A simultaneous, high-throughput and sensitive method for analysing 13 neonicotinoids and metabolites in urine using a liquid chromatography–tandem mass spectrometry

A simultaneous, high-throughput and sensitive method for analysing nine neonicotinoid pesticides (NEOs) and four metabolites (NEOms) in urine using a liquid chromatography-tandem mass spectrometry (LC–MSMS) was developed. The method detection limit (MDL) and lowest concentration minimum reporting limit (LCMRL) of the nine NEOs were 0.0013–0.048 ng/ml and 0.0050–0.17 ng/ml, respectively. The MDL and LCMRL of the four NEOms were 0.0052–0.52 ng/ml and 0.011–1.6 ng/ml, respectively. Intermediate precision for the nine NEOs and four NEOms was 7.5–12.5% and 7.4–10.9%, respectively. Accuracy for the nine NEOs and four NEOms was 3.83–5.60% and 3.01–29.2%, respectively. The developed method was applied to analyse urine samples collected from participants of a large-scale birth cohort study, namely, the Japan Environment and Children's Study (JECS). • The NEO and NEOm concentrations in 100 µl urine samples were analysed using a highly sensitive LC–MSMS.• Automated solid phase extraction in a 96-well plate was utilised to achieve high-throughput analysis.• Intermediate precision and accuracy were less than 12.5% and 94.8–99.1%, respectively.


a b s t r a c t
A simultaneous, high-throughput and sensitive method for analysing nine neonicotinoid pesticides (NEOs) and four metabolites (NEOms) in urine using a liquid chromatography-tandem mass spectrometry (LC-MSMS) was developed. The method detection limit (MDL) and lowest concentration minimum reporting limit (LCMRL) of the nine NEOs were 0.0013-0.048 ng/ml and 0.0050-0.17 ng/ml, respectively. The MDL and LCMRL of the four NEOms were 0.0052-0.52 ng/ml and 0.011-1.6 ng/ml, respectively. Intermediate precision for the nine NEOs and four NEOms was 7.5-12.5% and 7.4-10.9%, respectively. Accuracy for the nine NEOs and four NEOms was 3.83-5.60% and 3.01-29.2%, respectively. The developed method was applied to analyse urine samples collected from participants of a large-scale birth cohort study, namely, the Japan Environment and Children's Study (JECS).
• The NEO and NEOm concentrations in 100 μl urine samples were analysed using a highly sensitive LC-MSMS. • Automated solid phase extraction in a 96-well plate was utilised to achieve high-throughput analysis. • Intermediate precision and accuracy were less than 12.5% and 94.8-99.1%, respectively.

Sample preparation
The sample preparation method was based on our previous study [4] and modified to achieve high-throughput analysis. The major modification was automation of sample preparation using Microlab STAR (Hamilton Company, Reno, NV, USA) and EDR-384SX (BIOTEC Co., Ltd., Tokyo, Japan). First, 10 μl of IS solution and 10 μl of 50% methanol in water was added to 100 μl of each urine sample. Then, 600 μl of acetonitrile was added to precipitate proteins and centrifuged at 4°C, 2000 × g for 1 minute. An ISOLUTE® HYDRO DME + 400 mg plate (Biotage, Uppsala, Sweden) was prewashed with 100 μl of acetonitrile and centrifuged at 4°C, 1000 × g for 1 minute. The supernatants of samples were loaded onto the plate and centrifuged at 4°C, 1000 × g for 1 minute. The samples were evaporated to dryness with a centrifugal vacuum concentrator in combination with the TurboVap 96-well system (Biotage) at 45°C. Residues were dissolved with 200 μl of 5% methanol in 0.1% formic acid and 10 mM ammonium acetate and mixed for 30 seconds. From the final sample, 10 μl of each eluate was injected into a liquid chromatograph-tandem mass spectrometer (LC-MSMS) ( Fig. 1 ).

Instrument analysis and calculations
The Nexera X2 system (Shimadzu Corporation, Kyoto, Japan) was used for separation. A Triple Quad TM 6500 mass spectrometer (AB Sciex LLC, MA, USA) was used to detect and quantify target analytes. The MSMS system was operated using electrospray ionisation positive mode, and multiple reaction monitoring was performed. An ACQUITY UPLC HSS T3 (100 Å, 2.1 mm × 100 mm, 1.8 μm;       The calibration range is shown in Table 5 . Sulfoxaflor (SUL) was a diastereomer. The two isomers were quantified separately as SUL-A and SUL-B. All samples that fell outside the calibration range were reanalysed following further dilution.   NIT, nitenpyram; dm-ACE, acetamiprid-N-desmethyl; TCP-amid, thiacloprid-amide; dm-CLO, clothianidin-desmethyl; IMI-OF, imidacloprid-olefin; MDL, method detection limit; LCMRL, lowest concentration minimum reporting limit; IS, stable isotope labelled internal standard; R 2 , coeficient of determination.

Method validation/quality control (QC)
A pooled urine sample was prepared using maternal samples collected from volunteers to create QC samples. Levels of analytes varied in actual samples, so we tried to create the QC sample to reflect such concentrations. The QC samples had the concentration of dinotefuran around 2 ng/ml. To double the concentration, NEOs and NEOms were fortified such that the concentrations of ACE, TCP, SUL-A, SUL-B, FLN, THX, DIN, CLO, IMI, NIT, dm-ACE, TCP-amid, dm-CLO and IMI-OF were 0.5, 0.5, 0.5, 0.5, 10, 2.0, 5.0, 5.0, 5.0, 5.0, 5.0, 1.0, 20 and 100 ng/ml, respectively. The QC samples were subjected to the same procedure as the unknown samples, with five replicates analysed in each analytical sequence. The lowest concentration minimum reporting limit (LCMRL) was calculated following the U.S. Environmental Protection Agency's instructions [5] and summarised in Table 6 . The method detection limits (MDLs) in the current study were comparable with those in a previous study [6] , except that the MDLs of ACE, DIN and CLO were 2-100 times lower than those obtained using previous methods [ 6 , 7 ].
The eight-point calibration curves showed coefficients of determination (R 2 ) higher than 0.990. Repeatability and intermediate precision were determined based on ISO 5725:1994 and 27148:2010, with QC sample measurements (n = 100). QC for day-to-day analysis was determined using a Shewhart control chart ( ̄ -R control chart) according to ISO 7870. Intermediate precision was less than 12.5% ( Table 6 )  ACE, acetamiprid; TCP, thiacloprid; THX, thiamethoxam; CLO, clothianidin; SD, standard deviation; NMIJ, National Metrology Institute of Japan. 1 Certified concentration (expanded uncertainty concentrations) [ng/ml]: a unit of mass fraction of certified values was converted into concentration values using the value of specific gravity (1.0125 g/cm 3 ).
(0.47) ng/ml for ACE, CLO, TCP and THX, respectively, were analysed in nine replicates. All measured concentrations fell within the certified ranges ( Table 7 ).

Ethics statements
Not applicable.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.