Carbaryl and Chlorpyrifos pesticides quantification in honey by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Introduction

Honey has a characteristic sweet flavor with medicinal and therapeutic values has been consumed through ancient times across the world. The nectar collected through bees from flowers is regarded as honey (Codex, 2019; Wakgari & Yigezu, 2021a) [4,15] . The environmental impact for extensive farming has resulted in contamination of honey with pesticides. It may occur through either direct application of pesticide near beehive for treating diseases or the consumption of nectar, guttation fluid and pollen from pesticide contamination. The consumption of such pesticide-contaminated honey has been linked to causing neurological disorders in human beings and it has resulted in immunological disorders and carcinogenic stripping the therapeutic and medicinal of honey (Wakgari & Yigezu, 2021b) [16] . Pesticide exposure is an important consideration in apiculture because honeybees frequently forage in agricultural landscapes where insecticides, fungicides and herbicides are applied. Pesticides may reach bees through direct contact, contaminated nectar and pollen, water sources or environmental drift (Al-Waili et al., 2012; Drugs, 2020; Mukherjee, 2009; Verma & Bhardwaj, 2015) [2,6,10,14] . Residues can subsequently be transported into the hive and potentially become associated with bee products. Consequently, the analytical monitoring of pesticides in honey is relevant both to food safety and to understanding chemical exposure within agricultural ecosystems. Carbaryl and Chlorpyrifos are two insecticides of particular analytical interest. Carbaryl is a carbamate insecticide, whereas Chlorpyrifos belongs to the organophosphorus class. Both compounds are associated with inhibition of acetylcholinesterase and have been widely investigated in the context of pesticide exposure and residue monitoring.

The consumer safety aspect has led to establishing the maximum residue level (MRL) by the governing bodies to ensure permissible safe levels and to prevent the commercialization beyond the established limits (Codex, 2019) [4] . However, the determination of pesticides in honey presents several analytical challenges. Honey contains a high concentration of sugars together with a range of proteins, amino acids, organic acids, phenolic compounds, minerals and other minor constituents. These components may be co-extracted during sample preparation and may interfere with chromatographic separation or affect ionization in electrospray-based mass spectrometry. Consequently, analytical procedures intended for pesticide determination in honey require adequate selectivity and sensitivity. Liquid chromatography coupled with tandem mass spectrometry is particularly suitable for targeted pesticide analysis because chromatographic separation can be combined with compound-specific mass transitions (Tette et al., 2016) [12] . The use of multiple reaction monitoring (MRM) allows sensitive detection of target compounds in complex matrices and has become an established approach for pesticide residue analysis. The present study was therefore to investigate the quantitative response of carbaryl and chlorpyrifos in honey following controlled fortification at four concentration levels. An unfortified honey sample was first analyzed to establish the background response of the matrix. The fortified samples were subsequently analyzed by LC-MS/MS, and the measured concentrations were compared with the corresponding nominal concentrations to determine apparent recovery across a broad analytical range (da Silva et al., 2016) [12] .

Materials and Methods

Honey Sample

The multifloral honey sample was collected directly from a local beekeeper at Nettigere, Karnataka, India. The sample was collected immediately after harvesting, stored in sterile amber glass bottles, transported under ambient conditions, and maintained at 25 °C until analysis.

Chemicals

Analytical-grade carbaryl, chlorpyrifos acetonitrile (ACN), formic acid, ammonium formate, and methanol were obtained from Sigma-Aldrich. Ultrapure water was used throughout the study.

Study Design

The analytical experiment was designed as a controlled spiked study using honey as the sample matrix. The honey (HM1) was initially analyzed for the presence of crabaryl and chlorpyrifos and both were found to be in below quantifiable limits. Following the confirmation of the absence of quantifiable background residues, separate honey potioions were spiked with carbaryl and chlorpyrifos at concentrations of 0.01, 0.1, 1, and 10 ppm each respectively. The resulting analytical samples were designated HM2, HM3, HM4, and HM5 for carbaryl, HM6, HM7, HM8, and HM9 for chlopyrifos. The quantitative response for these four-level concentrations in honey were evaluated.

2.4

Liquid Chromatography-Tandem Mass Spectrometry Analysis A Ultra High Performance Liquid Chromatography (UHPLC) system by Shimadzu Corporation, Kyoto, Japan was used for the Liquid chromatography part of the system. This was comprised of two Shimadzu UHPLC: Nexera UHPLC system Column: Shim-pack (C18-120, 4.6 x 150 mm, 3 µm particle size). The column temperature was maintained at 40 °C and flow rate was 0.7 mL min -1 and injection volume being 10 μL (Tette et al., 2016) [12] . This was coupled with Mass Spectroscopy System (Make: Shimadzu Corporation, Kyoto, Japan, Model: LCMS 8040, Triple Quadrupole). The system has ion spray voltage: + 4.5 kV/-3.5 kV and dwell time of 5 msec with pause time being 1 msec comprising block temperature of 400º C, detector voltage of 1.3kv, nebulizer gas flow rate of 1.5 litre min -1 . This coupled system resulted in Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) system (FSSAI, 2016) [7] .

Sample Preparation for LC-MS/MS Analysis

A 2.5g sample was taken into a 50 mL centrifuge tube. To which 10 milliliters of ACN was added. Tubes were then capped and shaken by vortex for 5 min. To each tube, an Agilent Bond Elut QuEChERS EN extraction salt packet, containing 6 g anhydrous MgSO 4 , 1.5g sodium acetate was added directly (Anastassiades et al., 2003) [3] . Tubes were sealed tightly and shaken vigorously for 5 min by vortex to ensure that the solvent interacted well with the entire sample and crystalline agglomerates were broken up sufficiently. Sample tubes were centrifuged at 4000 rpm for 5 min. 5 mL aliquot of the upper ACN layer was transferred into a centrifuge tube and add Bond Elut powder; the 2 mL tube contained 25 mg of PSA and 150 mg of anhydrous MgSO 4 . The tubes were capped tightly and vortexed for 1 min. The tubes were centrifuged at 4000 rpm for 5 min. Then the supernatant was filtered through 0.22 µ filter and transferred into an autosampler vial. The samples were capped and vortexed thoroughly, to prepare for LC/MS/MS analysis (FSSAI, 2016; Gaweł et al., 2019) [7, 8, ] .

Calculation of apparent recovery

The apparent recovery at each spiked level was calculated by comparing the LC-MS/MS measured concentration with the spiked concentration into honey matrix, which is given by the Equation 1.

Apparent recovery = Measured concentration

Spiked concentration × 100 (Percentage). . .(Eq.1)

Results and Discussion

The initial analysis of the honey (HM1) showed below quantifiable results for both carbaryl and chlorpyrifos. Carbaryl was associated with a retention time of 10.6 min, while chlorpyrifos was associated with a retention time of 17.4 min. No quantifiable concentration was reported foe either analyte (Figure 1 & 2). This demonstrates that the concentrations subsequently measured in HM2-HM9 were associated with deliberate addition of the target pesticides rather than with quantifiable background residues in the honey matrix. The use of residue free matrix is an important component of pesticide residue analysis as it provides a basis for evaluating ananlytical response following spiking (Venu et al., 2019) [13] . Carbaryl was quantified at all four investigated spiked levels (Table 1). For the spiked concentration of 10 µg kg⁻¹, the LCMS/MS was able to quantify 7.84 µg kg⁻¹, giving an apparent recovery of 78.40%. The chromatographic retention time was 10.6 min (Almeida et al., 2020) [1] . At spiked level of 100 µg kg⁻¹, the measured concentration increased to 93.48 µg kg⁻¹, having an apparent recovery of 93.48%. The 1000 µg kg⁻¹ of spiking resulted in carbaryl concentation of 977.62 µg kg⁻¹, with recovery of 97.76% and for the spiking of 10000 µg kg⁻¹, the LC-MS/M yielded 91.45% of recovery (Figure 1). Similarly Chlorpyrifos was quantified at all four spiked levels (Table 1). For 10 µg kg⁻¹, the LC-MS/MS was able to quantify 6.55 µg kg⁻¹, resulting in an apparent recovery of 65.50%. However, the increase in the level of spiking concentration yielded higher recovery (Almeida et al., 2020; Hrynko et al., 2018) [1,9] . As it was observed for 100 µg kg⁻¹ of spiking recovery was 91.91 µg kg⁻¹. The higher spiking levels of 1000 and 10000 µg kg⁻¹ concentrations yielded recovery of 824.51 and 8759.82 µg kg⁻¹ respectively with apparent recovery of 82.45 and 87.60% (Figure 2). The spiking studies demonstrated unique quantitative responses for the two pesticides. Carbaryl exhibited an apparent recovery above 90% at three of the four spiked levels, whereas chlorpyrifos exceeded 90% at only 100 µg kg⁻¹ concentration level. Nevertheless, the two compounds showed broadly similar behavior at the higher concentration levels, with apparent recoveries generally falling between approximately 82 and 98%. The difference at the lowest spiked concentration level may be associated with the greater relative influence of analytical variability at lower analyte concnetrations (Gaweł et al., 2019) [8] . The mean apparent recovery obtained for carbaryl was 90.27%, compared with 81.87% for chlorpyrifos. These results were within the commonly accepted 70-120% recovery interval range at all four spike levels with exception of 10 µg kg⁻¹ concentration for chlorpyrifos (Pihlström et al., 2021 n.d.) [11] . The results demonstrate the feasibility of measuring pesticide concentrations in a high-sugar food matrix using LC-MS/MS (Hrynko et al., 2018) [9] . The honey sample matrix generated measurable and concentration dependent analytical responses. This design provides a direct comparison between the native matrix and pesticide-spiked matrix and minimizes the possibility of attributing the measured residues to naturally occurring contamination. This difference between nominal and measured concentration can therefore be used to evaluate the apparent recovery of the analytical procedure (Hrynko et al., 2018; Pihlström et al., 2021 n.d.) [9,11]

Conclusion

The present study demonstrated the applicability of LC-MS/MS for the targeted quantification of carbaryl and chlorpyrifos in a honey matrix following controlled spiking. The honey sample showed below quantifiable limit for both target pesticides, conforming the absence of quantifiable background residues in the matrix used for analysis. Following spiking, both compounds were successfully quantified over a range of 10-10000 µg kg⁻¹. Carbaryl showed measured concentrations of

References

1. Almeida MO, Oloris SCS, Faria VHF, Ribeiro MCM, Cantini DM, Soto-Blanco B. Optimization of method for pesticide detection in honey by using liquid and gas chromatography coupled with mass spectrometric detection. Foods. 2020;9(10):1368. doi:10.3390/foods9101368.
2. Al-Waili N, Salom K, Al-Ghamdi A, Ansari MJ. Antibiotic, pesticide, and microbial contaminants of honey: human health hazards. The Scientific World Journal. 2012;2012:1-9. doi:10.1100/2012/930849.
3. Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce. Journal of AOAC International. 2003;86(2):412-431.
4. Codex. Revised Codex standard for honey. In: CODEX STAN 12-1981. FAO/OMS; 2019.
5. da Silva PM, Gauche C, Gonzaga LV, Costa ACO, Fett R. Honey: chemical composition, stability and authenticity. Food Chemistry. 2016;196:309-323. doi:10.1016/j.foodchem.2015.09.051.
6. Drugs V. Pesticides and veterinary drugs. 2020.
7. Food Safety and Standards Authority of India. Manual of methods of analysis of foods: water. Food Safety and Standards Authority of India. Pesticide residue. 2016;18.
8. Gaweł M, Kiljanek T, Niewiadowska A, Semeniuk S, Goliszek M, Burek O, et al. Determination of neonicotinoids and 199 other pesticide residues in honey by liquid and gas chromatography coupled with tandem mass spectrometry. Food Chemistry. 2019;282:36-47. doi:10.1016/j.foodchem.2019.01.003.
9. Hrynko I, Łozowicka B, Kaczyński P. Liquid chromatographic MS/MS analysis of a large group of insecticides in honey by modified QuEChERS. Food Analytical Methods. 2018;11(8):2307-2319. doi:10.1007/s12161-018-1208-z.
10. Mukherjee I. Determination of pesticide residues in honey samples. 2009;(July 2008):818-821. doi:10.1007/s00128-009-9772-y.
11. Pihlström T, Fernández-Alba AR, Ferrer Amate C, Erecius Poulsen M, Lippold R, Carrasco Cabrera L, et al. Analytical quality control and method validation procedures for pesticide residues analysis in food and feed SANTE 11312/2021 v2. 2021.
12. Tette PAS, da Silva Oliveira FA, Pereira ENC, Silva G, de Abreu Glória MB, Fernandes C. Multiclass method for pesticides quantification in honey by means of modified QuEChERS and UHPLC-MS/MS. Food Chemistry. 2016;211:130-139. doi:10.1016/j.foodchem.2016.05.036.
13. Venu S, Santhi K, Rawson A, Paranthaman R, Sureshkumar K. Liquid chromatography-mass spectrometry/mass spectrometry method development for the determination of carbaryl residue in honey. Pharmacognosy Magazine. 2019;15(64):205. doi:10.4103/pm.pm_641_18.
14. Wakgari M, Yigezu G. Honeybee keeping constraints and future prospects. Cogent Food & Agriculture. 2021a;7(1). doi:10.1080/23311932.2021.1872192.
15. Wakgari M, Yigezu G. Honeybee keeping constraints and future prospects. Cogent Food & Agriculture. 2021b;7(1). doi:10.1080/23311932.2021.1872192.