Multi-Residue Method for Pesticides Determination in Dried Hops by Liquid Chromatography Tandem Mass Spectrometry

In this study a multi-residue determination method for 36 pesticides in dried hops was reported. The sample preparation procedure was based on the acetate buffered QuEChERS method. A few mixtures of dispersive solid phase extraction (dSPE) sorbents consisting PSA, C18, GCB, Z-Sep and Z-Sep+ were investigated to clean-up the supernatant and minimize matrix co-extractives. The degree of clean-up was assessed by gravimetric measurements, which showed the best results for mixtures containing the Z-Sep+ sorbent. This is the first study to apply Z-Sep+ sorbent for hops material and the first to improve the method for pesticide residues determination in hops. Samples were analysed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and the procedure was validated according to the SANTE/11813/2017 document at four concentration levels: 0.02, 0.05, 0.1 and 1 mg/kg. The limits of quantification (LOQ) were in the range of 0.02–0.1 mg/kg. For all active substances, the trueness (recovery) ranged from 70 to 120% and the precision (RSDr) value was <20%. Specificity, linearity and matrix effect were also evaluated. The validated method was applied to the analysis of 15 real dried hop samples and the relevant data on detected residues were included.


Introduction
Hop (Humulus lupulus L.) is a perennial climbing plant belonging to the same family as hemp (Cannabaceae L.) and the same order as nettles (Urticales L.). It is a dioecious plant producing both male and female inflorescences, characterized by twining vines that can reach from 3 m to 6 m in height. The leaves are arranged in opposite, with characteristic hairiness and spiky serration. Hop is widespread both in Europe (notably in Poland) and Central Asia. It is cultivated in many countries as an industrial plant used for brewing purposes. The habitat for wild hop includes moist thickets and river banks, as well as loamy, moist soils [1]. In the recent years, the hop production in the European Union has amounted to over 50,000 tons per year with Poland as the third largest producer of hop in Europe after Germany and Czechia [2].
Hop cones (hops) are a source of valuable bioactive compounds with, e.g., various phenolic compounds exhibiting antibacterial, antiviral and anticancer properties [3][4][5]. Due to the presence of alpha acids ( Figure 1) in cones, which are responsible for bitterness [6], hops are mainly used in beer production. Moreover, the use of hop cones in the medicine and cosmetics industry has been also reported [7]. Hops, like many other crops, are exposed to various diseases, lo and weed infestation. For this reason, a number of pesticides are us plant. However, the use of agricultural treatments generates probl consumer health due to the persistence of pesticide residues in hops f fore, it is important to control the level of pesticide residues to ensu sumers. As for that, the maximum residue levels (MRLs) of pesticid lished and regularly updated by European Commission [8].
The major components of hops are resins, essential oils, chlor compounds. Therefore, it is a complex matrix, for which an appropr tion and clean-up is required. In recent years, the QuEChERS metho popular for multi-residue pesticides' determination in food. This te acetonitrile extraction followed by a clean-up protocol with dispersiv tion (dSPE) on different sorbents, e.g., octadecylsilane (C18), prima (PSA), graphitized carbon black (GCB), and recently zirconia-based Sep+). The QuEChERS method was previously adopted by Dusek e termination of pesticide residues in hops by liquid chromatography c mass spectrometry.
The aim of the present study was to develop and validate an imp method for the determination of 36 pesticide residues in hop sampl For the clean-up protocol and to minimize matrix co-extractives sorbents, including Z-Sep+ (for the first time for hops matrix), we method was used for the determination of pesticide residues in 15 sa as a part of raw material quality control.

Optimization of Sample Preparation
Three versions of a QuEChERS extraction step, such as origina and acetate buffered are commonly used in labs for a wide range of various matrices with good results for most pesticides [11]. Howeve ods are suggested by authors of original QuEChERS as a good extra Hops, like many other crops, are exposed to various diseases, losses caused by pests and weed infestation. For this reason, a number of pesticides are used to protect the hop plant. However, the use of agricultural treatments generates problems connected with consumer health due to the persistence of pesticide residues in hops for a long time. Therefore, it is important to control the level of pesticide residues to ensure the safety of consumers. As for that, the maximum residue levels (MRLs) of pesticides have been established and regularly updated by European Commission [8].
The major components of hops are resins, essential oils, chlorophyll and phenolic compounds. Therefore, it is a complex matrix, for which an appropriate sample preparation and clean-up is required. In recent years, the QuEChERS method has been the most popular for multi-residue pesticides' determination in food. This technique is based on acetonitrile extraction followed by a clean-up protocol with dispersive solid phase extraction (dSPE) on different sorbents, e.g., octadecylsilane (C 18 ), primary secondary amine (PSA), graphitized carbon black (GCB), and recently zirconia-based sorbents (Z-Sep, Z-Sep+). The QuEChERS method was previously adopted by Dusek et al. [9,10] for the determination of pesticide residues in hops by liquid chromatography coupled with tandem mass spectrometry.
The aim of the present study was to develop and validate an improved multi-residue method for the determination of 36 pesticide residues in hop samples using LC-MS/MS. For the clean-up protocol and to minimize matrix co-extractives a few mixtures of sorbents, including Z-Sep+ (for the first time for hops matrix), were investigated. This method was used for the determination of pesticide residues in 15 sample batches of hops as a part of raw material quality control.

Optimization of Sample Preparation
Three versions of a QuEChERS extraction step, such as original unbuffered, citrate and acetate buffered are commonly used in labs for a wide range of pesticide residues in various matrices with good results for most pesticides [11]. However, the buffered methods are suggested by authors of original QuEChERS as a good extraction recovery of pH dependent analytes. An original unbuffered QuECHERS method [12] was published by Dusek et al. [9] for the determination of 48 pesticide residues in hops. The citrate buffered QuEChERS preparation protocol [13] for 56 pesticide residues in hops was published by the same authors [10]. In the proposed methodology in this paper, acetate buffered QuEChERS [14] was used for hop samples extraction.
The degree of dSPE clean-up was assessed by gravimetric measurements and then the content of co-extractives (corresponding to 1 mL in the final extract) was calculated.
In the first step, 3 mL of initial acetonitrile extract (in two repetitions) was transferred to pre-weighted glass vials. The moisture was removed by heating the vials for 1 h at 105 • C prior to weighing. After that, the extract was taken to dryness in nitrogen stream. Then, vials were heated again for 1 h at 105 • C to remove moisture and weighed. The same procedure was applied to final extracts cleaned by various sorbent mixtures. The difference in weight was assessed as the amount of co-extractives in the initial and final extracts. Table 1 presents the various mixtures used to optimize the dSPE clean-up step and obtained results. The Z-Sep sorbent is zirconium dioxide bonded to silica, whereas Z-Sep+ is a zirconium dioxide and C 18 dual bonded to silica. These sorbents were proposed as an alternative for PSA to lipids removal in high-content oil samples by Rajski et al. [15]. In this study, Z-Sep was shown to remove more co-extracted matrix components than PSA and C 18 . Other authors reported that PSA and C 18 in fish tissue sample removed most co-extractives by weight, however Z-Sep provided better trueness and precision [16]. The combination of PSA and Z-Sep+ was tested for dSPE clean-up in the determination of pesticide residues in honeybees [17]. The authors showed that clean-up mechanism of PSA is especially based on pH dependent ionic interactions, whereas Z-Sep+ is based on pH independent Lewis acid and hydrophobic interactions. The combination of these two sorbents in dSPE clean-up protocol of honeybee samples, consisting mainly of waxes and proteins, provided an excellent result. The combination of PSA, C 18 and Z-Sep was selected by Dusek et al. [9] for the sample preparation procedure of dried hops, but in the mentioned work the Z-Sep+ sorbent was not tested at all.
In the present study, several mixtures of sorbents were tested for the efficiency of removing co-extracted matrix components. The PSA and Z-Sep+ mixture (C) in the same amount as PSA and C 18 (A) showed almost 42% less matrix co-extractives. As compared to PSA and Z-Sep (B), the difference was also significant and equalled 34.5%.
The GCB sorbent is very effective for QuEChERS extracts clean-up, but retains pesticides with planar structures [14,18]. Due to the high content of chlorophyll in the hop samples, a small amount of GCB was used for an effective removal of this group of compounds. On the other hand, the procedural standard calibration was applied to compensate low extraction recoveries of planar pesticides such as thiabendazole. It should be mentioned that the extract after dSPE clean-up with GCB was better purified than in the absence of GCB sorbent, and the amount of co-extractives in this case was the lowest.
Finally, a mixture (D) of dSPE sorbents containing 50 mg MgSO 4 , 50 mg PSA, 50 mg Z-Sep+ and 5 mg GCB, which showed 47% less co-extractives than mixture (A), was chosen for the validation and routine analysis.

Validation of Analytical Procedure
The validation data, which are presented in Table 2, met the criteria of the SANTE/11813/2017 guidance document.
The LOQ values were in the range of 0.02-0.05 mg/kg for 89% of pesticides. Only for boscalid, hexythiazox, quinoxyfen and spirodiclofen the LOQs were equal to 0.1 mg/kg, but for all of the analytes these values were lower or equal to the MRLs.
A number of substances present in hops were co-extracted with the analytes during the sample preparation. Due to the co-extraction of matrix components in LC-MS/MS, the analyte signal might be enhanced and more often suppressed [19]. This phenomenon known as the matrix effect could affect the quality and performance of the LC-MS/MS analysis [20]. In the present study, the matrix effect was examined without an internal standard because there are reports that it may affect the results [11]. The signal enhancement was not observed for any analyte; however, suppression was demonstrated for all of the analytes. Negligible matrix effect (from −20% to 20%) was showed by 14% of compounds. Medium matrix effect (from 21% to 50% positive values and from −21% to −50% negative values) was proved for 53% of compounds, and a strong matrix effect (above 50% positive values and below −50% negative values) was observed for 33% of analytes. To overcome this phenomenon the procedural standard calibration was used.
The average precision represented as relative standard deviation (RSD r ) and trueness (recovery) were calculated at the levels of 0.02, 0.05, 0.1 and 1 mg/kg based on five replications for each level. For all of the analytes RSD r values were <20% and recovery ranged from 70 to 120%.
Calibration curves were prepared based on the procedural standard calibration. For this purpose, for each level, the blank samples before extraction were spiked in two repetitions with an appropriate amount of standard solution and internal standard for ensuring accuracy of the method. The data showed good linearity for all of the pesticides in the range of LOQ-4 mg/kg with a correlation coefficient (R 2 ) range 0.989-0.999.

Real Samples
All of the fifteen analysed dried hop samples contained pesticide residues. One or two pesticide residues were detected in six samples, three residues were found in seven samples and five residues in one sample. A maximum of six residues was also found in one sample ( Figure 2). However, in accordance with the European Commission limits [8], the MRLs were not exceeded in any case. In total, residues of twelve pesticides were detected in all samples. The highest detected residue level in one of the samples was for boscalid (9.4 mg/kg) but in this case the MRL for dried hops is equal to 80 mg/kg. In addition, in other samples, high concentrations of mandipropamid (7.3 mg/kg), dimethomorph (3.4 mg/kg), azoxystrobin (2.2 mg/kg) and pyraclostrobin (2.1 mg/kg) were detected, but the MRL limits for residues of these pesticides are 90, 80, 30 and 15 mg/kg, respectively. In the tested samples, residues of fungicides such as azoxystrobin, boscalid, pyraclostrobin and trifloxystrobin were the most dominant. Similar results were obtained in a study performed in Czechia, where 24 field samples of cone hops were analyzed [10]. The residues of fungicides such as mandipropamid, boscalid, azoxystrobin, pyraclostrobin, dimethomorph and ametoctradin were determined and the highest individual residue concentration was 31 mg/kg for mandipropamid [10]. The reason for the frequent detection of fungicide residues may be related to the fact that hop diseases caused by different types of fungi are among the most common in Europe [22]. Detailed data on the detected residues in analysed samples are presented in Table 3.

Instrumentation
For LC analysis, an Agilent 1200 Series (Waldbronn, Germany) liquid chromatograph equipped with a reversed phase C18 (Agilent Zorbax Eclipse XDB 4.6 mm × 150 mm, 5 µm particle size) column was applied. The column oven temperature was 40 °C and the analytes were separated using water (mobile phase A) and methanol (mobile phase B), both with 5 mM ammonium formate and 0.1% acetic acid. The flow rate was 0.5 mL/min and gradient program was made as follows: 20% B increased to 64% in 1 min, increased to 80% in 8 min, increased to 95% in 2 min and held for 10 min, decreased to 20% in 1 min and

Instrumentation
For LC analysis, an Agilent 1200 Series (Waldbronn, Germany) liquid chromatograph equipped with a reversed phase C 18 (Agilent Zorbax Eclipse XDB 4.6 mm × 150 mm, 5 µm particle size) column was applied. The column oven temperature was 40 • C and the analytes were separated using water (mobile phase A) and methanol (mobile phase B), both with 5 mM ammonium formate and 0.1% acetic acid. The flow rate was 0.5 mL/min and gradient program was made as follows: 20% B increased to 64% in 1 min, increased to 80% in 8 min, increased to 95% in 2 min and held for 10 min, decreased to 20% in 1 min and held for 10 min for column recondition. The total analysis run time was 32 min, and the injection volume was 10 µL. For MS/MS analysis, an AB Sciex 3200 QTRAP (Framingham, MA, USA) triple quadrupole mass spectrometer equipped with an electrospray ionisation source (ESI) was used. The ion source worked in positive ionisation mode and the scheduled multiple reaction monitoring (sMRM) was applied. The ion source settings were as follows: temperature-600 • C, ion spray voltage-5500 V, curtain gas-20 psi, collision gas-medium, ion source gas 1-45 psi, ion source gas 2-55 psi. The lC-MS/MS system was controlled by Analyst Software (version 1.5.2).

Sample Preparation
Homogenized dried hops (0.5 g) were weighed in a 50 mL centrifuge tube followed by the addition of 50 µL of the internal standard (10 µg/mL-atrazine D 5 ). Then, the sample was mixed with 5 mL of water using a vortex device for 30 s. In the next step, 10 mL of 1% acetic acid in acetonitrile was added and shaken manually for 1 min. After that, the sample was cooled in the freezer for 30 min. In the next step, a salting mixture (4 g of anhydrous MgSO 4 and 1 g of anhydrous C 2 H 3 NaO 2 ) was added, shaken manually for 1 min and centrifuged for 5 min at 4000 rpm. Then, 500 µL of supernatant was removed and cleaned by dispersive solid phase extraction mixture. The composition of the dSPE mixture, which was finally chosen, is as follows: 50 mg of anhydrous MgSO 4 , 50 mg of PSA, 50 mg of Z-Sep+ and 5 mg of GCB. The supernatant was shaken manually for 2 min and centrifuged again. Before injection into LC-MS/MS system the extract was filtered through a 0.2 µm syringe PTFE filter.

Validation of the Analytical Procedure
The initial full validation was conducted in accordance with the SANTE/11813/2017 guidelines, included on page 26 of this document [23]. Specificity, linearity, matrix effect, limit of quantification, trueness and precision were evaluated. The dried hop samples free from pesticide residues were used to prepare procedural standard calibration and used as blank in order to spike aliquots for validation studies. The limit of quantification (LOQ) was set as the minimum concentration that can be quantified with acceptable precision and trueness by spiking sample at this concentration level. Linearity was evaluated in duplicate at six concentration levels: 0.02, 0.05, 0.1, 1, 2 and 4 mg/kg. Spiked dried hop samples in five replicates for each level were used to estimate precision and trueness. The matrix effect (ME%) was examined by comparing the slopes of the calibration curves in pure solvent and matrix-matched without the internal standard using the following equation: ME% = slope of matrix matched calibration curve slope of calibration curve in pure solvent − 1 × 100 (1)

Real Samples
The developed and validated method was applied to analyse pesticide residues in 15 sample batches of Hallertauer Magnum variety dried hop, harvest year 2019, from Powiśle Lubelskie Region (southeast Poland). Subsequently, the supercritical CO 2 hop extracts were made from hops after pelleting.

Conclusions
Hop cones are the source of valuable bitter acids, essential oils and polyphenols [24]. On the other hand, they are also a complex matrix. As suggested by Hrnčič et al. [25], even the isolation of chemical compounds and their analyses are time-consuming and challenging due to the complexity of the hop cones matrix. Given the advantage of pesticide residue methods used so far, none of them tested Z-Sep+ for hop cones in order to remove matrix co-extractives.
The improved and validated method for determination of pesticide residues was successfully applied for dried hop samples by liquid chromatography coupled with tandem mass spectrometry. The combination of QuEChERS extraction and dSPE clean-up protocol with a mixture of PSA and Z-Sep+ enabled a decrease in co-extracted matrix components to a higher extent than with PSA and C 18, and PSA and Z-Sep. In addition, the use of a small amount of GCB effectively removed chlorophyll from the hop matrix, and the validation results showed satisfactory values for the precision and trueness.
The overall idea of pesticide residue testing is to assure end-users about the quality of product ingredients. According to the obtained results, all pesticides' residues (most belonging to fungicides) were detected at a level much lower than the allowable quantities. The proposed analytical method ensures consumer safety and brewing quality in further application steps and can be useful as an element of routine quality control of raw materials in the production process of supercritical CO 2 hop extract for brewing applications. Funding: This research was partially funded by THE MINISTRY OF EDUCATION for the project entitled "Development of the basis for the technology of biologically active extracts that lower blood sugar levels", grant number DWD/6/0467/2022 as well as the scientific activity of Prof. Grzegorz Woźniakowski which was funded by NATIONAL SCIENCE CENTRE for the project entitled: "The influence of natural plant extracts obtained by supercritical extraction on the replication inhibition of the most important corona and herpesviruses of poultry and swine", grant number UMO-2020/39/B/NZ7/00493.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in this manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Not applicable.