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High-throughput analysis of polyethoxylated tallow amine homologs in citrus using a modified QuEChERS-HILIC-MS method

A new method is described based on ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC) with electrospray mass spectrometry detection for comprehensive quantitative analysis of 66 polyethoxylated tallow amine (POE-tallowamine) homologs in citrus. Efficient separation, reduced band broadening, and high sensitivity were achieved by employing an acetonitrile-aqueous solution containing a 10 mM ammonium formate gradient on a hydrophilic interaction chromatography (HILIC) column with a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. The quantitative accuracy and precision of the method were improved by the use of matrix-matched calibration standards. At spiked levels of (50 + 250) μg/kg, (200 + 1000) μg/kg, and (500 + 2500) μg/kg POE-5 and POE-15 (1:5), the average recoveries of the POE-tallowamine homologs ranged from 71.9 to 112%, with RSDs < 16.6%. The limits of detection (LODs) and limits of quantification (LOQs) for POE-tallowamine homologs were 0.01–2.57 and 0.03–8.58 μg/kg, respectively. The method was successfully applied to determine POE-tallowamine in citrus samples from typical Chinese regions in 2021. POE-tallowamine was detected in all 54 samples, and the highest concentration (143 μg/kg) of POE-tallowamine was found in Jelly orange from Zhejiang Province, which might indicate a higher usage and demand of glyphosate herbicides in Zhejiang.


Introduction
Polyethoxylated tallow amine (POE-tallowamine) is a class of synthetic surfactants widely used in a variety of pesticides, especially in glyphosate-based herbicide formulations (1)(2)(3). POE-tallowamine can improve the plant coverage and penetration of glyphosate through surface tissues (4). However, POE-tallowamine has been demonstrated to have higher toxicity than its active substance glyphosate in a great number of studies (2, [5][6][7]. Considering the concerns about the toxicity of POE-tallowamine and its potential adverse effects on human health, POE-tallowamine has been listed as a co-formulant that is not accepted for inclusion in plant protection products by the European Union (EU) (8). The US Environmental Protection Agency (EPA) and Canada have recently limited the additive dose of POE-tallowamine to less than 25 and 20% in pesticide formulations, respectively (9,10). The increased attention focused on the occurrence of POE-tallowamine in agroecosystems necessitates the development of sensitive and robust analytical methods for determining these compounds in complex agroproduct matrices.
A typical POE-tallowamine comprises one linear aliphatic chain and two polyethoxylate chains attached to a single nitrogen atom with a range of 2-23 ethoxylate units (EO, n = 2-23, Supplementary Figure 1). The linear aliphatic chain is mainly derived from saturated palmitic acid (C16:0), saturated stearic acid (C18:0), and unsaturated oleic acid (C18:1, ω−9). The EO numbers in two ethoxylate chains of POE-tallowamine may equal or differ, rendering POEtallowamine analysis a major challenge. Reversed-phase liquid chromatography coupled with mass spectrometry (LC-MS) is suitable for the determination of POE-tallowamine as a mixture (11)(12)(13)(14)(15)(16). Wang et al. (13) and Rodriguez-Gil et al. (17) determined the total concentrations of a POE-tallowamine mixture in soil by a C18 column based on carbon chain length while lacking single ethoxylated POE-tallowamine homolog chromatographic separation and quantitation. The coelution of ethoxylated POE-tallowamine homologs will lead to competitive ionization suppression during the electrospray process and isobaric interferences between single-and double-charged POE-tallowamine ethoxymers (18)(19)(20)(21). Recently, hydrophilic interaction chromatography (HILIC) combined with mass spectrometry was used widely for the separation of polydisperse surfactants in petrochemicals that are difficult to separate in reversed-phase chromatography (19,(22)(23)(24)(25)(26). To the best of our knowledge, there are no reports of useful techniques for the determination of single ethoxylated POE-tallowamine in aro-product samples.
In this study, we developed a QuEChER-HILIC-MS method for quantification of trace levels of individual POE-tallowamine homologs in citrus samples. A matrix effect study was conducted, demonstrating that ion suppression from sample matrices was decreased. This method was then applied to characterize the distribution of different POE-tallowamine homologs in citrus samples, providing more foundation information for human health risk assessment.
Stock standard solutions of POE-2, POE-5, and POE-15 (10.0 g/L) were prepared in ACN, and the spiked solutions were a mixture of POE-5 and POE-15 (1:5). The working solutions were achieved by appropriate dilution with ACN. All of the solutions were stored at −20 • C. According to Tush et al. (15), Cz(s/u) EOn was used to describe the individual homologs, where z is the number of carbon atoms, s is a saturated tallow moiety, u is a monounsaturated tallow moiety, and n is the total number of EO units from two ethoxylate chains. It was assumed that C16s, C18s, and C18u accounted for 90% of the mixtures, and individual homologs obtained an equal instrument response, which was consistent with the analysis of nonylphenol ethoxylates (NPEOs) (27).

Sample collection and preparation
Fifty-four ripe citrus samples were collected from the main citrus production regions in China, including Guangxi province, Zhejiang province, Hunan province, and Chongqing municipality, which contained Shatang mandarin, Jelly orange, Bingtang sweet orange, Satsuma mandarin, Orah citrus, and W. Murcott Tangerine. Six to twelve parallel samples were collected for each species from different regions. All the samples were homogenized and stored at −20 • C before analysis.
Frozen citrus samples were allowed to thaw completely before being thoroughly mixed and subsampled for extraction. After thawing at room temperature, 10 g homogenized citrus samples were extracted with 10 mL of 1% formic acid in acetonitrile in a 50-mL polypropylene centrifuge tube. Each sample was vortexed for 1 min with a Vortex-Genie 2 (Scientific Industries Inc., Bohemia, NY, USA) and ultrasonically extracted by an ultrasonic cleaner (Model KQ-500DB, Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu, China) at room temperature for 20 min. Subsequently, 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride were added to the centrifuge tube and vortexed for 1 min. After centrifugation at 5,000 rpm for 10 min, 1 mL of the supernatant was pipetted into a 2.5-mL clean centrifuge tube that contained 50 mg PSA and 5 mg GCB, vortexed and centrifuged at 10,000 rpm for 5 min. Finally, the supernatant was filtered through a 0.22-µm membrane (Bonna-Agela Technologies Inc., Tianjin, China) and transferred into a 1.5-mL autosampler vial for direct analysis by ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS).

Instrumental conditions
Quantitative analyses of POE-tallowamine were conducted by a Shimadzu Triple Quadrupole LCMS-8050 system (Shimadzu, Kyoto, Japan) in positive electrospray ionization (ESI) mode. A Waters CQUITY UPLC R BEH HILIC column (2.1 × 100 mm, 1.7 µm particle size) was used to separate the analytes. A gradient procedure was performed using mobile phases A (water containing 10 mM ammonium formate) and B (ACN) at a flow rate of 0.30 mL/min. The injection volume was 5 µL, and the column temperature was maintained at 25 • C. The gradient elution program was as follows: mobile phase B was initiated with 92% (held for 1 min), followed by a linear decrease to 80% in 10 min and kept for 2 min. Then, it was increased to 92% to maintain the initial chromatographic condition within 4 min. The back pressure was 16.6 MPa. The column temperature was maintained at 25 • C. MS detection was carried out using a time-programmed selected ion monitoring mode with an LCMS-8050 tandem quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The ion source temperature was
Considering that the unfavorable separation and coelution of POE-tallowamine by reversed-phase chromatography column in previous studies (11)(12)(13)(14)(15)(16). POE-tallowamine was separated based on its hydrophilic moiety by a HILIC column. One of the main advantages of HILIC is the efficient separation of polar compounds in aqueous-organic mobile phases rich in organic solvents (usually ACN) (30). In general, the pH value and additives of the mobile phase have a significant impact on retention and selectivity in the chromatographic column (31)(32)(33). Figure 2 presents the chromatograms of standard mixtures containing 1 mg/L POE-5 and 5 mg/L POE-15 at different percentages of formic acid and ammonium formate. As exemplified by C16s in Figure 2A, the chromatographic peaks of C16s were not well separated between 1 and 6 min when the aqueous phase consisting of only formic acid was used, which displayed a reduction in retention and deterioration in peak shape. To improve the retention selectivity and peak profiles, the addition of ammonium formate to the mobile phase was also investigated. As shown in Figures 2B,C, the chromatographic peak shape was greatly improved, and the retention time was moved forward for POE-tallowamine with the increase in the ammonium formate concentration from 1 to 10 mM. The reason could be that salt could significantly affect the adsorption behavior of the stationary phase (30, 34). Compared to 1 mM ammonium acetate, sharper, and symmetrical peak shapes were observed by using a concentration of 10 mM ammonium acetate, with the band broadening reducing approximately 42-50% and the sensitivity increasing one-fold. This result suggested that the addition of ammonium formate also helped to the LC-electrolyte effects of POE-tallowamine. Similar effects of ammonium formate were reported in the separation of saccharide and antibiotics (34,35). Moreover, the effect of the addition of 0.2% formic acid and 10 mM ammonium formate was also evaluated (Figure 2D). The peak shapes of C16sEO6, C16sEO10, C16sEO11, and C16sEO15 were distinctly split, and C16sEO4-7 overlapped obviously, negatively impacting the quantitation of the target analytes. Consequently, 10 mM ammonium formate added to the mobile phase was ultimately selected in our study, providing optimum analytical separation.

Optimization of the sample preparation
ACN and ACN with different degrees of acidification were tested to optimize the extraction procedure, including 0.1, 0.5, 1, and 1.5% formic acid (FA) in ACN. As shown in

Frontiers in Nutrition
Supplementary Figure 3, the recoveries of different extraction solvents for POE-tallowamine homologs were in the range of 63.1-106%. However, the recoveries of the compounds showed improvement upon addition of 0.1-1.5% FA, which may be because the use of acidification during the extraction could improve the extraction efficiency of cationic surfactants (36,37). Satisfactory recoveries of antibiotics were also achieved by adding formic acid to adjust the pH (35). In comparison with C16sEO10 extracted by 1.5% FA in ACN (Figure 3A). Figure 3B shows obvious separation of C16sEO10 from impurity peaks with a good peak shape when extracted by 1% FA in ACN. The resolution of C16sEO10 was significantly increased by four-fold under 1% FA in ACN. As shown in Figures 3C,D, an adverse separation between C16sEO19 and impurity peaks under 0.1% FA in ACN and a refined separation of C16sEO19 under 1% FA in ACN were clearly observed. This might affect the hydrophobic interactions of the compounds as a result of their pH, leading to a better separation of POE-tallowamine homologs. Therefore, 1% FA in ACN treatment was ultimately selected as the extraction solvent.
To obtain high sensitivities and lower method limits, the dispersive solid-phase extraction (DSPE) method was used to purify the acetonitrile phase. Different DSPE sorbents were tested during optimization in this study. First, 50 mg PSA, 50 mg C18, and 10 mg GCB were evaluated by purifying crude citrus sample extracts spiked with POE-5 (200 µg/kg) and POE-15 (1000 µg/kg). As shown in Figures 4A-C, PSA, C18, and GCB showed good recoveries for all 66 POE-tallowamine homologs (PSA: 84.4-105%, C18: 73.1-102%, and GCB: 72.8-96.6%). The difference is that the analytical sensitivities for PSA were approximately four-fold higher than those for C18, as exemplified by C16sEO10 (Supplementary Figure 4). This might be because PSA has a greater capacity than aminopropyl sorbent based on the extra secondary amino group and provides adsorption properties for fatty acids (38-40). Additionally, previous studies indicated that GCB was highly effective in reducing coextracted pigments in QuEChERS (39, 41). Thus, different combinations of PSA and GCB sorbents were further optimized to improve adsorption effects. With the increase in GCB from 5 to 10 mg, the recoveries were observed to decrease from 64.6-112 to 31.8-89.1% (Figures 4D,E). Therefore, 50 mg PSA and 5 mg GCB were selected as the DSPE sorbent for all POE-tallowamine homologs in this study.

Method validation
To confirm the practicability of the present method, matrix effects were evaluated for POE-tallowamine by using the slope ratio of matrix-matched standard curves with a solvent standard calibration curve. Matrix-matched and solvent standard curves were created for POE-tallowamine with different spiked concentrations in the range of 5-1,000 ng/mL POE-5 and 25-5,000 ng/mL POE-15. As shown in Supplementary Figure 5, POE-tallowamine homologs in the tested citrus exhibited matrix suppression effects. Therefore, matrix-matched calibration curves were built for the POE-tallowamine analysis to reduce the influence of the matrix.
The recovery experiment was conducted to evaluate the method accuracy by spiking at three times the original concentration of the standard mixture into citrus samples. As shown in Table 1, at spiked levels of (50 + 250) µg/kg, (200 + 1,000) µg/kg, and (500 + 2,500) µg/kg POE-5 and POE-15 (1:5), the relative recoveries ranged from 71.9 to 112% with a relative standard deviation (RSD) < 16.6% in citrus samples. The correlation coefficients (R 2 ) of determination were typically greater than 0.9900, which indicated that linearity was acceptable for all target compounds over the citrusrelevant concentration range. Moreover, the limits of detection (LODs) and quantification (LOQs) of the POE-tallowamine homologs were estimated by analyzing spiked samples at low concentrations. LODs and LOQs were calculated on the basis of peak-peak signal-to-noise (S/N) values of 3 and 10, respectively (42-44). The obtained LODs and LOQs of the POE-tallowamine homologs were in the range of 0.01-2.57 and 0.03-8.58 µg/kg for citrus samples, respectively. These results demonstrated that the present QuEChERS-HILIC-MS method was suitable for the determination of POE-tallowamine homologs from various citrus matrices. It has been reported that a maximum of 15 homologs of C16s, C18s, and C18u were detected in previous analytical studies (12,14). However, the proposed method allowed us to simultaneously and selectively determine 66 POE-tallowamine homologs, significantly improving the highthroughput analysis of POE-tallowamine.

Concentrations and ethoxymer distribution of POE-tallowamine in citrus samples
A total of 54 citrus samples from four main production provinces were analyzed by the method described in this study. As shown in Figure 5, POE-tallowamine homologs were detected in all samples. The total concentrations of POEtallowamine ranged from 48.5 to 143 µg/kg, and the highest concentration was found in Zhejiang Province. For the six citrus species that we obtained, the highest concentration of POE-tallowamine was found in Jelly orange, with a range of 94.6-143 µg/kg. These results might indicate a higher usage and demand of glyphosate herbicides in Zhejiang Province than in other provinces.
The concentration profiles of POE-tallowamine homologs in citrus samples are shown in Supplementary Figure 6, with a concentration range of 0-59.5 µg/kg. Compared to POE-15 standards, which are widely used in glyphosate-based herbicide formulations (15), the contributions of EO units (n = 2-4) in citrus samples increased from 0 to 33.4%. The different distributions of POE-tallowamine homologs were possibly due to different POE-tallowamine technical mixtures applied in agroecosystems (14, 15, 45). Degradation that occurs similar to NPEOs may also contribute to the varying concentration profiles of POE-tallowamine homologs (46,47). It has been reported that NPEOs with a shorter chain could exhibit stronger toxicity and persistence than those with a longer chain. Therefore, more attention should be given to the toxicity and persistence of EO2-4, which might increase exposure risks to human health and the environment.

Conclusion
In the present study, we developed a highly sensitive UHPLC-MS method for the simultaneous determination of 66 POE-tallowamine homologs in citrus samples. HILIC achieved efficient separation of POE-tallowamine by a hydrophilic moiety. The validation test of the method demonstrated satisfactory linearity, method detection limit, and precision. This method was successfully applied to analyze typical citrus samples and provided a new reference for the rapid separation and analysis of POE-tallowamine homologs. Based on the developed method, further studies are needed to explore the occurrence and environmental fate of POE-tallowamine.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.