1. Introduction
The total consumption of livestock products is rising as the human population and personal incomes increase [
1]. For example, the global consumption of meat (in million metric tons) has increased steadily and rapidly since 1961, reaching approximately 340 million tons in 2018 [
2]. Livestock is exposed to pesticides on consumption of animal feeds made from agricultural by-products, such as cereal grains and straw [
3,
4,
5]. Therefore, greater consumption of livestock products leads to greater exposure to pesticides. Thus, analytical methods for monitoring pesticide residues in livestock products are required to ensure food safety.
Livestock products, such as meat, milk, and eggs, are complex matrices containing fatty materials that may interfere with pesticide extraction. Methods that eliminate interfering materials are critical to improve the efficiency of analytical tests of pesticide products in livestock [
6,
7]. Typical cleanup methods for fatty samples include matrix solid-phase dispersion, freezing and centrifugation, solvent–solvent partitioning, and solid-phase extraction (SPE) [
8,
9]. The quick, easy, cheap, effective, rugged, and safe (QuEChERS) method is a powerful tool for sample preparation and cleanup of agricultural and animal product matrices prior to instrumental analysis of pesticides [
10,
11,
12].
Fenpropimorph,
cis-4-[(
RS)-3-(4-
tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine, is a fungicide exhibiting systemic activity against the fungal diseases of various crops, such as grains and vegetables [
13]. Although fenpropimorph is primarily used for fungal control, unexpected adverse effects of morpholines on sterol biosynthesis have been reported in mammals and higher plants [
14,
15,
16]. These studies thus suggest that residues of fenpropimorph in edible animal and plant products should be monitored continuously to ensure public safety. Fenpropimorph is an enantioenriched chiral chemical that is stereoselectively metabolized by plants [
17,
18]. Although fenpropimorph can usually be metabolized to low-toxicity compounds in plants and livestock [
19], fenpropimorph acid, a typical metabolite, is included in the list of residues important for monitoring and risk assessment [
20]. Thus, evaluation of fenpropimorph residues in livestock products requires an analytical technique that simultaneously determines fenpropimorph and fenpropimorph acid levels.
A number of methods for determination of fenpropimorph in agricultural and insect products have been reported. Gas chromatography–tandem mass spectrometry (GC-MS/MS) has been used after application of modified QuEChERS methods to determine fenpropimorph in agricultural samples [
21,
22]; a buffered organic solvent was used for sample extraction. A GC-MS/MS method was also used for multi-residue analyses of pesticides (including fenpropimorph) in animal feed samples [
23]; organic solvent partitioning and SPE were used for sample preparation. A simpler method of sample preparation (followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS)) was used for multi-residue screening of pesticides (including fenpropimorph) in fruit and vegetable samples; the samples were frozen at −20 °C for 24 h and then subjected to cryogenic milling in dry ice [
24]. More recently, LC-MS/MS analyses coupled with modified QuEChERS methods have been used to complement GC/MS when performing multi-residue analyses of pesticides (including fenpropimorph) in insect and plant samples [
25,
26,
27].
The various methods for fenpropimorph determination were mainly derived based on agricultural samples; few studies employed animal products. Additionally, simultaneous determination of fenpropimorph and fenpropimorph acid in livestock products has not been described. Korea has several pesticide monitoring programs, operated by the Ministry of Food and Drug Safety (MFDS), for domestic and imported livestock products, but the MFDS does not simultaneously measure fenpropimorph and its acid residues. A method of fenpropimorph residue analysis should also be capable of detecting fenpropimorph acid residues because the acid is now included in the list of materials to be monitored.
In this study, we developed an analytical method for simultaneous determination of fenpropimorph and fenpropimorph acid in livestock products using LC-MS/MS. The samples were extracted and cleaned using a modified QuEChERS method to process six different livestock samples prior to LC-MS/MS analysis. The method was validated according to the CODEX guidelines. Inter-institutional validation was also conducted.
3. Materials and Methods
3.1. Chemicals and Reagents
Analytical standards of fenpropimorph (96.2%) and fenpropimorph acid (99.7%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and LGC Standards (Teddington, Middlesex, UK), respectively. All organic solvents used in this study were of HPLC grade, purchased from J.T Baker (Phillipsburg, NJ, USA). Other chemicals were of analytical grade, purchased from Junsel Chemical Co. (Chuo-ku, Tokyo, Japan). QuEChERS kits were obtained from Agilent Technologies (Santa Clara, CA, USA).
3.2. Sample Preparation
The methods for sample extraction and cleanup were developed by modifying a QuEChERS method recommended by the Ministry of Food and Drug Safety, Korea. Milk and egg samples were used as typical samples for establishment of the method. For sample extraction, livestock samples (1 kg) were homogenized thoroughly with dry ice and a portion (5 g) of the samples was transferred into a centrifuge tube (50 mL) containing 0.5 mL of ascorbate buffer solution and acetic acid in acetonitrile (10 mL). The ascorbate buffer solution was prepared by dissolving ascorbic acid (7.5 g) and sodium ascorbate (7.5 g) in water (100 mL). Acetic acid was used at ratios of 1.0–5.0% (v/v) in acetonitrile. The sample mixture was vortexed vigorously for 2 min, and anhydrous MgSO4 (4 g), sodium acetate (1 g), sodium citrate dehydrate (1.0 g) and sodium citrate dibasic sesquihydrate (1 g) were added, followed by shaking vigorously for 2 min. The mixture was then centrifuged at 3500 rpm for 5 min, and the supernatant (15 mL) was placed at −20 °C for 30 min, followed by centrifugation at 3500 rpm for 5 min. An aliquot (1.0 mL) was used for sample cleanup. The sample cleanup was performed using a QuEChERS kit consisting of anhydrous MgSO4 (150 mg), PSA (25 mg), and C18 (25 mg) in a dispersive SPE tube. The sample (1.0 mL) was mixed thoroughly with the kit for 1 min and centrifuged at 8000 rpm for 3 min. An aliquot (0.5 mL) of the supernatant was mixed with formate buffer solution (0.4 mL) and methanol (0.1 mL). The formate buffer solution was prepared by dissolving ammonium formate (100 mM) and formic acid (0.1%, v/v) in water. The sample solution was finally filtered through a fiber membrane filter (0.2 µm, PTFE-H) prior to LC-MS/MS analysis.
3.3. Method Validation
The method validation was conducted in terms of calibration linearity, sensitivity, accuracy, precision, and the matrix effect, according to the CODEX guidelines [
31]. Matrix-matched standard calibrations were performed for quantitative determination of fenpropimorph and fenpropimorph acid. The calibration solutions were prepared by adding blank matrix solutions to standard working solutions as follows: 0.5 mL of blank matrix solution, 0.4 mL of buffer solution, and 0.1 mL of standard working solution with six different concentrations in the range of 12.5 to 500 µg L
−1. The buffer solution consisted of ammonium formate (100 mM) and formic acid (0.1%,
v/
v) in water. The method sensitivity was investigated based on the limit of quantitation (LOQ) value. The LOQ was determined at a signal-to-noise (S/N) ratio of 10:1 and calculated as follows: LOQ (mg kg
−1) = (minimum detectable amount (ng) of analytes/sample injection volume (µL)) × (final sample volume (mL)/sample amount (g)). The accuracy and precision of the method were evaluated by the recovery tests. The recovery tests were performed in five replicates at levels of the LOQ, 2 × LOQ, and 10 × LOQ by investigating the concentration detected and fortified in the livestock samples. The coefficient of variation (CV) of the recovery tests was calculated by considering the average recovery and standard deviation, as previously described [
38]. The matrix effect (ME) of the sample matrices on the standard calibration was calculated as follows [
39,
40]: ME (%) = [(slope of calibration curve in matrix-matched standard slope of calibration curve in solvent standard)/(slope of calibration curve in solvent standard)] × 100.
The developed method was subjected to inter-institutional validation by an official institute of the government of Korea. The inter-institutional validation of the method was performed to examine whether the method could be used as an official method for simultaneous determination of fenpropimorph and fenpropimorph acid in livestock products. For this, the developed method was examined by EABRC.
3.4. Instruments
A Waters model Xevo TQD-MS triple quadrupole MS/MS spectrometer was used for simultaneous determination of fenpropimorph and fenpropimorph acid in the livestock samples. The MS/MS spectrometer was equipped with a Waters model ACQUITYTM UPLC system. The analytical column was a CAPCELL CORE C18 stainless column (Osaka Soda, 150 × 2.1 mm, 2.7 μm thickness). The mobile phase consisted of methanol and water containing 0.1% (v/v) formic acid, and it was flowed at 0.4 mL min−1 as follows: 30% methanol in isocratic conditions for 0.5 min, 60% methanol with a linear gradient for 3.0 min, 80% methanol with a linear gradient for 2.0 min, 80% methanol in isocratic conditions for 1.0 min. The electron spray ionization (ESI) method in positive ion mode was used for the MS/MS analysis. The LC-MS/MS conditions were optimized by adjusting instrumental parameters to obtain good resolution of the target ions.