# Formation of artifacts from simple phenolic compounds in SFC-UV-(HR)MS

Eliise Tammekivi, Karine Faure

► To cite this version:

Eliise Tammekivi, Karine Faure. Formation of artifacts from simple phenolic compounds in SFC-UV-(HR)MS. Analytical Chemistry, 2025, 97 (8), pp.4481-4489. 10.1021/acs.analchem.4c05941. hal-04995764

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# Formation of artifacts from simple phenolic compounds in SFC-UV-(HR)MS

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Eliise Tammekiviª and Karine Faureª*

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ªUniversite Claude Bernard Lyon 1, ISA UMR 5280, CNRS, 5 rue de la Doua, 69100 Villeurbanne, France

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*Corresponding author: karine.faure@isa-lyon.fr, Phone number +33-437423686

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## Abstract

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In this study, we present the formation of artifacts from simple phenolic compounds and derivatives in SFC-UV-MS analysis. These ions were detected only when the UV detector was turned on, demonstrating that the UV light is necessary for their formation. Based on high-resolution mass spectrometry (HRMS) analysis of 21 standards in negative electrospray ionization mode, the artifacts were annotated as ions where CO₂ or NO₂ had been added to the molecular ion or to an ion that had lost a functional group. In approximately half of the cases, the MS signal of the artifact was higher than the signal of the molecular ion. Although the formation of artifacts can complicate non-target analysis as the detected molecular ion does not match with the analyzed standard, we demonstrated that the phenomenon can aid with the structural identification of isomers due to the formation of specific ions. In addition, the overall MS signal increased when the UV was turned on, which can help with the detection of low-abundance compounds; and one compound – anisole – was detected only thanks to the artifact. Thus, the aim of this paper is to make researchers aware of the UV effect in SFC-UV-MS analysis together with the advantages and disadvantages of artifact formation.

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<figure><img src="image_2.png" /></figure>

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**Keywords:** phenols, sub/supercritical fluid chromatography, UV, mass spectrometry, artifacts

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**Abbreviations:** SFC: sub/supercritical fluid chromatography; HRMS: high-resolution mass spectrometry; LRMS: low-resolution mass spectrometry; ESI: electrospray ionization; Q-TOF: quadrupole time-of-flight mass spectrometer; SIM: selected ion monitoring; TIC: total ion current; EIC: extracted ion chromatogram; BPC: base peak chromatogram; BPR: backpressure regulator; BHT: 2,6-di-tert-butyl-4-methylphenol

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26 Introduction

27 Sub/supercritical fluid chromatography (SFC) coupled to a mass spectrometric detector (MS) is a
28 common instrumentation used in academia and industry. Often electrospray ionization (ESI) is used to
29 ionize the analytes prior to MS detection, yielding [M+H]$^+$ ions in positive mode and [M-H]$^-$ ions in
30 negative mode. However, depending on the analyte, eluent, eluent additives and other conditions, ESI
31 can produce various artifacts that are referred to as “adducts” when the formation of the specific ion is
32 widely known. In negative ESI mode (ESI-), examples include, chloride [M+Cl]$,^-$, formate
33 [M+HCOO]$,^-$, and acetate [M+CH$_3$COO]$^-$ adducts. It is important to consider the artifact or adduct
34 formation, as it can aid in the detection and characterization of an analyte but also affect the
35 quantitative analysis.¹ Although adduct formation has been studied more in liquid chromatography
36 (LC) than in SFC, likely due to the wider use of LC, adducts have also been detected in analyses
37 where SFC has been hyphenated to negative mode ESI-MS.² In addition, artifacts can be caused by the
38 electric noise of the mass spectrometer, such as “ringing” of the detector signal.³

39 To obtain more information about the analytes, a UV detector can also be connected after the SFC
40 column using a split or in-between the column and the MS detector. The technical aspects that are
41 considered when selecting and setting up of the system include the resulting pressure, control of
42 decompression, and whether all or part of the analytes reach both detectors.⁴,⁵ In this setup, the UV
43 detector is generally considered to be “non-destructive”; e.g., there should only be technical
44 constraints to overcome when connecting the UV to the MS. However, as was established by
45 Schweikart and Hulthe⁶, it is possible that the modern low-volume analytical UV flow cells may act as
46 photochemical reactors. They noted the partial transformation of analytes in the online HPLC-UV-MS
47 setup, where oxidation ions were formed from specific proteins, peptides, and amino acids. Therefore,
48 in addition to artifact formation caused by the ion source or mass spectrometer, artifacts can also
49 originate from degradation in the UV detector. To the best of our knowledge, the study by Schweikart
50 and Hulthe⁶ is the only one in which this kind of phenomenon has been demonstrated.

51 Here, we present the results of a discovery that intense artifacts can also be formed when using SFC-
52 UV-MS setup. As most commercial interfaces have UV connected upfront to MS,⁵ it is of utmost
53 importance to be aware of potential artifacts. Similarly to the adducts formed during ionization, these
54 new types of ions can give structural information about the analytes and improve their detection. In
55 contrast to the study by Schweikart and Hulthe⁶, the analytes for which the phenomenon was seen in
56 our work are simple phenolic compounds or derivatives (phenol, cresol, anisole, etc.), that are
57 commonly found in various samples, such as water, food, plastics, dyes, household chemicals,
58 biowaste, and fuel.⁷⁻¹¹ Potentially, some of these artifacts could be called “adducts”, but to facilitate
59 readability, here we use the general term “artifact” to refer to all of the ions formed due to the UV
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61 The formation of the same ions was observed with two entirely different SFC-UV-MS systems from
62 different suppliers, one using low-resolution single quadrupole MS (LRMS) and the other high-
63 resolution MS (HRMS). Although it has been previously noted that UV light can induce degradation
64 or chemical modification of phenolic compounds,¹²,¹³ to the best of our knowledge, this is the first
65 time a photochemical reaction leading to the formation of artifacts from phenolic compounds has been
66 detected in SFC-UV-MS analysis. Therefore, we present here the study of 30 phenolic compounds or
67 derivatives by SFC-UV-MS and SFC-MS together with the identification of the detected ions. In
68 addition, for selected compounds, the effect of varying the co-solvent, SFC flow rate, and gradient
69 slope on the artifact formation was investigated.

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71 Materials and methods

72 LC-MS grade methanol (MeOH) and ethanol (EtOH) of HPLC grade were purchased from Sigma- 73 Aldrich (Darmstadt, Germany). LC-MS grade acetonitrile (ACN) was purchased from Honeywell 74 Riedel-de-Haën (Seelze, Germany) and pressurised liquid CO₂ (99.995 %, N45 or ALPHAGAZ SFC) 75 from Air Liquide (Pierre Bénite, France).

76 Two different SFC-UV-MS instruments were used. First, for the accurate mass detection and artifact 77 identification, a Waters UPC² with an 8.4 µL analytical flow cell Acquity UPLC PDA detector 78 connected in series to an Agilent Jet stream ESI source installed on an Agilent 6560 Ion Mobility Q- 79 TOF was used. The HRMS was used in the “QTOF only acquisition mode”, i.e. no ion mobility 80 separation was applied. From now on, this setup will be referred to as the SFC-UV-HMRS. For the 81 SFC separation, a Waters Torus 1-aminoanthracene (1-AA) column (1.7 µm, 3 mm × 50 mm) was 82 used with CO₂ as the supercritical fluid and MeOH (marked as B) as the co-solvent. A flow rate of 1.4 83 mL min⁻¹ and the gradient program was the following: 5-50% of B in 4.00 min, then in 0.15 min back 84 to the starting conditions, and 1.00 min for equilibration, leading to a total run time of 5.15 min. Based 85 on Eq. (1), where C corresponds to %B, t₀ to the column dead time, and t_g to the gradient time, this 86 yields a normalized gradient slope (s) of 2%.

$$S = \frac{(C_{Final} - C_{Initial}) \times t_{0}}{t_{g}} \qquad (1)$$

88 For the HRMS, the following parameters were applied: ESI- mode, N₂ as nebulizer gas, nebulizer 89 pressure 40 psi, drying and sheath gas temperatures 350 °C and flow 11 L min⁻¹, capillary 3.5 kV, and 90 fragmentor 400 V. The scan range was 70-500 m/z with an acquisition rate of 5 spectra per second. As 91 the reference ion, trifluoroacetate (exact mass 112.9855 m/z) was introduced to the source using the 92 reference nebulizer during the run to improve the mass accuracy. Agilent MassHunter Qualitative 93 Analysis 10.0 software was used to study the chromatograms and mass spectra. For ion and formula 94 assignment, the Generate Formulas from Spectrum Peaks function was applied to the mass spectra of 95 each standard, where the mass spectrum of the baseline (background) extracted near the peak was 96 subtracted. The formula and ion species that had the highest overall match score are presented in this 97 paper.

98 Second, an Agilent 1260 Infinity II UHPSFC with a 13 µL Agilent 1260 DAD WR (G7115A) 99 connected in series to an Agilent InfinityLab LC/MSD with a single quadrupole in SIM mode was 100 used to study the trends in artifact formation. From now on, this setup will be referred to as SFC-UV- 101 LRMS. The following parameters were used: ESI- mode, N₂ as nebulizer gas, nebulizer pressure 35 102 psi, drying gas temperature 350 °C and flow 12 L min⁻¹, capillary 5.0 kV, and fragmentor 50 V. The 103 experiments were performed on another Waters Torus 1-AA column (1.7 µm, 3 mm × 50 mm) with a 104 gradient of 5-50% of co-solvent at two flow rates – 0.7 and 1.4 mL min⁻¹ – and at two normalized 105 gradient slopes – 2 and 10%. Three solvents were used as co-solvents: MeOH, EtOH, and ACN. When 106 ACN was used as a co-solvent for the analysis of hydroquinone and vanillyl alcohol, the final 107 composition was increased to 70% to ensure the elution of the compounds. The parameters for all of 108 the gradients mentioned in this paper are presented in S1 in the Supplementary Information (SI).

109 In both SFC-ESI-MS instrumentations, a preBPR splitter with a sheath pump⁵ was used to connect the 110 SFC to the ESI source. As the make-up solvent, MeOH was used at a flow rate of 0.5 mL min⁻¹. The 111 BPR pressure was 140 bar, oven temperature 40 °C, injection volume 1 µL, and UV signal was 112 registered at 210 and 254 nm. The 30 analyzed compounds (see section Characterization of the 113 artifacts or S2 in the SI) were standards purchased from Sigma-Aldrich, dissolved in MeOH with a 114 concentration of 0.2 mg mL⁻¹ or, in the case of vanillyl alcohol, 0.4 mg mL⁻¹. In all experiments, the 115 standard compounds were analyzed one at the time to ensure the correct assignment of the artifacts.
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The radical scavenger experiments were performed using the SFC-UV-HRMS setup with ascan range 70-500 *m/z* and an acquisition rate of 5 spectra per second. The HRMS parameters were the same as mentioned before. 2,6-di-*tert*-butyl-4-methylphenol (BHT) was purchased from Sigma-Aldrich and dissolved in MeOH or ACN to yield a concentration of 250 or 500 ppm. A flow of 1.4 mL min⁻¹ and 2% normalized slope was used to analyze the selected compounds one at the time. Therefore, for each compound, 6 experiments were performed where only the co-solvent was changed to either pure MeOH, MeOH + 250 ppm BHT, MeOH + 500 ppm BHT, pure ACN, ACN + 250 ppm BHT, or ACN + 500 ppm BHT.

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# Results and discussion

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## Characterization of the artifacts

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In Figure 1, the results for the injection of phenol in SFC-(UV)-HRMS are shown to showcase the UV effect on the mass spectrum. With the UV turned on, in addition to the molecular ion [M-H]⁻ with *m/z* of 93.0345, a very intense ion with *m/z* of 138.0196 corresponding to [M+NO₂-2H]⁻ is present in the mass spectrum (assignment of the ion is presented in the next paragraph). However, when the UV was turned off, only the molecular ion was detected – the extracted ion chromatogram (EIC) for the [M+NO₂-2H]⁻ ion showed no signal. Therefore, when UV is turned on, misidentification may occur as the most intense ion has also a higher *m/z* value, which may lead to the conclusion that the analyte is nitrophenol and that the *m/z* of 93.0345 is only a fragment. Fortunately, this misunderstanding can be avoided by simply turning off the UV light or using a split after the column to connect the detectors independently. On the contrary, artifact formation may potentially help with the detection of poorly ionizable compounds or low-level analytes. As shown in Figure 1, the intensity of the molecular ion was quite similar for both experiments; however, the formation of the artifact increased the area of the corresponding peak in the base peak chromatogram (BPC) by 2.5 times. Since the artifact exhibited the same retention time and peak shape, its presence could potentially be used to increase the MS peak area.
<figure><img src="image_3.png" /><figcaption>Figure 1. Example of the UV effect on the SFC-HRMS results. Detection of phenol when UV is off (first column) vs UV on (second column). Both mass spectra correspond to the signal registered from 0.57 to 0.60 min, where the background was manually subtracted.</figcaption></figure>

The Generate Formulas function in the MassHunter software was used to assign ion species and formulas. Figure 2 shows examples of ions that were detected for three different standards only when the UV was on. The ion assignment was made based on the overall match score (0-100%), which considers both mass and isotopic match between the measured data and values calculated for a formula candidate. In Figure 2, the match values for mass, isotopic abundance, and isotopic spacing are also presented separately. The formula with the highest overall match score was assigned to the detected ion. The parameters used for the Generate Formulas function, the formulas for matches with lower quality, and more details on the ion assignment are presented in S3 of the SI. In the software results, all ion species were identified as [M-H]⁻. The isotopic pattern of these three examples also confirms that the ions are singly charged, as the first isotopic satellite has a *m/z* value approximately 1.00335 Da higher than the monoisotopic ion. In the case of doubly charged ions, the mass difference would be divided by two (0.50168 Da).¹⁴ All of the ions presented in this study had an isotopic pattern consistent with singly charged ions.
<figure><img src="image_4.png" /><figcaption>Figure 2. Examples of assignments for the new ions that were detected for phenol, 4-ethylphenol, and syringol when the UV was turned on. The details and formulas for other matches with lower quality are presented in S3 in the SI.</figcaption></figure>

For example, the formula generation resulted in assigning the neutral formula of C₆H₅NO₃ for the ion detected during the analysis of phenol, molecular formula C₆H₆O. Therefore, the ion differs from the molecular formula by +NO₂-H, corresponding to the exact mass of 44.9851 Da. However, for some standards (like 4-ethylphenol in Figure 2), the difference corresponded to +CO₂, exact mass 43.9898 Da, suggesting the formation of another type of ion species. When discussing the assignment of ion species to the detected exact masses, we can present these ions as [M+NO₂-2H]⁻ and [M+CO₂-H]⁻. In addition to the [M+NO₂-2H]⁻ and [M+CO₂-H]⁻ ion assignments, another ion type was detected for some of the standards. These were grouped together as ions, which, together with the addition of NO₂, also exhibited a loss of a functional group. In the examples shown in Figure 2, this type of ion was only detected for syringol, where the difference between the molecular formula (C₈H₁₀O₃) and artifact formula with the highest overall match score (C₇H₇NO₅) corresponds to -CH₃+NO₂. This also is consistent with the structure of the three compounds – only syringol has a methoxy group in its structure from which the methyl group can easily leave. For other standards, which also had assignments involving the loss of a functional group, it was ensured that the structure of the standard was known to yield the corresponding leaving group.

The SFC-UV-HRMS results, together with proposed pathways for the artifact formation for 21 standards are shown in Table 1. The remaining 9 compounds, labeled as C22 to C30, are not shown in Table 1 because only the molecular ion and not the artifact was detected for them: methyl 4-hydroxybenzoate, acetosyringone, syringaldehyde, *p*-coumaric acid, 3,4-dimethylbenzoic acid, 4-hydroxybenzoic acid, 2,4,6-trimethylphenol, 4-hydroxyacetophenone, and 4-hydroxybenzaldehyde. As can be seen from Table 1, some compounds exhibited only [M+NO₂-2H]⁻ or [M+CO₂-H]⁻ ions, while others showed functional group loss along with artifact formation, such as [M-CH₃+NO₂-H]⁻ for guaiacol (C13). The loss of functional group was observed only with NO₂ addition, and not with CO₂ addition. The same specific ions were seen for the same compounds when using a completely different SFC-UV-LRMS instrumentation. For some compounds, such as caffeic acid and ferulic acid, the intensity of the artifact was low and may not interfere with the identification of the correctanalyte. However, in the case of 2,6-dimethylphenol and guaiacol, for example, very intense artifacts were observed.

<table><caption>Table 1. SFC-UV-HRMS measurement results of all standards for which also artifacts were detected at the same retention time. ¹The A/M represents the area ratio of the corresponding artifact to the molecular ion. ²Cannot be calculated, as molecular ion was not detected.</caption><thead><tr><td>ID</td><td>Standard compound</td><td>Detected</td><td>A/M<sup>1</sup></td><td>Identified ion</td><td>Error</td><td>Identification</td></tr></thead></table>
<table><thead><tr><th></th><th></th><th>m/z</th><th></th><th>ppm</th><th></th></tr></thead><tbody><tr><td rowspan="2">C1</td><td rowspan="2">phenol</td><td>93.0345</td><td>C<sub>6</sub>H<sub>6</sub>O-H</td><td>-1.48</td><td>[M-H]<sup>-</sup></td></tr><tr><td>138.0197</td><td>3.4</td><td>C<sub>6</sub>H<sub>5</sub>NO<sub>3</sub>-H</td><td>0.51</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td>C2</td><td>anisole</td><td>138.0197</td><td>NA<sup>2</sup></td><td>C<sub>6</sub>H<sub>5</sub>NO<sub>3</sub>-H</td><td>0.25</td><td>[M-CH<sub>3</sub>+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td rowspan="2">C3</td><td rowspan="2">m-cresol</td><td>107.0503</td><td></td><td>C<sub>7</sub>H<sub>8</sub>O-H</td><td>0.52</td><td>[M-H]<sup>-</sup></td></tr><tr><td>152.0354</td><td>1.2</td><td>C<sub>7</sub>H<sub>7</sub>NO<sub>3</sub>-H</td><td>0.82</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C4</td><td rowspan="2">o-cresol</td><td>107.0504</td><td></td><td>C<sub>7</sub>H<sub>8</sub>O-H</td><td>1.05</td><td>[M-H]<sup>-</sup></td></tr><tr><td>152.0355</td><td>5.7</td><td>C<sub>7</sub>H<sub>7</sub>NO<sub>3</sub>-H</td><td>1.00</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C5</td><td rowspan="2">p-cresol</td><td>107.0504</td><td></td><td>C<sub>7</sub>H<sub>8</sub>O-H</td><td>1.20</td><td>[M-H]<sup>-</sup></td></tr><tr><td>151.0401</td><td>0.3</td><td>C<sub>8</sub>H<sub>8</sub>O<sub>3</sub>-H</td><td>-0.06</td><td>[M+CO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td rowspan="2">C6</td><td rowspan="2">catechol</td><td>109.0295</td><td></td><td>C<sub>6</sub>H<sub>6</sub>O<sub>2</sub>-H</td><td>-0.10</td><td>[M-H]<sup>-</sup></td></tr><tr><td>154.0147</td><td>0.4</td><td>C<sub>6</sub>H<sub>5</sub>NO<sub>4</sub>-H</td><td>0.73</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C7</td><td rowspan="2">hydroquinone</td><td>109.0294</td><td></td><td>C<sub>6</sub>H<sub>6</sub>O<sub>2</sub>-H</td><td>-0.91</td><td>[M-H]<sup>-</sup></td></tr><tr><td>153.0192</td><td>0.5</td><td>C<sub>7</sub>H<sub>6</sub>O<sub>4</sub>-H</td><td>0.12</td><td>[M+CO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td rowspan="2">C8</td><td rowspan="2">2,5-dimethylphenol</td><td>121.0659</td><td></td><td>C<sub>8</sub>H<sub>10</sub>O-H</td><td>0.35</td><td>[M-H]<sup>-</sup></td></tr><tr><td>166.0510</td><td>1.7</td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>3</sub>-H</td><td>0.26</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C9</td><td rowspan="2">2,6-dimethylphenol</td><td>121.0660</td><td></td><td>C<sub>8</sub>H<sub>10</sub>O-H</td><td>0.62</td><td>[M-H]<sup>-</sup></td></tr><tr><td>166.0513</td><td>21.4</td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>3</sub>-H</td><td>2.12</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C10</td><td rowspan="2">2-ethylphenol</td><td>121.0659</td><td></td><td>C<sub>8</sub>H<sub>10</sub>O-H</td><td>0.26</td><td>[M-H]<sup>-</sup></td></tr><tr><td>166.0510</td><td>2.6</td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>3</sub>-H</td><td>0.43</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C11</td><td rowspan="2">4-ethylphenol</td><td>121.0659</td><td></td><td>C<sub>8</sub>H<sub>10</sub>O-H</td><td>0.19</td><td>[M-H]<sup>-</sup></td></tr><tr><td>165.0559</td><td>0.3</td><td>C<sub>9</sub>H<sub>10</sub>O<sub>3</sub>-H</td><td>0.96</td><td>[M+CO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td rowspan="2">C12</td><td rowspan="2">4-methylcatechol</td><td>123.0451</td><td></td><td>C<sub>7</sub>H<sub>8</sub>O<sub>2</sub>-H</td><td>-0.06</td><td>[M-H]<sup>-</sup></td></tr><tr><td>168.0304</td><td>0.2</td><td>C<sub>7</sub>H<sub>7</sub>NO<sub>4</sub>-H</td><td>0.80</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C13</td><td rowspan="2">guaiacol</td><td>123.0448</td><td></td><td>C<sub>7</sub>H<sub>8</sub>O<sub>2</sub>-H</td><td>-2.94</td><td>[M-H]<sup>-</sup></td></tr><tr><td>168.0302</td><td>20.8</td><td>C<sub>7</sub>H<sub>7</sub>NO<sub>4</sub>-H</td><td>-0.14</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C14</td><td rowspan="2">4-hydroxybenzyl alcohol</td><td>154.0146</td><td>85.9</td><td>C<sub>6</sub>H<sub>5</sub>NO<sub>4</sub>-H</td><td>0.00</td><td>[M-CH<sub>3</sub>+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td>123.0451</td><td></td><td>C<sub>7</sub>H<sub>8</sub>O<sub>2</sub>-H</td><td>-0.76</td><td>[M-H]<sup>-</sup></td></tr><tr><td rowspan="2">C15</td><td rowspan="2">pyrogallol</td><td>167.0350</td><td>6.4</td><td>C<sub>8</sub>H<sub>8</sub>O<sub>4</sub>-H</td><td>-0.57</td><td>[M+CO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td>138.0198</td><td>6.0</td><td>C<sub>6</sub>H<sub>5</sub>NO<sub>3</sub>-H</td><td>0.82</td><td>[M-CH<sub>2</sub>OH+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td rowspan="2">C16</td><td rowspan="2">syringol</td><td>125.0243</td><td></td><td>C<sub>6</sub>H<sub>6</sub>O<sub>3</sub>-H</td><td>-0.55</td><td>[M-H]<sup>-</sup></td></tr><tr><td>170.0097</td><td>0.2</td><td>C<sub>6</sub>H<sub>5</sub>NO<sub>5</sub>-H</td><td>1.44</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C17</td><td rowspan="2">vanillyl alcohol</td><td>198.0410</td><td>NA<sup>2</sup></td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>5</sub>-H</td><td>1.38</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td>184.0254</td><td>NA<sup>2</sup></td><td>C<sub>7</sub>H<sub>7</sub>NO<sub>5</sub>-H</td><td>1.55</td><td>[M-CH<sub>3</sub>+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td rowspan="2">C18</td><td rowspan="2">eugenol</td><td>169.0020</td><td>NA<sup>2</sup></td><td>C<sub>6</sub>H<sub>4</sub>NO<sub>5</sub>-H</td><td>2.20</td><td>[M-CH<sub>3</sub>-CH<sub>3</sub>+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td>153.0555</td><td></td><td>C<sub>8</sub>H<sub>10</sub>O<sub>3</sub>-H</td><td>-1.53</td><td>[M-H]<sup>-</sup></td></tr><tr><td rowspan="2">C19</td><td rowspan="2">coniferyl aldehyde</td><td>198.0406</td><td>1.3</td><td>C<sub>8</sub>H<sub>9</sub>NO<sub>5</sub>-H</td><td>-0.06</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td>184.0252</td><td>1.2</td><td>C<sub>7</sub>H<sub>7</sub>NO<sub>5</sub>-H</td><td>-0.13</td><td>[M-CH<sub>3</sub>+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td rowspan="2">C20</td><td rowspan="2">caffeic acid</td><td>168.0301</td><td>0.9</td><td>C<sub>7</sub>H<sub>7</sub>NO<sub>4</sub>-H</td><td>-0.47</td><td>[M-CH<sub>3</sub>O+NO<sub>2</sub>-H]<sup>-</sup> or [M-CH<sub>2</sub>OH+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td>163.0768</td><td></td><td>C<sub>10</sub>H<sub>12</sub>O<sub>2</sub>-H</td><td>-0.11</td><td>[M-H]<sup>-</sup></td></tr><tr><td rowspan="2">C21</td><td rowspan="2">ferulic acid</td><td>207.0665</td><td>0.7</td><td>C<sub>11</sub>H<sub>12</sub>O<sub>4</sub>-H</td><td>0.84</td><td>[M+CO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td>208.0628</td><td>0.5</td><td>C<sub>10</sub>H<sub>11</sub>NO<sub>4</sub>-H</td><td>7.14</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td rowspan="2">C22</td><td rowspan="2">coniferyl aldehyde</td><td>194.0463</td><td>2.2</td><td>C<sub>9</sub>H<sub>9</sub>NO<sub>4</sub>-H</td><td>3.00</td><td>[M-CH<sub>3</sub>+NO<sub>2</sub>-H]<sup>-</sup></td></tr><tr><td>177.0560</td><td></td><td>C<sub>10</sub>H<sub>10</sub>O<sub>3</sub>-H</td><td>1.51</td><td>[M-H]<sup>-</sup></td></tr><tr><td rowspan="2">C23</td><td rowspan="2">coniferyl aldehyde</td><td>222.0412</td><td>0.1</td><td>C<sub>10</sub>H<sub>9</sub>NO<sub>5</sub>-H</td><td>2.01</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td>179.0351</td><td></td><td>C<sub>9</sub>H<sub>8</sub>O<sub>4</sub>-H</td><td>-0.19</td><td>[M-H]<sup>-</sup></td></tr><tr><td rowspan="2">C24</td><td rowspan="2">coniferyl aldehyde</td><td>224.0202</td><td>0.02</td><td>C<sub>9</sub>H<sub>7</sub>NO<sub>6</sub>-H</td><td>-0.07</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr><tr><td>193.0508</td><td></td><td>C<sub>10</sub>H<sub>10</sub>O<sub>4</sub>-H</td><td>0.91</td><td>[M-H]<sup>-</sup></td></tr><tr><td rowspan="2">C25</td><td rowspan="2">coniferyl aldehyde</td><td>238.0372</td><td>0.02</td><td>C<sub>10</sub>H<sub>9</sub>NO<sub>6</sub>-H</td><td>5.26</td><td>[M+NO<sub>2</sub>-2H]<sup>-</sup></td></tr></tbody></table>

Here we can see some other advantages of using UV before the MS detection. For example, the molecular ion of anisole cannot be detected with the used instrumentation and parameters, regardless of whether UV is turned on or off. However, turning on the UV light enabled the detection of a very intense [M-CH<sub>3</sub>+NO<sub>2</sub>-H]<sup>-</sup> ion and thus, anisole was detected. The structure of the compound also affected the type of artifact detected (see the structures in S2). For example, the *m*- and *o*-isomers of
cresol had the [M+NO₂-2H]⁻ ion, while *p*-cresol had only the [M+CO₂-H]⁻ ion. Therefore, although the isomers have the same molecular ion, the detection of specific artifacts could aid in structural elucidation, by eliminating at least some of the isomers. The other standards that were also only *para*-substituted (C7, C11, and C14) showed the same [M+CO₂-H]⁻ ion as *p*-cresol. Interestingly, the only compound that also exhibited [M+CO₂-H]⁻ ion was eugenol (C18), even though the compound is *o*- and *p*-substituted. In addition, some compounds with a methoxy or hydroxymethyl group (such as C2, C14, C16, C17, and C18) showed a loss of the corresponding group together with the addition of NO₂. To illustrate the obtained mass spectra, the MS for vanillyl alcohol (C17) is shown in Figure 3. When the UV is off, the molecular ion and the in-source fragments are detected; however, when the UV is on, intense artifacts are produced. In both cases, the same intense in-source fragments were detected for which more information is given in S4 of the SI.

<figure><img src="image_5.png" /><figcaption>Figure 3. Example of the appearance of multiple artifacts in the mass spectrum of vanillyl alcohol (C17). Left mass spectrum – UV turned off; right mass spectrum – UV turned on. From both mass spectra, the background was manually subtracted. The molecular ion with 153.0555 *m/z* and main ion-source fragments were detected both with UV on and UV off. The most intense artifacts were 168.0302 *m/z* corresponding to [M-CH₃O+NO₂-H]⁻ or [M-CH₂OH+NO₂-H]⁻, 184.0250 *m/z* corresponding to [M-CH₃+NO₂-H]⁻, and 198.0408 *m/z* corresponding to [M+NO₂-2H]⁻.</figcaption></figure>

Although future studies are needed to understand the exact mechanism of the artifact formation, some hypotheses can already be made based on these results. Since the formation of artifacts from simple phenolic compounds by LC-UV-MS has not been reported, we presume that the subcritical fluid is needed for the photochemical reaction to take place. Although supercritical CO₂ is a known green solvent for chemical reactions, to the best of our knowledge, these kinds of reactions with phenols in the environment of supercritical CO₂ and co-solvent have not been studied. However, it is known that phenols can absorb light which can lead to the release of hydrogen and the formation of a phenolate anion, thus initiating a photochemical reaction.<sup>12,15</sup> Here, we also propose that the light-absorbing part may be the analyte molecule and not the solvent, as the highest artifact intensity can be seen at the peak maxima (Figure 1). The [M+CO₂-H]⁻ ion type had only the addition of the CO₂ molecule, which could imply that an adduct was formed. This was confirmed by performing a fragmentation study on the C7 standard (chromatograms and spectra are shown in S5). Even after applying 20 eV of collision energy, no new fragments were detected that could indicate a chemical reaction. Therefore, the ion species containing CO₂ are probably adducts.

However, the [M+NO₂-2H]⁻ ion always had one hydrogen less than the molecular ion, which could imply that the UV light initiated a substitution reaction in which the NO₂ group substituted a hydrogen on the benzene ring. This is consistent with observations that UV light can form reactive NO₂-type radicals capable of causing nitration of phenols.<sup>13</sup> These types of photonitration reactions are known to occur in the gas phase, in particles, and in atmospheric or surface waters.<sup>16,17</sup> In Figure 1, it can be seen that when phenol is analyzed with the UV light on, in addition to the artifact corresponding to [C₆H₅NO₃-H]⁻, there is an ion with 108.0217 *m/z* corresponding to [C₆H₅O₂-H]⁻. This is a known
fragment for deprotonated nitrophenol,¹⁸ which seems to have been formed by in-source fragmentation. Additional fragmentation experiments at 20 eV significantly increased the signal of this ion (spectrum shown in S5). The formation of this type of fragment was confirmed the analysis of C8 and C9, two isomers of dimethylphenol (C₈H₁₀O, chromatograms and spectra are shown in S5). Similar to phenol, both compounds had a fragment (C₈H₉O₂) corresponding to the loss of NO₂ from the NO₂-containing artifact (C₈H₉NO₃). When fragmentation was performed, already at 5 eV of collision energy, the signal of the mentioned fragment increased several folds, confirming the hypothesis that the NO₂-containing ion was formed by a chemical reaction.

Based on the analysis of *o*-, *m*-, and *p*-substituted cresols, we can propose that the formation of the [M+NO₂-2H]⁻ ion is favored when the *para*-position is available; if not, the formation of the [M+CO₂-H]⁻ adduct is more probable. For the other artifacts which also show demethylation or loss of other groups, the substitution of NO₂ could vary. In the case of anisole, the ion with [M-CH₃+NO₂-H]⁻ formula suggests that the compound was demethylated but nitrated in the aromatic ring. For syringol, also the nitration of the aromatic ring took probably place, as the [M+NO₂-2H]⁻ ion was detected. However, in addition, the [M-CH₃+NO₂-H]⁻ ion was detected, which matches the substitution of methyl at one of the methoxy groups with NO₂; thus, the aromatic ring was not directly nitrated. It may also be beneficial to mention that the detected artifacts always had only one CO₂ or NO₂ molecule added to their structure, no more. Nevertheless, more thorough studies focusing on the artifact formation mechanisms are needed in the future to propose precise reaction pathways.

258

## Study of trends in artifact formation

Nine standards were selected to study the trends of the artifact to molecular ion MS area ratios. Three of the compounds (C1, C8, C9) had only [M+NO₂-2H]⁻ artifact, three (C5, C7, C11) only [M+CO₂-H]⁻ adduct, and three (C16, C17, C18) had besides the [M+NO₂-2H]⁻ ion also at least one ion formed from a loss of functional group. All standards were analyzed with SFC-UV-LRMS using the SIM mode to follow the signal of the molecular ion, [M+CO₂-H]⁻, [M+NO₂-2H]⁻, and the specific artifacts identified previously with HRMS. All experiments were repeated with three co-solvents (MeOH, EtOH, ACN) at two flow rates (0.7 and 1.4 mL min⁻¹) and with two gradient slopes (2% and 10%).

In Figure 4, the results for the first six compounds are shown. For the compounds having only the [M+NO₂-2H]⁻ artifact (first row), the highest ratios were seen when ACN was used as the co-solvent. In all cases, this led to two to almost nine times higher artifact ion area compared to the molecular ion. Regarding MeOH and EtOH as the co-solvents, quite similar results were obtained; except for standard C9, where the ratio was much higher when MeOH was used. From this we can conclude that although with all co-solvents, the [M+NO₂-2H]⁻ ion was seen, ACN significantly enhanced the formation of this ion. For the other three standards (second row), no [M+CO₂-H]⁻ adduct was detected when ACN was the co-solvent. Again, quite similar ratios were obtained when MeOH or EtOH were used. We propose that these results could suggest that using aprotic or protic co-solvent affects the formation of the artifacts. The formation of [M+CO₂-H]⁻ adduct with protic co-solvent could suggest that hydrogen bonding is needed to stabilize the phenolate ion or some other intermediate needed for the adduct formation. Regarding the effect of gradient slope and flow rate, mainly higher ratios were obtained for [M+NO₂-2H]⁻ artifact when the lower flow rate was used. However, this was vice versa for the compounds having the [M+CO₂-H]⁻ adduct. The slope of the SFC gradient did not have a clear influence on the artifact formation.
<figure><img src="image_6.png" /><figcaption>Figure 4. MS signal area ratio of artifact to molecular ion obtained when different co-solvents, flow rates, and normalized gradient slopes were used. The purple bars represent MeOH, the blue bars EtOH, and the grey bars ACN as co-solvent. First row – three compounds (C1, C8, and C9) that had only [M+NO₂-2H]⁻ artifact; second row – three compounds (C5, C7, C11) that had only [M+CO₂-H]⁻ adduct.</figcaption></figure>

The same study was also performed with C16, C17, and C18 – compounds that, in addition to the [M+NO₂-2H]⁻ ion, had also an artifact formed together with the loss of a functional group. As the trends were similar but not as clear as for the compounds shown in Figure 4, the graphs are shown in S6 in the SI. It is likely that the formation of several types of artifacts for one standard happens together with competition with each other, making the study of the effect that the different co-solvents have on the ion formation more complex. However, the following conclusions could still be made. Although with ACN as the co-solvent still often the highest ratios were obtained for the NO₂-containing artifacts, for the ions including the loss of a group, quite similar results were obtained with the three co-solvents. Therefore, this implies that changing the co-solvent might not always cause a significant increase or decrease of a specific artifact. However, again, it was seen that with ACN as the co-solvent, no [M+CO₂-H]⁻ adduct could also be seen for the compounds that exhibited several artifacts.

### Effect of radical scavenger on the artifact formation

BHT is a widely used radical scavenger. In the work by Schweikart and Hulthe⁶, the authors used it to confirm their theory that the artifacts detected in the HPLC-UV-MS analysis are formed from the radical reactions caused by UV light. We also decided to study the effect of BHT in the co-solvent to see if the artifact formation is suppressed. For that, the six compounds shown in Figure 4 were analyzed with a flow of 1.4 mL min⁻¹ and a 2% normalized gradient slope. As the co-solvent, pure MeOH or ACN was used or BHT was dissolved in the eluent to yield a concentration of 250 or 500 ppm for the radical scavenger. The peak areas for the artifact and molecular ion were extracted from the data using their exact masses. The ratio of the artifact to molecular ion was calculated and plotted in Figure 5 using the logarithmic scale for better visualization. It was seen that the formation of [M+NO₂-2H]⁻ ions were suppressed significantly when 250 ppm of BHT was added to the co-solvent, especially when ACN was used. It must be kept in mind that the logarithmic scale is used in Figure 5; therefore, also with MeOH, the decrease in the ratio was significant. For example, for C9, the artifact to molecular ion ratio was 388 when pure MeOH was used as the co-solvent; however, the same value
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was 110 when 250 ppm of BHT was added to the MeOH co-solvent. Overall, the usage of 250 ppm BHT compared to pure eluent affected the ratio more than increasing the BHT concentration from 250 to 500 ppm in the co-solvent.

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In terms of the [M+CO₂-H]⁻ adduct, as was the case with pure ACN in Figure 4, this ion species was not detected when ACN+BHT was used as the co-solvent; therefore, the corresponding bar graph was not added to Figure 5. However, the ion was detected regardless of whether BHT was added to the co-solvent or not. Contrary to the NO₂-containing artifact, the ratio of [M+CO₂-H]⁻ to molecular ion was similar when pure MeOH was used as the co-solvent or BHT with a concentration of 250 or 500 ppm was added to MeOH. The results of the radical scavenger experiments suggest that the formation of the NO₂-containing ion species happens through radical reactions, as the addition of a radical scavenger decreased the ratio. However, as the same effect was not seen for the [M+CO₂-H]⁻ ion, it confirms that this ion is more likely an adduct formed through the phenolate ion. However, further studies are needed to understand more deeply the addition of CO₂ and nitration reactions leading to the formation of the detected artifacts.

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<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

Figure 5. Effect of radical scavenger on the artifact to molecular ion MS area ratio. The y-axis is in the logarithmic scale for better visualization. The purple and grey bars represent the results for MeOH and ACN as the co-solvent, respectively. The first darker color bars are the results for pure co-solvent while the lighter color bars correspond to co-solvent where BHT was added – the second bars represent the results for 250 ppm of BHT and the third bars for 500 ppm of BHT. The [M+CO₂-H]⁻ artifact results for ACN and ACN+BHT are not shown because the ion was not detected for the compounds in these conditions.

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Although in this paper, we have used the MS area ratio of artifact to molecular ion to study trends while changing the co-solvent, slope, or flow rate during a sequence, it is important to emphasize that the ratios changed when the instrumentation was changed, such as switching between the SFC-UV-LRMS and SFC-UV-HRMS. For example, when comparing the data for C1 analyzed with ACN as co-solvent, the ratio was found to be 4 and 453 with the respective instruments. One aspect that can potentially affect this ratio is the purity of the CO₂. For the CO₂ sources used in this work, the specification of one of them mentioned NO₂ by having that NO+NO₂ < 2 ppm. Although two different quality CO₂ cylinders were used in this work, including MS grade CO₂, the same ions were always detected. Therefore, we suspect that even low-level impurities can lead to artifact formation.

345

## Conclusions

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In this paper, we present how artifacts and adducts can be formed from simple phenolic compounds and derivatives only by turning the UV detector on in SFC-UV-(HR)MS analysis. For each compound, the same specific artifacts were obtained using two different SFC-UV-MS setups and were identified as [M+CO₂-H]⁻, [M+NO₂-2H]⁻, or addition of NO₂ with loss of functional group(s). Fragmentation of the standards suggested that the NO₂-containing ions were formed by chemical reaction, but that the [M+CO₂-H]⁻ ions could be more accurately classified as adducts. The radical
352 scavenger experiments confirmed that the NO₂-containing ions were suppressed when the radical
353 scavenger was added to the co-solvent, suggesting that radical reactions lead to the formation of these
354 nitrated analytes. For some compounds, the artifacts had several-fold higher signals compared to the
355 molecular ion. In several cases, the usage of protic or aprotic solvent as co-solvent affected the
356 formation of the artifacts; for example, with ACN, the [M+CO₂-H]⁻ ion was not detected, but the ratio
357 of the NO₂-containing ion to molecular ion was often the highest.

358 The formation of artifacts in SFC-UV-MS analysis must be acknowledged as it can hinder the
359 assignment of the correct molecular ions when performing non-target analysis of a mixture; however,
360 the solution is simple as the UV detector can be simply turned off. On the other hand, the formation of
361 the artifacts has the potential to be used to the advantage of the SFC-MS analysis. For example, we
362 demonstrated that the formation of specific ions can give valuable structural information on the
363 analytes, increase the MS signal, or even enable the detection of some otherwise non-ionizable
364 analytes. It is also important to note that only 30 phenolic compounds or derivatives were studied here,
365 so it is not yet known what type of other compounds might exhibit artifacts or adducts in SFC-UV-MS
366 analysis.

367

368 Acknowledgements

369 This work was funded by the French National Research Agency (project BIOPOLIOL, ANR-21-
370 CE43-0026). The authors would like to thank Dr. Ida Rahu and Dr. Henrik Hupatz for fruitful
371 discussions about nitration reactions. The authors would also like to thank Florent Rouvière for
372 performing the additional radical scavenger experiments. The graphical abstract was largely created
373 with BioRender.com.

374

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