High-throughput analysis of neonicotinoids and metabolites in urine by LC-MS

Protocol Steps

1. 1
   Relevance of this method development

   Neonicotinoid pesticides (NEOs) are agonists of neuronal nicotinic acetylcholine receptors [1]. Recently, many epidemiological studies have investigated the relationship between exposure to NEOs and neurodevelopmental delay [2,3]. However, most of these studies used questionnaires or assessed residential proximity to places of NEO use as exposure indices. For more detailed assessment of exposure, biomonitoring, i.e., chemical analysis of biological samples to reveal the internal burden, has been performed. Urinary concentrations can be used to assess NEO exposure. Published methods can capture some NEOs and their metabolites; however, they are not optimised for use in a large-scale cohort study involving hundreds of thousands of subjects. A high-throughput and highly sensitive method is required for the affordable measurements of urinary NEOs in such a study. We modified the currently available method [4] by modifying the SPE method and refining the separation of target compounds in the liquid chromatography, which resulted in the improved method performance including accuracy, precision and method reporting limits.

2. 2
   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).Fig. 1Flowchart of sample preparation.Figure 1

3. 3
   Instrument analysis and calculations

   The Nexera X2 system (Shimadzu Corporation, Kyoto, Japan) was used for separation. A Triple QuadTM 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; Waters, Ireland) was used as an analytical column. The LC and MSMS parameters are listed in Table 2, Table 3, Table 4. LC–MSMS run time was 24.5 minutes per sample. Multiple LC–MSMS systems were used parallelly to accelerate the analysis. The total analysis time was estimated to be 26.5 minutes per sample. Typical chromatograms are shown in Fig. 2.Table 2Liquid chromatography conditionsTable 2Time (minutes)Mobile phase A (%): 0.1% formic acid in 10 mM ammonium acetateMobile phase B (%): MethanolProgramme of mobile phase0.019554.0095518.00653518.5029820.5029820.5195524.50955Setting of valves switchingPosition0.00–5.00Waste5.00–19.00Load to tandem mass spectrometer19.00–24.50WasteFlow rate0.4 ml/minInjection volume10 µlSolvent for needle washMethanol:water = 7:3Temperature of column oven50°CTemperature of autosampler4°CRun time24.5 minutesTable 3Mass parameters and LC retention timeTable 3Target ion (m/z)Qualifier ion (m/z)Declustering potential (V)Collision energy (V)Dwell time (milliseconds)Retention time(min)PrecursorProductPrecursorProductTargetQualifierTargetQualifierACE223.2126.0223.299.0505028533015.0TCP253.0126.0253.090.1404027503017.0SUL-A174.0153.9174.0104.1404025373015.3SUL-B174.0153.9174.0104.1404025373015.7FLN230.1203.0230.1174.180802223408.5THX292.0211.0292.0181.1303017304010.1DIN203.0114.2203.0157.0353518112006.6CLO249.9169.2249.9132.0353517213012.5IMI256.3209.1256.3175.2353521263013.2NIT271.2237.1271.299.160602568409.2dm-ACE208.990.1208.999.1404042503014.3TCP-amid271.1126.0271.1228.0606032193014.1dm-CLO236.1132.0236.1155.1303017174010.3IMI-OF254.1204.9254.1171.1505020244011.0ACE-IS229.0132.2--50-30-3015.0TCP-IS259.0132.1--40-35-3017.0SUL-A-IS178.2157.1--40-26-3015.2SUL-B-IS178.2157.1--40-26-3015.7FLN-IS233.1206.2--80-25-408.5THX-IS297.0216.1--30-20-4010.1DIN-IS208.1132.3--35-18-2006.6CLO-IS255.0174.0--35-20-3012.5IMI-IS262.0215.1--35-26-3013.2NIT-IS276.0242.3--60-26-409.2dm-ACE-IS214.190.1--40-45-3014.3TCP-amid-IS277.0234.1--60-21-3014.0dm-CLO-IS241.1133.9--30-21-4010.3IMI-OF-IS259.1210.2--50-23-4011.0ACE, acetamiprid; TCP, thiacloprid; SUL, sulfoxaflor; FLN, flonicamid; THX, thiamethoxam; DIN, dinotefuran; CLO, clothianidin; IMI, imidacloprid; NIT, nitenpyram; IS, stable isotope labelled internal standard; dm-ACE, acetamiprid-N-desmethyl; TCP-amid, thiacloprid-amide; dm-CLO, clothianidin-desmethyl; IMI-OF, imidacloprid-olefin; LC, liquid chromatography.Table 4Ion source and collision cell conditionsTable 4ParameterSettingIonSpray voltage (V)5500Heating gas temperature (°C)450Nebulizer gas (psi)50Heating gas (psi)60Curtain gas flow (psi)20Collision gas pressure (psi)11Fig. 2Typical chromatograms of target compounds. a: native standard solution, b: stable isotope labeled internal standard solution.Figure 2

   1. a.The Nexera X2 system (Shimadzu Corporation, Kyoto, Japan) was used for separation. A Triple QuadTM 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. b.mm × 100 mm,
   3. c.µm; Waters, Ireland) was used as an analytical column. The LC and MSMS parameters are listed in Table 2, Table 3, Table 4. LC–MSMS run time was
   4. d.minutes per sample. Multiple LC–MSMS systems were used parallelly to accelerate the analysis. The total analysis time was estimated to be
   5. e.minutes per sample. Typical chromatograms are shown in Fig. 2.Table 2Liquid chromatography conditionsTable 2Time (minutes)Mobile phase A (%): 0.1% formic acid in 10 mM ammonium acetateMobile phase B (%): MethanolProgramme of mobile phase0.019554.0095518.00653518.5029820.5029820.5195524.50955Setting of valves switchingPosition0.00–5.00Waste5.00–19.00Load to tandem mass spectrometer19.00–24.50WasteFlow rate
   6. f.ml/minInjection volume10 µlSolvent for needle washMethanol:water = 7:3Temperature of column oven50°CTemperature of autosampler4°CRun time
   7. g.minutesTable 3Mass parameters and LC retention timeTable 3Target ion (m/z)Qualifier ion (m/z)Declustering potential (V)Collision energy (V)Dwell time (milliseconds)Retention time(min)PrecursorProductPrecursorProductTargetQualifierTargetQualifierACE223.2126.0223.299.0505028533015.0TCP253.0126.0253.090.1404027503017.0SUL-A174.0153.9174.0104.1404025373015.3SUL-B174.0153.9174.0104.1404025373015.7FLN230.1203.0230.1174.180802223408.5THX292.0211.0292.0181.1303017304010.1DIN203.0114.2203.0157.0353518112006.6CLO249.9169.2249.9132.0353517213012.5IMI256.3209.1256.3175.2353521263013.2NIT271.2237.1271.299.160602568409.2dm-ACE208.990.1208.999.1404042503014.3TCP-amid271.1126.0271.1228.0606032193014.1dm-CLO236.1132.0236.1155.1303017174010.3IMI-OF254.1204.9254.1171.1505020244011.0ACE-IS229.0132.2--50-30-3015.0TCP-IS259.0132.1--40-35-3017.0SUL-A-IS178.2157.1--40-26-3015.2SUL-B-IS178.2157.1--40-26-3015.7FLN-IS233.1206.2--80-25-408.5THX-IS297.0216.1--30-20-4010.1DIN-IS208.1132.3--35-18-2006.6CLO-IS255.0174.0--35-20-3012.5IMI-IS262.0215.1--35-26-3013.2NIT-IS276.0242.3--60-26-409.2dm-ACE-IS214.190.1--40-45-3014.3TCP-amid-IS277.0234.1--60-21-3014.0dm-CLO-IS241.1133.9--30-21-4010.3IMI-OF-IS259.1210.2--50-23-4011.0ACE, acetamiprid; TCP, thiacloprid; SUL, sulfoxaflor; FLN, flonicamid; THX, thiamethoxam; DIN, dinotefuran; CLO, clothianidin; IMI, imidacloprid; NIT, nitenpyram; IS, stable isotope labelled internal standard; dm-ACE, acetamiprid-N-desmethyl; TCP-amid, thiacloprid-amide; dm-CLO, clothianidin-desmethyl; IMI-OF, imidacloprid-olefin; LC, liquid chromatography.Table 4Ion source and collision cell conditionsTable 4ParameterSettingIonSpray voltage (V)5500Heating gas temperature (°C)450Nebulizer gas (psi)50Heating gas (psi)60Curtain gas flow (psi)20Collision gas pressure (psi)11Fig. 2Typical chromatograms of target compounds. a: native standard solution, b: stable isotope labeled internal standard solution.Figure 2
4. 4
   Instrument analysis and calculations

   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.Table 5Ranges of calibration curveTable 5Concentrations (ng/ml)ISIS concentrations (ng/ml)C0C1C2C3C4C5C6C7C8ACE00.00500.0100.0200.0500.100.200.501.0ACE-IS0.20TCP00.00500.0100.0200.0500.100.200.501.0TCP-IS0.20SUL-ASUL-B00.00500.0100.0200.0500.100.200.501.0SUL-A-IS0.4000.00500.0100.0200.0500.100.200.501.0SUL-B-IS0.40FLN00.100.200.401.02.04.01020FLN-IS8.0THX00.0200.0400.0800.200.400.802.04.0THX-IS0.80DIN00.0500.100.200.501.02.05.010DIN-IS2.0CLO00.0500.100.200.501.02.05.010CLO-IS2.0IMI00.0500.100.200.501.02.05.010IMI-IS2.0NIT00.0500.100.200.501.02.05.010NIT-IS2.0dm-ACE00.0500.100.200.501.02.05.010dm-ACE-IS4.0TCP-amid00.0100.0200.0400.100.200.401.02.0TCP-amid-IS0.40dm-CLO00.200.400.802.04.08.02040dm-CLO-IS16IMI-OF01.02.04.0102040100200IMI-OF-IS40ACE, acetamiprid; TCP, thiacloprid; SUL, sulfoxaflor; FLN, flonicamid; THX, thiamethoxam; DIN, dinotefuran; CLO, clothianidin; IMI, imidacloprid; NIT, nitenpyram; dm-ACE, acetamiprid-N-desmethyl; TCP-amid, thiacloprid-amide; dm-CLO, clothianidin-desmethyl; IMI-OF, imidacloprid-olefin; IS, stable isotope labelled internal standard.

Materials & Reagents

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