# Quantification of chlorinated paraffins by chromatography coupled to high-resolution mass spectrometry – Part A: Influence of gas chromatography and ionisation source parameters

Cherine Amoura*, Frédéric Larvor, Philippe Marchand, Bruno Le Bizec, Ronan Cariou, Emmanuelle Bichon**

Oniris, INRAE, LABERCA, F-44300, Nantes, France

<figure><img src="image_1.png" /></figure>

## HIGHLIGHTS

* PCAs were detected up to C₂₀ using the optimised GC-ECNI-HRMS method.

* Influence of GC and ECNI parameters on PCA patterns were revealed.

* Ion source temperatures above 250 °C lead to possible formation of PCOs.

* Degradation of heaviest PCAs may occur at elevated inlet source temperatures.

## GRAPHICAL ABSTRACT

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

## ARTICLE INFO

Handling editor: Leondios Leondiadis

**Keywords:**

Chlorinated paraffins
Polychlorinated alkanes
GC-ECNI-HRMS
Homologue group pattern
Quantification

## ABSTRACT

The analysis of chlorinated paraffins (CPs) has become a major analytical challenge. GC-ECNI-HRMS coupling is often used to analyse and quantify them. However, the influence of certain GC and ECNI parameters on the responses of polychlorinated *n*-alkanes (PCAs), the dominant components of CPs, has hardly been studied. In this paper, we investigated not only the influence of GC column characteristics, but also oven, GC inlet and source temperatures for simultaneous analysis of PCAs with chain-length ranging from 10 up to 20 carbon atoms (PCAs–C₁₀–₂₀). Particular attention was paid to the absolute response and PCA homologue group pattern obtained for a CP technical mixture. The optimum conditions for a wide homologue group determination were GC inlet, final gradient and ion source temperatures set at 220–240 °C, 340 °C and 200 °C. At the same time, a higher response was obtained with the Optima 5HT column compared to Optima 1 column, and with a length and film thickness of 12.5 m and 0.25 µm, respectively. The homologue group pattern of the technical mixture studied was significantly modified as a function of the source and GC inlet temperatures, film thickness and composition

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: laberca@oniris-nantes.fr (C. Amoura), emmanuelle.bichon@oniris-nantes.fr (E. Bichon).</footnote>
of the stationary phase. Here we recommend conditions that will improve the overall PCA pattern, in order to better characterise their occurrence in future environmental monitoring and exposure assessment.

# 1. Introduction

Chlorinated paraffin (CP) blends are produced industrially by reacting chlorine with certain fractions of paraffins (n-alkanes) from petroleum refining, under forced conditions (high temperatures and/or under UV/visible radiation). This non-stereospecific reaction leads to complex mixtures composed of thousands of polychlorinated n-alkanes (PCAs) isomers of general formula $C_nH_{2n+2-x}Cl_x$ (n ∈ [10,30], 40-70% Cl $w/w$) (Yuan et al., 2020). Based on the carbon chain length range in the initial bulk mixture, CPs are classified into 3 subclasses: short-chain (SCCPs, $C_{10}-C_{13}$), medium-chain (MCCPs, $C_{14}-C_{17}$) and long-chain (LCCPs, $C_{\ge 18}$) chlorinated paraffins. In the present article, we refer to Fernandes et al. (2023) for terms and abbreviations related to CPs and PCAs.

CPs have been produced since the 1930s mainly for their properties as flame retardants (rubber, paints, adhesives), plasticisers and high temperature lubricants for metallurgy. In 1969, 28,000 tons of CPs were produced in the United States, putting CP manufacturing in third place after low-molecular-weight hydrocarbons (tetrachloromethane, chloroform, etc.) and polychlorinated biphenyls (PCBs) (Zikto and Arsenault, 1974). At present, the production volume of CPs is estimated at over 2 million tons per year (De Boer, 2019), with China being the world's leading producer. This production has resulted in a significant release of CPs into the total environment, mainly from industrial activities (washing of facilities, incorrect disposal of used lubricants) (Canadian Environmental Protection Act, 1993).

With regard to their persistence in the total environment, bioamplification and biomagnification in organisms, toxicity and ability to be transported over long distances, SCCPs have been classified as Persistent Organic Pollutants (POPs) by the Stockholm Convention since May 2017 (United Nations Environment Programme (UNEP) 2017). Due to restrictions on the production and use of SCCPs, recourse to the MCCPs and LCCPs has already been reported in many applications (Glüge et al., 2018). Consequently, a shift in environmental occurrence from PCAs-$C_{10-13}$ to PCAs-$C_{14-17}$ and PCAs-$C_{\ge 18}$ has been observed (Glüge et al., 2018; Yuan et al., 2022). For example, Yuan et al. (2022) recently showed that concentrations of PCAs-$C_{\ge 18}$ are higher than those of other PCAs in 40% of the biota samples analysed. They also showed an increase in the level of PCAs-$C_{\ge 18}$ from 1995 to 2019, while during this period, the level of PCAs-$C_{10-13}$ decreased. MCCPs are currently under review for inclusion on the Stockholm Convention list and are also candidates for classification as substances of very high concern (SVHC) by the European chemical agency (ECHA).

It is worth noting that acquiring reliable data on the occurrence of PCAs in the total environment and in foodstuffs remains a challenge due to the analytical difficulties encountered in quantifying them. One of the main limitations is the use of technical mixtures as semi-quantification standards. These mixtures are characterised only by a range of carbon chain lengths and average chlorine content, while their exact composition in PCA congeners is not known. At the same time, Tomy et al. (1998) and Coelhan et al. (2000) have already demonstrated that when the chlorine level of the technical CP mixture is different from that of the sample, the quantification errors can differ by several orders of magnitude. Another significant limitation stems from the difficulty to obtain a consensual value between laboratories in the context of interlaboratory studies (ILs) (Krätschmer and Schächtele, 2019). The choice of instrument, calibration standards and data processing methods are the main factors influencing the quantification (Krätschmer and Schächtele, 2019).

Since the 1990s, PCAs have generally been analysed using gas or liquid chromatography (GC and LC, respectively) coupled to low- or

high-resolution mass spectrometry (HRMS). The use of electron capture negative ionisation (ECNI) combined to HRMS enables each homologue group of PCAs to be determined. However, GC-ECNI-(HR)MS has several limitations that need to be considered. Firstly, this approach is not suitable for PCAs-$C_{\ge 18}$ because of their low vapour pressure (Drouillard, 1996; Glüge et al., 2013; van Mourik et al., 2016). Secondly, the chlorine content of CP mixtures is overestimated using ECNI. Indeed, lower chlorinated PCAs-$Cl_{\le 5}$ are not detected. Moreover, although GC-MS is the most widely used analytical technique today, the instrumental parameters that influence PCA response and homologue group patterns are not well understood.

This study therefore examined the impact of instrumental parameters on the homologue group patterns. The influence of temperature in the ion source as well as in the GC inlet and oven was assessed on the one hand, while the properties of the chromatographic column were also investigated on the other, working on column length, film thickness and stationary phase composition. The aim of this study was to obtain the optimum response and the widest range of homologue groups detected on the GC-ECNI-HRMS system, in order to better characterise these mixtures. Similar issues were addressed for LC-HRMS coupling in a companion paper (Amoura et al., 2024).

# 2. Material and methods

## 2.1. Chemical and reagents

Dichloromethane (DCM), hexane and magnesium sulphate (Florisil) were purchased from LGC Promochem (Picograde quality, Wesel, Germany). Sulphuric acid (98%) was provided by Panreac (Barcelona, Spain). Silica gel (70/230 mesh, Fluka, Buchs, Switzerland) was purchased from Merck (Darmstadt, Germany). LCMS grade acetonitrile (ACN) was obtained from Carlo Erba (Rodano, Italy). $^{13}$C$_6$-hexachlorobenzene ($^{13}$C-HCB, Cluzeau Info Labo, Sainte-Foy-la-Grande, France) was used as a recovery standard.

CP standard mixture A was prepared in cyclohexane by combining four technical standards from Dr. Ehrenstorfer (Augsburg, Germany) at equal proportions, including two SCCP standard mixtures (51.5% Cl and 63% Cl) and two MCCP standard mixtures (42% Cl and 57% Cl). Each standard concentration was 5 ng µL$^{-1}$ in the working solution.

In order to go further in PCA analysis and to be more representative of environmentally contaminated samples, CP standard mixture B was prepared in cyclohexane by combining six technical standards from Dr. Ehrenstorfer (Augsburg, Germany) at equal proportions, including the four from mixture A plus LCCP standard mixtures ($C_{18-20}$, 36% Cl and 49% Cl). Each standard concentration was 4 ng µL$^{-1}$ in the working solution.

Comparisons of the different studied parameters were done on the absolute responses of PCAs and on their relative responses by normalising the area of each homologue group toward the global ΣPCAs.

## 2.2. Samples

Samples from four interlaboratory studies (ILs, 2017; ILs, 2018; ILs, 2019; ILs, 2021) organised by the European Union reference laboratory for halogenated persistent organic pollutants in feed and food (EURL POPs) were analysed. Overall, 5 coconut fat samples, 5 lard samples, 1 pork sausage sample and 1 infant milk sample were considered. For each sample, the preparation and analysis were performed in triplicate. A detailed description of the coconut fat and lard samples can be found in the publication of Krätschmer and Schächtele (2019). A summary is presented in Table S1.
## 2.3. Sample preparation

Samples were prepared as described by Mézière et al. (2020). Briefly, freeze-dried pork sausage and infant milk were extracted by a pressurised liquid extraction device (SpeedExtractor E-914, Büchi), using 3 cycles of 5 min at 120 °C and at 100 bar. The extraction solvent used was a mixture of toluene/acetone (7:3, v/v). For all samples, purification was performed using acidic silica gel chromatography and then deactivated Florisil.

## 2.4. Instrumentation

The analyses were carried out using a Trace GC 1310 device equipped with a TriPlus RSH autosampler and coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific, San José, CA, USA).

For the GC part, the influence of the column length (30 m, 15 m, 12.5 m and 10 m) was investigated on an Optima 5 HT column (5% phenyl-95% dimethylpolysixolane, Macherey-Nagel). The influence of the film thickness (0.25 µm vs. 0.10 µm) was assessed on an Optima 5 HT column vs. a Zebron 5-HT (5% phenyl-95% dimethylpolysixolane, Phenomenex) column, respectively. Then, the influence of the composition of the stationary phase was studied by comparing an Optima 1 column (100% dimethylpolysiloxane) with the Optima 5 HT column.

Helium was used as carrier gas with a flow rate of 1.4 mL/min. The oven program temperature began at 60 °C during 2 min. Then, the temperature increased by 50 °C/min until 325 °C or 340 °C, and was maintained during 8 min. The injector was used in the splitless surge mode at six different temperatures: 220 °C, 240 °C, 260 °C, 280 °C, 300 °C and 320 °C.

The settings of the mass spectrometer parameters are those used by Krätschmer et al. (2018) with some modifications. The transfer line was set at 340 °C. The ionisation of PCAs was carried out in ECNI. Methane was used as the reagent gas at a flow rate of 1.2 mL/min. The monitored ions were [M – Cl]⁻. The source temperature was assessed from 110 to 300 °C. Data were acquired in full scan mode over the *m/z* range 150–1100 at a resolving power set to 120,000 FWHM at *m/z* 200. Perfluorotributylamine was used to calibrate the instrument. The automatic gain control target value was set to 1 × 10⁶ ions with a maximum injection time set at 200 ms.

## 2.5. Semi-quantitative calibration method

The semi-quantification of ΣPCAs was inspired by the methods developed by Reth et al. (2005) and then by McGrath et al. (2020), with some modifications. Indeed, a double regression was realised taking into account the concentration and the chlorine content of the technical mixtures used as calibration standards. All the terms used (relative total area, total response factor) refer to the formulas which can be found in the publication of McGrath et al. (2020). See Table S2 and Fig. S1 for more details about the calibration method.

## 2.6. Post-acquisition data treatment

The GC-ECNI-HRMS raw data files were converted into the *mxXML* format using MSConvert (v3.0.22091–15385b9). Then, an automated data processing was carried out for PCAs and polychlorinated *n*-olefins (PCOs) using the in-house and open-source CP-Seeker software (v1.1) dedicated to the analysis of PCAs and related chemical classes. A detailed description has been provided by McGrath et al., (under review). Briefly, it involves signal deconvolution using the enviPat package (Loos et al., 2015) with the mass analyser setting to obtain a list of the homologue group isotopic patterns in the ranges of C₆.₃₆ and Cl₃.₃₀. Non-Gaussian peaks of spectrometric peaks above 1% of the base peak are then automatically integrated. The output comprises the sum area of homologue groups and, for identification confidence, a pattern score and an average mass deviation.

In the present work, only the [M – Cl]⁻ ions were considered. Other ions have been reported in the literature using GC-ECNI-MS, such as the nonspecific ions [Cl₂]⁻ and [HCl₂]⁻ ions (Tomy et al., 1998) or the [M – HCl]⁻ ions (McGrath et al., 2020), but they were not considered in this study. Extracted ion chromatograms were searched at ± 5 ppm. For identification, a minimum pattern score between the observed and theoretical patterns was arbitrarily set at 70%.

# 3. Results and discussion

## 3.1. Influence of the source temperature on the PCA response

Few studies highlight the influence of the ionisation source temperature on PCA analysis (Tomy et al., 1998; Gao et al., 2016; McGrath et al., 2021). Tomy et al., 1998 analysed a SCCP mixture (PCAs–C₁₀–₁₁Cl₄.₇) for ionisation source temperatures ranging from 120 °C to 220 °C. They showed that abundance of the [M – Cl]⁻, relative to the nonspecific [Cl₂]⁻ and [HCl₂]⁻ ions, decreases when the ion source temperature increases. Therefore, they recommended to work at 120 °C in order to maximise the relative abundance of the characteristic [M – Cl]⁻ ions. Those results were confirmed later on two PCA isomers (Gao et al., 2016). McGrath et al., 2021 analysed technical mixtures of SCCPs and MCCPs separately from 140 °C to 250 °C. They showed an optimal formation of the [M – Cl]⁻ ions at 170 °C. In our study, we analysed mixture B containing SCCPs, MCCPs and LCCPs at six source temperatures ranging from 110 °C to 300 °C. The analyses were performed on the Optima 5 HT column (12.5 m × 0.25 mm, 0.10 µm) with a GC inlet and an oven end temperature of 280 °C and 340 °C, respectively. The responses were compared within a single day during which the MS tuning did not vary significantly (tune values are given in Fig. S2).

The maximum abundance was observed for a source temperature of 200 °C for each PCA subclass (Table 1). The sum of each subclass decreased down to 27–78% of their maximum abundance for the two extreme ion source temperatures. In general, in the studies analysing PCAs–C₁₀–₁₃ and PCAs–C₁₄–₁₇ by GC-ECNI-MS, the ion source temperature is set at 150 °C.

The total ion chromatograms (TICs) obtained at ion source temperatures of 110 °C and 300 °C for the same mixture did not superimpose, revealing differential behaviours between PCAs (Fig. 1a). Indeed, the left part of the humps was relatively more intense at 110 °C while the right part was relatively more intense at 300 °C. The retention time window at 5.2–5.4 min corresponded to the elution of the PCAs–C₁₀–₁₁Cl₆.₇ while the one at 6.5–7.8 min corresponded to the elution of PCAs–C₁₈–₂₀ and higher chlorinated PCAs–Cl≥₁₀.

In more details, the relative abundance of PCAs–C₁₀–₁₂Cl₅.₈ were significantly higher at 110 °C (Fig. 2) whereas the relative abundance of PCAs–C₁₄–₂₀ was significantly higher at a source temperature of 300 °C. The detailed impact of the ion source temperature on each homologue group response is reflected in a heat map (Tables S3 and S4). It confirms that shorter chain lengths and lower chlorinated PCAs (C₁₀–₁₄Cl₅, C₁₁–₁₃Cl₆) are better revealed at 110 °C, relative to the respective maximum source temperature value (200 °C). In contrast, higher chlorinated PCAs (C₁₉Cl₈, C₁₇–₁₉Cl₉, C₁₆–₁₉Cl₁₀, C₁₅–₁₉Cl₁₁) were better revealed at higher ionisation source temperature (above 250 °C). The other PCAs were better characterised at 200 °C. Indeed, working at a low source temperature enhanced the relative abundance of short-chain and lower chlorinated PCAs to the detriment of the heavier ones, whereas the opposite tendency was observed at a higher source temperature. These results are consistent with the ones published by Tomy et al., 1998 with a higher abundance of the [M – Cl]⁻ ions reported at 120 °C for lower chlorinated PCAs–C₁₀–₁₁Cl≤₆. However, further comparison with their results will not be possible due to the difference in the calculated relative abundance of the [M – Cl]⁻ ions and also because the other instrumental parameters are slightly different between our studies.

The decrease in the relative abundance of the short and lower chlorinated PCAs at 300 °C can be explained by an increase in the
<caption>**Table 1**</caption>

Areas (× 10⁶) obtained for the different PCA subclasses according to each tested condition. The gain factor was determined for the optimal condition. For the column length parameter, the gain factor was determined compared to the 15 m values.

<table><thead><tr><th></th><th>ΣPCAs-C<sub>10</sub><br>13</th><th>ΣPCAs-C<sub>14</sub><br>17</th><th>ΣPCAs-C<sub>18</sub><br>20</th><th>ΣPCAs-C<sub>10</sub><br>20</th></tr></thead><tbody><tr><td><strong>Source temperature</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>(°C)</td><td></td><td></td><td></td><td></td></tr><tr><td>110</td><td>420</td><td>130</td><td>7.4</td><td>557</td></tr><tr><td>150</td><td>540</td><td>190</td><td>12</td><td>742</td></tr><tr><td>180</td><td>460</td><td>180</td><td>12</td><td>652</td></tr><tr><td>200</td><td>690</td><td>300</td><td>27</td><td>1017</td></tr><tr><td>250</td><td>470</td><td>250</td><td>26</td><td>746</td></tr><tr><td>300</td><td>330</td><td>180</td><td>21</td><td>531</td></tr><tr><td><strong>Inlet temperature</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>(°C)</td><td></td><td></td><td></td><td></td></tr><tr><td>220</td><td>74</td><td>35</td><td>4.2</td><td>113</td></tr><tr><td>240</td><td>72</td><td>33</td><td>4.1</td><td>109</td></tr><tr><td>260</td><td>68</td><td>30</td><td>3.5</td><td>102</td></tr><tr><td>280</td><td>62</td><td>26</td><td>2.7</td><td>90</td></tr><tr><td>300</td><td>54</td><td>21</td><td>2.0</td><td>77</td></tr><tr><td>320</td><td>55</td><td>23</td><td>2.3</td><td>80</td></tr><tr><td><strong>Oven end temperature (°C)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>325</td><td>580</td><td>250</td><td>210</td><td>851</td></tr><tr><td>340</td><td>690<br>(+19%)</td><td>300<br>(+20%)</td><td>270<br>(+29%)</td><td>102<br>(+20%)</td></tr><tr><td><strong>Column film thickness (μm)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>0.25</td><td>27</td><td>10</td><td>0.7</td><td>38</td></tr><tr><td>0.1</td><td>8.7<br>(-67%)</td><td>3.2<br>(-70%)</td><td>0.3 (-70%)</td><td>12.3<br>(-68%)</td></tr><tr><td><strong>Column length (m)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>30</td><td>133</td><td>38</td><td>/</td><td>171</td></tr><tr><td>15</td><td>445<br>(+234%)</td><td>117<br>(+207%)</td><td>/</td><td>562<br>(+228%)</td></tr><tr><td><strong>Column length (m)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>15</td><td>420</td><td>160</td><td>10</td><td>590</td></tr><tr><td>12.5</td><td>690<br>(+64%)</td><td>300<br>(+88%)</td><td>27<br>(+170%)</td><td>1002<br>(+72%)</td></tr><tr><td>10</td><td>560</td><td>220</td><td>16</td><td>796</td></tr><tr><td><strong>Column chemistry</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Optima 1</td><td>396</td><td>114</td><td>4.0</td><td>514</td></tr><tr><td>Optima 5 HT</td><td>422 (+7%)</td><td>156<br>(+37%)</td><td>10.1<br>(+153%)</td><td>588<br>(+14%)</td></tr></tbody></table>

fragmentation of corresponding ions at a higher source temperature. PCA degradation products have already been observed when subjected to high temperatures (Bergman et al., 1984; Schinkel et al., 2017, 2018; Xin et al., 2019). For example, Schinkel et al., 2018 observed the formation of PCOs by in-source fragmentation of PCAs. In the present study, we observed an increase in the proportion of PCOs above 250 °C (See Fig. S3a). As the PCOs are most likely produced in the ionisation source, this could partly explain the shift to higher retention time of the PCA chromatographic hump observed at 300 °C (Fig. 1a).

To conclude on the source temperature, for the global determination of PCAs in a single injection (PCAs–C10–17 or PCAs–C10–20), we suggest to set the ion source temperature at 200 °C, as the optimal value. This source temperature is higher than the ones set in other studies for the determination of PCAs–C10–17 (150 °C: Tomasko et al., 2021; McGrath et al., 2020; 180 °C: Krätschmer et al., 2018). In terms of relative response, the source temperature influences the observed PCA pattern. Choosing a lower source temperature (110–150 °C) will better reveal PCAs–C10–13 and PCAs–Cl<sub>≤6</sub>) compared to the other PCAs. On the other hand, working at a higher source temperature (250–300 °C) will better reveal PCAs–C18–20 and PCAs–Cl<sub>≥10</sub> on an overall PCA pattern. Therefore, special attention should be paid to this parameter when PCA patterns are compared between studies.

## 3.2. Influence of the GC inlet temperature on the PCA response

The influence of the GC inlet temperature (from 220 °C to 320 °C) on the volatilisation was studied, in particular for PCAs–C18–20 and higher chlorinated PCAs.

The sensitivity and the homologue group patterns were assessed on mixture B injected on the Optima 5 HT column (12.5 m × 0.25 mm, 0.10 µm) with source and final oven temperatures of 200 °C and 340 °C, respectively. The influence of the GC inlet temperature was illustrated with PCAs–C12 (most abundant C-chain length in the mixture) for readability purposes (Fig. 3), as these groups appeared representative of other PCAs (Fig. S4).

As displayed in Fig. 3 and Table 1, the highest response was globally obtained for a GC inlet temperature of 220–240 °C.

From the relative abundance perspective, lower chlorinated PCAs seemed to be slightly better revealed at elevated GC inlet temperature (320 °C) (Fig. S4b; Tables S5 and S6). For higher chlorinated PCAs–Cl<sub>≥8</sub>, the optimal relative response appeared to be at a relatively low GC inlet temperature (220 °C).

As the relative abundance of longer and higher chlorinated PCAs was

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Total ion chromatograms (TIC) obtained by GC-ECNI-HRMS for mixture B a) at source temperatures of 110 °C (green) or 300 °C (black), after separation according to the same parameters and b) with film thicknesses of 0.1 µm (black) or 0.25 µm (red). Column geometry: 12.5 m × 0.25 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Influence of the source temperature on the relative response of homologue groups of a) PCAs–C₁₀–₁₃, b) PCAs–C₁₄–₁₇ and c) PCAs–C₁₈–₂₀ (mixture B; n = 1). Each relative response was normalised towards the global ΣPCAs.</figcaption></figure>

decreasing when the GC inlet temperature increased, we hypothesised that the heaviest PCAs were degraded at a higher injection temperature, which was already reported in the literature. Indeed, Schinkel et al. (2018) proposed that PCOs could be formed in the GC inlet or in the column. In this study, we detected PCOs as impurities in mixture B. However, their relative abundance did not increase when the temperature of the GC inlet increased (Fig. S3b), so our hypothesis was rejected.

Finally, we do not recommend increasing the GC inlet temperature for PCA analysis. For the global determination of PCAs in a single injection (PCAs–C₁₀–₁₇ or PCAs–C₁₀–₂₀), we therefore suggest to set the GC inlet temperature at 220–240 °C. This optimal value is lower than the one set in the majority of studies dealing with the analysis of PCAs by GC-MS (280 °C: Krätschmer et al., 2018; Xu et al., 2019). The gain in sensitivity, working at 220 °C GC inlet temperature was +19%, +35% and +56% for the ΣPCAs–C₁₀–₁₃, ΣPCAs–C₁₄–₁₇ and the ΣPCAs–C₁₈–₂₀, respectively, compared to a GC inlet temperature set at 280 °C (Table 1). In terms of relative response, the GC inlet temperature also influences the observed homologue group pattern. A higher GC inlet temperature

better reveals lower chlorinated PCAs–C₁₀–₁₂ whereas a lower GC inlet temperature better reveals the longer and higher chlorinated PCAs. Therefore, as for the source temperature, this setting of this parameter could influence an observed PCA pattern.

### 3.3. Influence of the final oven temperature on the homologue group responses

Limitations regarding the elution of the heaviest PCAs, in particular PCAs–C₁₈–₂₀, from the GC column are expected, because of their relatively low volatility. The oven final gradient temperature appears as a key parameter for improving the detection of the latter, as described hereafter.

The influence of the oven final temperature (325 °C vs. 340 °C) on the sensitivity and the PCA pattern was assessed based on mixture B separation using the Optima 5 HT column (12.5 m × 0.25 mm, 0.10 µm) with GC inlet and source temperatures of 280 °C and 200 °C, respectively. The abundances obtained for the ΣPCA–C₁₀–₁₃, ΣPCA–C₁₄–₁₇ and ΣPCA-
<figure><img src="image_5.png" /><figcaption>Fig. 3. Influence of the GC inlet temperature on the response of PCAs–C₁₂ (mixture B; n = 3).</figcaption></figure>

C₁₈-₂₀ at each oven final temperature are given in Table 1. An incidence was expected on the heaviest PCAs, but a similar tendency was observed for the other homologue groups (Fig. S5). As displayed, the maximal abundances were obtained for an oven final temperature of 340 °C. The sensitivity was increased by around 20% for ΣPCA-C₁₀-₁₃ and ΣPCA-C₁₄-₁₇, and by around 28% for ΣPCA-C₁₈-₂₀ compared to the abundances obtained at 325 °C (Table 1). Therefore, increasing the oven final temperature led to a better response but, according to the relative responses, it did not influence the homologue group pattern (Fig. S5b).

Working at 340 °C seems to improve the detection of PCAs, in particular for ultra-trace analysis purposes. Moreover, increasing the oven temperature does not seem to lead to the degradation of PCAs. Indeed, PCOs proportions remained similar at both temperatures (Fig. S3c).

## 3.4. Comparison of chromatographic columns

### 3.4.1. Influence of column film thickness

The film thickness is a column parameter that has been studied for other families of flame retardants such as polybrominated diphenyl ethers (PBDEs). Björklund et al. (2004) showed that a higher film thickness (0.25 µm vs. 0.10 µm) led to a decrease in the relative area of the hepta-to deca-BDEs. Therefore, the objective of this study was to determine whether, as for PBDEs, the film thickness has an influence on the response and the relative areas of homologue groups. To this end, mixture B was analysed in triplicate using two 5% phenyl-95% dimethylpolysiloxane columns (Optima 5-HT and Zebron 5-HT) with distinct film thicknesses (0.10 µm vs. 0.25 µm, respectively). The assays were performed with the following instrumental conditions: GC inlet, oven final and source temperatures of 280 °C, 340 °C and 200 °C, respectively.

In terms of retention, it appeared that the film thickness plays an important role (Fig. 1b). Indeed, PCAs present a shorter retention time on the 0.10 µm film thickness column (5.5–7.5 min) compared to the 0.25 µm one (6–8 min).

In general, a better response sensitivity was obtained with the 0.25 µm film thickness (+68% for the ΣPCAs, Table 1), except for the higher chlorinated PCAs–Cl≥₁₀ for which a higher abundance was observed with the 0.1 µm column film thickness (Fig. S6 and Table S7).

According to Björklund et al. (2004), a column with a thicker stationary phase increases the elution temperature of PBDEs. Therefore, PBDEs stay longer and at a higher temperature in the column, leading to a decrease of the relative areas of highly brominated PBDEs. It seemed to be also the case for the higher chlorinated homologue groups (C₁₈-₁₉Cl₁₀, C₁₅-₁₆Cl₁₁, C₁₃Cl₁₂) which were better revealed with the 0.1

µm column thickness (Fig. S6). Indeed, as shown in section 3.2, PCAs–C₁₈-₂₀ are more temperature sensitive. Therefore, working with a thinner stationary phase appeared to be more adequate for these PCA chain lengths. For this reason, a column film thickness of 0.1 µm has been selected for the next development steps.

### 3.4.2. Influence of the column length

#### 3.4.2.1. 30 m vs. 15 m. Coelhan (1999) showed that using a shorter column can lead to an increase in the absolute response of PCAs by a factor of 100. Moreover, they obtained a separation of a 63% chlorine undecane mixture using a 0.65 m as effective as with a 30 m column length, due to thinnest peak width. Later, McGrath et al., 2021 compared a 15 vs. a 30 m DB-5MS column with GC-ECNI-LRMS, and they did not observe better separation of the co-eluting PCAs-C₁₀-₁₇ using the longer column. They also concluded that the shorter column increases the PCA response. In a first assay, we intended to confirm these results using GC-ECNI-HRMS. Therefore, we evaluated the impact of the column length (30 m vs. 15 m) on the absolute response and on the homologue group patterns of PCAS-C₁₀-₁₇. For this, mixture A was analysed on Optima 5HT columns (0.10 µm film thickness) in the following conditions: GC inlet, oven final gradient and source temperatures of 280 °C, 325 °C and 180 °C, respectively.

The impact of the column length on the absolute response and on the pattern is shown in Fig. 4. The highest sensitivities were reached with the 15 m length column (Table 1 and Fig. S7a), which led to an increase of 234%, 207% and 228% for ΣPCAs-C₁₀-₁₃, ΣPCAs-C₁₄-₁₇ and ΣPCAs-C₁₀-₂₀, respectively (see Table 1). No influence was observed on the homologue group patterns (Fig. S7b). These results are consistent with those published by Coelhan (1999) and McGrath et al. (2021).

#### 3.4.2.2. 15 m vs. shorter. To further investigate the column length influence on the PCA responses, two shorter column lengths (12.5 m and 10 m) were tested with mixture B in order to better reflect environmental and biota contamination patterns. The analysis was carried out on the Optima 5HT column (0.1 µm film thickness) in the same conditions (GC inlet, oven final gradient and source temperatures of 280 °C, 340 °C and 200 °C, respectively).

The highest sensitivity was obtained with the 12.5 m column (Fig. 5; Fig. S8 for a complete illustration). Moreover, using the 12.5 m column seemed to better reveal the PCAs–C₁₆-₂₀ relative to the global ΣPCAs (Fig. 5).

The use of 10 m column led to a decrease in the response of all homologue groups detected. This is likely a limit of the instrument because it was difficult to stabilise the purge flow due to the possible backpressure exerted by the column. Moreover, using short columns with a 0.25 mm diameter can destabilise the void into the MS. Because of these technical limits, the investigation on even shorter columns was not performed.

In conclusion, the length of the chromatographic column strongly influences the detection sensitivity but not the homologue group patterns (except for the higher chlorinated PCAs–C₁₆-₁₈). Therefore, the choice of a 12.5 m column seems to be the most appropriate for the analysis of PCAs under optimal conditions for the considered instrument.

### 3.4.3. Influence of the stationary phase composition on the determination of PCAs

The majority of studies dealing with the analysis of PCAs involve a 5 MS GC column (5% phenyl-95% dimethylpolysiloxane). Additional studies used columns containing higher rates of phenyl groups like a 35MS (Zencak et al., 2004; Reth et al., 2005). In GC × GC, the coupling of a DB-1 (100% dimethylpolysiloxane) with a 007-65HT column (65% phenyl methylpolysiloxane) gave the clearest separation of homologue groups (Korytar et al., 2005), showing the influence of the GC column
<figure><img src="image_6.png" /><figcaption>Fig. 4. Response of PCA homologue groups obtained according to the column length: 30 m vs. 15 m (mixture A; n = 3).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. Response of PCA homologue groups obtained according to the column length: 15 m vs. 12.5 m vs. 10 m (mixture B; n = 1).</figcaption></figure>

polarity on the separation of PCAs.

Regarding PCA isomer retention, Hammer et al. (2021) demonstrated that retention index (RI) on GC polar columns are greater than on non-polar ones, highlighting the polar interaction abilities of PCAs. In our study, two stationary phase chemistries with little polar and non-polar interactions were compared, i.e. the Optima 5HT and Optima 1 columns. On the Optima 5HT column, phenyl groups have been grafted onto the stationary phase of the dimethylpolysiloxane polymer, in order to enhance the column polarity, whereas Optima 1 has been built with PDMS only. The analysis was done on mixture B using GC inlet, oven final gradient and source temperatures of 280 °C, 325 °C and 180 °C, respectively.

Looking at the TICs obtained in each condition, no retention

improvement was observed with the Optima 5HT column compared to the Optima 1 (Fig. S9), highlighting no stronger interaction of this column with PCAs.

Concerning the responses, a global enhancement was observed on Optima 5HT, with a non-significant improvement for $C_{\ge 12}$ chain lengths (Fig. S10). The absolute abundance increased by around 7%, 37% and 143% for the ΣPCAs-$C_{10-13}$, ΣPCAs-$C_{14-17}$ and ΣPCAs-$C_{18-20}$, respectively (Table 1).

The choice of the stationary phase influences the PCA homologue group patterns. While PCAs–$C_{10-12}$ were better revealed using the Optima 1 column, a slight increase in the relative areas of PCAs–$C_{14-20}$ was observed with the Optima 5HT column. Following these observations, the Optima 5HT column was selected for quantification purposes, in
accordance with the majority of published methods for PCA analysis.

## 3.5. Recommendations for PCA analysis: summary of the findings

Various instrumental and chromatographic parameters were studied in order to enhance the response of PCAs and their detection range. Concerning the instrumental parameters, the best compromise for the simultaneous analysis of all homologue groups was obtained with GC inlet, oven final gradient and source temperatures of 220 °C, 340 °C and 200 °C, respectively. For the chromatographic parameters, a 12.5 m column and a film thickness of 0.25 µm gave the best responses. Regarding the polarity, the more polar column, Optima 5HT, also gave the best response. The setting of these parameters appeared to be the most appropriate in the case of PCA trace analysis. However, the source temperature, the column length, the film thickness and the polarity of the stationary phase could be selected more appropriately whenever the specific detection of a subclass of interest is required. Indeed, this study demonstrates that those parameters significantly influence the homologue group pattern. Therefore, for PCAs–C₁₀–₁₃ only, a source temperature between 110 °C and 150 °C seems to be a better compromise. For PCAs–C₁₄–₁₇ and PCAs–C₁₈–₂₀, better compromises seem to be 200 °C and 250 °C, respectively. In general, for PCAs–C₁₄–₂₀, a shorter column length (12.5 m) and a thinner stationary phase (0.1 µm) will better reveal these PCA subclasses. For the detection of PCAs–C₁₀–₁₃, these parameters seem to have less influence.

Following these observations, the choice of the 12.5 m × 0.25 mm, 0.25 µm column will give the highest sensitivity in PCA analysis. This column will be interesting for routine analysis of PCAs–C₁₀–₁₇. However, if the objective is to broaden the detection range of PCAs, with a particular focus on PCAs–C₁₈–₂₀ and/or higher chlorinated PCAs–Cl≥₁₀, a shorter residence time in the column is recommended. The choice of the 12.5 m × 0.25 mm, 0.10 µm column will be thus more interesting. This last one was then chosen in our study for the semi-quantification of PCAs in samples from ILs.

## 3.6. Investigation of ΣPCAs-C₁₀–₁₃ and ΣPCAs-C₁₄–₁₇ in ILs samples

Quantification of PCAs was carried out on samples from interlaboratory studies organised by the EURL. The optimised GC-ECNI-HRMS conditions were used to quantitate ILs samples. The results were compared with the assigned values determined by the organiser. Only ΣPCAs-C₁₀–₁₃ and ΣPCAs-C₁₄–₁₇ values were reported here, as ΣPCAs-C₁₈–₂₀ were not considered in these ILs in terms of quantification. However, their proportions in all samples, relative to the global ΣPCA areas, was determined.

### 3.6.1. PCA homologue group patterns

The obtained homologue group patterns of the enriched samples were similar to those already published in other studies for technical mixtures (Bogdal et al., 2015; Yuan et al., 2017; Li et al., 2018). Indeed, there was a Gaussian-like distribution of the chlorine content for a given chain length, with a dominance of PCAs–Cl₇.₉ (Figs. S11, S12 and S13). Looking at the pork sausage sample, which was naturally contaminated, the general homologue group pattern looked different compared to fortified samples. Indeed, the chlorine distribution for a given chain length was more expanded (e.g., PCAs–C₁₂Cl₅–₁₃) with a lesser Gaussian-like distribution of the homologue groups (Fig. S14). These differences can be due to the different stages that can affect a sample. Indeed, it has already been shown that preferential bioaccumulation and biomagnification lead to PCA pattern modifications (Mézière et al., 2021).

The presence of PCAs–C₁₈–₂₀Cl₇.₉ was also observed in all the enriched samples (coconut oil, lard and infant milk) at proportions <4%. A higher proportion of PCAs–C₁₈–₂₀ was found in the naturally contaminated sample (pork sausage) with PCAs–C₁₈Cl₉ representing around 18% of the overall proportion of PCAs (Fig. S14). Although

PCAs–C≥₁₈ are hardly detectable by GC-based couplings, the developed method allows detecting PCAs up to C₂₀.

### 3.6.2. Quantification of ILs samples

In this part, the goal was to apply the optimised parameters to the quantification of PCAs in food matrices from ILs studies. Calibration curves were constructed to integrate all enriched samples, as the enrichment concentrations were known. For the enriched samples (coconut fat, lard), the assigned values correspond to the amount of fortification. For the fortified infant milk and the naturally contaminated pork sausage sample, the assigned value corresponds to the robust mean of the participants. EURL results evaluation was based on a relative standard deviation (RSD) defined as 25% and a calculated Z-score value for proficiency assessment. To fall into the acceptability criteria, the absolute value of the Z-score should be equal or below 2. For values between 2 and 3, the results are defined as questionable. Then, for values above 3, the results are defined as unsatisfactory (Krätschmer and Schächtele, 2019). The results are shown in Table 2.

First, the concentrations of ΣPCAs-C₁₀–₁₃ determined in the lard and the infant milk samples was consistent with the values assigned by the EURL (|Z-score| ≤ 2, Fig. S15). In general, this was also the case for the other laboratories, which quantified correctly the ΣPCAs-C₁₀–₁₃, with a tendency to underestimate in the case of the lard sample (Figs. S15a to S15d). For the infant milk sample, only 3 results have been considered to calculate the robust mean, therefore the assigned value of ΣPCAs-C₁₀–₁₃ was seen as provisional. The calculated value fell into the acceptability criteria and was very close to the assigned value, as for the laboratories number 4 and 7 (Fig. S15e). However, interpretation remains difficult due to the very low number of results (n = 3). In the case of ΣPCAs-C₁₄–₁₇, the concentration was in general overestimated compared to the assigned value and compared to the other participants for the two fortified lard samples (Fig. S16). For the infant milk sample, the results fell outside the calibration range for ΣPCAs-C₁₄–₁₇, so that no quantification was possible.

Concerning the pork sausage sample, the chlorine content of PCAs–C₁₀–₁₃ was outside the chlorine content calibration range. Therefore, the quantification of ΣPCAs-C₁₀–₁₃ was not possible for this sample. For the ΣPCAs-C₁₄–₁₇, the calculated value fell within the acceptability criteria (|Z-score| ≤ 2, Fig. S16c). However, interpretations remain difficult due to the low number and wide distribution of results (n = 7).

Last, all enriched coconut fat samples fell outside the concentration range (1–10 ng µL⁻¹) for respective fortification sub-groups (Table 2). The concentrations of ΣPCAs-C₁₀–₁₃ and ΣPCAs-C₁₄–₁₇ in the enriched coconut fat samples were overestimated in the majority of cases (>10 ng µL⁻¹, except for ΣPCAs-C₁₄–₁₇ in sample CFE at < 1 ng µL⁻¹). Therefore, it was not possible to determine the level of PCA subclasses in those samples.

According to Krätschmer and Schächtele (2019), ILs results are mainly influenced by the choice of the instrument, the data processing method and/or the choice of calibration standards. However, because all laboratories included in these ILs studies used their appropriate analytical method, highlighting which analytical part influences the most the results was not possible. However, the choice of the recovery standard ¹³C-HCB (correction of analytical deviations) was mentioned as being unsuitable for matrix effects correction. Indeed, a significant increase in the ¹³C-HCB area was observed in samples containing matrices compared to the calibration standards, highlighting ion enhancement. The influence of some internal and recovery standards for quantification of ΣPCAs-C₁₀–₁₃ and ΣPCAs-C₁₄–₁₇ has already been published McGrath et al. (2021). For example, they showed that using ε-hexachlorocyclohexane as recovery standard, they observed an overestimation of ΣPCAs-C₁₄–₁₇ for the EURL samples while the quantification of ΣPCAs-C₁₀–₁₃ fell within the acceptability criteria. The same observation was made for the lard and the infant milk samples in our study using ¹³C-HCB. For the pork sausage sample, the determination of ΣPCAs-C₁₄–₁₇ fell into the acceptability criteria. In that case, a possible
<caption>**Table 2**</caption>

Average chlorine content (%), concentrations of ΣPCAs-C₁₀-13 and ΣPCAs-C₁₄-17, standard deviation (SD) and associated mean Z-scores from analysis of EURL ILs samples, using the optimised parameters (except for the inlet temperature, which was set to 280 °C).

<table><thead><tr><th rowspan="2">EURL sample</th><th rowspan="2">Matrix</th><th colspan="3">ΣPCAs-C<sub>10-13</sub></th><th colspan="3">ΣPCAs-C<sub>14-17</sub></th></tr><tr><th>Average chlorine content (%) ± SD</th><th>Concentration in ng g<sup>-1</sup>lipids ± SD (consensus value)</th><th>Z-score (σ = 25%)</th><th>Average chlorine content (%) ± SD</th><th>Concentration in ng g<sup>-1</sup> lipids ± SD (consensus value)</th><th>Z-score (σ = 25%)</th></tr></thead><tbody><tr><td>CFA</td><td rowspan="5">Coconut fat</td><td>63.2 ± 0.4</td><td>25.8 ± 6.2</td><td>/</td><td>58.0 ± 0.3</td><td>34.8 ± 3.7</td><td>/</td></tr><tr><td>CFB</td><td>62.5 ± 0.4</td><td>/</td><td>/</td><td>59.4 ± 0.2</td><td>49.4 ± 23.3</td><td>/</td></tr><tr><td>CFC</td><td>62.9 ± 0.04</td><td>39.8 ± 10.0</td><td>/</td><td>60.4 ± 0.01</td><td>/</td><td>/</td></tr><tr><td>CFD</td><td>62.5 ± 0.04</td><td>/</td><td>/</td><td>60.4 ± 0.03</td><td>/</td><td>/</td></tr><tr><td>CFE</td><td>67.3 ± 3.0</td><td>/</td><td>/</td><td>60.5 ± 0.1</td><td>/</td><td>/</td></tr><tr><td>LAA</td><td rowspan="5">Lard</td><td>68.7 ± 0.5</td><td>/</td><td>/</td><td>59.6 ± 0.4</td><td>/</td><td>/</td></tr><tr><td>LAB</td><td>64.6 ± 0.1</td><td>53 ± 9 (69)</td><td>-0.93</td><td>58.9 ± 0.1</td><td>153 ± 46 (56)</td><td>6.9</td></tr><tr><td>LAC</td><td>64.4 ± 0.1</td><td>65 ± 12 (67)</td><td>-0.14</td><td>58.9 ± 0.03</td><td>/</td><td>/</td></tr><tr><td>LAD</td><td>65.5 ± 0.2</td><td>21 ± 4 (31)</td><td>-1.2</td><td>59.7 ± 1.1</td><td>/</td><td>/</td></tr><tr><td>LAE</td><td>64.0 ± 0.1</td><td>127 ± 27 (149)</td><td>-0.57</td><td>58.9 ± 0.1</td><td>177 ± 40 (121)</td><td>1.8</td></tr><tr><td>1905-PO</td><td rowspan="2">Pork sausage</td><td>68</td><td>/</td><td>/</td><td>59</td><td>26.22 (48.4)</td><td>-1.8</td></tr><tr><td>IFB</td><td>67.2 ± 0.02</td><td>124 ± 7 (121)</td><td>0.1</td><td>60.5 ± 0.1</td><td>/</td><td>/</td></tr></tbody></table>

lesser matrix effect from this sample could explain this better quantification of ΣPCAs-C₁₄-17. Then, the analysis of the coconut fat samples with ¹³C-HCB as recovery standard showed that this molecule is not appropriate for PCA analysis. Indeed, this molecule seems to have an analytical behaviour that is not mimetic of PCAs.

Consequently, the use of another recovery standard, for example 6-MeO-BDE-157 as recommended in McGrath et al. (2021) study, could bring more accurate results for PCA quantification. More interestingly, the recently released third generation standards, the ¹³C₃-labeled standards from the Chloffin project (Chiron, Trondheim, Norway), could be a real step forward in a more accurate quantification of PCAs.

## 4. Conclusion

In this study, we compared GC-ECNI-HRMS parameters for the analysis of PCAs. In the end, the selected stationary phase was an Optima 5HT column of geometry 12.5 m × 0.25 mm, 0.10 µm. As optimised temperature values, we recommend 220–240 °C, 340 °C and 200 °C for the inlet, the final plateau in the oven and the ion source, respectively. We were also able to highlight the key parameters that influence the homologue group pattern. These parameters are the GC inlet and the ionisation source temperature, the length of the stationary column, the film thickness and the composition of the stationary phase. Consequently, in the absence of a characterised quantification standard (known proportion of each homologue group in the mixture), it is necessary to take these instrumental parameters into account when comparing quantification results. In order to improve the quantification of PCAs, further developments are required, such as the choice of a suitable internal standard for the analysis of PCAs.

## Funding

The authors acknowledge the French Ministry of Agriculture and Food, General Directorate for Food (DGAl) for its financial support.

## CRediT authorship contribution statement

**Cherine Amoura:** Conceptualization, Formal analysis, Methodology, Writing – original draft. **Frédéric Larvor:** Formal analysis. **Philippe Marchand:** Conceptualization. **Bruno Le Bizec:** Funding acquisition. **Ronan Cariou:** Conceptualization, Methodology, Project administration, Supervision, Writing – review & editing. **Emmanuelle Bichon:** Conceptualization, Methodology, Project administration, Supervision, Writing – review & editing.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Data will be made available on request.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2024.141400.

## References

* Amoura, C., Larvor, F., Marchand, P., Le Bizec, B., Cariou, R., Bichon, E., 2024. Quantification of chlorinated paraffins by chromatography coupled to high-resolution mass spectrometry - Part B: influence of liquid chromatography separation. Chemosphere (in press).

* Bergman, A., Hagman, A., Jacobsson, S., Jansson, B., Ahlman, M., 1984. Thermal degradation of polychlorinated alkanes. Chemosphere 13, 237–250. https://doi.org/10.1016/0045-6535(84)90130-9.

* Björklund, J., Tollbäck, P., Hiärne, C., Dyremark, E., Östman, C., 2004. Influence of the injection technique and the column system on gas chromatographic determination of polybrominated diphenyl ethers. J. Chromatogr. A 1041, 201–210. https://doi.org/10.1016/j.chroma.2004.04.025.

* Bogdal, C., Alsberg, T., Diefenbacher, P.S., MacLeod, M., Berger, U., 2015. Fast quantification of chlorinated paraffins in environmental samples by direct injection high-resolution mass spectrometry with pattern deconvolution. Anal. Chem. 87, 2852–2860. https://doi.org/10.1021/ac504444d.

* Canadian Environmental Protection Act, 1993. Chlorinated Paraffins (Priority Substances List Assessment Report).

* Coelhan, M., 1999. Determination of short-chain polychlorinated paraffins in fish samples by short-column GC/ECNI-MS. Anal. Chem. 71, 4498–4505. https://doi.org/10.1021/ac9904359.

* Coelhan, M., Saraci, M., Parlar, H., 2000. A comparative study of polychlorinated alkanes as standards for the determination of C10–C13 polychlorinated paraffines in fish samples. Chemosphere 40, 685–689. https://doi.org/10.1016/S0045-6535(99)00444-0.

* De Boer, J., 2019. Current status of analytical methods for chlorinated paraffins. In: Of the International Symposium on Halogenated Persistent Organic Pollutants, 39th Edition. Kyoto, Japan.

* Drouillard, K.G., 1996. Physico-Chemical Property Determinations of Chlorinated N-Alkanes (C10 to C12). Parameters for Estimation of the Environmental Fate of Chlorinated N-Paraffins. M.Sc. Thesis. University of Manitoba.

* Fernandes, A.R., Krätschmer, K., McGrath, T.J., Yuan, B., Brandsma, S., Brits, M., Cariou, R., Letcher, R.J., Mueller, J., Muir, D., Vetter, W., Wang, T., Yu, G., Bergman, Å., 2023. Recommended terms and abbreviations for polychlorinated alkanes (PCAs) as the predominant component of chlorinated paraffins (CPs). Trac-Trend Anal Chem 169, 117363. https://doi.org/10.1016/j.trac.2023.117363.

* Gao, W., Wu, J., Wang, Y., Jiang, G., 2016. Quantification of short- and medium-chain chlorinated paraffins in environmental samples by gas chromatography quadrupole
time-of-flight mass spectrometry. J. Chromatogr. A 1452, 98-106. https://doi.org/10.1016/j.chroma.2016.04.081.

Glüge, J., Bogdal, C., Scheringer, M., Buser, A.M., Hungerbühler, K., 2013. Calculation of physicochemical properties for short- and medium-chain chlorinated paraffins. J. Phys. Chem. Ref. Data 42, 023103. https://doi.org/10.1063/1.4802693.

Glüge, J., Schinkel, L., Hungerbühler, K., Cariou, R., Bogdal, C., 2018. Environmental risks of medium-chain chlorinated paraffins (MCCPS): a review. Environ. Sci. Technol. 52, 6743-6760. https://doi.org/10.1021/acs.est.7b06459.

Hammer, J., Matsukami, H., Endo, S., 2021. Congener-specific partition properties of chlorinated paraffins evaluated with COSMOtherm and gas chromatographic retention indices. Sci. Rep. 11, 4426. https://doi.org/10.1038/s41598-021-84040-z.

Korytar, P., Parera, J., Leonards, P.E., Santos, F.J., de Boer, J., Brinkman, U.A., 2005. Characterization of polychlorinated n-alkanes using comprehensive two-dimensional gas chromatography-electron-capture negative ionisation time-of-flight mass spectrometry. J. Chromatogr. A 1086, 71-82. https://doi.org/10.1016/j.chroma.2005.05.003.

Krätschmer, K., Cojocariu, C., Schächtele, A., Malisch, R., Vetter, W., 2018. Chlorinated paraffin analysis by gas chromatography Orbitrap high-resolution mass spectrometry: method performance, investigation of possible interferences and analysis of fish samples. J. Chromatogr. A 1539, 53-61. https://doi.org/10.1016/j.chroma.2018.01.034.

Krätschmer, K., Schächtele, A., 2019. Interlaboratory studies on chlorinated paraffins: evaluation of different methods for food matrices. Chemosphere 234, 252-259. https://doi.org/10.1016/j.chemosphere.2019.06.022.

Li, T., Gao, S., Ben, Y., Zhang, H., Kang, Q., Wan, Y., 2018. Screening of chlorinated paraffins and unsaturated analogues in commercial mixtures: confirmation of their occurrences in the atmosphere. Environ. Sci. Technol. 52, 1862-1870. https://doi.org/10.1021/acs.est.7b04761.

Loos, M., Gerber, C., Corona, F., Hollender, J., Singer, H., 2015. Accelerated isotope fine structure calculation using pruned transition trees. Anal. Chem. 87, 5738-5744. https://doi.org/10.1021/acs.analchem.5b00941.

McGrath, T.J., Poma, G., Bombeke, J., Covaci, A., 2020. A simplified screening method for short- and medium-chain chlorinated paraffins in food by gas chromatography-low resolution mass spectrometry. J. Chromatogr. A 1631, 461574. https://doi.org/10.1016/j.chroma.2020.461574.

McGrath, T.J., Poma, G., Bombeke, J., Limonier, F., Van Hoeck, E., Joly, L., Covaci, A., 2021. Optimization and validation of an analytical method for the quantification of short- and medium-chained chlorinated paraffins in food by gas chromatography-mass spectrometry. Food Control 119, 107463. https://doi.org/10.1016/j.foodcont.2020.107463.

McGrath, T. J., Saint-Vanne, J., Hutinet, S., Vetter, W., Poma, G., Fujii, Y., Dodson, R., Johnson-Restrepo, B., Muenhor, D., Le Bizec, B., Dervilly, G., Covaci, A. and Cariou, R., Detection of Bromochloro Alkanes in Indoor Dust Using a Novel CP-Seeker Data Integration Tool. ..

Mézière, M., Cariou, R., Larvor, F., Bichon, E., Guitton, Y., Marchand, P., Dervilly, G., Le Bizec, B., 2020. Optimized characterization of short-, medium, and long-chain chlorinated paraffins in liquid chromatography-high resolution mass spectrometry. J. Chromatogr. A 1619, 460927. https://doi.org/10.1016/j.chroma.2020.460927.

Mézière, M., Marchand, P., Hutinet, S., Larvor, F., Baéza, E., Le Bizec, B., Dervilly, G., Cariou, R., 2021. Transfer of short-, medium-, and long-chain chlorinated paraffins to eggs of laying hens after dietary exposure. Food Chem. 343, 128491 https://doi.org/10.1016/j.foodchem.2020.128491.

Reth, M., Zencak, Z., Oehme, M., 2005. New quantification procedure for the analysis of chlorinated paraffins using electron capture negative ionization mass spectrometry. J. Chromatogr. A 1081, 225-231. https://doi.org/10.1016/j.chroma.2005.05.061.

Schinkel, L., Lehner, S., Heeb, N.V., Lienemann, P., McNeill, K., Bogdal, C., 2017. Deconvolution of mass spectral interferences of chlorinated alkanes and their thermal degradation products: chlorinated alkenes. Anal. Chem. 89, 5923-5931. https://doi.org/10.1021/acs.analchem.7b00331.

Schinkel, L., Lehner, S., Heeb, N.V., Marchand, P., Cariou, R., McNeill, K., Bogdal, C., 2018. Dealing with strong mass interferences of chlorinated paraffins and their transformation products: an analytical guide. Trac-Trend Anal Chem 106, 116-124. https://doi.org/10.1016/j.trac.2018.07.002.

Tomasko, J., Stupak, M., Hajslova, J., Pulkrabova, J., 2021. Application of the GC-HRMS based method for monitoring of short- and medium-chain chlorinated paraffins in vegetable oils and fish. Food Chem. 355, 129640. https://doi.org/10.1016/j.foodchem.2021.129640.

Tomy, G.T., Tittlemier, S.A., Stern, G.A., Muir, D.C.G., Westmore, J.B., 1998. Effects of temperature and sample amount on the electron capture negative ion mass spectra of polychloro-n-alkanes. Chemosphere 37, 1395-1410. https://doi.org/10.1016/S0045-6535(98)00130-1.

United Nations Environment Programme (UNEP), 2017. " listing of short-chain chlorinated paraffins." from. http://chm.pops.int/Portals/0/download.aspx?d=UNEP-POPS-COP.8-SC-8-11.English.pdf.

van Mourik, L.M., Gaus, C., Leonards, P.E.G., de Boer, J., 2016. Chlorinated paraffins in the environment: a review on their production, fate, levels and trends between 2010 and 2015. Chemosphere 155, 415-428. https://doi.org/10.1016/j.chemosphere.2016.04.037.

Xin, S., Gao, W., Cao, D., Lv, K., Liu, Y., Zhao, C., Wang, Y., Jiang, G., 2019. The thermal transformation mechanism of chlorinated paraffins: an experimental and density functional theory study. J. Environ. Sci. 75, 378-387. https://doi.org/10.1016/j.jes.2018.05.022.

Xu, J., Guo, W., Wei, L., Gao, Y., Zhang, H., Zhang, Y., Sun, M., Chen, J., 2019. Validation of a HRGC-ECNI/LRMS method to monitor short-chain chlorinated paraffins in human plasma. J. Environ. Sci. (China) 75, 289-295. https://doi.org/10.1016/j.jes.2018.04.004.

Yuan, B., Bogdal, C., Berger, U., MacLeod, M., Gebbink, W.A., Alsberg, T., de Wit, C.A., 2017. Quantifying short-chain chlorinated paraffin congener groups. Environ. Sci. Technol. 51, 10633-10641. https://doi.org/10.1021/acs.est.7b02269.

Yuan, B., Lysak, D.H., Soong, R., Haddad, A., Hisatsune, A., Moser, A., Golotvin, S., Argyropoulos, D., Simpson, A.J., Muir, D.C.G., 2020. Chlorines are not evenly substituted in chlorinated paraffins: a predicted NMR pattern matching framework for isomeric discrimination in complex contaminant mixtures. Environ. Sci. Technol. Lett. 7, 496-503. https://doi.org/10.1021/acs.estlett.0c00244.

Yuan, B., Rüdel, H., de Wit, C.A., Koschorreck, J., 2022. Identifying emerging environmental concerns from long-chain chlorinated paraffins towards German ecosystems. J. Hazard Mater. 424, 127607 https://doi.org/10.1016/j.jhazmat.2021.127607.

Zencak, Z., Reth, M., Oehme, M., 2004. Determination of total polychlorinated n-alkane concentration in biota by electron ionization-MS/MS. Anal. Chem. 76, 1957-1962. https://doi.org/10.1021/ac0353381.

Zikto, V., Arsenault, E., 1974. Chlorinated Paraffins : Properties, Uses and Pollution Potential, vol. 27. Environment Canada.
Supplementary information

Quantification of chlorinated paraffins by chromatography coupled to high-resolution mass spectrometry –

Part A: influence of gas chromatography and ionisation source parameters

Cherine AMOURA, Frédéric LARVOR, Philippe MARCHAND, Bruno LE BIZEC, Ronan CARIOU, Emmanuelle BICHON

Oniris, INRAE, LABERCA, F-44300 Nantes, France
<table><caption>**Table S1.** Summary of the fortification carried out by the EURL POPs for ILs samples (Krätschmer and Schächtele, 2019). The natural contamination was assessed only for the pork sausage sample.</caption><thead><tr><th>Matrix</th><th>Sample</th><th></th></tr></thead><tbody><tr><td rowspan="6">Coconut fat</td><td>ILs - 2017</td><td>Fortification</td></tr><tr><td>CFA</td><td>Not fortified</td></tr><tr><td>CFB</td><td>SCCPs 55.5% CI – Dr. Ehrenstorfer</td></tr><tr><td>CFC</td><td>MCCPs 57% CI – Dr. Ehrenstorfer</td></tr><tr><td>CFD</td><td>SCCPs 55.5% CI and MCCPs 57% CI – Dr. Ehrenstorfer</td></tr><tr><td>CFE</td><td>SCCPs 51.5% CI and 63% CI – Dr. Ehrenstorfer</td></tr><tr><td rowspan="6">Lard</td><td>ILs - 2018</td><td>Fortification</td></tr><tr><td>LAA</td><td>Not fortified</td></tr><tr><td>LAB</td><td>PCAs-C<sub>10</sub> 61% CI; C<sub>11</sub> 59% CI; C<sub>12</sub> 60% CI; C<sub>13</sub> 60% CI</td></tr><tr><td>LAC</td><td>PCAs-C<sub>14</sub> 53% CI; C<sub>15</sub> 51% CI; C<sub>16</sub> 53% CI; C<sub>17</sub> 51% CI – Hohenheim</td></tr><tr><td>LAD</td><td>+ Aroclor 1248</td></tr><tr><td>LAE</td><td>+ TBDE-79</td></tr><tr><td rowspan="2">Pork sausage</td><td>ILs - 2019</td><td>Natural contamination</td></tr><tr><td>1905-PO</td><td>SCCPs + MCCPs</td></tr><tr><td rowspan="2">Infant milk</td><td>ILs - 2021</td><td>Fortification</td></tr><tr><td>IFB</td><td>SCCPs + MCCPs</td></tr></tbody></table>
# Semi-quantification method

## Calibration curves

The calibration curves were produced separately for SCCPs and MCCPs technical mixtures. For each of these two subclasses, twenty calibration standards were prepared. The calibration range includes four concentrations ranging from 1 to 10 ng/µL and, for each concentration, five solutions were prepared at different chlorine levels (see the detailed composition of each calibration solution in Table S2).

First, for each chlorine level mixture, linear regressions were carried out between the relative total area of ΣPCAs-C₁₀-₁₃ or ΣPCAs-C₁₄-₁₇ according to the concentration of the considered mixture. Then, a second regression was realised by taking the slope of each linear regression according to the chlorine content of the mixtures. These calibration points fit with an exponential curve with an R² > 0.98 for ΣPCAs-C₁₀-₁₃ and ΣPCAs-C₁₄-₁₇ (Figure S1).
<table><caption>**Table S2.** Summary table of concentrations used in ng µL⁻¹ for each SCCPs and MCCPs technical mixtures. Quantification based on chlorine-content calibration from (McGrath et al., 2020).</caption><thead><tr><th>SCCPs</th><th>51.5% CI</th><th>51.5% CI + 55.5% CI</th><th>55.5% CI</th><th>55.5% CI + 63% CI</th><th>63% CI</th></tr></thead><tbody><tr><td>SCCP A</td><td>1</td><td>0.5 + 0.5</td><td>1</td><td>0.5 + 0.5</td><td>1</td></tr><tr><td>SCCP B</td><td>2.5</td><td>1.25 + 1.25</td><td>2.5</td><td>1.25 + 1.25</td><td>2.5</td></tr><tr><td>SCCP C</td><td>5</td><td>2.5 + 2.5</td><td>5</td><td>2.5 + 2.5</td><td>5</td></tr><tr><td>SCCP D</td><td>10</td><td>5 + 5</td><td>10</td><td>5 + 5</td><td>10</td></tr><tr><td>SCCP E</td><td>20</td><td>10 + 10</td><td>20</td><td>10 + 10</td><td>20</td></tr></tbody><thead><tr><th>MCCPs</th><th>42% CI</th><th>42% CI + 52% CI</th><th>52% CI</th><th>52% CI + 57% CI</th><th>57% CI</th></tr></thead><tbody><tr><td>MCCP A</td><td>1</td><td>0.5 + 0.5</td><td>1</td><td>0.5 + 0.5</td><td>1</td></tr><tr><td>MCCP-B</td><td>2.5</td><td>1.25 + 1.25</td><td>2.5</td><td>1.25 + 1.25</td><td>2.5</td></tr><tr><td>MCCP-C</td><td>5</td><td>2.5 + 2.5</td><td>5</td><td>2.5 + 2.5</td><td>5</td></tr><tr><td>MCCP-D</td><td>10</td><td>5 + 5</td><td>10</td><td>5 + 5</td><td>10</td></tr><tr><td>MCCP-E</td><td>20</td><td>10 + 10</td><td>20</td><td>10 + 10</td><td>20</td></tr></tbody></table>
<figure><img src="image_8.png" /></figure>

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

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure S1.** Double regression curves with the relative total area of a) ΣPCAs-C₁₀-₁₃ or c) ΣPCAs-C₁₄-₁₇ according to the concentration of a) SCCPs or c) MCCPs, and the exponential regression between the total response factor of b) ΣPCAs-C₁₀-₁₃ or d) ΣPCAs-C₁₄-₁₇ according to the chlorine content of each mixture.</figcaption></figure>
Q Exactive GC Tune Report

Thermo
SCIENTIFIC

Date of Tune Report: 2022-02-21 11:54:54
Last Mass Calibration: 2022-02-10 12:52:48
Instrument Identification: Exactive Series slot 143 (4C52621FB059)
Software Version: 2.11-211200/2.11.0.3007
Data System User: GC_QEXACTIVE\instrument(INSTRUMENT)
Tune File: C:\Xcalibur\methods\NCI SCCP Kerstin.mstune - not saved

m/z 282.98593
Intensity: 1.007e+07
Accuracy: -0.45 ppm
Resolution: 59,840

m/z 451.97467
Intensity: 4.285e+08
Accuracy: -0.46 ppm
Resolution: 47,319

m/z 632.96332
Intensity: 4.205e+08
Accuracy: -0.61 ppm
Resolution: 39,652

<figure><img src="image_12.png" /></figure>

Q Exactive GC Tune Report

Thermo
SCIENTIFIC

Date of Tune Report: 2022-02-22 14:41:03
Last Mass Calibration: 2022-02-10 12:52:47
Instrument Identification: Exactive Series slot 143 (4C52621FB059)
Software Version: 2.11-211200/2.11.0.3007
Data System User: GC_QEXACTIVE\instrument(INSTRUMENT)
Tune File: C:\Xcalibur\methods\FMNCI.mstune - not saved

m/z 282.98591
Intensity: 1.754e+07
Accuracy: -0.53 ppm
Resolution: 119,832

m/z 451.97462
Intensity: 5.435e+08
Accuracy: -0.56 ppm
Resolution: 95,112

m/z 632.96317
Intensity: 4.322e+08
Accuracy: -0.83 ppm
Resolution: 79,531

<figure><img src="image_13.png" /></figure>

Q Exactive GC Tune Report

Thermo
SCIENTIFIC

Date of Tune Report: 2022-02-23 11:43:38
Last Mass Calibration: 2022-02-10 12:52:45
Instrument Identification: Exactive Series slot 143 (4C52621FB059)
Software Version: 2.11-211200/2.11.0.3007
Data System User: GC_QEXACTIVE\instrument(INSTRUMENT)
Tune File: C:\Xcalibur\methods\FMNCI.mstune - not saved

m/z 282.98587
Intensity: 2.161e+07
Accuracy: -0.65 ppm
Resolution: 120,232

m/z 451.97458
Intensity: 7.197e+08
Accuracy: -0.66 ppm
Resolution: 93,889

m/z 632.96321
Intensity: 2.397e+08
Accuracy: -0.78 ppm
Resolution: 79,577

<figure><img src="image_14.png" /><figcaption>**Figure S2.** GC-ECNI-HRMS tune reports obtained for the three days of analysis.</figcaption></figure>
<table><caption>**Table S3.** Influence of the ion source temperature on the response of homologue groups, relative to respective maximum value (presented according to C-chain length first).</caption><thead><tr><th rowspan="3">Ion source temperature (°C)</th><th colspan="14">PCAs-C<sub>10-13</sub></th></tr><tr><th colspan="4">C<sub>10</sub></th><th colspan="4">C<sub>11</sub></th><th colspan="4">C<sub>12</sub></th><th colspan="4">C<sub>13</sub></th></tr><tr><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th></tr></thead><tbody><tr><td>110</td><td>0</td><td>100</td><td>96</td><td>83</td><td>73</td><td>67</td><td>0</td><td>0</td><td>100</td><td>100</td><td>85</td><td>75</td><td>66</td><td>56</td><td>47</td><td>0</td><td>100</td><td>92</td><td>75</td><td>66</td><td>55</td><td>47</td><td>41</td><td>0</td><td>100</td><td>80</td><td>67</td><td>58</td><td>49</td><td>40</td><td>33</td><td>23</td></tr><tr><td>150</td><td>0</td><td>93</td><td>98</td><td>94</td><td>88</td><td>81</td><td>0</td><td>0</td><td>82</td><td>98</td><td>96</td><td>90</td><td>82</td><td>76</td><td>62</td><td>0</td><td>73</td><td>100</td><td>91</td><td>83</td><td>74</td><td>67</td><td>60</td><td>0</td><td>68</td><td>97</td><td>86</td><td>77</td><td>68</td><td>60</td><td>51</td><td>38</td></tr><tr><td>180</td><td>0</td><td>76</td><td>83</td><td>78</td><td>70</td><td>51</td><td>0</td><td>0</td><td>56</td><td>81</td><td>82</td><td>77</td><td>67</td><td>56</td><td>42</td><td>0</td><td>56</td><td>84</td><td>81</td><td>74</td><td>62</td><td>52</td><td>42</td><td>0</td><td>70</td><td>84</td><td>78</td><td>70</td><td>59</td><td>47</td><td>36</td><td>22</td></tr><tr><td>200</td><td>0</td><td>86</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>64</td><td>93</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>58</td><td>97</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>51</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td></tr><tr><td>250</td><td>0</td><td>40</td><td>54</td><td>59</td><td>62</td><td>65</td><td>0</td><td>0</td><td>20</td><td>45</td><td>55</td><td>61</td><td>67</td><td>70</td><td>68</td><td>0</td><td>13</td><td>49</td><td>60</td><td>67</td><td>72</td><td>75</td><td>80</td><td>88</td><td>0</td><td>52</td><td>64</td><td>72</td><td>77</td><td>82</td><td>86</td><td>90</td></tr><tr><td>300</td><td>0</td><td>24</td><td>33</td><td>38</td><td>39</td><td>41</td><td>0</td><td>0</td><td>0</td><td>26</td><td>35</td><td>40</td><td>46</td><td>48</td><td>37</td><td>0</td><td>0</td><td>29</td><td>38</td><td>45</td><td>51</td><td>52</td><td>55</td><td>67</td><td>0</td><td>30</td><td>42</td><td>51</td><td>55</td><td>60</td><td>62</td><td>66</td></tr></tbody></table>

<table><thead><tr><th rowspan="3">Ion source temperature (°C)</th><th colspan="14">PCAs-C<sub>14-17</sub></th></tr><tr><th colspan="4">C<sub>14</sub></th><th colspan="4">C<sub>15</sub></th><th colspan="4">C<sub>16</sub></th><th colspan="4">C<sub>17</sub></th></tr><tr><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th></tr></thead><tbody><tr><td>110</td><td>100</td><td>76</td><td>58</td><td>49</td><td>41</td><td>32</td><td>25</td><td>0</td><td>0</td><td>60</td><td>51</td><td>44</td><td>35</td><td>27</td><td>17</td><td>0</td><td>0</td><td>0</td><td>45</td><td>38</td><td>29</td><td>21</td><td>11</td><td>0</td><td>0</td><td>41</td><td>41</td><td>32</td><td>25</td><td>15</td><td>0</td><td>0</td></tr><tr><td>150</td><td>80</td><td>95</td><td>80</td><td>70</td><td>62</td><td>51</td><td>41</td><td>0</td><td>0</td><td>90</td><td>73</td><td>65</td><td>54</td><td>44</td><td>34</td><td>0</td><td>0</td><td>0</td><td>67</td><td>59</td><td>46</td><td>36</td><td>23</td><td>0</td><td>0</td><td>78</td><td>64</td><td>51</td><td>41</td><td>28</td><td>16</td><td>0</td></tr><tr><td>180</td><td>62</td><td>85</td><td>74</td><td>65</td><td>55</td><td>42</td><td>30</td><td>0</td><td>0</td><td>81</td><td>69</td><td>61</td><td>49</td><td>38</td><td>25</td><td>0</td><td>0</td><td>79</td><td>67</td><td>57</td><td>44</td><td>31</td><td>18</td><td>0</td><td>0</td><td>77</td><td>63</td><td>50</td><td>39</td><td>26</td><td>13</td><td>0</td></tr><tr><td>200</td><td>61</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>95</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>94</td><td>91</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>98</td><td>91</td><td>87</td><td>0</td></tr><tr><td>250</td><td>16</td><td>56</td><td>69</td><td>78</td><td>84</td><td>91</td><td>93</td><td>91</td><td>0</td><td>61</td><td>73</td><td>82</td><td>90</td><td>96</td><td>100</td><td>0</td><td>0</td><td>59</td><td>77</td><td>87</td><td>96</td><td>100</td><td>100</td><td>0</td><td>0</td><td>71</td><td>81</td><td>93</td><td>100</td><td>100</td><td>100</td><td>0</td></tr><tr><td>300</td><td>0</td><td>34</td><td>47</td><td>57</td><td>64</td><td>69</td><td>70</td><td>77</td><td>0</td><td>37</td><td>50</td><td>61</td><td>70</td><td>77</td><td>80</td><td>0</td><td>0</td><td>36</td><td>53</td><td>67</td><td>77</td><td>81</td><td>84</td><td>0</td><td>0</td><td>46</td><td>57</td><td>73</td><td>84</td><td>87</td><td>86</td><td>0</td></tr></tbody></table>

<table><thead><tr><th rowspan="3">Ion source temperature (°C)</th><th colspan="14">PCAs-C<sub>18-20</sub></th></tr><tr><th colspan="4">C<sub>18</sub></th><th colspan="4">C<sub>19</sub></th><th colspan="4">C<sub>20</sub></th><th colspan="4"></th></tr><tr><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th></tr></thead><tbody><tr><td>110</td><td>0</td><td>41</td><td>36</td><td>29</td><td>19</td><td>11</td><td>0</td><td>0</td><td>0</td><td>31</td><td>32</td><td>24</td><td>14</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>17</td><td>15</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>150</td><td>0</td><td>69</td><td>57</td><td>46</td><td>32</td><td>22</td><td>0</td><td>0</td><td>0</td><td>63</td><td>51</td><td>39</td><td>27</td><td>16</td><td>0</td><td>0</td><td>0</td><td>0</td><td>38</td><td>31</td><td>17</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>180</td><td>0</td><td>67</td><td>57</td><td>46</td><td>32</td><td>20</td><td>0</td><td>0</td><td>0</td><td>60</td><td>52</td><td>40</td><td>26</td><td>14</td><td>0</td><td>0</td><td>0</td><td>0</td><td>43</td><td>32</td><td>18</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>200</td><td>0</td><td>100</td><td>100</td><td>100</td><td>91</td><td>84</td><td>69</td><td>0</td><td>0</td><td>100</td><td>100</td><td>98</td><td>87</td><td>78</td><td>0</td><td>0</td><td>0</td><td>0</td><td>100</td><td>93</td><td>84</td><td>73</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>250</td><td>0</td><td>72</td><td>85</td><td>96</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>67</td><td>89</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td><td>0</td><td>92</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>300</td><td>0</td><td>43</td><td>62</td><td>79</td><td>86</td><td>87</td><td>91</td><td>0</td><td>0</td><td>51</td><td>65</td><td>83</td><td>87</td><td>92</td><td>0</td><td>0</td><td>0</td><td>0</td><td>67</td><td>88</td><td>88</td><td>92</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table S4.** Influence of the ion source temperature on the response of homologue groups, relative to respective maximum value (presented according to Cl number first).</caption><thead><tr><th rowspan="2">Ion source temperature (°C)</th><th colspan="10">PCAs-Cl<sub>5</sub></th><th colspan="10">PCAs-Cl<sub>6</sub></th><th colspan="10">PCAs-Cl<sub>7</sub></th><th colspan="10">PCAs-Cl<sub>8</sub></th></tr><tr><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th></tr></thead><tbody><tr><td>110</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>100</td><td>100</td><td>92</td><td>80</td><td>76</td><td>60</td><td>0</td><td>41</td><td>41</td><td>31</td><td>0</td><td>96</td><td>85</td><td>75</td><td>67</td><td>58</td><td>51</td><td>45</td><td>41</td><td>36</td><td>32</td><td>17</td><td>83</td><td>75</td><td>66</td><td>58</td><td>49</td><td>44</td><td>38</td><td>32</td><td>29</td><td>24</td><td>15</td></tr><tr><td>150</td><td>0</td><td>82</td><td>73</td><td>68</td><td>80</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>93</td><td>98</td><td>100</td><td>97</td><td>95</td><td>90</td><td>0</td><td>78</td><td>69</td><td>63</td><td>0</td><td>98</td><td>96</td><td>91</td><td>86</td><td>80</td><td>73</td><td>67</td><td>64</td><td>57</td><td>51</td><td>38</td><td>94</td><td>90</td><td>83</td><td>77</td><td>70</td><td>65</td><td>59</td><td>51</td><td>46</td><td>39</td><td>31</td></tr><tr><td>180</td><td>0</td><td>56</td><td>56</td><td>70</td><td>62</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>76</td><td>81</td><td>84</td><td>84</td><td>85</td><td>81</td><td>79</td><td>77</td><td>67</td><td>60</td><td>0</td><td>83</td><td>82</td><td>81</td><td>78</td><td>74</td><td>69</td><td>67</td><td>63</td><td>57</td><td>52</td><td>43</td><td>78</td><td>77</td><td>74</td><td>70</td><td>65</td><td>61</td><td>57</td><td>50</td><td>46</td><td>40</td><td>32</td></tr><tr><td>200</td><td>0</td><td>64</td><td>58</td><td>51</td><td>61</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>86</td><td>93</td><td>97</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>98</td><td>93</td></tr><tr><td>250</td><td>0</td><td>20</td><td>13</td><td>0</td><td>16</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>40</td><td>45</td><td>49</td><td>52</td><td>56</td><td>61</td><td>59</td><td>71</td><td>72</td><td>67</td><td>0</td><td>54</td><td>55</td><td>60</td><td>64</td><td>69</td><td>73</td><td>77</td><td>81</td><td>85</td><td>89</td><td>92</td><td>59</td><td>61</td><td>67</td><td>72</td><td>78</td><td>82</td><td>87</td><td>93</td><td>96</td><td>100</td><td>100</td></tr><tr><td>300</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>24</td><td>26</td><td>29</td><td>30</td><td>34</td><td>37</td><td>36</td><td>46</td><td>43</td><td>51</td><td>0</td><td>33</td><td>35</td><td>38</td><td>42</td><td>47</td><td>50</td><td>53</td><td>57</td><td>62</td><td>65</td><td>67</td><td>38</td><td>40</td><td>45</td><td>51</td><td>57</td><td>61</td><td>67</td><td>73</td><td>79</td><td>83</td><td>88</td></tr></tbody></table>

<table><thead><tr><th rowspan="2">Ion source temperature (°C)</th><th colspan="10">PCAs-Cl<sub>9</sub></th><th colspan="10">PCAs-Cl<sub>10</sub></th><th colspan="10">PCAs-Cl<sub>11</sub></th><th colspan="10">PCAs-Cl<sub>12</sub></th></tr><tr><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th></tr></thead><tbody><tr><td>110</td><td>73</td><td>66</td><td>55</td><td>49</td><td>41</td><td>35</td><td>29</td><td>25</td><td>19</td><td>14</td><td>0</td><td>67</td><td>56</td><td>47</td><td>40</td><td>32</td><td>27</td><td>21</td><td>15</td><td>11</td><td>0</td><td>0</td><td>0</td><td>47</td><td>41</td><td>33</td><td>25</td><td>17</td><td>11</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>150</td><td>88</td><td>82</td><td>74</td><td>68</td><td>62</td><td>54</td><td>46</td><td>41</td><td>32</td><td>27</td><td>17</td><td>81</td><td>76</td><td>67</td><td>60</td><td>51</td><td>44</td><td>36</td><td>28</td><td>22</td><td>16</td><td>0</td><td>0</td><td>62</td><td>60</td><td>51</td><td>41</td><td>34</td><td>23</td><td>16</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>180</td><td>70</td><td>67</td><td>62</td><td>59</td><td>55</td><td>49</td><td>44</td><td>39</td><td>32</td><td>26</td><td>18</td><td>51</td><td>56</td><td>52</td><td>47</td><td>42</td><td>38</td><td>31</td><td>26</td><td>20</td><td>14</td><td>0</td><td>0</td><td>42</td><td>42</td><td>36</td><td>30</td><td>25</td><td>18</td><td>13</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>200</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>98</td><td>91</td><td>87</td><td>84</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>94</td><td>91</td><td>84</td><td>78</td><td>73</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>95</td><td>91</td><td>87</td><td>69</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>250</td><td>62</td><td>67</td><td>72</td><td>77</td><td>84</td><td>90</td><td>96</td><td>100</td><td>100</td><td>100</td><td>100</td><td>65</td><td>70</td><td>75</td><td>82</td><td>91</td><td>96</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>68</td><td>80</td><td>86</td><td>93</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td><td>88</td><td>90</td><td>91</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>300</td><td>39</td><td>46</td><td>51</td><td>55</td><td>64</td><td>70</td><td>77</td><td>84</td><td>86</td><td>87</td><td>88</td><td>41</td><td>48</td><td>52</td><td>60</td><td>69</td><td>77</td><td>81</td><td>87</td><td>87</td><td>92</td><td>92</td><td>0</td><td>37</td><td>55</td><td>62</td><td>70</td><td>80</td><td>84</td><td>86</td><td>91</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<figure><img src="image_15.png" /><figcaption>**Figure S3.** Influence of a) the ion source temperature (n = 1), b) the inlet temperature (n = 3) and c) the oven end temperature (n = 1) on the response of PCAs-C₁₀-₁₃ (blue) and PCOs-C₁₀-₁₃ (orange), relative to respective maximum value.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Figure S4. Influence of the inlet temperature on a) the response and b) the pattern for the 12.5 m × 0.25 mm, 0.10 µm column (mixture B; n = 3).</figcaption></figure>
<table><caption>**Table S5.** Influence of the inlet temperature on the response of homologue groups, relative to respective maximum value (presented according to C-chain length first).</caption><thead><tr><th rowspan="3">Inlet temperature (°C)</th><th colspan="12">PCAs-C<sub>10-13</sub></th><th colspan="12">PCAs-C<sub>14-17</sub></th><th colspan="12">PCAs-C<sub>18-20</sub></th></tr><tr><th colspan="6">C<sub>10</sub></th><th colspan="6">C<sub>11</sub></th><th colspan="6">C<sub>12</sub></th><th colspan="6">C<sub>13</sub></th><th colspan="6">C<sub>14-17</sub></th><th colspan="6">C<sub>18-20</sub></th></tr><tr><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th></tr></thead><tbody><tr><td>220</td><td>0</td><td>92</td><td>97</td><td>98</td><td>97</td><td>100</td><td>0</td><td>0</td><td>79</td><td>90</td><td>99</td><td>99</td><td>100</td><td>100</td><td>100</td><td>0</td><td>76</td><td>89</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>84</td><td>92</td><td>99</td><td>100</td><td>100</td><td>100</td><td>100</td><td>84</td></tr><tr><td>240</td><td>0</td><td>96</td><td>100</td><td>100</td><td>100</td><td>91</td><td>0</td><td>0</td><td>86</td><td>93</td><td>100</td><td>100</td><td>98</td><td>99</td><td>89</td><td>0</td><td>84</td><td>93</td><td>99</td><td>100</td><td>98</td><td>95</td><td>93</td><td>90</td><td>94</td><td>93</td><td>100</td><td>97</td><td>97</td><td>91</td><td>87</td><td>83</td></tr><tr><td>260</td><td>0</td><td>97</td><td>99</td><td>96</td><td>91</td><td>98</td><td>0</td><td>0</td><td>89</td><td>91</td><td>98</td><td>97</td><td>94</td><td>91</td><td>82</td><td>0</td><td>82</td><td>92</td><td>96</td><td>97</td><td>91</td><td>86</td><td>78</td><td>75</td><td>82</td><td>93</td><td>96</td><td>95</td><td>90</td><td>84</td><td>74</td><td>65</td></tr><tr><td>280</td><td>0</td><td>88</td><td>94</td><td>90</td><td>82</td><td>79</td><td>0</td><td>0</td><td>82</td><td>87</td><td>94</td><td>92</td><td>85</td><td>76</td><td>71</td><td>0</td><td>82</td><td>88</td><td>92</td><td>91</td><td>82</td><td>72</td><td>59</td><td>53</td><td>77</td><td>89</td><td>91</td><td>86</td><td>78</td><td>68</td><td>54</td><td>42</td></tr><tr><td>300</td><td>0</td><td>89</td><td>87</td><td>82</td><td>74</td><td>77</td><td>0</td><td>0</td><td>83</td><td>88</td><td>89</td><td>84</td><td>73</td><td>60</td><td>49</td><td>0</td><td>80</td><td>86</td><td>89</td><td>83</td><td>70</td><td>56</td><td>43</td><td>29</td><td>87</td><td>88</td><td>84</td><td>78</td><td>66</td><td>53</td><td>39</td><td>26</td></tr><tr><td>320</td><td>0</td><td>100</td><td>94</td><td>84</td><td>71</td><td>67</td><td>0</td><td>0</td><td>100</td><td>100</td><td>98</td><td>89</td><td>72</td><td>53</td><td>48</td><td>0</td><td>100</td><td>100</td><td>99</td><td>89</td><td>72</td><td>52</td><td>35</td><td>0</td><td>100</td><td>100</td><td>99</td><td>84</td><td>66</td><td>48</td><td>32</td><td>19</td></tr></tbody></table>
<table><caption>**Table S6.** Influence of the inlet temperature on the response of homologue groups, relative to respective maximum value (presented according to Cl number first).</caption><thead><tr><th rowspan="2">Inlet temperature (°C)</th><th colspan="8">PCAs-Cl<sub>5</sub></th><th colspan="8">PCAs-Cl<sub>6</sub></th><th colspan="8">PCAs-Cl<sub>7</sub></th><th colspan="8">PCAs-Cl<sub>8</sub></th></tr><tr><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th></tr></thead><tbody><tr><td>220</td><td>0</td><td>79</td><td>76</td><td>84</td><td>88</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>92</td><td>90</td><td>89</td><td>92</td><td>90</td><td>100</td><td>0</td><td>100</td><td>92</td><td>100</td><td>0</td><td>97</td><td>99</td><td>100</td><td>99</td><td>100</td><td>98</td><td>96</td><td>100</td><td>100</td><td>100</td><td>100</td><td>98</td><td>99</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>90</td></tr><tr><td>240</td><td>0</td><td>86</td><td>84</td><td>94</td><td>88</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>96</td><td>93</td><td>93</td><td>93</td><td>90</td><td>90</td><td>0</td><td>94</td><td>100</td><td>87</td><td>0</td><td>100</td><td>100</td><td>99</td><td>100</td><td>98</td><td>100</td><td>100</td><td>93</td><td>91</td><td>91</td><td>98</td><td>100</td><td>100</td><td>97</td><td>99</td><td>95</td><td>91</td><td>87</td><td>90</td><td>97</td><td>100</td></tr><tr><td>260</td><td>0</td><td>89</td><td>82</td><td>82</td><td>83</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>97</td><td>91</td><td>92</td><td>93</td><td>89</td><td>86</td><td>0</td><td>91</td><td>94</td><td>83</td><td>0</td><td>99</td><td>98</td><td>96</td><td>96</td><td>95</td><td>100</td><td>93</td><td>88</td><td>83</td><td>77</td><td>80</td><td>96</td><td>97</td><td>97</td><td>95</td><td>93</td><td>90</td><td>84</td><td>78</td><td>76</td><td>81</td><td>84</td></tr><tr><td>280</td><td>0</td><td>82</td><td>82</td><td>77</td><td>86</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>88</td><td>87</td><td>88</td><td>89</td><td>86</td><td>87</td><td>0</td><td>78</td><td>78</td><td>66</td><td>0</td><td>94</td><td>94</td><td>92</td><td>91</td><td>89</td><td>86</td><td>82</td><td>76</td><td>70</td><td>62</td><td>62</td><td>90</td><td>92</td><td>91</td><td>86</td><td>84</td><td>80</td><td>72</td><td>65</td><td>60</td><td>61</td><td>64</td></tr><tr><td>300</td><td>0</td><td>83</td><td>80</td><td>87</td><td>83</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>89</td><td>88</td><td>86</td><td>88</td><td>83</td><td>78</td><td>0</td><td>68</td><td>66</td><td>56</td><td>0</td><td>87</td><td>89</td><td>89</td><td>84</td><td>86</td><td>77</td><td>70</td><td>63</td><td>56</td><td>49</td><td>47</td><td>82</td><td>84</td><td>83</td><td>78</td><td>72</td><td>69</td><td>61</td><td>52</td><td>47</td><td>45</td><td>44</td></tr><tr><td>320</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>94</td><td>0</td><td>80</td><td>77</td><td>67</td><td>0</td><td>94</td><td>98</td><td>99</td><td>99</td><td>96</td><td>89</td><td>85</td><td>76</td><td>67</td><td>60</td><td>56</td><td>84</td><td>89</td><td>89</td><td>84</td><td>80</td><td>76</td><td>67</td><td>59</td><td>53</td><td>52</td><td>50</td></tr></tbody></table>

<table><thead><tr><th rowspan="2">Inlet temperature (°C)</th><th colspan="8">PCAs-Cl<sub>9</sub></th><th colspan="8">PCAs-Cl<sub>10</sub></th><th colspan="8">PCAs-Cl<sub>11</sub></th><th colspan="8">PCAs-Cl<sub>12</sub></th></tr><tr><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th><th>C<sub>10</sub></th><th>C<sub>11</sub></th><th>C<sub>12</sub></th><th>C<sub>13</sub></th><th>C<sub>14</sub></th><th>C<sub>15</sub></th><th>C<sub>16</sub></th><th>C<sub>17</sub></th><th>C<sub>18</sub></th><th>C<sub>19</sub></th><th>C<sub>20</sub></th></tr></thead><tbody><tr><td>220</td><td>97</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>84</td><td>58</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>93</td><td>76</td><td>61</td><td>0</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>76</td><td>60</td><td>0</td><td>0</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>94</td><td>84</td><td>0</td><td>0</td><td>0</td></tr><tr><td>240</td><td>100</td><td>98</td><td>98</td><td>97</td><td>93</td><td>89</td><td>89</td><td>94</td><td>98</td><td>100</td><td>100</td><td>91</td><td>99</td><td>95</td><td>91</td><td>89</td><td>88</td><td>90</td><td>100</td><td>100</td><td>100</td><td>98</td><td>0</td><td>0</td><td>0</td><td>93</td><td>87</td><td>85</td><td>86</td><td>98</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td><td>0</td><td>90</td><td>83</td><td>87</td><td>88</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td></tr><tr><td>260</td><td>91</td><td>94</td><td>91</td><td>90</td><td>85</td><td>80</td><td>76</td><td>76</td><td>79</td><td>80</td><td>87</td><td>98</td><td>91</td><td>86</td><td>84</td><td>79</td><td>73</td><td>72</td><td>77</td><td>82</td><td>84</td><td>100</td><td>0</td><td>0</td><td>0</td><td>78</td><td>74</td><td>70</td><td>68</td><td>76</td><td>77</td><td>87</td><td>0</td><td>0</td><td>0</td><td>0</td><td>75</td><td>65</td><td>67</td><td>70</td><td>81</td><td>94</td><td>0</td><td>0</td><td>0</td></tr><tr><td>280</td><td>82</td><td>85</td><td>82</td><td>78</td><td>72</td><td>66</td><td>61</td><td>57</td><td>58</td><td>57</td><td>61</td><td>79</td><td>76</td><td>72</td><td>68</td><td>62</td><td>53</td><td>52</td><td>54</td><td>54</td><td>56</td><td>68</td><td>0</td><td>0</td><td>0</td><td>59</td><td>54</td><td>50</td><td>45</td><td>48</td><td>47</td><td>53</td><td>0</td><td>0</td><td>0</td><td>0</td><td>53</td><td>42</td><td>47</td><td>50</td><td>57</td><td>0</td><td>0</td><td>0</td></tr><tr><td>300</td><td>74</td><td>73</td><td>70</td><td>66</td><td>59</td><td>51</td><td>45</td><td>42</td><td>41</td><td>40</td><td>39</td><td>77</td><td>60</td><td>56</td><td>53</td><td>45</td><td>40</td><td>35</td><td>35</td><td>34</td><td>35</td><td>50</td><td>0</td><td>0</td><td>0</td><td>43</td><td>39</td><td>34</td><td>29</td><td>31</td><td>29</td><td>34</td><td>0</td><td>0</td><td>0</td><td>0</td><td>29</td><td>26</td><td>35</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>320</td><td>71</td><td>72</td><td>72</td><td>66</td><td>60</td><td>52</td><td>48</td><td>44</td><td>43</td><td>42</td><td>41</td><td>67</td><td>53</td><td>52</td><td>48</td><td>41</td><td>37</td><td>32</td><td>33</td><td>31</td><td>32</td><td>48</td><td>0</td><td>0</td><td>0</td><td>35</td><td>32</td><td>28</td><td>23</td><td>24</td><td>23</td><td>29</td><td>0</td><td>0</td><td>0</td><td>0</td><td>19</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<figure><img src="image_17.png" /><figcaption>**Figure S5.** Influence of the final oven temperature on a) the response and b) the pattern for the 12.5 m × 0.25 mm, 0.10 µm column (mixture B; n = 1).</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**Figure S6:** Influence of the film thickness on the relative area of homologue groups (ratio between the area determined with 0.1 µm vs. 0.25 µm film thicknesses). Homologue groups are classified according to a) the chlorine number and b) the C-chain length for the 12.5 m × 0.25 mm, 0.10 µm column (mixture B; n = 3).</figcaption></figure>
<table><caption>**Table S7.** Influence of the film thickness on the response of homologue groups, relative to respective maximum value (presented according to the C-chain length first).</caption><thead><tr><th rowspan="3">Chromatographic column</th><th colspan="15">PCAs-C<sub>10-13</sub></th></tr><tr><th colspan="5">C<sub>10</sub></th><th colspan="5">C<sub>11</sub></th><th colspan="5">C<sub>12</sub></th><th colspan="5">C<sub>13</sub></th></tr><tr><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th></tr></thead><tbody><tr><td>30 m x 0.25 mm, 0.25 µm</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>86</td></tr><tr><td>30 m x 0.25 mm, 0.10 µm</td><td>0</td><td>43</td><td>43</td><td>40</td><td>37</td><td>52</td><td>0</td><td>0</td><td>42</td><td>38</td><td>32</td><td>34</td><td>41</td><td>53</td><td>0</td><td>0</td><td>43</td><td>31</td><td>30</td><td>30</td><td>36</td><td>57</td><td>100</td><td>0</td><td>40</td><td>29</td><td>26</td><td>30</td><td>38</td><td>55</td><td>100</td></tr></tbody></table><table><thead><tr><th rowspan="3">Chromatographic column</th><th colspan="15">PCAs-C<sub>14-17</sub></th></tr><tr><th colspan="5">C<sub>14</sub></th><th colspan="5">C<sub>15</sub></th><th colspan="5">C<sub>16</sub></th><th colspan="5">C<sub>17</sub></th></tr><tr><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th></tr></thead><tbody><tr><td>30 m x 0.25 mm, 0.25 µm</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>97</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>77</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>0</td><td>0</td></tr><tr><td>30 m x 0.25 mm, 0.10 µm</td><td>0</td><td>31</td><td>27</td><td>27</td><td>30</td><td>41</td><td>76</td><td>0</td><td>27</td><td>26</td><td>26</td><td>33</td><td>53</td><td>100</td><td>0</td><td>0</td><td>27</td><td>29</td><td>40</td><td>74</td><td>100</td><td>0</td><td>0</td><td>29</td><td>28</td><td>32</td><td>52</td><td>96</td><td>100</td><td>0</td></tr></tbody></table><table><thead><tr><th rowspan="3">Chromatographic column</th><th colspan="15">PCAs-C<sub>18-20</sub></th></tr><tr><th colspan="5">C<sub>18</sub></th><th colspan="5">C<sub>19</sub></th><th colspan="5">C<sub>20</sub></th><th colspan="5"></th></tr><tr><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th><th>Cl<sub>5</sub></th><th>Cl<sub>6</sub></th><th>Cl<sub>7</sub></th><th>Cl<sub>8</sub></th><th>Cl<sub>9</sub></th><th>Cl<sub>10</sub></th><th>Cl<sub>11</sub></th><th>Cl<sub>12</sub></th></tr></thead><tbody><tr><td>30 m x 0.25 mm, 0.25 µm</td><td>0</td><td>100</td><td>100</td><td>100</td><td>100</td><td>85</td><td>0</td><td>0</td><td>100</td><td>100</td><td>100</td><td>80</td><td>0</td><td>0</td><td>0</td><td>0</td><td>100</td><td>100</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>30 m x 0.25 mm, 0.10 µm</td><td>0</td><td>33</td><td>30</td><td>41</td><td>67</td><td>100</td><td>0</td><td>0</td><td>38</td><td>53</td><td>85</td><td>100</td><td>0</td><td>0</td><td>0</td><td>0</td><td>78</td><td>89</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<figure><img src="image_19.png" /><figcaption>Figure S7. Influence of column length on a) the response and b) the homologue group pattern (mixture A; n = 3).</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>Figure S8. Influence of column length on a) the response and b) the homologue group pattern (mixture B; n = 1).</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>**Figure S9.** Superposition of the TICs obtained for mixture B with the Optima 5HT column (black) and with the Optima 1 column (red).</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**Figure S10.** Influence of the column composition on a) the response and b) the homologue group pattern (mixture B; n = 1).</figcaption></figure>
<figure><img src="image_23.png" /><figcaption>**Figure S11.** Relative area, toward the global ΣPCAs, and relative standard deviations (n = 3) for each homologue group detected for a) CFB, b) CFC, c) CFD and d) CFE samples.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>**Figure S12.** Relative area, toward the global ΣPCAs, and relative standard (n = 3) deviations for each homologue group detected for a) LAB, b) LAC, c) LAD and d) LAE samples.</figcaption></figure>
<figure><img src="image_25.png" /><figcaption>**Figure S13.** Relative area, toward the global ΣPCAs, and relative standard deviations for each homologue group detected for the infant milk sample (n = 3).</figcaption></figure>
<figure><img src="image_26.png" /><figcaption>**Figure S14.** Relative area of each homologue group detected in the naturally contaminated pork sausage sample provided by the EURL POP (n = 2).</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>**Figure S15.** Spider chart representing the projection of the concentration of ΣPCAs-C₁₀-₁₃ for a) LAB, b) LAC, c) LAD, d) LAE and e) IFB samples. The red circle corresponds to the assigned value. The green circles delimit the acceptability zone (|Z-score| ≤ 2). The calculated value is represented by the dark dot. The results from the laboratories included in the proficiency test are represented by the blue dots.</figcaption></figure>
<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /><figcaption>**Figure S16.** Spider chart representing the projection of the concentration of ΣPCAs-C₁₄-₁₇ for a) LAB, b) LAE and c) 1905-PO samples. The red circle corresponds to the assigned value. The green circles delimit the acceptability zone (|Z-score| ≤ 2). The calculated value is represented by the dark dot. The results from the laboratories included in the proficiency test are represented by the blue dots.</figcaption></figure>
References

Krätschmer, K. and Schächtele, A., 2019. Interlaboratory studies on chlorinated paraffins: Evaluation of different methods for food matrices. Chemosphere 234, 252-259.
https://doi.org/10.1016/j.chemosphere.2019.06.022.

McGrath, T. J., Poma, G., Bombeke, J. and Covaci, A., 2020. A simplified screening method for short- and medium-chain chlorinated paraffins in food by gas chromatography-low resolution mass spectrometry. J Chromatogr A 1631, 461574. https://doi.org/10.1016/j.chroma.2020.461574.