Fragmentation Pathway of Organophosphorus Flame Retardants by Liquid Chromatography–Orbitrap-Based High-Resolution Mass Spectrometry

Organophosphorus flame retardants (OPFRs) have been widely used in polymeric materials owing to their flame retardant and plasticizing effects. Investigating the fragmentation pathway of OPFRs is of great necessity for further discovering and identifying novel pollutants using orbitrap-based high-resolution mass spectrometry (HRMS). A total of 25 OPFRs, including alkyl, halogenated, and aromatic types, were analyzed in this study. The fragmentation pathways of the OPFRs were investigated using orbitrap-based HRMS with high-energy collision dissociation (HCD) in positive mode. The major fragmentation pathways for the three types of OPFRs are greatly affected by the substituents. In detail, the alkyl and halogenated OPFRs underwent three McLafferty hydrogen rearrangements, wherein the substituents were gradually cleaved to form the structurally stable [H4PO4]+ (m/z = 98.9845) ions. In contrast, the aromatic OPFRs would cleave not only the C-O bond but also the P-O bond, depending on the substituents, to form fragment ions such as [C6H7O]+ (m/z = 95.0495) or [C7H7]+ (m/z = 91.0530), among others. Using HRMS improved the accuracy of fragment ion identification, and the pathway became more evident. These fragmentation laws can provide identification information in pollutant screening work and theoretical references for the structural characterization of compounds with diverse substituent structures.

At present, the detection of OPFRs mainly relies on gas or liquid chromatography-mass spectrometry techniques, wherein the qualitative and quantitative analysis of a limited range of targets can be carried out with reference materials [17][18][19].However, with thousands of OPFRs and derivatives currently available and continuing to expand, it is crucial to perform both suspect and non-target screening, while the requirements for mass spectrometry information related to OPFRs are becoming increasingly stringent.There are many types of organophosphorus flame retardants (OPFRs), mainly including organophosphate esters, organophosphonates, organophosphate salts, phosphine oxides, and organophosphate heterocycles compounds.Organophosphate esters with P(=O)(OR)3 as their characteristic structure account for 84.2% of the available OPFRs monomers [20].These compounds may produce the same characteristic fragment ions during mass spectrometry cleavage because they contain the same phosphate skeleton, and these characteristic ions can be used as a basis for non-target screening of organophosphorus compounds.There are some variations in the type and abundance of fragment ions produced by different types of organophosphorus compounds, and these differential features are a tool for further differentiation and identification of the target.According to the substituents, OPFRs are currently classified as alkyl, halogenated, and aromatic [21]: alkyl OPFRs substituents are mainly straight or branched composed of C and H elements, halogenated OPFRs substituents are usually H substituted by chlorine or bromine on alkane chains, and aromatic OPFRs substituents generally contain one or more aromatic rings.Lesage et al. [22] used a deuterium label to study the fragmentation of tri-n-butyl phosphate.Deuterium labeling makes the fragmentation mechanism more obvious.However, it is still difficult to obtain relevant deuterium-labeled products for the increasing variety of organophosphorus flame retardants.Ma et al. [23] used gas chromatography tandem mass spectrometry to explain the common fragmentation pathways of 13 OPFRs under electron ionization (EI) sources.They found that alkyl OPFRs follow a rearrangement reaction from the precursor [M] + to [M − R + 2H] + , [M − 2R + 3H] + , and [M − 3R + 4H] + patterns.Yang et al. [24] optimized the ionization energy of the EI source and summarized the fragmentation pathways and characteristic fragment ion database of 17 OPFRs, which provided a basis for determining OPFRs in complex matrix samples.The high-resolution mass spectrometry (HRMS) screening technique can provide higher index parameters in terms of screening accuracy and sensitivity, improve the accuracy of fragment ion identification, effectively perform high-throughput detection, and reduce false positive and false negative rates.Ye et al. [25] used liquid chromatography tandem HRMS with an atmospheric pressure chemical ionization (APCI) source for target, suspect, and non-target screening of OPFRs in Taihu sediments, using characteristic fragment ions for qualitative analysis of emerging OPFRs, which significantly simplifies the information processing during non-target screening but also requires the researcher to learn the target well enough to remove interferences during subsequent analysis effectively.Since there are many different types of OPFRs substituents and the physicochemical properties of the various OPFRs vary widely [24], the study of the fragmentation pathways of the target compounds and the summary of the relevant mass spectrometry information are a crucial step before the screening process.
This study aims to analyze the main characteristic fragment ions generated during the fragmentation of OPFRs by LC-Orbitrap-HRMS technology using an electrospray ionization (ESI) source.The selected OPFRs include alkyl, halogenated, and aromatic classes, which are widely representative considering the chain length and substituent structure.The fragmentation laws were investigated, and the effective information was applied to the screening of OPFRs in rice samples to provide a basis for detecting and accurately characterizing the targets.

Fragmentation Pathway and Characteristic Ions of Three Types of OPFRs
To analyze the fragmentation pathway of the three types of OPFRs, each compound was injected separately at 200 µg/kg, and precursor ions were fragmented under the stepcollision energy by HCD.

Fragmentation Pathway and Characteristic Ions of Alkyl OPFRs
In this study, nine representative alkyl OPFRs were selected (Table 1), including straight chain substituents (TMP, TEP, TnPP, TnBP, TPeP, TBOEP) and branched substituents (TiPP, TiBP, TEHP), corresponding to the substituent groups in the order of methyl, ethyl, n-propyl, n-butyl, n-pentyl, 2-butoxyethyl, isopropyl, isobutyl, and 2-ethylhexyl.Among them, TiPP and TnPP, TiBP and TnBP are two pairs of isomers.(m/z = 98.9847) ions.The abundance ratio and accurate mass of fragment ions were effective bases for qualitative analysis.Among the alkyl OPFRs, most of the compounds had [H 4 PO 4 ] + (m/z = 98.9847) as the base peak, which was a common characteristic fragment ion of alkyl OPFRs.It was very stable in the mass spectrum and could be used as an identifying feature for alkyl OPFRs during screening.However, for TBOEP (Figure 2a), the precursor ion [M + H] + was the base peak.This may be due to the fact that the substituent group of TBOEP was 2-butoxyethyl, and the TBOEP precursor ion was stabilized by the presence of oxygen in the side chain, which led to difficulty in McLafferty hydrogen rearrangement.This feature, in combination with the abundance of fragment ions, could be a basis for screening TBOEP.High-resolution mass spectrometry can provide the accurate mass of the characteristic fragment ions, which provides a guarantee for eliminating interferences and accurately matching molecular formulae during the screening process.
McLafferty hydrogen rearrangement.This feature, in combination with the abundance of fragment ions, could be a basis for screening TBOEP.High-resolution mass spectrometry can provide the accurate mass of the characteristic fragment ions, which provides a guarantee for eliminating interferences and accurately matching molecular formulae during the screening process.McLafferty hydrogen rearrangement.This feature, in combination with the abundance of fragment ions, could be a basis for screening TBOEP.High-resolution mass spectrometry can provide the accurate mass of the characteristic fragment ions, which provides a guarantee for eliminating interferences and accurately matching molecular formulae during the screening process.Isomers are difficult to distinguish in qualitative analysis due to their similar structures and same molecular weights but some isomers differ in the type or abundance of fragment ions, which can be used as tools for further identification.Among the alkyl OPFRs, TnBP and TiBP, TnPP and TiPP are two pairs of isomers (Figure 2b 1.This may be because branched alkanes are more stable than straight-chain alkanes, and when used as substituents, the rearrangement reaction of branched OPFRs mainly occurs in the oxygen atoms connecting the substituent groups, while the energy of straight-chain compounds is dispersed during fragmentation, so the branched compounds are more prone to the formation of [M − 3R + 4H] + , and the proportion of the fragments produced is relatively higher.This conclusion can provide a reference for distinguishing isomers during the qualification of compounds.
Due to the high natural abundance of chlorine and bromine isotopes, halogenated OPFRs produce unique ion clusters during ionization.According to the relative abundance ratios of the isotope peaks in the MS 1 spectrum (Figure S5), the type and amount of halogens can be deduced, which facilitates the characterization of the compounds.The two halogenated OPFRs, TDCIPP and T23DBPP, both had distinctive isotope peaks in the MS 1   Isomers are difficult to distinguish in qualitative analysis due to their similar structures and same molecular weights but some isomers differ in the type or abundance of fragment ions, which can be used as tools for further identification.Among the alkyl OPFRs, TnBP and TiBP, TnPP and TiPP are two pairs of isomers (Figure 2b This may be because branched alkanes are more stable than straight-chain alkanes, and when used as substituents, the rearrangement reaction of branched OPFRs mainly occurs in the oxygen atoms connecting the substituent groups, while the energy of straight-chain compounds is dispersed during fragmentation, so the branched compounds are more prone to the formation of [M − 3R + 4H] + , and the proportion of the fragments produced is relatively higher.This conclusion can provide a reference for distinguishing isomers during the qualification of compounds.
Due to the high natural abundance of chlorine and bromine isotopes, halogenated OPFRs produce unique ion clusters during ionization.According to the relative abundance ratios of the isotope peaks in the MS 1 spectrum (Figure S5), the type and amount of halogens can be deduced, which facilitates the characterization of the compounds.The two halogenated OPFRs, TDCIPP and T23DBPP, both had distinctive isotope peaks in the MS 1 spectrum.For TDCIPP, the relative abundance ratio of the isotope peaks  of the isotope peaks is 5.00:30.00:75.00:100.00:75.00:30.00:5.00.This also matches the experimental isotopic peaks produced by T23DBPP.In the MS 1 spectrum of TDCIPP, the highest isotopic abundance was observed for the molecular formula C9H15Cl

Fragmentation Pathway and Characteristic Ions of Aromatic OPFRs
The structures of aromatic OPFRs usually contain one or more aromatic Different from the above-mentioned two types of OPFRs, the substituent types of aro OPFRs are more abundant.The experiment involved dBPhP and BdPhP with a be ring and n-butyl; EHDPP with a benzene ring and 2-ethylhexyl; IDDP with iso CDPP with benzene and toluene; TPHP with a benzene ring; T35DMPP with xylen isomers o-TCP, m-TCP, and p-TCP with toluene.
Experiments have shown that the aromatic ring had an influence o fragmentation of the compound, and its fragmentation pathways and charact fragments were different from those of alkyl and halogenated OPFRs.OPFRs contained an alkane chain, the alkane chain was very easily cleaved, an structure with a benzene ring was relatively stable, which could be used as charact fragment ions in screening.
For aromatic OPFRs in which the three substituents were all aromatic ring compounds had a stable structure due to the conjugation effect.Therefore, the prec peak was the base peak in the MS 2 spectrum.If the substituent was a benzene ring(T

Fragmentation Pathway and Characteristic Ions of Aromatic OPFRs
The structures of aromatic OPFRs usually contain one or more aromatic rings.Different from the above-mentioned two types of OPFRs, the substituent types of aromatic OPFRs are more abundant.The experiment involved dBPhP and BdPhP with a benzene ring and n-butyl; EHDPP with a benzene ring and 2-ethylhexyl; IDDP with isodecyl; CDPP with benzene and toluene; TPHP with a benzene ring; T35DMPP with xylene; and isomers o-TCP, m-TCP, and p-TCP with toluene.
Experiments have shown that the aromatic ring had an influence on the fragmentation of the compound, and its fragmentation pathways and characteristic fragments were different from those of alkyl and halogenated OPFRs.For aromatic OPFRs containing alkane chains in the substituents, such compounds would preferentially cleave the C-O bond connected to the alkane chain during the fragmentation process to form a stable structure.The structure of dBPhP contains one benzene ring and two alkane chains, and the possible fragmentation process and MS 2 spectra are shown in Figures 5 and 6a ) ions in the subsequent fragmentation process.It was observed that when the substituent of aromatic OPFRs contained an alkane chain, the alkane chain was very easily cleaved, and the structure with a benzene ring was relatively stable, which could be used as characteristic fragment ions in screening.
For aromatic OPFRs in which the three substituents were all aromatic rings, the compounds had a stable structure due to the conjugation effect.Therefore, the precursor peak was the base peak in the MS 2 spectrum.If the substituent was a benzene ring(TPHP),

Comparison with Fragmentation Pathways under the EI Source
The full MS spectra of OPFRs under the EI source are shown in Figures 7a and S6.This paper compared the precursor and fragment ions of OPFRs under the two instruments to study the differences in their ionization and fragmentation characteristics.

Comparison with Fragmentation Pathways under the EI Source
The full MS spectra of OPFRs under the EI source are shown in Figures 7a and S6 This paper compared the precursor and fragment ions of OPFRs under the two instruments to study the differences in their ionization and fragmentation characteristics

Comparison with Fragmentation Pathways under the EI Source
The full MS spectra of OPFRs under the EI source are shown in Figures 7a and S6.This paper compared the precursor and fragment ions of OPFRs under the two instruments to study the differences in their ionization and fragmentation characteristics.It was found that after ESI ionization, alkyl and aromatic OPFRs would generate precursors [M + H] + , while some halogenated OPFRs would generate [M + NH 4 ] + in addition to [M + H] + .This may be because the proton affinity of this compound is close to that of ammonium.[M + Na] + was found in the MS 1 spectrum of most OPFRs, usually due to impurities in the sample vial, liquid phase line, or solvent [26].The ion clusters characteristic of the halogenated OPFRs made the [M + NH 4 ] + and [M + Na] + more prominent.The ionization voltage of the EI source was usually 70 eV, and the precursors were often fragmented by ionization.So, the precursor ion information could not be presented in the mass spectrum.By comparing the fragment ions under the two instruments, it was found that the fragmentation pathways of most alkyl and halogenated OPFRs were similar, and usually underwent McLafferty hydrogen rearrangement after generating precursors.[M − R + 2H] + , [M − 2R + 3H] + , [M − 3R + 4H] + fragment ions were formed after the chemical bond was cleaved.However, there were still significant differences in the ionization and fragmentation of some compounds between the two instruments, such as TMP (Figure 7

Screening of Organophosphorus Flame Retardants in Actual Samples
Mass spectrometry information that can be used for qualitative analysis was obtained by exploring the fragmentation pathways of organophosphorus compounds under LC-HRMS, and in order to preliminarily verify the suitability of the relevant mass spectrometry information, an experiment was conducted to screen OPFR in rice samples under the same chromatographic and mass spectrometry conditions as those described in Section 3.2 in the text.Firstly, the target chromatographic peaks were extracted from the tested rice samples by the accurate mass of the precursor ions.Then, the extracted precursor ions were sent to the HCD collision cell for fragmentation, and the MS 2 spectrum was matched to the standard solution spectrum.Compounds with less than two characteristic fragmentation ions were identified as false positives and further analyzed based on the mass spectrometry information generated by fragmentation.
As shown in Figure 8, two undetermined compounds, TEP (m/z = 183.0779)and In general, due to the different ionization methods and fragmentation principles, there will be some differences in the fragmentation pathways and fragment ions generated under the two instruments.The fragmentation spectrum of the EI source usually cannot display the precursor ion information, which makes some references missing in the screening process.However, this ion source is easy to use and obtains abundant fragment ions, often used to establish a standard spectrum.Compared with the EI ionization and fragmentation, the ESI ionization and HCD fragmentation have wider applicability.The soft ionization method can retain the information of the precursor ions, acquire the accurate mass of the precursor ions in high-resolution mass spectrometry to achieve the accurate matching of molecular formulae, and combine with the HCD, which can be fragmented at different collision energies, to further confirm the structure of the compounds by fragment ions and provide a complete basis for the screening experiments.Although the ion fragmentation through the collision cell increases the complexity of the experimental operation, it is beneficial for the experimenter to discover more rules by adjusting the parameters.The comparison of the two instruments has greatly contributed to the target mass spectrum database, providing strong support for the screening of new pollutants.

Screening of Organophosphorus Flame Retardants in Actual Samples
Mass spectrometry information that can be used for qualitative analysis was obtained by exploring the fragmentation pathways of organophosphorus compounds under LC-HRMS, and in order to preliminarily verify the suitability of the relevant mass spectrometry information, an experiment was conducted to screen OPFR in rice samples under the same chromatographic and mass spectrometry conditions as those described in Section 3.2 in the text.Firstly, the target chromatographic peaks were extracted from the tested rice samples by the accurate mass of the precursor ions.Then, the extracted precursor ions were sent to the HCD collision cell for fragmentation, and the MS 2 spectrum was matched to the standard solution spectrum.Compounds with less than two characteristic fragmentation ions were identified as false positives and further analyzed based on the mass spectrometry information generated by fragmentation.
As shown in Figure 8, two undetermined compounds, TEP (m/z = 183.0779)and CDPP (m/z = 341.0936),were extracted from rice samples by the accurate mass of precursor ions.The four characteristic ions in the MS 2 spectrum of TEP are consistent with the standard solution spectrum, and the mass deviation of the characteristic ions is less than 5 ppm, with a relative abundance deviation of less than 20%, indicating the actual detected compound.However, according to the MS 2 spectrum, the undetermined CDPP cannot generate corresponding fragment ions in HCD, making it difficult to accurately identify.Even with the use of HRMS, screening based solely on the accurate mass of precursor ions may still result in some false positives.In order to make more accurate judgments, it is necessary to study the fragment pathways and fragment ions of compounds.

Main Materials and Reagents
Basic information on OPFRs is provided in Table 1.The structure of these OPFR shown in Figure S1 in the Supporting Information (SI).Standards of the 25 OPFRs w that the collision stability of alkyl OPFRs was the worst, followed by halogenated OPFRs and aromatic OPFRs.In terms of characteristic fragment ions, both alkyl and halogenated types could form structurally stable [H 4 PO 4 ] + (m/z = 98.9845) ions, but aromatic OPFRs produced characteristic fragment ions such as [C 6 H 8 PO 4 ] + (m/z = 175.0155),[C 12 H 12 PO 4 ] + (m/z = 251.0465),and [C 12 H 8 ] + (m/z = 152.0620)according to different substituents.These characteristic fragment ions provide identification signals in follow-up screening work and theoretical references for the rapid recognition of OPFRs' targets.Through the analysis of several isomers, it was found that in alkyl OPFR compounds, the relative abundance ratio of [M − 3R + 4H] + to [M − 2R + 3H] + fragment ions produced by compounds with branched substituents was higher, which was about 6:1.In contrast, the ratio of [M − 3R + 4H] + to [M − 2R + 3H] + fragment ions with straight substituents was lower, about 2:1.This ratio can be used as an index to distinguish several kinds of isomers in the subsequent screening process, assist in judging the structural types of substituents, and make more detailed qualitative analysis of the compounds found.The application of OPFRs' fragmentation laws in food and environmental samples will be further investigated to expand the scope of our research in the future.
For aromatic O containing alkane chains in the substituents, such compounds would preferentially the C-O bond connected to the alkane chain during the fragmentation process to f stable structure.The structure of dBPhP contains one benzene ring and two alkane c and the possible fragmentation process and MS 2 spectra are shown in Figures 5 a The characteristic fragment ions [C10H16PO4] + (m/z = 231.0778)and [C6H8PO4] + ( 175.0154) were formed by cleavage of the C-O bonds on the two alkane chains, fol by cleavage of the P-O bonds to form the ion [C6H7O] + (m/z = 95.0496).The structures of BdPhP, EHDPP, and IDDP contain two benzene rings and an a chain.These compounds formed characteristic fragment ions [C12H12PO4] + (m/z = 251 with two benzene rings by the cleavage of the C-O bond on the alkane chain gradually formed [C12H8] + (m/z = 152.0620)ions and [C6H7O] + (m/z = 95.0496)ions subsequent fragmentation process.It was observed that when the substituent of aro [C12H8] + (m/z = 152.0620)and [C6H7O] + (m/z = 95.0496)characteristic fragment ions w be generated; if the aromatic ring was toluene(o-TCP, m-TCP, p-TCP), [C13H9] + 165.0698), [C7H7] + (m/z = 91.0547)and [C5H5] + (m/z = 65.0393)characteristic fragmen would be generated; for compounds containing both toluene and benzene rings like
. The characteristic fragment ions [C 10 H 16 PO 4 ] + (m/z = 231.0778)and [C 6 H 8 PO 4 ] + (m/z = 175.0154)were formed by cleavage of the C-O bonds on the two alkane chains, followed by cleavage of the P-O bonds to form the ion [C 6 H 7 O] + (m/z = 95.0496).The structures of BdPhP, EHDPP, and IDDP contain two benzene rings and an alkane chain.These compounds formed characteristic fragment ions [C 12 H 12 PO 4 ] + (m/z = 251.0465)with two benzene rings by the cleavage of the C-O bond on the alkane chain, and gradually formed [C 12 H 8 ] + (m/z = 152.0620)ions and [C 6 H 7 O] + (m/z = 95.0496
), which followed the above rearrangement rules under the ESI ionization and HCD fragmentation, producing [C 2 H 8 PO 4 ] + (m/z = 127.0156),[CH 6 PO 4 ] + (m/z = 113.0001),and [H 4 PO 4 ] + (m/z = 98.9846) fragment ions.While under the EI ionization and fragmentation, TMP lost the methyl group by the C-O bond cleavage to form [CH 3 PO 4 ] + (m/z = 110.0124)and [PO 4 ] + (m/z 94.9559) fragment ions.For aromatic OPFRs, due to the relatively stable structure, precursor peaks could be observed in the fragmentation spectrum under the EI source.The characteristic fragment ion of OPFRs with a benzene ring as the substituent was [C 6 H 7 O] + (m/z = 95.0495)under the ESI ionization and HCD fragmentation, and [C 6 H 6 O] + (m/z = 94.0409)under the EI source; the OPFR compound whose substituent was toluene would produce characteristic fragment ions of [C 7 H 7 ] + (m/z = 91.0530)and [C 5 H 5 ] + (m/z = 65.0393) in both fragmentation modes.Molecules 2024, 29, x FOR PEER REVIEW 10 of 14

Figure 7 .
Figure 7. Mass spectra of TMP under two instruments.

Figure 7 .
Figure 7. Mass spectra of TMP under two instruments.

Molecules 2024 , 11 Figure 8 .
Figure 8. Matching of MS 2 spectra of (a) TEP and (b) CDPP in standard solutions and rice sam 3. Materials and Methods

Figure 8 .
Figure 8. Matching of MS 2 spectra of (a) TEP and (b) CDPP in standard solutions and rice samples.