Laboratories-Mass Spectrometry Facility and Department of Biochemistry, College of Agriculture,

Food and Natural Resources, University of Missouri-Columbia, Columbia, M O 65211, USA

GC/MS method was employed for the separation and identification of resin acids from pulp mill effluents as their pentafluorobenzyl (PFB) esters. The mass spectra of standards and emuent samples were analyzed and fragmenta- tion patterns were elncidated for 13 resin acids. Characteristic fragment ions and the mass spectra of 13 resin acids are presented, namely pimaric, sandaracopimaric, isopimaric, 8,15-isopimaric, 8,15-pimaric, abietic, neoabietic, palustric, levopimaric, dehydroabietic, 7-oxodebydroabietic, O-methylpodwarpic and 8(14)-abietenic acid.

KEYWORDS : resin acids; pentafluorobenzyl ester; gas chromatography/mass spectrometry; fragmentation

INTRODUCTION

Resin acids are tricyclic diterpenoids which occur natu- rally in conifers. Softwood species are widely used as raw material in the pulp and paper industry. Resin acids are released from the wood during the manufacture of pulp and paper. They are very resistant to chemical degradation and survive the pulping and bleaching process. When effluents remain untreated, they are dis- charged into the environment. Resin acids of pulp mill effluent are considered to be major contributors to the toxicity of f i~h . l -~

Resin acids must be derivatized before analysis by capillary gas ~hromatographyl.~-~ and methylation with diazomethane has usually been applied.4~9~'0 In recent years, pentafluorobenzyl bromide (PFBBr), a less toxic and harzardous derivatizing agent than diazo- methane, has been used for the derivatization of the acids.3111 In contrast to diazomethane, this agent is commercially available, safe in handling and the PFB derivatives are more stable than the methyl esters. Fur- thermore, 'native' methyl esters can be analyzed together with acids. Gas chromatography coupled with mass spectrometry (GC/MS) has always been applied for the reliable identification of individual com- pounds. 1.3-6,8,10-12 F undamental investigations of the fragmentation of diterpenoids including resin acid methyl esters have been carried out by Biemann,I3 Audier and c o - ~ o r k e r s ' ~ ~ ' ~ and Chang et a1.16

In this paper, we report the GC separation of and MS data for 13 resin acid PFB esters. The interpreta- tion of the mass spectra of the resin acid PFB esters is based on the important investigations of the authors mentioned above.

The method has also been applied to a pulp mill EOP bleaching process water (EOP = alkaline extrac- tion, oxygen and peroxide, the chemicals used in the bleaching process). The results have been published ele- sewhere."

REAGENTS AND MATERIALS

All solvents were of analytical grade. Methanol and toluene were purchased from Riedel-de Haen (Seelze, Germany). Triethylamine was obtained from Merck (Darmstadt, Germany) and pentafluorobenzyl bromide from Aldrich (Steinheim, Germany). RP-C, , solid-phase material was purchased from Baker (Deventer, The Netherlands).

All resin acids were obtained with a purity between 90 and 99% from Helix Biotechnologies (Richmond, BC, Canada): 8( 14)-abietenic, isopimaric, 8,154~0- pimaric, abietic, palustric, levopimaric, neoabietic, dehydroabietic, 7-oxodehydroabietic and O-methyl- podocarpic acid. Pimaric acid was supplied at a purity of 85% and accompanied by 15% sandaracopimaric acid; 8,15-pimaric acid was supplied at a purity of 79%, accompanied by 8J5-isopimaric (16%) and pimaric acid (3%). They all were used without further purification.

EXPERIMENTAL

Solid-phase extraction (SPE) with RP-C18 adsorbent

Glass cartridges filled with 1 g of RP-C,, adsorbent were used for SPE. The solid-phase columns were con- ditioned by rinsing with 6 ml of methanol followed by

CCC 1076-5174/96/101163-06 0 1996 by John Wiley & Sons, Ltd.

Received 20 January 1996 Accepted 4 July 1996

1164 F. DETHLEFS, K. 0. GERHARDT AND H.-J. STAN

6(14)-Abietenic acid la(l4).s.betM-lh*)

@ "COOH

Levopimaric acid (8(14) 1MbidDdOn-IBQLeI

Pimaric acid (a(15) 1 Spirnsr.dl.n-lll-de)

@'I 'COOH

5ff COOH

8,lS-lsopmanc acid (8.15i50plmnradlen 18-aate)

Dehydmabtetic aud 18 11 lUblehtnehl8Q.(CI

@ "COOH

,;_.c;r COOH

' ' 1 1 ,

8.15-Pimanc acid (8 15prnr~len- laode)

& C W H

0-Methylpodcarpic acid

@ Palustric "COOH acid

18.13-akI.d~en-l~Fopo

lsopimaric acid (7.1 asopimandicn-1-e)

'COW 7-Oxodehydmabieiic acid (7-xod.11 , l3..~~mn-l8-olts)

Figure 1. Formulae of the 13 resin acids.

8 ml of a mixture of deionized water adjusted to pH 8-9 with 10% of methanol, corresponding to the content of methanol in the sample. The spiked water sample (10 ml) and the EOP emuent sample (10 ml) were allowed to percolate through the column. The car- tridges were dried, first by applying low vacuum and then with a gentle stream of nitrogen. Resin acids were eluted with 8 ml of methanol into a pointed-bottom flask and the extracts were evaporated to dryness using a rotary evaporator.

Derivatization with PFBBr

A volume of 200 p1 of a PFBBr solution (2% in toluene) and 10 p1 of triethylamine were added to the dry residue of extracts in a pointed-bottom flask. The flask was sealed and kept for 1 h at 90 "C, then excess reagent was removed by a stream of nitrogen. The PFB deriv- atives were dissolved in 1 ml of toluene and analyzed by GC/MS. The derivatized extracts of EOP effluent were injected directly.

13 1

Figure 2. Total ion chrornatograrns (TIC) of (a) 2.5 pI of a stan- dard mixture of resin acids (50 ng of each substance, san- daracopimaric acid 3.75 ng) and b) an extract of 10 ml of EOP ffluent. 1 = Pirnaric acid; 2 = sandaracopimaric acid; 3 = isopimaric acid; 4 = palustric acid; 5 = 8(14)-abietenic acid; 6 = levopimaric acid; 7 = abietic acid; 8 = dehydroabietic acid; 9 = 0-rnethylpodocarpic acid (ISTD); 10 = neoabietic acid; 11 = 7-oxodehydroabietic acid; 12 = secodehydroabietic acid; 13 = 8.1 5-isopimaric acid; 14 = 8.1 5-pimaric acid; 15 = triene resin acid; 16 = monoene resin acid; 17 = diene resin acid; 18 = 6,8,11,13-abietatetraenic acid. The numbers correspond to those in Table 1.

Instrumental analysis

A gas chromatograph from Hewlett-Packard (HP5890 Series 11) equipped with an HP 7673 autosampler and a Gerstel KAS3 injector was coupled to the mass spectro- meter MS-Engine HP 5989A operating in the electron impact (EI) mode (70 eV) at a source temperature of 240 "C. Data acquisition over the mass range 50-500 u was carried out with an HP 59944C MS ChemStation. An QV-17 fused-silica column (25 m x 0.32 mm id.) with a film thickness of 0.25 pm was used with helium grade 5.0 (purity 99.999%) as the carrier gas. Addi- tionally, a deactivated fused-silica column (1 m x 0.32 mm id.) was used as a retention gap. The injection port temperature program was initially 150 "C, increased at 12°C s-' to 260"C, held for 1 min, then increased at 12°C s - ' to 300"C, held for 5 min; the splitless time was 1.8 min. A 2.5 p1 sample was injected with the autosampler. The temperature program of the GC oven was started at 90°C, held for 3 min, then increased at 4 "C min- to 260 "C, held for 20 min. The temperature of the transfer line was 190 "C.

GC/MS OF RESIN ACID PFB ESTERS 1165

467

IT 911m l- I I

icwpmats Ud

Abu- 91

181 1 3

90 “I m I 146 I

3.31

2Fb s! m l 703 181

RESULTS AND DISCUSSION

Figure 3. Mass spectra of the 13 resin acid PFB esters.

Figure 1 shows the structures of the 13 resin acids inves- tigated. They can be readily determined after derivatiza- tion with PFBBr to their pentafluorobenzyl (PFB) ester^.^.'^ This method has been successfully applied to the analysis of resin acids in a pulp mill EOP bleaching process emuent after isolation by solid-phase or liquid- liquid extraction. A detailed description of the analyti- cal procedure and the application to EOPemuent has been published by Dethlefs and Stan.”

Figure 2 presents a representative chromatogram of resin acids in a standard mixture and of one extract of an EOP eMuent sample. Of the 13 resin acids discussed, 11 can be identified in the chromatogram of the EOP emuent. 0-Methylpodocarpic acid was used as an inter- nal standard because it had not been found in pulp mill

effluents. This substance has also been used by others as a surrogate or internal standard because of its structur- al similarity to the resin In addition to the clearly identified resin acids, another five compounds could be assigned to the resin acids based on their molecular ion and certain characteristic fragments.

The mass spectra of the PFB esters of the 13 resin acids are presented in Fig. 3. The complexity of frag- mentation suggested presenting the mass spectra as bar graphs rather than in tabulated form. All low-resolution spectra were obtained by GS/MS with (EI) ionization (70 eV). A careful background subtraction was per- formed. The resin acids belong to the tricyclic (& ter- penoids and show some structural similarities. All have 4P-and l0P-methyl and 4a-carboxyl-PFB substitution. The isomers differ in number and location of double bonds and stereochemistry, e.g. epimerism. Whereas the abietane series has an isopropyl substituent at C-13, the

1166 F. DETHLEFS, K. 0. GERHARDT AND H.-J. STAN

I kpmmc W

241 Atund.M

181

50 im 150 200 250 300 350 ~ D D 11s~

468

181 1 171

31 3

(4 Figure 3. Continued.

gous to those of the methyl esters elucidated by the previous workers. Our emphasis was to establish a ref- erence collection of the mass spectra of the PFB deriv- atives from resin acids which are of utmost interest to environmental chemists.

A very important characteristic feature is that all compounds show a clearly recognizable molecular ion ranging in relative abundance from 7 to 67%. Obvi- ously the cyclic structure is a stabilizing factor. The characteristic ions supporting the identification of the resin acids are formed by loss of the PFB moiety ([M - 1811). Another characteristic ion as for all PFB esters is found at m/z 181, representing the highly stabil- ized pentafluorobenzyl/tropylium ion which is always observed at high abundance. However, the carboxyl group is simultaneously or consecutively eliminated as COz, HCOz or HCOZH, resulting in [M - 2251 ([M - 181 - 44]), [M - 2261 ([M - 181 - 451) or [M - 2271 ([M - 181 - 461) ions, respectively. The relative abundances of these ions are given in Table 1. The loss of a methyl radical is frequently observed ([M - 151). The formation of [M - 2411 results from the combined loss of the methyl, pentafluorobenzyl and formate radicals. This fragmentation pathway is obvi- ously favoured by many resin acid esters and results in seven of the studied compounds in the base peak. The fragmentation was derived from basic interpretations of tricyclic diterpenoids published by the workers men- tioned previously and following general fragmentation rules as given by McLafferty and Turqek.’

In analogy the mass spectra of all the compounds investigated were interpreted and the ions assigned plausible structures. Seven resin acids were found in the EOP effluent that were not available as standard sub- stances (peaks12-18). Characteristic fragments in their mass spectra allowed them to be recognized as resin acids. The two minor peaks 12 and 18 were identified as secodehydroabietic and 6,8,11,13-abietatetraenic acid. Confirmation was achieved by comparison of the mass spectra of the methyl esters with entries in the Wiley mass spectral library.’O The two major peaks 13 and 14 were identified as the two epimers 8,15-isopimaric and 8,15-pimaric acid from their PFB ester mass spectra, which are almost identical. The two compounds were therefore synthesized on commission (Helix Biotech- nologies, Richmond, BC, Canada) in order to confirm their identity and to distinguish the epimers chromato- graphically.

pimarane and isopimarane series have epimeric vinyl and methyl groups at the same position.l6,’*

Thirty years ago, fundamental studies on the frag- mentation of the methyl esters of the resin acids were carried out by Biemann,13 Audier and c o - w o r k e r ~ ’ ~ ~ ’ ~ and Chang et al.’6 In this study, we found the fragmen- tation patterns of the resin acid PFB esters to be analo-

Acknowledgements

The authors thank the Deutsche Forschungsgemeinschaft (DFG) for financial support through the Sonderforschungsbereich 193 (Sfb 193) of the Technical University of Berlin.

~~

Tabl

e 1.

Fra

gmen

tatio

n pa

ttern

s of t

he 1

3 re

sin a

cid

PF

B es

ters

: m/e

with

rel

ativ

e abu

ndan

ce (“h

) in p

aren

thes

es

Res

in a

cid

8(14

)-A

biet

enic

aci

d 8,

15-ls

opim

aric

aci

d-1 3

-40.

83

8.1

5- P

imar

ic a

cid

Pim

aric

aci

d S

anda

raco

pim

aric

aci

d ls

opim

aric

aci

d P

alus

tric

acid

Le

vopi

mar

ic a

cid

Abi

etic

aci

d N

eoab

ietic

aci

d D

ehyd

roab

ietic

aci

d 7-

Oxo

dehy

droa

biet

ic ac

id

0- M

ethy

lpod

ocar

pic a

cid

Peak

N

o. 5 13

14 1 2 3 4 6 7 10 8 11

9

RT

(min

) M

+

43.3

1 48

4 (8

) 40

.83

482

(9)

41.3

9 48

2 (9

) 41

.75

482

(18)

42

.08

482

(23)

42

.62

482

(38)

43

.18

482

(62)

43

.65

482

(34)

44

.54

482

(70)

46

.68

482

(36)

44

.77

480

(9)

51.1

9

494

(1 8)

45.1

8 46

8 (8

7)

[M-151

469

(1)

467

(10)

46

7 (1

2)

467

(19)

46

7 (2

3)

467

(12)

46

7 (88)

467

(2)

467

(5)

467

(1 j

465

(1 2)

47

9 (2

) 45

3 (4

)

[M-1

811

303

(4)

301 (8)

301 (8)

301

(13)

30

1 (1

0)

301

(76)

30

1 (7

) 30

1 (1

0)

301

(100

) 30

1 (6

) 29

9 (-

) 31

3 (4

0)

287(

4)

[M - 2

251

259

(1 6)

25

7 (2

5)

257

(1 8)

257

(28)

25

7 (2

1 )

257

(51)

25

7 (8

) 25

7 (8)

257

(26)

25

7 (3

) 25

5 (1

) 26

9 (6

) 24

3 (3

)

[M - 22

61

[M - 22

71

[M - 24

11

258

(4)

257

(3)

243

(1 2)

25

6 (1

0)

255

(9)

241

(100

) 25

6 (9

) 25

5 (8)

241

(100

) 25

6 (6

) 25

5 (5

) 24

1 (1

3)

256

(3)

255

(4)

241

(20)

25

6 (6

7)

255

(72)

24

1 (1

00)

256

(4)

255

(4)

241

(100

) 25

6 (6

) 25

5 (1

4)

241

(4)

256

(88)

25

5 (3

0)

241

(46)

25

6 (3

) 25

5 (2

) 24

1 (4

) 25

4 (-

) 25

3 (-

) 23

9 (1

00)

268

(10)

26

7 (2

4)

253

(100

) 24

2 (3

) 24

1 (1

1)

227(

100)

Base

pea

k 2 $

241

E? 12

1 2

21 5

24

1 P *

121

241

241 91

30

1 m

8 cd

.rl

135

El 23

9 3

253

6 27

7

1168 F. DETHLEFS, K. 0. GERHARDT AND H.-J. STAN

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