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Journal of Chromatography A, 1218 (2011) 7264– 7274

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

j our na l ho me p ag e: www.elsev ier .com/ locate /chroma

ultistep derivatization method for the determination of multifunctionalxidation products from the reaction of �-pinene with ozone

onrad Kowalewski, Tomasz Gierczak ∗

aculty of Chemistry, Warsaw University, al. Zwirki i Wigury 101, 02-089 Warsaw, Poland

r t i c l e i n f o

rticle history:eceived 19 April 2011eceived in revised form 18 August 2011ccepted 19 August 2011vailable online 25 August 2011

a b s t r a c t

A novel three-step analytical method was developed which enables the simultaneous detection and iden-tification of multifunctional oxygenated products resulting from the reaction of �-pinene with ozone.The method consists of the following steps: conversion of carbonyl groups to methyloximes using methy-loxyamine, conversion of carboxylic acids to methyl esters using trimethylsilyldiazomethane (TMSD), andconversion of alcohols to trimethylsilyl ethers using N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA).

eywords:econdary organic aerosol-Pineneultifunctional oxygenated compoundserivatizationC–EI-MS

The derivatization procedure at each stage was optimized yielding the appropriate amount of deriva-tization reagent, reaction temperature and time. The newly developed analytical procedure manageswithout processes of extraction and evaporation to dryness at any stage. Total time for sample analysisis short ca. 3 h. The characteristic ions of derivatives and common pattern for ion fragmentation in capil-lary gas chromatography electron impact mass spectrometry (GC–EI–MS) analysis were elucidated anddiscussed.

. Introduction

An active interest in the secondary organic aerosols composi-ion and mechanism of formation has been stimulated when itranspired that SOA can directly counteract the global warmingffect through the reflection of solar radiation [1,2]. Additionally,t has been shown that SOA also alters the physicochemical prop-rties of clouds, indirectly affecting the planet’s climate. The SOAormation occurs via oxidation of volatile organic compounds inhe atmosphere [3]. The sources of large emissions of volatilerganic compounds into the atmosphere are presumed to origi-ate from anthropogenic and biogenic sources. The anthropogenicource includes the vehicle and fossil-fueled power plant emis-ions, solvent usage, landfills and hazardous waste facilities [4].he biogenic source is thought to arise from vegetation whichmits large amounts of isoprene and terpenes [5–7]. It is esti-ated that world-wide emissions of volatile organic compounds

excluding methane) are 60–140 million tons per year from anthro-ogenic sources and ∼1150 million tons (of carbon) per year fromiogenic sources [8]. These compounds emitted from biogenicources belong to different organic families: alkanes, alkenes (espe-

ially terpenes), alcohols, aldehydes, ketones, ethers and esters.hey are oxidized to semi- and low-volatile compounds in theresence of hydroxyl radical, ozone and nitrate radical [9–13].

∗ Corresponding author.E-mail address: gierczak@chem.uw.edu.pl (T. Gierczak).

021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2011.08.061

© 2011 Elsevier B.V. All rights reserved.

Generally, as a result of oxidation of gaseous precursors mono-and multifunctional oxygenated compounds are formed, such asmono- and dicarboxylic acids, alcohols and carbonyl compounds.The presence of functional groups makes a molecule heavier andmore polar, hence oxidized compounds are characterized by lowervolatility. These oxidized compounds must exhibit sufficiently lowvapour pressures at particular atmospheric conditions to condenseand form secondary organic aerosol particles. However, a major-ity of aldehydes, alcohols, ethers and monocarboxylic acids stillremains too volatile to form aerosol. They act mainly as interme-diate products which are further oxidized. The final products arestrongly polar and might be dicarboxylic acids, polyhydric alcohols,amino acids and other multifunctional compounds. These con-dense easily contributing to secondary organic aerosol formation[14–20].

�-Pinene, which is the most abundant terpene in nature, playsa significant role in the formation of SOA [8]. In 1998 Jang andKamens [21] investigated the reaction of �-pinene with ozone,obtaining nor-pinonic acid, pinonic acid, 2,2-dimethylcyclobutane-1,3-dicarboxylic acid, pinic acid and pinonaldehyde as majorproducts (see Fig. 1). Identification of the dicarboxylic acids sug-gested that they can act as nuclei of biogenic aerosols due to theirlow vapour pressures [22]. However, not all products and pre-cise mechanisms of the atmospheric oxidation of monoterpenes

have been found so far [23]. This is why, there are problems inanalyzing them, in addition to their extreme complexity and diver-sity and the lack of diagnostic standards. The knowledge of thesecondary aerosols composition will shed light on the aerosol

K. Kowalewski, T. Gierczak / J. Chroma

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moqdptchsiprst

Fig. 1. Simplified diagram of �-pinene ozonolysis.

ormation mechanisms and answer to what degree the SOA affectshe climate, atmospheric chemistry and human health [24,25].

The long term objective of our research is the investigation ofechanisms for the reaction of unsaturated hydrocarbons with

zone leading to the formation of secondary organic aerosol. Conse-uently, we need the appropriate analytical tools to simultaneouslyetect, separate and identify the oxygenated products, especiallyroducts containing several oxygenated functional groups. In ordero separate components of a complex mixture superbly efficientapillary gas chromatography (GC) has been applied. However,ighly polar compounds require derivatization prior to GC analy-is. Several derivatization analytical methods have been describedn the literature and these are based on the derivatization of single

olar group followed by the subsequent gas chromatography sepa-ation [26–34]. In order to analyze several polar groups within oneample the multistep derivatization procedure coupled with GCechnique was proposed in this work. The method described herein,

togr. A 1218 (2011) 7264– 7274 7265

is designed to identify within one sample, the oxidation prod-ucts based on the specific derivatization of different oxygenatedpolar groups to identify multifunctional polar organic compounds.Only a few methods, like the one presented in this rationale,have been described in the literature and these require severaloperations and are time-consuming [35–38]. In addition, some ofthese operations can lead to the unaccounted loss of analytes. Yuet al. [35] suggested a two-step derivatization for the identificationof the hydrocarbons oxidation products. They used O-(2,3,4,5,6-pentafluorobenzyl)hydroxyamine (PFBHA) for the derivatizationof carbonyl groups and BSTFA for the derivatization of carboxyland hydroxyl groups. Similarly, Kleindienst et al. [36] carried outderivatization with the use of the same reagents to determinethe photooxidation products of toluene. In both above mentionedworks, PFBHA was added as aqueous solution. This required consec-utive evaporation to dryness before addition of BSTFA. Additionally,the reaction time ranged from 16 up to 24 h to ensure the complete-ness of the reaction with PFBHA. Jang and Kamens [21] analyzedseparately carbonyl products and carboxylic acids of SOA usingPFBHA and pentafluorobenzyl bromide (PFBBr), respectively [37].The conversion of acids took 2–3 h at 40–60 ◦C, whereas the deriva-tization procedure for carbonyl compounds lasted up to 24 h. Thederivatization products were extracted with a mixture of hexaneand dichloromethane (DCM), and concentrated to the volume of1 mL. In another research [38] three different types of derivatiza-tion reagents were used in order to identify hydroxydicarboxylicacids: N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) with1% trimethylchlorosilane (TMCS) for silylation, diazomethane formethylation and ethanol with TMCS for ethylation. The silylationpotential of MSTFA is similar to that of BSTFA, while the use ofdiazomethane for derivatization of acids is not currently recom-mended on account of its extreme toxicity and explosive propertieswhen handled inappropriately.

The main aim of this work was to develop a multistepderivatization procedure allowing the simultaneous detectionand identification of different SOA oxygenated compounds. Athree-step derivatization procedure is proposed which consistsof converting the carbonyl groups into methyloximes, this is fol-lowed by conversion of the carboxyl groups into methyl esters, andfinally the hydroxyl groups into trimethylsilyl ethers. Therefore, weused respectively O-methyloxyamine hydrochloride, 2 M solutionof TMSD in hexane, and BSTFA with TMCS in a ratio 99:1. The elim-ination of evaporation and solvent extraction was a high priority inorder to prevent loss of the analytes and to avoid laborious processof sample preparation.

2. Experimental

2.1. Optimization of the derivatization procedures

The optimization of every single derivatization step was per-formed to find the most selective reagent volume corresponding tospecific given concentration of analytes as well as the best reactiontime and temperature. To optimize particular derivatization step,the compounds bearing specific functional group were only used,e.g. aldehydes and ketones to optimize the reaction with methy-loxyamine.

2.1.1. Alkylation with methyloxyamineThe reaction of aldehydes and ketones with methyloxyamine

solution was optimized in terms of reaction times and concen-

trations of reagent. Additionally, the reaction temperature wasinvestigated for the optimal concentration of methyloxyamine atregular intervals of time. 500 �L mixtures containing nine com-pounds (acrolein, pentanal, benzaldehyde, nonanal, butan-2-one,

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266 K. Kowalewski, T. Gierczak / J. C

entan-2-one, 4-methylpentan-2-one, heptan-4-one, undecan-2-ne) in DCM (0.12 mg/mL), and 500 �L methyloxyamine solutionn acetonitrile (ACN) of 1, 5 and 10 mg/mL concentration wererepared. The course of reactions was observed in time peri-ds extending up to 78 h. Experiments described above wereonducted in neutral solutions and at room temperature. Then,t was checked what influence temperature exerts on a react-ng mixture. Therefore, 300 �L of methyloxyamine solution of

mg/mL in ACN was added to 200 �L of sample (0.12 mg/mL inCN/CH2Cl2 (1:1,v:v)) containing 5 ketones (butan-2-one, pentan--one, 4-methylpentan-2-one, heptan-4-one and undecan-2-one),

aldehydes (pentanal, benzaldehyde) and undecane as internaltandard. Reactions were conducted for 0.5 h, 1 h, 2 h, 3 h, 4 h and

h at the following temperatures: 45 ◦C, 60 ◦C, 70 ◦C and 80 ◦C.

.1.2. Methylation with TMSDThe second step, conversion of carboxylic acids to methyl esters,

as optimized taking into account the TMSD solution and methanololumes. The following eight carboxylic acids were chosen forptimization: pentanoic, ethanedioic, propanedioic, heptanoic,exanedioic, octanedioic, citric and decanedioic. Samples of 200 �L

n volume containing these acids and internal standard (propylben-ene) of 40 �g/mL each dissolved in a mixture of ACN/CH2Cl2 (1:1,:v) were prepared. Then, they were treated with increasing vol-mes of TMSD solution (ranging from 1 �L to 200 �L) and methanolfrom 0 �L to 25 �L). Such samples were sonicated for 20 min tollow derivatization to occur.

.1.3. Silylation with BSTFAThe optimization of the silylation reaction relied on check-

ng kinetics of the reaction and finding the right volume ofSTFA with TMCS in relation to the amount of hydroxycompoundssed. Twelve alcohols were chosen: izopropanol, propan-1-ol,ert-butanol, butan-1-ol, 2-metoxyethanol, pentan-1-ol, cyclopen-anol, propane-1,2-diol, benzyl alcohol, octan-1-ol, nonan-1-ol andecan-1-ol. At the time of optimization, to 200 �L of 50 �g/mL solu-ions in CH2Cl2 0.1; 1; 2; 5 and 10 �L of BSTFA were added. Silylationas conducted for 2 h at 70 ◦C in a water bath. Reaction times were

nvestigated for 200 �L sample of alcohols prepared exactly in theame way, adding 2 �L of BSTFA and carrying out derivatization for0, 45, 60 and 120 min at 70 ◦C.

.2. Model compounds derivatization

18 model compounds containing various oxygenated groupsere used to prove the effectiveness of the three-step derivati-

ation method developed in this work. They were dissolved in aixture of CH2Cl2 and ACN (1:1) to obtain a solution of 0.12 mg/mL

f each compound. At first, 260 �L of the heated acetonitrile solu-ion of methyloxyamine hydrochloride (1 mg/mL) were added to00 �L of the heated solution of 18 model compounds. The vialontaining these two solutions was sealed and left in a water batht 70 ◦C for about 2 h. Then, at the second stage of derivatization,0 �L of TMSD solution and 10 �L of methanol were added to theixture and the vial was placed in an ultrasonic bath for 20 min.t the third stage of derivatization, an excess of BSTFA was added

n order to neutralize methanol. In this case, 155 �L of BSTFA weresed. However, only 2–5 �L of BSTFA reacted with hydroxyl groupsf the model compounds. The sample was heated at 70 ◦C for 1 h.hen cooled to room temperature, 1 �L of the sample was injected

nto the GC column.

.3. GC–EI–MS analysis

Shimadzu gas chromatograph (GC-17A) coupled with auadrupole mass spectrometer (QP-5000) with EI mode was

togr. A 1218 (2011) 7264– 7274

used. The injection port was heated to 300 ◦C. A ZB-5ms column(30 m × 0.25 mm × 0.50 �m) was used to separate derivatives. Theoven temperature program was set to 40 ◦C for 3 min, then tem-perature increased to 160 ◦C at a rate of 6 ◦C min−1, and to 300 ◦Cat a rate of 25 ◦C min−1. Temperature of 300 ◦C was held for 3 min.1 �L of a sample was injected. The only exception to the chromato-graphic conditions described above took place when temperatureeffect on ketones derivatization was investigated (Fig. S6). In thiscase, the column initial temperature was kept at 35 ◦C for 3 min,subsequently temperature rose to 160 ◦C at a rate of 6 ◦C min−1,and to 260 ◦C at a rate of 25 ◦C min−1, next was held at 260 ◦C for3 min. The injector and transfer line temperatures were set to 200 ◦Cand 260 ◦C, respectively. The sample injection volume remained thesame.

2.4. Chemicals and solvents

All compounds and reagents were purchased fromSigma–Aldrich and were characterized as being of GC or 98%purity except trans-norpinic acid and pinic acid, whose purityand identity have not been verified. All chemicals were usedwithout further purification. BSTFA contained 1% of TMCS, actingas catalyst. Dichloromethane, acetonitrile and methanol wereobtained from Sigma–Aldrich as GC quality solvents. Glasswarewas washed, rinsed with hot water and left to dry at room temper-ature. Just before use, the glassware was rinsed three times withdichloromethane and dried at 100 ◦C.

2.5. Model compounds

18 model compounds were selected to confirm effectiveness ofthe worked out multistep derivatization method. These compoundswere characterized by various functional groups and molecu-lar masses higher than 100 amu to resemble oxidation productsof �-pinene. They were composed of aldehydes (benzaldehyde,nonanal), ketones (heptan-4-one, 4-methylpentan-2-one), alco-hols (nonan-1-ol, decan-1-ol), mono- and dicarboxylic acids(hexanoic acid, heptanoic acid, hexanedioic acid, octanedioic acid,decanedioic acid), multifunctional compounds (3-hydroxybenzoicacid, tartaric acid, citric acid), among them biogenic products of �-pinene oxidation (cis-pinonic acid, pinic acid, trans-norpinic acid,2-hydroxy-3-pinanone). The model compounds were derivatizedin accordance with the procedures described above and analyzedusing GC–EI-MS. The structures of model compounds and their finalderivatives are listed in Table S1 in Appendix A.

3. Results and discussion

The major aim of this study was to develop a multistepderivatization method for the simultaneous analysis of multifunc-tional oxygenated compounds containing carbonyl, carboxyl andhydroxyl groups, without the necessity of extraction or solventevaporation. The necessary steps required to develop such a pro-cedure are described in the following paragraphs.

3.1. Multistep derivatization

The selection of derivatizing agents was defined by three cru-cial factors: (i) the addition of reagent’s moiety small enough so thatthe access to adjacent groups undergoing derivatization in the nextsteps is not impeded, (ii) miscibility of reagents in the set of applied

solvents, and (iii) the avoidance of artifacts (BSTFA preferable toN,O-bis(trimethylsilyl)acetamide, BSA) [39]. The appropriate sol-vents should not only mix with reagents, but they should dissolveanalytes of various polarity and remain inert when derivatization

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K. Kowalewski, T. Gierczak / J. Ch

roceeds at high temperature. Whereas octan-1-ol or nonanal dis-olve in dichloromethane indefinitely, a few milligrams of tartariccid or citric acid are not soluble completely in 50 mL of this sol-ent. Therefore, a mixture of dichloromethane and acetonitrile inqual volumes has been applied.

For the quantification analysis purposes, it was found that therder of derivatization reactions could not be changed as it wastrictly controlled by several factors. Firstly, the ketones contain-ng at least one hydrogen atom attached to � carbon atom adjacento carbonyl group may undergo isomerization to the enol groupsnd these, when mixed with BSTFA, will form ethers. This phe-omenon was observed by Jaoui et al. [40]. Secondly, aldehydes andetones react with alcohols (methanol) in the presence of acidicatalyst, giving acetals. Raising temperature makes the reactionore efficient. It is a competing reaction when compared to methy-

oxyamine. Lastly, BSTFA derivatizes both alcohols and carboxyliccids. In order to perform quantitative analyses and facilitate distin-uishing hydroxyl group from carboxyl group, derivatization withethyloxyamine should be prior to methylation and silylation, andethylation should be followed by silylation.Yu et al. [35] reported the derivatization of carbonyl groups

ith PFBHA·HCl dissolved in ACN with the addition of a minimummount of water. In our work, O-methyloxyamine hydrochlorideas used, a white solid soluble in polar solvents. However, 5 mg of

he compound do not dissolve completely in 5 mL of ACN at roomemperature, giving a murky solution. The addition of water is notdvisable owing to moisture sensitivity of BSTFA used at the thirdtage of derivatization process. It was found that methyloxyamineissolved in the proportion mentioned earlier when heated toigher temperature (70 ◦C) until the solution became clear. Thus,he presence of water in the system and extraction were avoided.

.2. Optimization

The aim of the optimization performance was to minimizehe formation of artifacts by finding optimal quantitative ratiosetween the amount of analytes and a corresponding derivatiza-ion agent and fixing reaction times for the functional groups ofnterest. (Results for optimization experiments presented in theollowing paragraphs are shown in Figs. S1–S13 in Appendix A.he standard deviations shown there were calculated from mea-urements performed using three identically derivatized standardolutions. Standard deviation values were marked for two selectedompounds to make diagrams legible.)

.2.1. Conversion of carbonyl groupsReactions of carbonyl compounds with methyloxyamine did

ot reach an end point even after 8 h at room temperature whichs shown in Figs. S1–S3. Peak areas of methyloximes have stilleen increasing attaining maximum values after ca. 50 h. When the

argest peak areas for individual concentrations of methyloxyaminere compared (Fig. S4 in Appendix A), the optimal reagent con-entration is 1 mg/mL. However, in this case the reaction takesp 56 h. The result is not satisfactory because it makes the firsttage of worked out analytical method time-consuming. Addi-ionally, ketones react with methyloxyamine so slowly that we

ay observe their peaks on chromatograms after 77 h from theeginning of reaction, which makes the derivatization reactionon-quantitative. Therefore, the impact of temperature on deriva-ization rate was investigated. The results presented in Fig. S5 showhat relative areas of derivatives fluctuate around similar values for

ll investigated reaction temperatures (except for methyloximes of-methylpentan-2-one and heptan-4-one whose areas seem to be

ower for shorter reaction times at 45 ◦C and 60 ◦C). However, theisappearance of peaks of ketones occurs only after 5 h at 45 ◦C

togr. A 1218 (2011) 7264– 7274 7267

and after 2 h at other temperatures (see Fig. S6). To sum up, raisingtemperature shortens sample preparation time significantly.

Methyloxyamine reacts with unsymmetrical carbonyl com-pound forming two isomers (syn and anti) of methyloximewhich form two “twin” derivative peaks observed on the GC-chromatogram. In case of ketone methyloximes, the first peak issmaller than the second, whereas for methyloximes of aldehy-des there is a reverse relation. A single peak of derivative appearsfor symmetrical heptan-4-one. It can be calculated that one moleof carbonyl compound with one functional group attached cor-responds to one mole of methyloxyamine. Jang and Kamens [21]reported also the formation of geometric isomers of oximes at roomtemperature in 24 h-reaction with PFBHA diluted with methanol.In our study, none of the solvents containing active hydrogen wasused to prevent artifacts formation. Additionally, reaction time wasdecreased 12-fold comparing to 24 h [21].

3.2.2. Conversion of acidsMethylation of acids by means of TMSD triggers off two compet-

ing reactions: methylation and silylation. Methanol addition has agreat impact on the quantitative response of methylation reaction.A lack of methanol favours the formation of silylated derivatives,whose peaks are larger than the peaks of methyl esters. The resultsof addition of increasing volumes of methanol (0, 5, 10, 25 �L) arepresented in Figs. S7–S10. As more methanol was added, it wasnoted that the peak areas of the methyl esters increased signifi-cantly and reached a maximum for 25 �L of methanol as shown inFig. S11. The disappearance of silylated derivatives peak is clearlyseen on chromatograms when sufficient volume of methanol ispresent. The amount of 90–170 moles of TMSD and 600–1500moles of methanol constitutes a proper dose for the derivatiza-tion of one mole of dicarboxylic acid. It is not recommended toadd more than 100 �L of reagent as it affects the clearness of chro-matograms through a decrease in S/N ratio and artifacts formation.This significantly worsens both detection and identification.

Similar esterification to methyl esters was described by Jaoui etal. [40] with the use of BF3–methanol, where the reaction lasted20 min at 65 ◦C. After derivatization, the neutralization of BF3 wasrequired by the addition of water saturated with sodium chloride,followed by triple extraction and the time-consuming drying pro-cedure. In our work, TMSD was applied which does not need tobe neutralized prior to consecutive derivatization or injection on acolumn.

3.2.3. Conversion of hydroxyl groupsFor the third stage of multistep procedure, the silylation of alco-

hols, the optimal amount of reagent has been estimated at a 10-foldmolar excess in relation to monohydroxyl alcohol. Moreover, apartfrom TMS derivative of tert-butanol, insufficient amount of reagentresults in a decrease of the reaction yield by 90%, while at sub-stantial reagent excess the yield fluctuates between 30% and 95%(Fig. S12). This yield for the derivatization reaction depends onthe molecular structure of alcohol as well. A derivative of tertiarytert-butanol has been formed to a much lesser degree than anyother derivative of primary alcohol. BSTFA is deemed as an effectivederivatizing reagent. The experiment, whose results are present inFig. S13, showed 2 h reaction time to be unnecessary, half of thistime is enough to complete the reaction.

3.3. Model compounds analysis

In the final step of this work the performance of the three-

step procedure developed here was checked. 18 model compoundswere analyzed. These compounds represent all major types of theoxygenated compounds, possibly forming the secondary organicaerosol. Fig. 2 presents GC/MS chromatograms performed after

7268 K. Kowalewski, T. Gierczak / J. Chromatogr. A 1218 (2011) 7264– 7274

Fig. 2. Three-step analysis. (1) methyloxime of 4-methylpentan-2-one; (2) methyl hexanoate; (3) methyloxime of heptan-4-one; (4) hexanoic acid; (5) methyl heptanoate;(6) heptanoic acid; (7) methyloxime of benzaldehyde; (8) nonan-1-ol; (9) methyloxime of nonanal; (10) methyloxime of 2-hydroxy-3-pinanone; (11) decan-1-ol; (12)derivative of cis-pinonic acid with both carbonyl and carboxyl groups derivatized; (13) methyloxime of cis-pinonic acid; (14 and15) BSTFA; (16) dimethyl hexanedioate(dimethyl adipinate); (17 and 18) dimethyl derivatives of norpinic acid, cis and trans isomers; (19) contamination from BSTFA reagent; (20 and 21) dimethyl derivativesof pinic acid, cis and trans isomers; (22) methyl ester of 3-hydroxybenzoic acid; (23) dimethyl octanedioate (dimethyl suberate); (24) methyl 3-trimethylsilyloxybenzoate;(25) trimethyl ester of 2-hydroxypropane-1,2,3-tricarboxylic acid (trimethyl citrate); (26) dimethyl decanedioate (dimethyl sebacate); (27) BSTFA; (28) contamination; (29)1-trimethylsilyloxynonane (trimethylnonyloxysilane); (30) 1-trimethylsilyloxydecane (trimethyldecyloxysilane); (31) methylated and silylated derivative of dihydroxybu-tanedioic acid (tartaric acid).

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Table 1GC–EI-MS data for model compounds.

Peak number Derivative of a compound Ions present in EI spectrum Molecular formulaof a compound

MW1 of acompound

Molecularformula of aderivative

MW2 of aderivative

[MW2–OCH3]+ [MW2–CH3]+

1 Methyloxime of4-methylpentan-2-one

129,114,98,87,72,57,56,42,41 C6H12O 100 C7H15NO 129 98 114

2 Methyl hexanoate 101,99,74,59,55,43 C6H12O2 116 C7H14O2 130 99 –3 Methyloxime of

heptan-4-one143,128,115,100,87,70,57,41 C7H14O 114 C8H17NO 143 – 128

5 Methyl heptanoate 113,101,74,59,55,43 C7H14O2 130 C8H16O2 144 113 –7 Methyloxime of

benzaldehyde135,108,104,89,77,65,51,39 C7H6O 106 C8H9NO 135 104 –

9 Methyloxime of nonanal 140,86,73,69,55 C9H18O 142 C10H21NO 171 140 –16 Dimethyl adipinate 143,114,101,83,74,59,55,43 C6H10O4 146 C8H14O4 174 143 –10 Methyloxime of

2-hydroxy-3-pinanone126,125,111,99,79,71,55,43 C10H16O2 168 C11H19NO2 197 – –

29 Nonyloxytrimethylsilane 201,143,129,115,103,89,83,75,73,69 C9H20O 144 C12H28OSi 216 – 20117,18 Dimethyl esters of

norpinic acid169,140,114,99,83,67,55,39 C8H12O4 172 C10H16O4 200 169 –

30 Decyloxytrimethylsilane 230,217,215,115,103,101,97,89,75,61 C10H22O 158 C13H30OSi 230 – 21520,21 Dimethyl esters of pinic

acid183,182,154,139,128,114,96,83,69,55,41,39 C9H14O4 186 C11H18O4 214 183 –

22 Methyl3-hydroxybenzoate

152,121,107,93,77,65,53,39 C7H6O3 138 C8H8O3 152 121 –

23 Dimethyl suberate 171,138,129,111,97,83,74,69,59,55,43,41 C8H14O4 174 C10H18O4 202 171 –24 Methyl

3-trimethylsilyloxy-benzoate

224,209,193,177,166,149,135,121,105,91,89,73,59,45 C7H6O3 138 C11H16O3Si 224 193 209

31 Derivative of tartaric acid,2,3-bis(trimethyl-silyloxy)dimethyl ester ofbutanedioic acid

307,263,247,234,217,175,161,147,133,119,103,89,73,59,45 C4H6O6 150 C12H26O6Si2 322 – 307

25 Trimethyl citrate 175,153,143,111,101,84,69,59,43 C6H8O7 192 C9H14O7 234 – –12 Derivative of cis-pinonic

acid with derivatized–C O and –COOH groups

196,154,136,128,108,100,99,81,69,68,58,41 C10H16O3 184 C12H21NO3 227 196 –

26 Dimethyl sebacate 199,166,157,138,125,98,97,84,83,74,59,55,43,41 C10H18O4 202 C12H22O4 230 199 –

Characteristic ions are written in bold, base peaks are underlined.

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270 K. Kowalewski, T. Gierczak / J. C

ach stage of the multistep derivatization method studied here.he identification was based on the characteristic and specific frag-ent ions present in EI mass spectra of obtained derivatives. In this

aragraph we assigned a molecular weight of a model compound asW1 and its derivative as MW2. The molecular weights of the ana-

yte derivatives and their characteristic fragment ions are presentedn Table 1.

Carbonyl groups. The characteristic ions of methyloximes facili-ating structure confirmation are [MW2–CH3]+ and [MW2–OCH3]+.

ethyloximes are imines containing –C N– double bond with thedjacent methoxy group. Such unsaturated functionality may leado the McLafferty rearrangement through a six-membered ring,hich gives an explanation for the unexpected fragment ions. Ana-

yzing the mass spectrum of cis-pinonic acid derivative (ketoaciderivative, MW2 = 227) shown in Fig. 3, the origin of the m/z 154 ionight be explained by the elimination of •CH2COOCH3 radical from

molecular ion (typical fragmentation for methyl esters), whereashe presence of the m/z 100 ion suggests the McLafferty rearrange-

ent with the participation of nitrogen atom which results in allylictabilization of unpaired electron and the formation of very stablentermediate [41]. The m/z 196 characteristic ion is also present.he structural details of fragmentation mechanisms are given inig. 4. By contrast, in the mass spectrum of 2-hydroxy-3-pinanoneerivative (MW2 = 197) there are not any fragment ions observedhich would form by simple elimination of methyl or methoxy rad-

cal. The highest-mass ion, crucial for identification, is at m/z 126hose formation might result from cyclohexane ring cleavage of

he molecular ion. Another ion at m/z 99 forms via the McLaffertyearrangement of the molecular ion with subsequent hydrogenearrangement leading to cyclobutane ring opening. Then the elim-nation of 4-methylpenta-1,3-dien-2-ol gives rise to the m/z 99 ionsee Fig. S14). Therefore, it might be inferred that a fragmentationattern of any derivative should be considered individually with areat attention to details.

Carboxyl groups. TMSD reacts with carboxylic acids to yieldethyl esters and by-products (artifacts), mainly trimethylsilyl

sters and trimethylsilylmethyl esters. After methanol addition,ethylation reaction is favoured. In the mass spectra of methyl

sters, ions [MW2–OCH3]+ and [MW2–CH3OH]+• are common.dditionally, esters of long-chain acids, both monocarboxylic andicarboxylic, are characterized by the m/z 74 ion (present in spectraf methyl hexanoate and heptanoate, dimethyl adipinate, suberatend sebacate), formed as a result of the McLafferty rearrangement41]. If this method was to be used to analyze the real samples, thenhe m/z 74 ion would serve as a good indicator for distinguishingetween acidic �-pinene oxidation products and long-chain acids.hese straight aliphatic acids were found in the real samples ofM2.5 aerosol [42].

Citric and tartaric acids represent monohydroxy tricarboxylicnd dihydroxy dicarboxylic acids, respectively. Their derivativeragmentation patterns exhibit some common features. In the spec-rum of trimethyl citrate (MW2 = 234) the m/z 175 ion is obtained as

result of •COOCH3 radical loss from a molecular ion, whereas therigin of the m/z 143 ion might be explained by the loss of •OCH3adical from the molecular ion with subsequent loss of •COOCH3nd •H radicals from the newly formed fragment ion (m/z 203).ragmentation pathway of trimethyl citrate is presented in Fig. S15.imilarly, in the spectrum of tartaric acid derivative (MW2 = 322,ee Fig. 3), the loss of •COOCH3 radical from a molecular ion resultsn the formation of the m/z 263 ion. Ion at m/z 247 might be derivedndirectly from the molecular ion through the loss of methyl radicalCH3 (m/z 307), followed by the loss of •COOCH3 and •H radi-

als, which is shown in details in Fig. 5 [38,43]. Looking at thepectrum of dimethyl norpinate (MW2 = 200) in Fig. 3, it mighte concluded that the cation radical at m/z 140 is formed fromMW2–OCH3]+ ion at m/z 169 by the loss of CO with •H, and fur-

togr. A 1218 (2011) 7264– 7274

ther as a result of cyclobutane ring fragmentation (loss of ethenemolecule, C2H2) the cation radical at m/z 114 forms. When methoxyradical •OCH3 is eliminated from this fragment ion, the ion at m/z83 is obtained. However, another fragmentation pathway existsleading to the formation of the m/z 140 ion. The McLafferty rear-rangement may happen resulting in the opening of cyclobutanering. Then consecutive loss of CH3OH and CO results in the for-mation of m/z 140 ion. Fig. 6 depicts the proposed fragmentationmechanisms of dimethyl norpinate. For the homologue of dimethylnorpinate, dimethyl pinate, assuming the same fragmentation pro-cess as described above [MW2–OCH3–CO + H–C2H2]+, ions at m/z183, 154 and 128 are obtained (see Fig. S16). Koch et al. [44] andChistoffersen et al. [22] presented the same EI spectrum of dimethylester of norpinic acid and dimethyl ester of pinic acid, respectively.

The peaks of two isomeric derivatives (cis and trans) for norpinicand pinic acids are present. At the second stage of the three-stepanalysis the competing silylated derivates of organic acids havehardly appeared, when TMSD was used with sufficient amount ofmethanol. With reference to fragmentation, it is not advisable tointerpret the presence of low-mass ions, for instance ions at m/z41, 43, as indicators of carboxyl groups in methyl esters [40]. In ourwork, although these ions were present together or singly in themass spectra of methyl esters, the m/z 41 ion was also a base peakfor methyloxime of heptan-4-one and the m/z 43 ion was a basepeak for methyloxime of 2-hydroxy-3-pinanone. However, the m/z59 ion is useful being present in the mass spectra of methyl estersof carboxylic acids and hydroxycarboxylic acids exclusively.

In future, the multistep analytical procedure presented in thiswork could, perhaps, be applied for field measurements. Hence,benzaldehyde and 3-hydroxybenzoic acid were also included intothe model compounds. These compounds were chosen to mimicsome other aromatic compounds (for instance vanillic acid orsyringic acid) possibly present in real environmental samples [45].The identification of benzaldehyde and 3-hydroxybenzoic acidderivatives was relatively simple, due to the presence of molecularions, distinct fragmentation pattern of benzene ring and char-acteristic ions. These derivatives will be readily identified anddistinguished among the peaks of �-pinene oxidation products fornatural aerosol sample analysis.

Hydroxyl groups. BSTFA reacts with every functional grouppossessing an active hydrogen. After the methylation ofcarboxyl groups, BSTFA derivatizes only hydroxyl groups.Characteristic ions of silylated derivatives of hydroxycom-pounds make: [Si(CH3)3]+ at m/z 73, [HO Si(CH3)2]+ at m/z75, and [MW2–CH3]+, [MW2–Si(CH3)3]+, [MW2–OSi(CH3)3]+,[MW2–(CH3)2Si OSi(CH3)3]+ [35,43,46–49]. In silylated ethers ofalcohols, there is evident that both fragmentation pathways of amolecular ions take place, initiated either by the charge located onoxygen atom or initiated by a radical. In the first case [Si(CH3)3]+

ion at m/z 73 is formed, whereas in the second case [MW2–CH3]+

ion and [H2C O-Si(CH3)3]+ ion at m/z 103 are present in themass spectra of 1-trimethylsilyloxynonane (see Fig. S17) or 1-trimethylsilyloxydecane and also in the mass spectrum of tartaricacid derivative (dihydroxy dicarboxylic acid derivative). The m/z103 ion may suggest the position of TMSoxy group bonded withaliphatic carbon atom rather than bonded directly with a ringstructure.

Similar deduction might be presented for[(CH3)2Si OSi(CH3)3]+ ion at m/z 147 present in the spectrumof tartaric acid derivative with methylated carboxyl groups andsilylated hydroxyl groups. The formation mechanism of this ion isshown in Fig. 5. The m/z 147 ion constitutes a marker for the iden-

tification of derivatives possessing two TMS groups in a moleculesituated closely enough to interact together. This relationship wasobserved by Yu et al. [35] for dicarboxylic acids, hydroxy carboxylicacids and dihydroxy aldehydes when derivatized with BSTFA.

K. Kowalewski, T. Gierczak / J. Chromatogr. A 1218 (2011) 7264– 7274 7271

Fig. 3. Mass spectra of: (a) cis-pinonic acid derivative (MW2 = 227), (b) tartaric acid derivative (MW2 = 322) and (c) dimethyl norpinate (MW2 = 200).

7272 K. Kowalewski, T. Gierczak / J. Chromatogr. A 1218 (2011) 7264– 7274

Fig. 4. The proposed fragmentation of cis-pinonic acid derivative.

of tar

ddtoaacad

Fig. 5. Fragmentation

Trimethyl citrate and methyloxime of 2-hydroxy-3-pinanoneo not undergo silylation, probably on account of the steric hin-rance. The trimethylsilyl group occupies too much space to reachhe hydroxyl group and substitute hydrogen atom. Therefore, peaksf trimethyl citrate and methyloxime of 2-hydroxy-3-pinanonere also present on the third chromatogram for the three-step

nalysis (Fig. 2). A small peak of tartaric acid derivative may indi-ate incomplete derivatization of the hydroxyl groups of tartariccid which resulted in signal decrease if compared with othererivatives.

taric acid derivative.

The results obtained prove that the multistep derivatiza-tion analytical method is capable to analyze all used modelcompounds irrespective of their polarity or functionality. Asshown by the EI mass spectra molecular ions are not present(except aromatic compounds) or are of low relative intensitiesto be taken into consideration. However, characteristic ions and

advanced fragmentation of a molecule provide the opportunityof elucidating structure of the molecule if initial knowledge onexpected oxidation products or mechanisms of their formation isavailable.

K. Kowalewski, T. Gierczak / J. Chromatogr. A 1218 (2011) 7264– 7274 7273

entat

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cknowledgements

Financial support of the Polish Committee for Scientific Researchnder grant 3 TO9A 019 27 is acknowledged. The authors would likeo thank reviewers for their critical comments on the manuscript.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.chroma.2011.08.061.

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