Molecular characterization of aldehydes and ketones in particle phase of mainstream and sidestream cigarette smoke

Aldehydes and ketones (AKs) in cigarette smoke are risk to humans and environment. Due to the complexity of itself and the interference of the smoke tar matrix, the aldehydes and ketones in particle phase (AKPs) of mainstream smoke (MSS) and sidestream smoke (SSS) have not been well investigated. In this study, the AKPs of MSS and SSS were derivatized into polar products by reaction with Girard T reagent. The derivatives were isolated rapidly by column chromatography and analysed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Fifteen species of aldehydes and ketones were detected by positive ion electrospray ionization (ESI) FT-ICR MS: O1–6, N1O1–4, N2O1–3 and N3O2–3. The total number of AKPs obtained by ESI FT-ICR MS in MSS and SSS is about 1100 and 970, respectively. After hydrolysis, the original AKPs were obtained and 63 carbonyls were identified and quantified by gas chromatography–mass spectrometry (GCMS). The nitrogen-containing and high-oxygen AKPs were further characterized by Orbitrap mass spectrometry. Structures of compounds with high relative abundance in the mass spectrum were speculated (e.g. a series of degradants of cembrenediol) by comparison with the results of GCMS.

ion at higher m/z than on the FT ICR MS, this means that a part of the AKs, which are derivatizated by Girard's T reagent are not detected. Consequently, the assertion of the authors concerning the same ionization efficiency for all compounds after reaction of AKs with Girard's reactant is totally wrong. The comparison of the DBE distribution for a given class compound (O1 for example) is significantly different why? This deserves to be deeper discuss. What about the compounds which contain two or more carbonyl chemical groups? One other important issue is relative to the detection NxOy compounds by ESI-FT ICR MS: how are the author sure that these species are associated with derivatizated MSS/or SSS compounds. A large part of cigarette smoke simultaneously contains nitrogen and oxygen see the paper corresponding to ref 4 and the other papers of the same group. In the same way how are the author sure that only AKs are present in the so-called AKs fraction? Without this confirmation the obtained conclusions are highly disputable. What about the cotinine behavior which is a well-known cigarette smoke tracer? Why the authors used paper filters? More adequate filters are quartz filter for which it can be considered that it cannot interact with the smoke sample. Why collect the sample for 20 cigarettes in the same time. Interaction and chemical reaction may happen between the still collected particles and the compounds (in gas and PM phase). This may have a significant importance after the 20 smoking procedure. Details of the extraction step have to be given. The non-controlled evaporation of the solvent may lead to the evaporation of the more volatile cigarette smoke components. The mass resolution of FT ICR MS measurement has to be given. Details of the "tentative identification and quantification" page 3 line 57/60. The quantification aspect is not discussed in the results and discussion section. The figures are too small and very hard to be clearly examined. What's mean MK and CK in Title: Molecular characterization of aldehydes and ketones in particle phase of mainstream and sidestream cigarette smoke Manuscript ID: RSOS-181832 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Reviewer: 2
Comments to the Author(s) This paper presents an interesting piece of work dealing with the investigation of aldehydes and ketones (AKs) in complex mixture by mass spectrometry using a derivatization step by the Girard's T reagent. This methodology is used to compare the AK composition of two different cigarette smoke the MSS and the SSS. In spite of the interest of this work significant major revision are required for different reasons. The introduction requires to be improved and modified. It has to be more clearly focused on the analysis of cigarette smoke by non-targeted mass spectrometry. Significant references are missing as those of the Zimmerman group and those of other groups. In the last five years, a number of papers reported the analysis of AKs in complex mixture by HR MS after a reduction or a derivitization step. The papers have to be cited and discussed in the introduction (and compared to the proposed methodology in the conclusion). One important issue in this paper is the use of two HR MS instruments. The authors justified the use of orbitrap according to the low mass cut-off of FT ICRMS which is too high to observe the "naked" AKs. This is correct, but the orbitrap instrument has the capability to investigate both the low mass and the high mass range. Moreover, authors observed on the orbitrap mass spectra ion at higher m/z than on the FT ICR MS, this means that a part of the AKs, which are derivatizated by Girard's T reagent are not detected. Consequently, the assertion of the authors concerning the same ionization efficiency for all compounds after reaction of AKs with Girard's reactant is totally wrong. The comparison of the DBE distribution for a given class compound (O1 for example) is significantly different why? This deserves to be deeper discuss. What about the compounds which contain two or more carbonyl chemical groups? One other important issue is relative to the detection NxOy compounds by ESI-FT ICR MS: how are the author sure that these species are associated with derivatizated MSS/or SSS compounds. A large part of cigarette smoke simultaneously contains nitrogen and oxygen see the paper corresponding to ref 4 and the other papers of the same group. In the same way how are the author sure that only AKs are present in the so-called AKs fraction? Without this confirmation the obtained conclusions are highly disputable. What about the cotinine behavior which is a well-known cigarette smoke tracer? Why the authors used paper filters? More adequate filters are quartz filter for which it can be considered that it cannot interact with the smoke sample.
Why collect the sample for 20 cigarettes in the same time. Interaction and chemical reaction may happen between the still collected particles and the compounds (in gas and PM phase). This may have a significant importance after the 20 smoking procedure. Details of the extraction step have to be given. The non-controlled evaporation of the solvent may lead to the evaporation of the more volatile cigarette smoke components. The mass resolution of FT ICR MS measurement has to be given. Details of the "tentative identification and quantification" page 3 line 57/60. The quantification aspect is not discussed in the results and discussion section. The figures are too small and very hard to be clearly examined. What's mean MK and CK in

RSOS-181832 -review
In this manuscript the authors use a well-established technique for the, mainly, qualitative analysis of carbonyl compounds in cigarette smoke. They demonstrate that a good amount of quantitative data can be gained this way on a complex mixture of related compounds. This gives the work the value that warrants a publication of it.
The text is written in good English but it should be polished, in some places it is somewhat bumpy or simply incorrect -one example (line 38 on page 2): "After reacted with the Girard T reagent, the polarity of carbonyls produced quarternary ammonium salt derivatives is much higher than other components in the neutral fraction and is easily separated." It would be a pity not to correct such expressions.
Some further questions that should be cleared up are: -Although the expression "Girard's reagent" is known, it is my sense that "Girard reagent" is much more common -What detector was used in gas chromatography? In the section "Instrument conditions" I don't find any explicit mentioning of this.
-Why did the authors used both Orbitrap and FT-ICR MS? In the section mentioned above I don't see any rationale for this. If the reason was the better ion transmission for Orbitrap in the lower molecular weight region, why then use FT-ICR at all? And is this assumption borne out by the results (e.g. Figure 7)? -Quantification. This is a mine field and it is simply impossible to deduce quantitative relationships from the signal heights for two compounds, as is done in GC. Despite this, the authors use a quantitation for the Girard derivatives by postulating, without further justification, that since they all have the same permanent ionic structure, their "ionization efficiens are basically the same" (line 31, page 4). Is this assumption really true? Do the authors not neglect the hydrophobicity effect, described by John Fenn himself, that is known to have a major effect on the signal height in ESI-MS? This effect has been described in the literature for compounds of a very similar kind to that used here, namely in ref. 18 and also in DOI 10.1021/ef5028108 It was shown that compounds present in equimolar amounts easily gave MS signals that differed by a factor of 2.
There are also uncertainties growing out of the data presented by the authors. Take as an example figure 7. In the FT O1 Kendrick plot, the two largest dots are of approximately the same size. However, in the gas chromatogram, that of the open structure is ca twice as large as that of the cyclohexeneone. In view of this, how do the authors justify quantifying based only on MS signal heights? Furthermore, how do the authors justify the abundance percentages for different compound classes (Figure 2, Figure 5) when knowing that N containing compounds usually have a much higher ionization efficiency in ESI than other compounds, thus producing considerably higher signal heights for the same molar amounts?
The authors state on line 6, page 2: "The pre-charged property of Girard's reagent can enhance and uniform ionization efficiency of the derivatives in ESI-MS analysis.
[36]". I certainly agree with the enhancement but the quoted sentence is not supported by the reference indicated; this reference is mainly an overview of different reagents that have been used for LC-MS, including carbonyl compounds.
-Lines 33-35 on page 2: The first two sentences in this paragraph seem to be identical.
- Table 1: Do the standard deviations justify four significant figures in the percentage numbers?
- Table 2: The first letter in the compound names should be either capitalized or not capitalized throughout, not mixed Misspelling of "7-methly-1-indanone" "4,7,9-Megastigmatrien-3-one" -obviously there are several isomers, thus it would be appropriate to indicate this here and on each line add "or isomer". The same for "12-Isopropyl-1,5,9-trimethyl-4,8,13-cyclotetradecatriene-1,3-diol" The compound at 16.98 minutes: There are two names here, please separate them, perhaps by putting the long name in parenthesis. Please don't use capital C in cyclohexen.
Figure 1: In the first "round-bottom flask", the amounts of the different compounds is given but not that of the Girard reagent. Is that intentional?

Appendix B
We would like to thank the reviewer for providing constructive comments and help in improving the contents of this manuscript. We have revised the manuscript according to the reviewer's suggestions.
We have responded to each comment in detail as shown below. Our responses are formatted in italics.

RSOS-181832 -review1
In this manuscript the authors use a well-established technique for the, mainly, qualitative analysis of carbonyl compounds in cigarette smoke. They demonstrate that a good amount of quantitative data can be gained this way on a complex mixture of related compounds. This gives the work the value that warrants a publication of it.
We thank the reviewer for these comments.
The text is written in good English but it should be polished, in some places it is somewhat bumpy or simply incorrectone example (line 38 on page 2): "After reacted with the Girard T reagent, the polarity of carbonyls produced quarternary ammonium salt derivatives is much higher than other components in the neutral fraction and is easily separated." It would be a pity not to correct such expressions.
Response: Thanks very much for your comments, which are very helpful for us to improve the manuscript, and our language should be improved. After carefully check, we found many grammar and sentence errors, and have modified the manuscript. Furthermore, we have invited several English teachers help correct grammar and sentences, and we hope the revised paper will be more clear and accurate on expressions.
For example, the sentence have been revised to: "After reacted with the Girard's reagent T, the resulting aldehyde or ketone derivative is very easy to separate. This is because the polarity of the quaternary ammonium salt is much higher than the other components in the neutral fraction." Some further questions that should be cleared up are: -Although the expression "Girard's reagent" is known, it is my sense that "Girard reagent" is much more common Response: Thanks for the comment. We have changed 'Girard's reagent' to 'Girard reagent' (but we retained the term "Girard's reagent T" according to other literature).
-What detector was used in gas chromatography? In the section "Instrument conditions" I don't find any explicit mentioning of this.
Response: We used mass spectrometry as the gas chromatographic detector. We have add the following sentence in the revised manuscript: "The mass spectrometry (MS) was used as chromatographic detector. The electron impact (EI) ionization source was operated under 70 eV ionization energy. The MS ion source was at 230 °C. The mass range was 35-420 m/z with a 0.5 s scan period." -Why did the authors used both Orbitrap and FT-ICR MS? In the section mentioned above I don't see any rationale for this. If the reason was the better ion transmission for Orbitrap in the lower molecular weight region, why then use FT-ICR at all? And is this assumption borne out by the results (e.g. Figure  7)?
Response: The Orbitrap MS has better ion transmission both in the lower and higher molecular weight region, and it is suitable for the analysis of original AKPs. The FT-ICR MS has higher resolution at high molecular weight ends (i.e. at 400 m/z ) compared to Orbitrap MS, and it is more suitable for the analysis of GirT derivatives.
In fact, we found that for cigarette samples, the resolution of the Orbitrap MS on the high molecular weight ends is sufficient. But we want to compare these two results, so we finally chose to list the results obtained by the two mass spectrometers. We have added the following sentence in the revised manuscript: "To obtain a comprehensive result, the high resolution mass spectrometry is used to analyze the sample. Comparing with the FT-ICR MS, The Orbitrap MS has better ion transmission both in the lower and higher molecular weight region. It's considered that the FT-ICR MS has higher resolution at high molecular weight region. Since the molecular weight of original AKPs is much lower than the corresponding GirT derivatives, the Orbitrap MS was selected to analyse the original AKPs." -Quantification. This is a mine field and it is simply impossible to deduce quantitative relationships from the signal heights for two compounds, as is done in GC. Despite this, the authors use a quantitation for the Girard derivatives by postulating, without further justification, that since they all have the same permanent ionic structure, their "ionization efficiens are basically the same" (line 31, page 4). Is this assumption really true? Do the authors not neglect the hydrophobicity effect, described by John Fenn himself, that is known to have a major effect on the signal height in ESI-MS? This effect has been described in the literature for compounds of a very similar kind to that used here, namely in ref. 18 and also in DOI 10.1021/ef5028108 It was shown that compounds present in equimolar amounts easily gave MS signals that differed by a factor of 2. There are also uncertainties growing out of the data presented by the authors. Take as an example figure 7. In the FT O1 Kendrick plot, the two largest dots are of approximately the same size. However, in the gas chromatogram, that of the open structure is ca twice as large as that of the cyclohexeneone. In view of this, how do the authors justify quantifying based only on MS signal heights?
Response: We are sorry that we have made a mistake. We did not fully considered the hydrophobicity effect to ionization efficiency. Our expression "Since the highly conversion of AKs reaction with Girard's reagent, and the ionized groups after derivatization are all quaternary ammonium groups (the ionization efficiencies are basically the same)" is totally wrong. We have removed this sentence in the revised manuscript. The related content has also been modified accordingly. Furthermore, we have removed and revised other statements about 'justify quantifying based only on MS signal heights' in the revised manuscript.
Furthermore, how do the authors justify the abundance percentages for different compound classes (Figure 2, Figure 5) when knowing that N containing compounds usually have a much higher ionization efficiency in ESI than other compounds, thus producing considerably higher signal heights for the same molar amounts?
Response: We are sorry that we have caused a misunderstanding.
The data shown in Figure 2 and Figure 5 are the summed mass spectrometric intensities but do not quantitatively reflect the abundance of species in the sample. We have added the following sentence in the revised manuscript: "The relative abundance of each class and type (DBE) is calculated and shown in Figure 2(b). Relative abundance is defined as the magnitude of each peak divided by the sum of the magnitudes of all identified peaks (exclude the isotopic peaks) in the MS spectrum.
The data shown in Figure 2 are the summed mass spectrometric intensities but do not quantitatively reflect the abundance of species in the sample. The O 1 class AKPs with the most relative abundance in MSS and SSS, followed by the O 2 , O 3 , and O 4 class. Compared to SSS, the MSS with a little higher relative abundance of high oxygenate. This result is consistent with the elemental analysis result in Table 1." The authors state on line 6, page 2: "The pre-charged property of Girard's reagent can enhance and uniform ionization efficiency of the derivatives in ESI-MS analysis.
[36]". I certainly agree with the enhancement but the quoted sentence is not supported by the reference indicated; this reference is mainly an overview of different reagents that have been used for LC-MS, including carbonyl compounds.
Response: According to your suggestion, we have revised the text: "The pre-charged property of Girard's reagent can enhance the ionization efficiency of the derivatives in ESI-MS analysis.
[36]" -Lines 33-35 on page 2: The first two sentences in this paragraph seem to be identical.
Response: According to your suggestion, we have revised the text.  Table 2: The first letter in the compound names should be either capitalized or not capitalized throughout, not mixed Misspelling of "7-methly-1-indanone" "4,7,9-Megastigmatrien-3-one"obviously there are several isomers, thus it would be appropriate to indicate this here and on each line add "or isomer". The same for " 5,8,3diol" The compound at 16.98 minutes: There are two names here, please separate them, perhaps by putting the long name in parenthesis. Please don't use capital C in cyclohexen.
Response: According to your suggestion, we have revised the text. Figure 1: In the first "round-bottom flask", the amounts of the different compounds is given but not that of the Girard reagent. Is that intentional?
Response: It is our negligence that the amount of Girard reagent is not given in Figure 1. The amount of Girard reagent is given in the "Materials and Methods section-Derivatization and separation of aldehydes and ketones", and we also add this data in Figure 1 in the revised manuscript.
We would like to thank the reviewer for providing constructive comments and help in improving the contents of this manuscript. We have revised the manuscript according to the reviewer's suggestions.
We have responded to each comment in detail as shown below. Our responses are formatted in italics.
Comments to the Author(s)-2 This paper presents an interesting piece of work dealing with the investigation of aldehydes and ketones (AKs) in complex mixture by mass spectrometry using a derivatization step by the Girard's T reagent. This methodology is used to compare the AK composition of two different cigarette smoke the MSS and the SSS. In spite of the interest of this work significant major revision are required for different reasons.
The introduction requires to be improved and modified. It has to be more clearly focused on the analysis of cigarette smoke by non-targeted mass spectrometry. Significant references are missing as those of the Zimmerman group and those of other groups. In the last five years, a number of papers reported the analysis of AKs in complex mixture by HR MS after a reduction or a derivitization step. The papers have to be cited and discussed in the introduction (and compared to the proposed methodology in the conclusion).
Response: Thanks for your suggestion. According to your suggestion, we have added the following text in the introduction.
"The AKs in the complex matrix can be directly analyzed by gas chromatography-mass spectrometry (GCMS) [11,12]  One important issue in this paper is the use of two HR MS instruments. The authors justified the use of orbitrap according to the low mass cut-off of FT ICRMS which is too high to observe the "naked" AKs. This is correct, but the orbitrap instrument has the capability to investigate both the low mass and the high mass range. Moreover, authors observed on the orbitrap mass spectra ion at higher m/z than on the FT ICR MS, this means that a part of the AKs, which are derivatizated by Girard's T reagent are not detected. Consequently, the assertion of the authors concerning the same ionization efficiency for all compounds after reaction of AKs with Girard's reactant is totally wrong.
Response: According to your suggestion and the comment from another reviewer, our expression "Since the highly conversion of AKs reaction with Girard's reagent, and the ionized groups after derivatization are all quaternary ammonium groups (the ionization efficiencies are basically the same)" is totally wrong. We have removed this sentence in the revised manuscript. The related content has also been modified accordingly. Furthermore, we have removed and revised other statements about 'justify quantifying based only on MS signal heights' in the revised manuscript.
The comparison of the DBE distribution for a given class compound (O1 for example) is significantly different why? This deserves to be deeper discuss. What about the compounds which contain two or more carbonyl chemical groups?
Response: According to your suggestion, the following text were revised: " Figure 3 shows the iso-abundance plots of DBE as a function of carbon number for O1 ~ O3 species. The O1 and O3 class species has a DBE value ranges (1−13) and carbon number (6-35). Since the carbonyl group contributes to an unsaturation, DBE = 1 series are saturated AKs. The AKs with DBE=2-14 may contain unsaturated bond, naphthenic and/or aromatic ring. O2 species with DBE = 1 are saturated AKs but contained a hydroxyl or an ether group. O2 species with DBE > 1 may contained two carbonyl group or unsaturated bond.
O3 species almost not contained DBE=1 series, suggest that at least contained two carbonyl groups.
The abundance percentages of O1-3 species with DBE values of 1-4 and greater than 4 for MSS and SSS were calculated, respectively.
The results indicated that the SSS contain more unsaturated and condensed AKPs than MSS. This may be due to the low level of oxygen in the production of SSS facilitates the pyrolysis process. This consists with the H/C ratio shown in Table 1." One other important issue is relative to the detection NxOy compounds by ESI-FT ICR MS: how are the author sure that these species are associated with derivatizated MSS/or SSS compounds. A large part of cigarette smoke simultaneously contains nitrogen and oxygen see the paper corresponding to ref 4 and the other papers of the same group. In the same way how are the author sure that only AKs are present in the so-called AKs fraction? Without this confirmation the obtained conclusions are highly disputable. What about the cotinine behavior which is a well-known cigarette smoke tracer?

Response
Response: Cotinine with a structure: .Cotinine is a neutral compound (lactam), but not an aldehyde or ketone compound.
It was eluted by DCM / methanol = 20/1 in the second step and not be study by this work.
Why the authors used paper filters? More adequate filters are quartz filter for which it can be considered that it cannot interact with the smoke sample.
Response: Thanks for the comment. That is true, we should use the quartz filter. We bought the inappropriate filter, but we think this type filter will not affect the analysis results of this study. This is because the aldehyde and ketone cannot react with the filter; and the appropriate solvent was used to completely elute the compound from the filter, and the physical adsorption of the compound by the filter was excluded. The model of the Cambridge filter pad we bought is F319-04 (paper filters), which meets the requirements of Standard ISO3308:2000 (Routine analytical cigarette-smoking machine-definitions and standard conditions). But now the ISO3308:2000 has been withdrawn and revised by ISO 3308:2012. We will remove the model of Cambridge filter pad (F319-04) from this article so as not to mislead other researchers. In future research we will use the quartz filter. Thanks again.
Why collect the sample for 20 cigarettes in the same time. Interaction and chemical reaction may happen between the still collected particles and the compounds (in gas and PM phase). This may have a significant importance after the 20 smoking procedure.
Response: We are sorry that we have caused a misunderstanding.
This is the original sentence in the article: "The particle phase of MSS of 20 cigarettes was collected on whatman fiber pads." We mean that we totally collected 20 cigarettes' particle phase to do the next experiment. In this procedure, five cigarettes are smoked consecutively under carefully controlled conditions. Then the particle phase on four fiber pads were combined. We have revised the text: "20 cigarettes were divided into four groups on average. The particulate phase of MSS and SSS of each group was collected on fiber pads under a set of internationally agreed standard smoking conditions The particle phase on four fiber pads were eluted by DCM/MeOH = 3/1, combined and stored at -20 °C." Details of the extraction step have to be given.
Response: According to your suggestion, we have added the were removed very cleanly. The GirT derivatives still retain on the silica gel and cannot be eluted due to its high polarity. Then a much higher polarity eluent, DCM / methanol = 3/2 was used to elute the GirT derivatives. The huge difference in polarity between these two parts ensure only AKs are present in the AKs fraction." "1 g of anhydrous sodium sulfate, 2 g of KOH-modified silica gel, 1 g of anhydrous sodium sulfate, 4 g of HCl acid-modified silica gel and 1 g anhydrous sodium sulfate were sequentially packed into a 1 cm diameter column A. 10 g of ordinary silica gel, 1 g of anhydrous sodium sulfate and 0.5 g of KOH-modified silica gel were sequentially packed into another 1 cm diameter column B. The obtained cigarette smoke particle phase (shown in Table 1) was dissolved in a 4 mL of DCM/THF = 7/3 (v/v) mixture, and the solution was subjected to the chromatography A using 25 mL of a DCM/THF = 7/3 (v/v) mixture as the eluent to elute the neutral fraction. The polarity of the mixed solvent is high enough to elute the aldehydes and ketones in the neutral fraction." The non-controlled evaporation of the solvent may lead to the evaporation of the more volatile cigarette smoke components.
Response: Agree. The solvent for neutral fraction and aldehyde/ketone fraction is THF/DCM and DCM, respectively. When evaporate the solvent by rotary evaporator, the bath temperature is always controlled below 10 o C to prevent some smoke components from volatilizing. Despite this, there is still some loss of volatile components. This is the drawback of chemical derivatization method compared to non-targeted method.
We have added the sentence in the revised manuscript: "After removing the solvent below 10 o C by a rotary evaporator, the neutral fraction was obtained." "Then the DCM layers were combined in a flask, the solvent was removed by a rotary evaporator below 10 o C." "The post-treatment process may result in the loss of volatile components, so some of small carbonyl specie cannot be observed in the Figure 4." The mass resolution of FT ICR MS measurement has to be given.
Response: According to your suggestion, we have added the sentence in the revised manuscript: "The peaks with a 380000 resolving power at m/z = 326." Details of the "tentative identification and quantification" page 3 line 57/60. The quantification aspect is not discussed in the results and discussion section.
Response: According to your suggestion, we have added the following sentence in the revised manuscript: "The quantitative results are listed in Table 2. Figure 4 and Table   2 shows that the AKPs in MSS and SSS are very close, but the content is different. In addition, the content of specific carbonyl is very different to each other. The content of high-carbon linear aldehydes (Henicosanal to Pentacosanal) in MSS is higher than that of SSS, but the content of some low-carbon aromatic-contained aldehydes (trans-Cinnamaldehyde, alpha-Methylcinnamaldehyde) in sidestream smoke is higher than that of mainstream smoke. This may be due to the fact that MSS and SSS are formed in different ways of combustion. The