Validation of short run time GC-EI (+) MS/MS method for pesticide residues determination in dry Hibiscus syriacus L. via application of d-MRM and end-point backflush quantification techniques

ABSTRACT In this paper, the short injection method using GC-EI (+) MS/MS with end-point back-flush and dynamic-multi-reaction mechanism technique was validated for simultaneous determination of pesticide residues in dry Hibiscus syriacus L. applying QuEChERS extraction covering 176 GC-amenable pesticide residues. The GC optimum temperature program achieved a short run time and it was 20 minutes. The linearity of method was evaluated using matrix matched calibration standards at five concentration levels and the results showed that the coefficient of determination (r2) of each analyte was more than 0.990. The LODs was considered to be the lowest concentration that could be detected under optimal experimental conditions and it was 2 μg/kg (S/N = 3) and the LOQs was 10 μg/kg (S/N = 10). Acceptable recoveries of all the selected pesticides at three fortification levels were in the range from 60% to 102% with relative standard deviations ≤20% (n = 6) and matrix effect ranged from −31% to 6%‎. The method expanded measurement uncertainty was estimated as 40% (coverage factor k = 2, confidence level 95%). The validation results prove that it fit the intended use in accordance with the EC guidelines (SANTE/12682/2019) and could successfully expand the detection efficiency of pesticide residues in dry Hibiscus syriacus L. and other dry commodities.


Introduction
Dry Hibiscus syriacus L. is one of Hibiscus tropical wild plant classes rich in organic acids, polyphenols, anthocyanin, polysaccharides and volatile constituents [1].Nowadays, Hibiscus water extract of the leaves used worldwide for alternative medicine due to its anti-cancer [2], anti-oxidative [3], anti-ageing [4], anti-mycobacterial activity antidiabetic activity and neuroprotective activity [5].Dry hibiscus belongs to the dry herbal plants food group that may contain as well as water extract pesticide residues at higher levels compared to other food classes [6].
The term pesticide applies to herbicides, fungicides, and various other substances used to control pests, which consist of different groups that are associated with various chemical classes depending on their effect on pest control.However, the bioaccumulation of these chemical compound residues in crops have negative effects on human health; such as geno-toxicity, mutagenicity, inhibition of acetyl cholinesterase activity, hepatic and renal toxicity [7].
Currently, gas chromatography equipped with different detectors is one of the main techniques that are used for the determination of pesticide residues in agriculture products, where the sensitivity and selectivity will depend on type of detector used.Due to the complexity of matrix effect in different fruits, vegetables and dry matter that interferes with the target residue and may cause reporting false positive results using different selective detectors like Electron capture (ECD) and Flame Thermionic Detector (FTD) used as advanced technique for detection [8].
Therefore, the presence of co-extractive interfaces accompanied with pesticides to GC may either result in a response enhancement or suppression due to interactions with different parts of GC system like active sites of the liner, column and detector, which void pesticides losses by concepts of thermal degradation or by adsorption, leading to increment of detected pesticides signals [9,10].Despite the advanced detection technique of GC coupled with mass spectrometry detection (GC-MS/MS) being successfully used for the analysis of pesticide residues.The main challenge is unavoidable in presence of more complex samples even after the thorough cleaning step [11].At present, there have been many different combinations of QuEChERS (quick, easy, cheap, effective, rugged and safe) reagents and sample preparation combined with common d-SPE sorbents graphitised carbon black and primary secondary amine in AOAC and EN 15662 Official QuEChERS method to quantitate wide range of pesticide groups [12].Consequently, realising the role of impurity removal and purification has gradually become the most popular pretreatment method for multi-component pesticide residue determination [13].Among them [14,15], the application of QuEChERS method combined with GC-MS/MS for pesticide residue analysis in agreements with the recent regulations of maximum residues level (MRL).
QuEChERS sample preparation for pesticide residue analysis applied by both AOAC and EN 15562 methods to identify and characterise matrix components during different instruments including GC-MS/MS.It was found that the major components of the matrix are terpenoids, fatty acids and fatty acid esters.Also, the trend of residues analysis of pesticide in difficult matrices combined with the efficiency and the quantitative measurements of target residues has been developed to decrease this effect [16].In addition to sample preparation techniques such as the QuEChERS method, where GC-MS/MS exhibited more co-extractive interference effect, the use of an IS with matrix-matching also showed a substantial increase in GC-quantitative accuracy [17][18][19].
A modified QuEChERS method using 10 mg of graphitised carbon black (GCB) as d-SPE in combination with primary secondary amine (PSA) for decreasing co-extract to detect 66 pesticide residues in spinach using GC-MSD selected-ion monitoring mode (SIM).This yielded average recoveries ranging from 66% to 104%, with a relative standard deviation below 16% and the limits of quantitation (LOQ) were 50 µg/kg [20].Also, using the extract of the blank sample matrix for preparation of matrix matched calibration recommended via [21,22] can help investigate the matrix effect but cannot be easily applicable in routine analysis.Considering the inconsistency of matrix effects where the matrix effects are often unpredictable especially when food matrices are highly complex.Significant matrix effect was detected at lower pesticide concentration levels in different matrices such as deltamethrin and cymermthrin in melon and eggplant, respectively [23,24].
A study was performed on the effective extraction of matrix interferences in acetonitrile solution where the results found that the removal of matrix interferences using traditional solid-phase dispersive extraction cleanup agents of primary secondary amine is not sufficient.However, even when using matrix-matched calibration, a variation in intensity was detected between solvent-based and matrix-based calibration [25,26].Therefore, the present study aimed to validate multi-residues method using GC-MS/MS with dynamic multi reaction mechanism (d-MRM) detection technique in combination with modified QuEChERS method for the quantification of selected pesticide residues in dry Hibiscus syriacus L., following the EC guidelines (SANTE/12682/2019) [21].In addition, evaluations of matrix effect as well as calculation of the expanded uncertainty were also demonstrated in this study.

Pesticides reference standards
All tested pesticide active ingredients were purchased from Dr. Ehrenstorfer-LGC GmbH (Augsburg, Germany) with a purity of more than 95%.Stock solutions for all pesticides were individually prepared to a concentration of 1000 µg/mL dissolved in toluene and stored at −20°C.Three intermediate mixtures were prepared at a level of 10 µg/mL then calibration mixtures at 2, 10, 50, 100 and 500 µg/L were prepared in toluene from serial dilution of the working solution.Chlorpyrifos-D10 used as an internal standard maintained at level 100 µg/L in all calibrations, and stored in a refrigerator at −4°C.

Apparatus
Geno/Grinder 2010-SPEX Sample Prep (UK) was used for automated shaking of sample in both extraction and d-SPE, centrifuge up to 4500 rpm (Sigma, Germany 3-16KL).Hiedolph rotary evaporator (Hei-VAP Core, Heidolph Instruments GmbH & CO.KG) was used for sample evaporation at 40°C.The volumetric flasks and pipettes used were from class A glassware, as well as calibrated micropipettes (Hirschman Laborgerate-Germany) for preparations of calibration in ranges (10-100, 100-100 µL) were used.In addition to Grindomix Knife Mills GM 300 grinding machine (Retsch GmbH, Germany), water purification system MilliQ UF-Plus system (Millipore, Germany) and a solvent dispenser with a 10 mL capacity (Hirschman Laborgerate, Germany).

GC-MS/MS conditions
Agilent benchtop GC-7890 equipped with MS-7010B triple quad system and highefficiency (HES-EI) ion source, end-point backflush accompanied with Agilent GC system was used.Helium of purity 99.9999% was used as the carrier gas at a constant flow rate of 0.7 mL/min.The injection volume was 1 µL in the split-less mode.The temperature of multimode inlet on split-splitless mode was maintained at 250°C including inert liner Agilent 5181-3316I (Split less, single taper, inert).End-point backflush technique was used for GC separation where the first capillary column J&W DB-35 ms UI (20 m length, 0.18 mm id, 0.18 µm film thicknesses) connected from inlet to inert purged ultimate union assay.Then, a second pre-column of 70 cm inert fused silica of id-0.15 mm connected from inert purged ultimate union assay to MS allows transfer of components via the transfer line to MS-7010B unit.The column oven temperature was held at 70°C for 1.3 min after injection, ramped at 70°C/min to 150°C, ramped at 12°C/min to 270°C, then ramped at 18°C/min to 310°C, and held for 6.3 min.Post-run end-point backflush at 310°C for 2 minutes and negative pressure of 40 psi was applied in order to thermally clean the first part of the column from accumulated matrices.The ion source, quads and transfer line temperatures were 320, 150 and 320°C, respectively.The helium quench gas and the nitrogen of purity (99.999%) for collision gas were set at standard manufacturing value of 1.5 mL/min and 2.25 mL/min, respectively.Dynamic multiple reaction monitoring (d-MRM) mode was adopted for the quantitative analysis using Agilent Mass Hunter Data Acquisition 10.0.The mass spectrometer MS-7010B triple quad system was operated in electron impact ionisation mode where the temperature of ion source maintained at 300°C, Quadruple 180°C, and filament current 100 µA.Moreover, the system performs auto-tune using perfluorotributylamine (PFTBA) as a system suitability test for MS-System.

Sample handling and homogenisation
Organic samples of dry Hibiscus syriacus L. approximately 500 g were grinded a homogenised powder to obtain 300 μm particular size to get homogeneous sample using knife mill Grindomix Model GM 300 and confirmed free from target analyte via quantitative analysis to confirm that they are free of target pesticides used as a blank then stored at −20°C.

Sample extraction
A 5 g of homogenous sample was weighted into 50 mL polypropylene centrifuge tube followed by the addition of 10 mL of de-Ionised water, shaken the tube and then centrifuged for 30 s to decant the sample, applying QuEChERs [14] E7-extraction module where the sample was mixed before and after salts addition with Acetonitrile in Geno-Grinder for 2 minutes at 700 rpm then centrifuge at 4500 rpm for 5 minutes followed by C3-dSPE sample clean-up module where the aliquot was transferred to PSA-dSPE tube.The d-SPE tubes were shaken for 30 s and then centrifuged at 4500 rpm for 2 minutes, 2 mL of the acetonitrile layer was transferred into 50 mL flask.They were then evaporated to near dryness at 40°C and diluted with 2 mL of Chlorpyrifos-d10 at 100 µg/L, and finally filtered through 0.45 µm syringe filter membrane into clear vial injected into GC-MS/MS.
The validation study was applied to dry Hibiscus syriacus L., where a set of six replicates at three spiking level of 10, 50, 100 µg/kg and blank samples were weighted, fortified and extracted applying the workflow for sample extraction and then transferred the vial to GC-MS/MS analysis.

Optimisation of dynamic multi reaction monitoring (d-MRM) of GC-MS/MS conditions
First, the proper transitions for each individual pesticide were selected from EU Reference Laboratories (EURLs) Data-Pool website [27] to build up the acquisition method was performed, and then each pesticide was injected individually on scan mode applying GCtemperature programme was obtained to select a minimum of two transitions for each compound.Second, 176 dissolved pesticide mixtures were injected into the toluene solvent to improve the application of d-MRM to tackle large multianalyte assays and for accurate quantification.
After using d-MRM, where ion transitions and the retention time window for each compound were stored in a certain method, each target pesticide was simultaneously monitored when they eluted from the GC to MS, there were remarkable increases in cycle/ second, number of data points and decrease in dwell time that enhance the sensitivity obtained for each compound necessary for reliable quantitation.
Acquisition of data for all pesticides were monitored by applying SANTE-12682/2019 guidelines for identification requirements for different MS techniques where minimum two product ions were used except for those pesticides defined with bold font in Supplementary data Table 1.This was performed to prevent any false positive or false negative results due to matrix effect may be found during sample analysis and results evaluation of dry Hibiscus l., as it does not effect on the sensitivity of other compounds.
In Figure 1, the d-MRM separation profile for 176 pesticides using Chlorpyrifos-d10 as internal standards at 18.5 min for pure solvent with average separation of 10 compounds per min in order to get the optimum separation of GC conditions over run time.
Separation and detection of 176 pesticides applying Dynamic MRM technique using Chlorpyrifos-d10 as internal standards in 18.5 min where the data was generated with an Agilent Mass Hunter software GC-7890B and 7010B Triple Quad.The accompanying figure of the temperature program and ramps during injection, at the end of the 2 min endpoint backflush was applied to thermally clean the front part of the capillary column with negative flow that increases column lifetime, and the total run-time was 20.97 min.

Method validation
The results of validation were evaluated in agreement with SANTE-12682/2019 guidelines, via testing method linearity in matrix matched calibration solutions, trueness via recovery of six replicates at three different fortification levels, and precision via calculation of relative standard deviation (RSD%).

Method trueness and precision
The validity and accuracy of the method were evaluated from the recovery, precision and repeatability data sorted with the retention time and calculated relative standard deviation (RSD %) for six replicates at 10, 50, 100 µg/kg fortification level of dry HibiscusL.(Table 1).The LODs were selected as the lowest calibration concentration of the target component that could be detected under optimal experimental conditions at 2 µg/kg, where it must be lower than the MRLs and LOQs.The LOQs of the target pesticides were evaluated via injecting of six fortified blank at 10 µg/kg ≤MRLs level that produce signal, which could be effectively quantified through a precise analytical method and covered via calibration linear range.
The result shows that about 89% of the pesticides recovery ranged from 70% to 120%, while the recovery for 11% of the tested pesticides was less than 70% and 8% with recoveries more than 120% at LOQ of level 10 µg/kg with RSD <20% which was in a good agreement with SANTE-guideline where in the exceptional case due to analyte distribution in the partition step the mean recovery from initial validation outside the range of 70-120% can be accepted if RSD ≤20% [21].

Matrix effect (ME)
The matrix effects of all pesticides over linear range of the calibration set were estimated using matrix matched calibrations and applying the equation using slope data obtained from each five-level calibrations, one of them found that the matrix effect ranged from −31% to 6% for dry Hibiscus syriacus L. In Table S2 summarised the linearity parameters (range, the equation, r 2 ) obtained by using pesticide standards in toluene and dry Hibiscus syriacus L. extracts along with evaluation of the matrix effects measured as 100% × (1slope toluene/slope dry Hibiscus syriacus L. ME% = 100%_ (1 -slope Toluene/slope matrix) [18]

Measurement uncertainty
From the data obtained for Qtype % (average recovery of the three levels of concentration) and pooled RSD % in Table 1, where a significance test was applied to test if the recovery is significantly different from 100.Reproducibility was estimated by pooling the variances of the three different levels of concentration [28].
RSD = Relative standard deviation RSD pooled ¼ ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffiffi The estimation of measurement uncertainty was carried out by applying the approaches that major part of uncertainty could be estimated form recovery and precision calculated form validation data using the following equations [29,30].6.53 is greater than t tab = 2.57), the recovery is statistically significantly different from 100, but in the normal application of the method no correction is applied.The uncertainty should be increased to account for the fact that the recovery has not been corrected.The estimation of measurement uncertainty was carried arising from RSD% (precision) and mean recovery % (trueness) of the method including matrix effects shows that: Combined Uncertainty (U c ) = 19.8%.Therefore, the expanded uncertainty was calculated at a confidence level of 95% and k = 2 and found that Expanded Uncertainty (U exp ) = 40% which did not exceed 50% as ISO/IEC 17025-2017.

Conclusion
Based on the results of this study, a short injection method using GC-EI (+) MS/MS with endpoint back-flush and dynamic-multi-reaction mechanism technique for the simultaneous determination of 176 GC-amenable pesticide residues in dry Hibiscus syriacus L. was achieved and validated.The method linear range was from 2 to 500 μg/L with linear curve regression factor (r 2 ) more than 0.990.The LODs and LOQs (S/N = 10) were 2 μg/kg and 10 μg/kg, respectively.The tested pesticides provide acceptable recoveries ranging from 60% to 102% with relative standard deviations of ≤20% (n = 6) and matrix effect ranged from −31% to 6%.The method expanded measurement uncertainty was estimated to be 40% (coverage factor k = 2, confidence level 95%).The validation results prove that it fit the intended use in accordance with the EC guidelines (SANTE/12682/2019) and can successfully expand the detection efficiency of pesticide residues in dry Hibiscus syriacus L and other dry commodities.

Figure 1 .
Figure 1.Total ion counts for (TIC) dynamic MRM analysis for 176 pesticides at 500 μg/L calibration level in toluene solvent using internal standards.

rn=
Number of samples.

Figure 2 .Figure 3 .
Figure 2. Shows matrix-matched five level calibration data of 176 target pesticides in dry Hibiscus L.

Table 1 .
Performance characteristics: average recovery (Rec± RSD%), relative standard deviation, relative standard deviation pooled (RSD pooled %) and the average recovery (Q.Type%) for three fortification levels of dry Hibiscus syriacus L.