LC-MS/MS
119
120
Materials and Methods
Chemicals and Consumables
HPLC grade methanol (MeOH) and acetonitrile (ACN) was purchased from Fisher Scientific (Seoul, South Korea), whereas formic acid (purity > 99.7%) and ammonium formate (purity ≥ 99%) were from Sigma-Aldrich (St. Louis, MO, USA). QuEChERS salts packets containing 4 g of magnesium sulfate (MgSO4) and 1 g of sodium chloride (NaCl), dispersive SPE tubes containing 25 mg of primary and secondary amine (PSA) and 150 mg of MgSO4, and dSPE containing GCB (2.5 and 7.5 mg) were obtained from Restek (Bellefonte, PA, USA).
High purity of analytical reference (309 compounds) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA), Chemservice (West Chester, PA, USA), Wako (Osaka, Japan), Dr. Ehrenstorfer (Augsburg, Germany), and Ultra Scientific (North Kingstown, RI, USA). Individual pesticide stock solutions of 1000 µg/mL (100 µg/mL for carbendazim) were prepared in ACN or MeOH, considering each of the purity. After making a composite standard mixture containing 309 pesticides at concentration of 5 µg/mL by combining aliquot of individual stock solution, working standard solutions at the concentration of 1, 2, 5, 10, 20, 50, 100, 500, and 1000 µg/mL were prepared by serial dilution using ACN. For multiple reaction mornitoring (MRM) optimization, 1 µg/mL of individual standard solutions were also prepared in ACN. All standard solutions were kept at -20°C.
LC-MS/MS instrumentation
LC-MS/MS analysis was performed on a Shimadzu LCMS-8050 triple-quadrupole mass spectrometer (Kyoto, Japan) coupled with Nexera X2
ultra-121
high pressure liquid chromatograph. A Phenomenex Kinetex C18 analytical column (10 cm × 2.1 mm i.d., 2.6 µm particle size) with 40 °C of column oven temperature was used for separation. The methanol-based mobile phase consisted of water (A) and methanol (B) containing 5mM ammonium formate and 0.1% formic acid was compared with acetonitrile-based mobile phase consisted of water (A) and acetonitrile (B) containing 0.1% formic acid with the following gradient program. Initially, the organic solvent mobile phase (B) was hold at 5% for 0.5 min, ramped to 55% B in 0.5 min, followed by a linearly increased to 95% B over 7 min, held for 3 min. Finally, it was ramped again to 100% B over 1 min, decreased to 5% B in 0.1min and maintained for 2.9 min (A total run time was 15 min). The flow rate was 0.2 mL/min and injection volume was 5 µL.
A scheduled multiple reaction monitoring mode using fast switching between positive and negative mode in electrospray ionization was employed to apply a large number of LC-MS/MS-amenable pesticides. The temperature parameters for heated ESI were interface temperature of 300°C, desolvation line (DL) temperature of 250°C, and heat-bock temperature of 400°C. The flow rate parameters for heating (air), nebulizing (N2), and drying gas (N2) were 10, 3, and 10 L/min (air), respectively. After automatic optimization procedure of MRM transitions, the best quantifier, qualifier ion, and collision energies (eV) were optimized by injections of individual compounds (1 µg/mL).
Final sample preparation procedure
The modified QuEChERS method based on ACN containing 0.1% formic acid for extraction solvent, that was previously validated was used for sample preparation procedure (Lee et al., 2017). An amount (10.0 ± 0.1 g) of homogenized samples (brown rice, orange, and spinach) by dry ice were
122
weighed into 50 mL of centrifuge tube. For brown rice, 5.0 ± 0.1 g were used and 5 mL of deionized water was added, and then soaking for 30 min. ACN (10 mL) containing 0.1% formic acid was added for extraction and vigorously shaken for 1 min on a Geno Grinder (1600 miniG SPEX Sample Prep, Metuchen, NJ, USA) at 1500 rpm. To minimize the heat release causing by moisture absorbing with MgSO4, the tubes were cooled in an ice bath for a while. Furthermore, 4 g of anhydrous MgSO4 and 1 g of NaCl were added into the tube and shaken for another 1 min. After centrifuged at 3500 rpm (5 min), the supernatant (1 mL) was transferred into a dispersive SPE tube (2 mL) containing 150 mg of anhydrous MgSO4 and 25 mg of PSA sorbent. The tubes was mixed on vortex mixer for 1 min before centrifugation at 15000 rpm (5 min). The supernatant (400 µL) were transferred into 2 mL of amber vial and added ACN (100 µL) for LC-MS/MS injection.
Validation of analytical method
Recovery experiments were carried out to validate the analytical method on brown rice, orange, and spinach sample. The Five replicates at two concentrations (10 and 50 ng/g) were conducted by fortifying pesticide mixture on each of three commodities. The trueness and precision of the optimized method were determined using average recovery rate (%) and relative standard deviation (RSD, %) respectively. Concentrations of each analytes were calculated by matrix-matched calibration to compensate matrix-induced signal enhancement or suppression. The matrix-matched standards for calibration (1, 2, 5, 10, 20, 50, and 100 ng/g) were prepared by adding the solvent standard solution to blank extracts, which was prepared with same procedure. Limit of quantitation (LOQ) was defined as the minimum concentration achieving the signal-to-noise (S/N) ratio of above 10 for quantifier ion in the solvent-only
123
standard calibration curve. Linearity of calibration curves were also evaluated by matrix-matched calibration.
Matrix effect
Matrix effects (ME, %) were was calculated by comparing the peak response of 100 ng/g within the matrix-matched standards (brown rice, orange, and spinach) and solvent-only standards using the following equation:
ME, % = �peak area of matrix matched standard
peak area of solvent − only standard − 1� × 100
A negative value of matrix effect indicates signal suppression, a positive value indicates signal enhancement in matrix contained environment.
124
Results and discussion
MRM optimization
To achieve best signal intensity in LC-MS/MS, the MRM transitions were optimized by injection of individual standard solution (1000 ng/g) without passing through an analytical column. First of all, a full scan spectrums of each compounds were obtained in the mass range of 50 to 1000 m/z using quadrupole 3 (Q3) scan with the switching positive/negative ionization. Considering the signal intensity, the most abundant ion were selected as a precursor ion for each of analytes. Most of the pesticides were easily ionized by positive mode, forming (M+H)+ ion, whereas ammonium adduct form of (M+NH4)+ were chosen as precursor ion in the ten pesticides (e.g., oxamyl, flumiclorac-pentyl, butafenacil, and deltamethrin). The sodium adducts forming (M+Na)+ were only found for two pesticides (butocarboxim and pyribenzoxim). Eighteen compounds (e.g., bentazone, haloxyfop, lufenuron, and hexaflumuron) were more suitable for negative ionization mode than positive mode. Then, different collision energies were automatically tested to obtain the corresponding product ions with higher sensitivity at the range of 0-50 eV. The highest transition in sensitivity was used for quantifier and the second most selective transition for qualifier. The detail MRM transitions of each pesticides including retention times are listed in Table S4 in the supplemental data online.
125
Selection of mobile phase
Ammonium formate has been widely used mobile phase additives as a donor of ammonium ion. With the limitation that low solubility in acetonitrile, most of method use ammonium formate with methanol for mobile phase. Even though it has been reported that ammounium formate with acetonitrile is capable to apply for mobile phase (Bordin et al., 2017), the heating is needed to dissolve ammonium formate. Also, from our experience, the insoluble ammonium formate residue could potentially block spray needle in LC-MS/MS, leading to increase the maintain cost. On the other hand, acetonitrile is often used for mobile phase with advantage of strong elution strength and providing lower pressure in analytical column.
Because methanol is capable of applying ammonium formate and acetonitrile is not, the effects on peak sensitivity in different combinations of mobile phase were studied. The result was compared by relative peak area as shown in Figure 18. Many compounds showed higher intensities in methanol-based mobile phase, whereas only 44 compounds had higher peak area in acetonitrile-based mobile phase. Average 250% of peak area were increased in based mobile phase. Relatively high peak response of the methanol-based mobile phase could be attributed to increased ionization efficiency by the addition of ammonium formate. As expected, the molecules that was ionized to ammonium adducts showed greatly reduced peak area by the absence of ammonium formate. Moreover, four pesticides, i.e. cycloprothrin, deltamethrin, famoxadonen, and lactofen were not even detected in acetonitrile-based mobile phase. On the basis of these results, methanol and water containing 5 mM annonium formate and 0.1% formic acid each were selected as mobile phase for LC-MS/MS analysis.
126
Optimization of injection volume
The relationship between injection volume and precision was studied to acquire reliable quantitation. To eliminate deviation on data processing, representative 180 compounds that gave symmetric peak with distinct signal intensity were selected. Figure 19 summarizes the average peak area of target compounds and repeatability results obtained from different concentration and injection volumes. Higher precision with low relative standard deviation (RSD) were observed as the injection volume and concentration increased. In the injection of 0.5 ng/g, relatively higher RSD were observed owing to sensitivity problem and only 10 µl injections were generally satisfied the RSD value under 10. Most of peak area increased as much as injection volume increased, but not at the same proportion in 10 uL injection. The peak area were increased by 2.5 times when the injection volume of 2 uL increased to 5 uL, whereas the peak area in 10 uL injection were not increased by 2 times than those of 5 uL injections. The results were likely due to ion suppression phenomenon caused by large amount of ions in ionization process (Stahnke et al., 2012). Although large injection volume is one of the effective approach to provide low detection limit, it could lead to frequent maintenance of LC-MS/MS like cleaning source unit. We selected the injection volume of 5 uL in order to eliminate possibility of poor linearity in calibration curve as well as avoid carry-over problem that was previously discussed(Charalampous et al., 2015; Hughes et al., 2007).
127
Figure 18. Relative peak area of the methanol-based mobile phase (A) compared with acetonitrile-based mobile phase (B) at solvent standard mixture of 100 ng/g (n = 5, 100 ng/g). The graph were plotted against the number of pesticides ranked by relative peak area of (A).
128
0 100 200 300 400 500 600 700 800 900 1000
1 51 101 151 201 251 301
Realative peak area, %