Part 2: Forensic attribution profiling of Russian VX in food using liquid chromatography-mass spectrometry.

This work is part two of a three-part series in this issue of a Sweden-United States collaborative effort towards the understanding of the chemical attribution signatures of Russian VX (VR) in synthesized samples and complex food matrices. In this study, we describe the sourcing of VR present in food based on chemical analysis of attribution signatures by liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with multivariate data analysis. Analytical data was acquired from seven different foods spiked with VR batches that were synthesized via six different routes in two separate laboratories. The synthesis products were spiked at a lethal dose into seven food matrices: water, orange juice, apple purée, baby food, pea purée, liquid eggs and hot dog. After acetonitrile sample extraction, the samples were analyzed by LC-MS/MS operated in MRM mode. A multivariate statistical calibration model was built on the chemical attribution profiles from 118 VR spiked food samples. Using the model, an external test-set of the six synthesis routes employed for VR production was correctly identified with no observable major impact of the food matrices to the classification. The overall performance of the statistical models was found to be exceptional (94%) for the test set samples retrospectively classified to their synthesis routes.

A ricin forensic profiling approach based on a complex set of biomarkers.

A forensic method for the retrospective determination of preparation methods used for illicit ricin toxin production was developed. The method was based on a complex set of biomarkers, including carbohydrates, fatty acids, seed storage proteins, in combination with data on ricin and Ricinus communis agglutinin. The analyses were performed on samples prepared from four castor bean plant (R. communis) cultivars by four different sample preparation methods (PM1-PM4) ranging from simple disintegration of the castor beans to multi-step preparation methods including different protein precipitation methods. Comprehensive analytical data was collected by use of a range of analytical methods and robust orthogonal partial least squares-discriminant analysis- models (OPLS-DA) were constructed based on the calibration set. By the use of a decision tree and two OPLS-DA models, the sample preparation methods of test set samples were determined. The model statistics of the two models were good and a 100% rate of correct predictions of the test set was achieved.

Deamidation in ricin studied by capillary zone electrophoresis- and liquid chromatography-mass spectrometry.

Deamidation in ricin, a toxin present in castor beans from the plant Ricinus communis, was investigated using capillary zone electrophoresis (CZE) and liquid chromatography coupled to high resolution mass spectrometry. Potential sites for deamidation, converting asparagine (Asn) into aspartic or isoaspartic acid (Asp or isoAsp), were identified in silico based on the protein sequence motifs and tertiary structure. In parallel, CZE- and LC-MS-based screening were performed on the digested toxin to detect deamidated peptides. The use of CZE-MS was critical for the separation of small native/deamidated peptide pairs. Selected peptides were subjected to a detailed analysis by tandem mass spectrometry to verify the presence of deamidation and determine its exact position. In the ricin preparation studied, deamidation was confirmed and located to three asparagine residues: Asn54 in the A-chain, and Asn42 and Asn60 in the B-chain. Possible in vitro deamidation occurring during sample preparation was monitored using a synthetic peptide with a known and rapid rate of deamidation. Finally, we showed that the isoelectric diversity previously reported in ricin is related to the level of deamidation.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Direct derivatization and gas chromatography-tandem mass spectrometry identification of nerve agent biomarkers in urine samples.

Rapid determination of nerve agent biomarkers at low-ppb levels in urine samples was achieved by direct derivatization and sample analysis using gas chromatography-tandem mass spectrometry. The studied biomarkers were alkylphosphonic acids (APAs), as they are specific hydrolysis products of organophosphorus nerve agents that can be used to verify nerve agent exposure. The sample preparation technique employed involves rapid direct derivatization (5min) of acidified urine samples (25µL) using a highly fluorinated phenyldiazomethane reagent [1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene]. The derivatization conditions were optimized using statistical experimental design and multivariate data analysis. The APA derivatives were analyzed by GC-MS and MS/MS using negative ion chemical ionization. The selectivity and sensitivity of analyses performed by low and high resolution single ion monitoring MS-mode were compared with those performed by multiple reaction monitoring MS/MS-mode. The MS/MS technique offered the greatest sensitivity and selectivity of the tested mass spectrometric techniques, with limits of detection ranging from 0.5 to 1ng APAs/mL of urine. The method's robustness was evaluated using urine samples from the OPCW 2nd biomedical confidence building exercise and all APAs present in the samples were conclusively identified. The method thus offers excellent performance and is viable for the simultaneous trace determination of a wide range of nerve agent markers.

A concept study on identification and attribution profiling of chemical threat agents using liquid chromatography-mass spectrometry applied to Amanita toxins in food.

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and ß-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.

Determination of S-2-(N,N-diisopropylaminoethyl)- and S-2-(N,N-diethylaminoethyl) methylphosphonothiolate, nerve agent markers, in water samples using strong anion-exchange disk extraction, in vial trimethylsilylation, and gas chromatography-mass spectrometry analysis.

Since the establishment of the Chemical Weapons Convention in 1997, the development of analytical methods for unambiguous identification of large numbers of chemicals related to chemical warfare agents has attracted increased interest. The analytically challenging, zwitterionic S-2-(N,N-diisopropylaminoethyl) methylphosphonothiolate (EA-2192), a highly toxic degradation marker of the nerve agent VX, has been reported to resist trimethylsilylation or to result in an unacceptably high limit of detection in GC-MS analysis. In the present study, the problem is demonstrated to be associated with the presence of salt, which hinders trimethysilylation. EA-2192 was extracted from aqueous samples by use of a strong anion-exchange disk, derivatized as a trimethylsilyl derivative via in vial solid-phase trimethylsilylation and identified by GC-MS. The limits of detection were 10 ng/mL and 100 ng/mL (in a water sample) for SIM and SCAN mode respectively. The analytical method was found to be repeatable with relative standard deviation <10%. The performance of the method was evaluated using a proficiency test sample and environmental samples (spiked river water and Baltic Bay water) and compared with the commonly used evaporation-silylation method. The disk method displayed good tolerance to the presence of salt and the spiked EA-2192 was conclusively identified in all matrices. In addition, the applicability of the method was further demonstrated for other selected hydrolysis products of VX and Russian VX, namely S-2-(N,N-diethylaminoethyl) methylphosphonothiolate, ethyl methylphosphonic acid, methylphosphonic acid, and isobutyl methylphosphonic acid. For the synthesis of reference compounds, EA-2192 and its analog from degradation of the Russian VX isomer, the present methods were improved by using a polymer-bound base, resulting in >90% purity based on (1)H NMR. Based on the current results and earlier work on alkylphosphonic acids using the same method, we conclude that the method is a viable choice for the simultaneous determination of a wide range of degradation products of nerve agents - zwitterionic, monoacid, diacid, and monothioacid chemicals - with excellent performance.

An isomer-specific high-energy collision-induced dissociation MS/MS database for forensic applications: a proof-of-concept on chemical warfare agent markers.

Spectra database search has become the most popular technique for the identification of unknown chemicals, minimizing the need for authentic reference chemicals. In the present study, an isomer-specific high-energy collision-induced dissociation (CID) MS/MS spectra database of 12 isomeric O-hexyl methylphosphonic acids (degradation markers of nerve agents) was created. Phosphonate anions were produced by the electrospray ionization of phosphonic acids or negative-ion chemical ionization of their fluorinated derivatives and were analysed in a hybrid magnetic-sector-time-of-flight tandem mass spectrometer. A centre-of-mass energy (E(com)) of 65 eV led to an optimal sequential carbon-carbon bond breakage, which was interpreted in terms of charge remote fragmentation. The proposed mechanism is discussed in comparison with the routinely used low-energy CID MS/MS. Even-mass (odd-electron) charge remote fragmentation ion series were diagnostic of the O-alkyl chain structure and can be used to interpret unknown spectra. Together with the odd-mass ion series, they formed highly reproducible, isomer-specific spectra that gave significantly higher database matches and probability factors (by 1.5 times) than did the EI MS spectra of the trimethylsilyl derivatives of the same isomers. In addition, ionization by negative-ion chemical ionization and electrospray ionization resulted in similar spectra, which further highlights the general potential of the high-energy CID MS/MS technique.

Direct derivatization and rapid GC-MS screening of nerve agent markers in aqueous samples.

A rapid screening and identification method based on derivatization and gas chromatography mass spectrometry (GC-MS) has been developed for the detection of alkylphosphonic acids (APAs), the degradation products of organophosphorus nerve agents. The novel method described involves rapid (5 min) and direct derivatization of 25 microL aqueous sample using highly fluorinated phenyldiazomethane reagents (e.g., 1-(diazomethyl)-3,5-bis(trifluoromethyl)benzene). The APA derivatives are then screened by GC-MS negative ion chemical ionization (NICI) and identified by electron ionization (EI) mode. The conditions for the derivatization were optimized using statistical experimental design and multivariate data analysis. Method robustness was evaluated using aqueous samples from an official OPCW Proficiency Test and all APAs present in the sample were conclusively identified. Limits of detection for rapid screening using SIM NICI were between 5 and 10 ng/mL APA in aqueous sample, and for identification using full scan EI 100 ng/mL.

Combination of solid phase extraction and in vial solid phase derivatization using a strong anion exchange disk for the determination of nerve agent markers.

Alkylphosphonic acids (APAs) are degradation products and chemical markers of organophosphorous (OP) nerve agents (chemical warfare agents). Anion exchange disk-based solid phase extraction (SPE) has been combined with in vial solid phase derivatization (SPD) and GC-MS analysis for the determination of APAs in aqueous samples. The optimization of critical method parameters, such as the SPD reaction, was achieved using statistical experimental design and multivariate data analysis. The optimized method achieved quantitative recoveries in the range from 83% to 101% (n=13, RSD from 4% to 10%). The method was sensitive, with LODs in SIM mode of 0.14 ppb, and demonstrated excellent linearity with an average R(2)>or=0.99 over the concentration range of 0.07-1.4 ppm in full scan mode and from 0.14 ppb to 14 ppb in SIM mode. For forensic applications, aqueous samples containing APAs at concentrations exceeding 14 ppb were concentrated and target analytes were successfully identified by spectral library and retention index matching. Method robustness was evaluated using aqueous samples from the official OPCW Proficiency Test (round 19) and all APAs present in the sample were conclusively identified. The SPE disk retained the underivatized APAs in a stable condition for extended periods of time. No significant losses of APAs from the disk were observed over a 36-day period. Overall, the method is well suited to the qualitative and quantitative analysis of degradation markers of OP nerve agents in aqueous matrices with simplicity, a low risk of cross-contamination and trace level sensitivity.

De novo sequencing of RCB-1 to -3: peptide biomarkers from the castor bean plant Ricinus communis.

Ricinus communis (also know as the castor bean plant) whose forbears escaped from suburban gardens or commercial cultivation grow wild in many countries. In temperate and tropical climates seeds will develop to maturity, and plants may be perennial. In Australia these plants have become widespread and are regarded as noxious weeds in many localities. The seeds of R. communis contain ricin, a protein toxin which can easily be extracted into an aqueous solution. Ricin is toxic by ingestion, inhalation, and injection. The history of terrorist and anarchist interest in the use of seeds from R. communis has driven the development of strategies for determination of cultivar and geographic location of the source of an extract of wild-grown castor bean seed. This forensic information is of considerable interest to law enforcement and intelligence organizations. During forensic studies of both the metabolome and proteome of extracts from eight specimens of six different cultivars of R. communis ("zanzibariensis" collected from Kenya and Tanzania, "gibsonii", "impala", "dehradun", "carmencita", and "sanguineus" collected from Spain and Tanzania), three peptide biomarkers (designated Ricinus communis biomarkers, or RCB) were identified in both the MALDI and electrospray LC-MS spectra. Two of these peptides (RCB-1 and RCB-2) were present in varying amounts in all cultivars, while RCB-3 was present only in the "carmencita" cultivar. The amino acid sequences of RCB-1 to -3 were determined using LC-MS(n) fragmentation and de novo sequencing on both the intact and the carbamidomethyl modified peptides. The connectivity of the two disulfide bonds that were present in all three RCB were determined using a strategy of partial reduction and differential alkylation using tris-(2-carboxyethyl)phosphine with N-ethylmaleimide to reduce and alkylate the most accessible disulfide bond, followed by reduction and alkylation of the remaining disulfide bond with dithiolthreitol and iodoacetamide. The possible functional role of RCB-1 to -3 in R. communis seeds is also discussed.

Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic.

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.

Solvent-assisted trypsin digestion of ricin for forensic identification by LC-ESI MS/MS.

The castor bean plant (Ricinus communis) is used in large quantities for oil production and is also a common ornamental garden plant. However, the beans contain 1-3% of the highly toxic protein ricin, a type II ribosome-inactivating protein that is covered by the Chemical Weapons Convention, and there have been a number of reports concerning the use, or alleged use, of the toxin in terrorist and criminal activities. In the study reported here, we investigated the potential utility of organic solvent-assisted trypsin digestion of crude extracts containing the closely related toxins ricin or abrin to prepare samples for peptide analysis by liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Diagnostic tryptic fragments of the toxins were detected and unambiguously identified by this procedure. The sample preparation protocol substantially reduces the sample preparation time, from overnight to an hour, and thus greatly reduces the total time required for analyses, to less than 2 h. Furthermore, the reported procedure leaves the disulfide bonds in the protein intact. This is highly relevant in the context of the Chemical Weapons Convention, since the disulfide bond connecting the two chains of ricin indicates the presence of an intact toxin and provides additional forensic evidence for the analytical results.

PCDD/F in source-sorted waste fractions and emissions from their co-combustion with reed canary-grass.

The dry combustible fraction of source-sorted household waste, including material that would otherwise be recycled, was mixed with the energy crop reed canary-grass (Phalaris arundinacea L.), and combusted as briquettes in 150 and 600 kW biofuel-boilers without advanced cleaning systems. The source-sorted waste was further sorted and characterized according to its material and chemical contents. The bulk of the waste's chlorine content came from the non-package plastic fraction, whereas 90-95% of summation operator PCDD/F (74-90% of WHO-TEQ) originated from the textile fraction. The sources of the dioxins in the waste fractions are discussed. The balance of dioxin levels was negative, i.e., the amounts of dioxins output in the flue gas were lower than those input in the fuel, except when there were operational disturbances in the combustion. In one of the combustion trials the total levels of dioxins in the flue-gas and ashes were also lower than the input levels. The use of additional cleaning equipment will be needed to ensure that emissions of dioxins and hydrochloric acid will be below legal limits.

Gas chromatography-mass spectrometry determination of the pentafluorobenzoyl derivative of methylhydrazine in false morel (Gyromitra esculenta) as a monitor for the content of the toxin gyromitrin.

The main toxic compound found in false morel (Gyromitra esculenta) is acetaldehyde-N-methyl-N-formylhydrazone (gyromitrin). This paper describes a method of determining the total hydrazones content based on acid hydrolysis of gyromitrin and other related hydrazones in air-dried false morel followed by derivatisation of methylhydrazine with pentafluorobenzoyl chloride. The derivative, tris-pentafluorobenzoyl methylhydrazine (tris-PFB-MH) is analyzed by gas chromatography-mass spectrometry. The overall precision of the method is better than 10% (relative standard deviation) for 0.5 ng/microl methylhydrazine in solution. The minimum detectable concentration of methylhydrazine (tris-PFB-MH) by this method is estimated to be approximately 12 pg/microl, which is equal to 0.3 microg/g dry matter (DM) of false morel.

Emissions of polychlorinated dibenzodioxins and dibenzofurans and polychlorinated biphenyls from uncontrolled burning of garden and domestic waste (backyard burning).

To assess emissions of dioxins (chlorinated dibenzodioxins and dibenzofurans) and PCB from uncontrolled domestic combustion of waste ("backyard burning"), test combustions in barrels and open fires were monitored. The waste fuels used were garden waste, paper, paper and plastic packaging, refuse-derived fuel (RDF), PVC, and electronic scrap. Combustions including PVC and electronic scrap emitted several orders of magnitude more dioxins than the other waste fuels. Emissions from the other fuels had considerable variations, but the levels were difficult to relate to waste composition. Emission factors of PCDD/F and PCB from the backyard burning ranged from 2.2 to 13 000 ng (WHO-TEQ)/kg. The levels found in ash usually were less than 5% of the total. For assessment of total emissions of dioxins and PCB from backyard burning of low and moderately contaminated wastes, an emission factor range of 4-72 ng (WHO-TEQ)/kg is suggested. These figures implythat combusting waste in the backyard could contribute substantially to total emissions, even if the amounts of fuel involved are equivalent to just a few tenths of a percent of the amounts combusted in municipal waste incinerators.

Emissions from small-scale energy production using co-combustion of biofuel and the dry fraction of household waste.

In sparsely populated rural areas, recycling of household waste might not always be the most environmentally advantageous solution due to the total amount of transport involved. In this study, an alternative approach to recycling has been tested using efficient small-scale biofuel boilers for co-combustion of biofuel and high-energy waste. The dry combustible fraction of source-sorted household waste was mixed with the energy crop reed canary-grass (Phalaris Arundinacea L.), and combusted in both a 5-kW pilot scale reactor and a biofuel boiler with 140-180 kW output capacity, in the form of pellets and briquettes, respectively. The chlorine content of the waste fraction was 0.2%, most of which originated from plastics. The HCl emissions exceeded levels stipulated in new EU-directives, but levels of equal magnitude were also generated from combustion of the pure biofuel. Addition of waste to the biofuel did not give any apparent increase in emissions of organic compounds. Dioxin levels were close to stipulated limits. With further refinement of combustion equipment, small-scale co-combustion systems have the potential to comply with emission regulations.

Forensic identification of neat ricin and of ricin from crude castor bean extracts by mass spectrometry.

The protein toxin ricin, which originates from the seeds of Ricinus communis plants, has been the subject of increased interest, due to its potential terrorist use. Exceptionally, this toxin is also subject to the Chemical Weapons Convention. In this paper, it is shown that mass spectrometry can be used to unambiguously verify the presence of ricin in crude toxin preparations. It is demonstrated that MALDI MS can be used for screening, either by direct analysis or by trypsin digestion and peptide mapping. Purified ricin from several varieties of R. communis was characterized by LC-ES MS(/MS). A crude ricin preparation from a single bean was similarly characterized. An LC method was set up with product ion MS/MS detection of selected marker peptides specific for ricin: T5, T7, T11, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. This method was then used to unambiguously identify ricin in a crude preparation of ricin. The MALDI MS molecular weight analysis and the marker peptides LC-ES MS/MS analysis give a forensic level of identification of ricin when combined with activity testing.

An experimental comparison of a kinetic model for the reaction of alpha-pinene and Delta(3)-carene with ozone and nitrogen oxides.

UNLABELLED: A kinetic model was compiled to simulate reactions of the monoterpenes, alpha-pinene and Delta(3)-carene, with O(3), NO(2) and NO. The influence of different initial settings of O(3), NO(2) and NO on the monoterpene reaction was evaluated. At initial levels of 75 p.p.b. of O(3), NO(2) and NO each, 1.5% of alpha-pinene and 1.1% of Delta(3)-carene were calculated to react after 215 s. The corresponding experimental results showed that 9.3-12.2% of alpha-pinene and 9.9-11.7% of Delta(3)-carene reacted. The calculated levels of O(3), NO(2) and NO were compared to experimental measurements and were shown to correspond well. However, comparison of the amount of monoterpene reacted between calculated and experimental results, demonstrated that the calculations underestimated the amount of monoterpene reacted in the experimental chamber. The difference between experimental and calculated results could, e.g., be the effect of surfaces and the presence of water, which are parameters not included in the kinetic model known to have influence on these reactions. PRACTICAL IMPLICATIONS: A compiled kinetic model could be used to predict concentrations of O(3), NO(2), NO and to predict the effects on the monoterpene reaction in a Teflon reaction chamber when changing the initial concentrations of O(3), NO(2). However, the results in the present work show that surface and moisture effects have to be settled and the effects included in future models.

Emissions of aldehydes and ketones from a two-stroke engine using ethanol and ethanol-blended gasoline as fuel.

Besides aliphatic gasoline, ethanol-blended gasoline intended for use in small utility engines was recently introduced on the Swedish market. For small utility engines, little data is available showing the effects of these fuels on exhaust emissions, especially concerning aldehydes and ketones (carbonyls). The objective of the present investigation was to study carbonyl emissions and regulated emissions from a two-stroke chain saw engine using ethanol, gasoline, and ethanol-blended gasoline as fuel (0%, 15%, 50%, 85%, and 100% ethanol). The effects of the ethanol-blending level and mechanical changes of the relative air/fuel ratio, lambda, on exhaust emissions was investigated, both for aliphatic and regular gasoline. Formaldehyde, acetaldehyde, and aromatic aldehydes were the most abundant carbonyls in the exhaust. Acetaldehyde dominated for all ethanol-blended fuels (1.2-12 g/kWh, depending on the fuel and lambda), and formaldehyde dominated for gasoline (0.74-2.3 g/kWh, depending on the type of gasoline and lambda). The main effects of ethanol blending were increased acetaldehyde emissions (30-44 times for pure ethanol), reduced emissions of all other carbonyls exceptformaldehyde and acrolein (which showed a more complex relation to the ethanol content), reduced carbon monoxide (CO) and ntirogen oxide (NO) emissions, and increased hydrocarbon (HC) and nitrogen dixodie (NO2) emissions. The main effects of increasing lambda were increased emissions of carbonyls and nitrogen oxides (NOx) and reduced CO and HC emissions. When the two types of gasoline are considered, benzaldehyde and tolualdehyde could be directly related to the gasoline content of aromatics or olefins, but also acrolein, propanal, crotonaldehyde, and methyl ethyl ketone mainly originated from aromatics or olefins, while the main source for formaldehyde, acetaldehyde, acetone, methacrolein, and butanal was saturated aliphatic hydrocarbons.