Journal of Chromatography B, 972 (2014) 102–110

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Ultra performance liquid chromatography-tandem mass spectrometry for the determination of amicarthiazol residues in soil and water samples Wen-jun Guia , Jie Tianb , Chun-xia Tiana , Shu-ying Lia , You-ning Maa , Guo-nian Zhua,∗ a b

Institute of Pesticide and Environmental Toxicology, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, Zhejiang, China Zhenjiang Institute of Termite Control, 65 Yunhe Road, Zhengjiang 212003, China

a r t i c l e

i n f o

Article history: Received 7 December 2013 Accepted 21 September 2014 Available online 28 September 2014 Keywords: Amicarthiazol Solid phase extraction Ultra performance liquid chromatography-tandem mass spectrometry Soil Water

a b s t r a c t A reliable and rapid method has been optimized to determine the residue of amicarthiazol in soil and environmental water samples. After extraction and evaporation, the extraction was carried out with solid phase extraction (SPE) cleanup using HLB cartridge (only soil samples) and for the quantitative determination by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The resulting residues of amicarthiazol were analyzed by a gradient separation performed on a UPLC system with a C18 column, methanol and water containing 0.1% (v v−1 ) formic acid as the mobile phase in the mode of electrospray positive ionization (ESI+ ) and multiple reaction monitoring (MRM). Results showed that the recoveries for spiked samples were 74.4–97.1% and 72.1–109.9% for soil and water, respectively, with the relative standard deviation (RSD) less than 10.2% when fortified at 10, 100 and 1000 ␮g L−1 . The limits of detection (LODs) and the limits of quantification (LOQs) for matrix matched standards ranged from 0.073–0.425 ␮g L−1 and 0.243–1.42 ␮g L−1 . The intra-day precision (n = 5) and the inter-day precision over 10 days (n = 10) for the amicarthiazol in soils and water samples spiked at 100 ␮g L−1 was 7.9% and 15.9%, respectively. Results indicated that the developed method could be a helpful tool for the controlling and monitoring of the risks posed by amicarthiazol to human health and environment safety. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Amicarthiazol (2-amino-4-methyl-carboxanilithiazol) (Fig. 1), a kind of amide fungicides (bactericide), contains both groups of carboxylamide and thiazole, possessing not only the biological activities of carboxylamide fungicides for controlling basidiomycetous diseases but also those of thiazole bactericides against bacterial diseases [1]. As a systemic fungicide, amicarthiazol is widely applied on rice, wheat, cotton, citrus and tobacco in China [2]. This fungicide was generally applied as seed treating agent, despite that several formulations of it were registrated as plant spraying agents recently [3]. Frequent application of this fungicide poses a potential risk to either environmental bio-species [4] or human beings. However, to the best of our knowledge, the maximum residue limits (MRLs) of amicarthiazol is still not wellestablished globally.

∗ Corresponding author. Tel.: +86 57186971220; fax: +86 57186430193. E-mail address: [email protected] (G.-n. Zhu). http://dx.doi.org/10.1016/j.jchromb.2014.09.022 1570-0232/© 2014 Elsevier B.V. All rights reserved.

Traditionally, the analytical methods for the analysis of residual amicarthiazol in capsicum, soil [5,6] and tobacco seedlings [2] samples were developed using high performance liquid chromatography (HPLC), requiring several steps and large quantities of solvents for sample preparation and extraction of the analytes. Recently, Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MSMS) was frequently adopted to analyze trace amounts of pesticides in environmental matrices. Neverthless, studies with respect to analysis of amicarthiazol by this high-performance technique is poorly reported. Solid-phase-extraction (SPE) method, as is known, can be used to extract and pre-concentrate a wide array of compounds [7] and reduce matrix effects (signal suppression or enhancement) in a single fashion for the UPLC-MS/MS analysis [8]. Another advantage of SPE is that it can be automated, either off-line [7] or on-line [9,10], greatly reducing the involvement of manual operation and thus increasing the reproducibility of extractions through tight control of variables, such as flow rates, solvent volumes, and equilibration and drying times [11].

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Table 2 Gradient elution of UPLC.

Fig. 1. Chemical structure of amicarthiazol.

Time (min)

Flow rate (mL/min)

Eluent Aa (%)

Eluent Bb (%)

Curvec

Initial 2.5 3.0 3.5

0.2 0.2 0.2 0.2

40 20 20 40

60 80 80 60

Initial 1 6 6

a

Eluent A: 0.1% (v/v) formic acid in ultrapurefied water. Eluent B: methanol. Mixing curve of mobile phases for Waters HPLC system, 1 means “immedieately goes to specified conditions” and 6 is a default value which means “linear effect”. b c

Nowadays, LC-MS/MS is becoming one of the powerful techniques for the residue analysis of polar, ionic or low volatility fungicides in fruits and vegetables [12]. Especially HPLC-MS/MS has proven to be a powerful tool for trace analysis due to its high selectivity, precision and sensitivity [13]. Among the several ionization modes, electrospray ionization (ESI) has proven to be a reliable, robust and sensitive mode [14–16]. In MS/MS the use of multiple reactions monitoring (MRM) mode permits a significant decrease in the detection limits, owing to the increased signal-to-noise ratio. In contrast to conventional HPLC, a recently developed technology termed UPLC has been applied to pesticide residue detection, and provided a higher peak capacity and a faster speed of analysis [17,18]. Here, a rapid, sensitive and reliable method for analysis of amicarthiazol residues in soil and water was developed. As far as we know, this was the first report of quantitative analysis of amicarthiazol residues in the environment using UPLC-MS/MS.

2. Experimental 2.1. Reagents and materials Analytical standard of amicarthiazol (99.5% purity) was obtained from Dr. Ehrenstorfer (Augsburg, Germany). HPLC-grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Formic acid was obtained from Tedia (Fairfield, USA). Water was processed with a Milli-Q water purification system (Millipore Corp, USA) and used to prepare all aqueous solutions. Five soil and five water samples from different locations were used in the present study. All samples were free of amicarthiazol residue by HPLC. Five soil samples were collected from Wuxi (Jiangsu province, paddy soil), Quzhou (Zhejiang province, red soil), Harbin (Heilongjiang province, black soil), Jiaxing (Zhejiang province, moisture soil) and Hangzhou (Zhejiang province, powder soil). Samples were air dried and then passed through a 2 mm sieve for removal of particles and non-decomposed plant residues. The prepared soil samples were stored at room temperature during the studies. The properties of the five soil series are presented in Table 1. Five water samples were taken in different regions: lake water (from West Lake, Hangzhou, pH 6.5), river water (from Tiesha river, Hangzhou, pH 6.0), pond water (from Qizhen lake, Hangzhou, pH 6.0), spring water (from Hupao Spring, Hangzhou, pH 5.5), and tap water (pH 6.8). All water samples were collected in amber polyethylene terephthalate (PET) bottles and transported to the laboratory at 4 ◦ C in the dark.

2.2. Instruments Centrifugation was performed in an Anke DL-5-B centrifuge (Shanghai flying pigeon company, China), Mechanical shaking extraction was used a constant temperature incubator shaker (Shanghai zhicheng analysis instrument manufacturing Co., Ltd., China). The rotary evaporator RE-2000 was purchased from Yarong biochemical instrument plant (Shanghai, China). 2.3. Preparation of standard solutions Standard stock solution (1000 mg L−1 ) was prepared by accurately dissolving 10.0 mg of the analyte in 10.0 mL of methanol and stored at 4 ◦ C for no more than three months. The working solutions of amicarthiazol were prepared weekly by series dilution of stock solution in methanol at the concentrations ranging from 5 ␮g L−1 to 100 mg L−1 for sample fortification or optimization of experimental conditions. In order to optimize the extraction conditions and in the validation study in different concentration levels from 5 ␮g L−1 to 1000 ␮g L−1 , the calibration standards at the concentrations 5, 10, 50, 100, 200, 500 and 1000 ␮g L−1 were constructed by re-dissolution of matrix extracts of control samples in working standards directly. 2.4. UPLC-MS/MS analysis Chromatographic analyses were conducted by using an Acquity Ultra Performance LC (Waters, USA). The analyte were separated on an ACQUITY UPLC® HSS T3 column (2.1 mm × 100 mm, with a 1.8 ␮m particle size. Waters, USA). The mobile phases consisting of eluent A (0.1% formic acid in ultrapure water) and eluent B (methanol) were used with a gradient elution (shown in Table 2). The injection volume was 10 ␮L, and the column temperature was maintained at 30 ◦ C. Mass spectrometric detection was carried out by using a Triple quadrupole 5500 mass spectrometer (Applied Biosystems Sciex, USA) in positive multiple reaction modes (MRM). The instrument was equipped with an electrospray (ESI) ionization source. Typical ESI parameters were used as follows: ion spray voltage (IS), 3500 V; Atomization air pressure (GS1), 40.00 psi; Auxiliary gas (GS2), 50.00 psi; Curtain gas (CUR), 20.00 psi; Ion source temperature (TEM), 450 ◦ C; Collision activated dissociation (CAD), 5.00 V; Entrance potential (EP). 3.00 V; Declustering potential (DP), 10.00 V and product ions at m/z 115 and 141, optimum collision energies were 28 eV, 30 eV, respectively. Instrument control and data

Table 1 Characterization for the five soils. Resource

Texture class

pH

Sand (%)

Silt (%)

Clay (%)

Organic carbon (%)

CECa (cmol kg−1 )

Harbin Wuxi Jiaxing Hangzhou Quzhou

Black soil Paddy soil Moisture soil Powder soil Red soil

7.80 6.18 7.52 6.80 4.20

39.7 10.1 7.6 21.5 44.8

43.9 80.7 69.3 71.1 44.0

16.4 9.2 23.1 7.4 11.2

2.94 2.95 5.02 3.10 0.93

22.2 14.8 21.3 10.6 21.4

a

CEC: cation exchange capacity.

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acquisition and evaluation were performed with the AB Sciex Analyst 1.6 software (Applied Biosystems, USA). 2.5. Sample pretreatment 2.5.1. Water sample To a mixing cylinder with stopper which was pre-loaded with 2 g of NaCl and 8 g of MgSO4 , 20 mL of filtrated water sample was transferred. Then, 20 mL of acetonitrile was added and shaken vigorously for 3 min and kept still for stratification. Subsequently,

10 mL of clarified supernatant (acetonitrile) was separated and decompressed evaporated to dryness at 45 ◦ C on a water bath. The residue was re-dissolved by 2 mL of methanol, then filtered through a 0.22 ␮m one-off filter membrane and undergone for UPLC-MS/MS analysis.

2.5.2. Soil samples To 20 g of soil sample 70 mL of mixed solution (methanol/water = 5/1, v v−1 ) which containing 0.03% (v v−1 )

Fig. 2. Full scan and MS/MS spectra corresponding to different collision energies at 100 ␮g L−1 of amicarthiazol (Product ion spectrum of the [M + H]+ ion of amicarthiazol (precursor ion m/z 235), collision energy from top left to bottom right: 10, 15, 20, 25, 28, 30, 35 and 40 eV).

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ammonia was added. After shaken for 30 min using a horizontal shaker (Shanghai zhicheng analysis instrument manufacturing Co., Ltd., China)) at 180 rpm (revolutions per minute) in room temperature, the mixture was centrifuged (Anke DL-5-B, Shanghai flying pigeon company, China) at 4000 rpm for 5 min. An aliquot of 35 mL of the supernatant was separated and transferred into a 250 mL separatory funnel which was preconditioned with 10 mL 10% sodium chloride (w v−1 ) and extracted three times with ethyl acetate (50, 40, 30 mL). The resulting supernatant was mergered and decompressed evaporated to dryness on a water bath at 45 ◦ C. The residue was dissolved in 10 mL of mixed solution (methanol/water, 2:8, v v−1 )) to prepare the crude extract and then subjected to SPE clean-up.

2.6. Solid-phase extraction In order to obtain the best recoveries, the following sorbents were tested: C18 (3 mL × 200 mg, Waters, USA), Cleanert ODS C18-N (3 mL × 200 mg, Agela Technologies, USA), OASIS HLB (3 mL × 60 mg, Waters, USA), Strata-X-C (3 mL × 60 mg, Phenomenex, USA), Cleanert COOH (3 mL × 60 mg, Agela

105

Technologies, USA), HyperSep Retain-CX (3 mL × 60 mg, Thermo Scientific, USA). 2.6.1. Initially selected SPE protocol C18, C18-N, HLB cartridges were conditioned with 3 mL of methanol and equilibrated with 3 mL of 20% (v v−1 ) methanol/water. Strata-X-C, Cleanert COOH, HyperSep Retain-CX cartridges were conditioned and equilibrated with the same procedure except that the water contained 1% (v v−1 ) of additional formic acid. To study the retention capacity of the amicarthiazol on the various sorbents, the break-through recoveries were tested, which were carried out at 1000 ␮g L−1 . The loading of cartridges were performed using standard solution which was diluted with 20% (v v−1 ) of methanol/water and 20% (v v−1 ) of methanol/water containing 1% (v v−1 ) formic acid, respectively. 2.6.2. Final SPE protocol After initial optimization, an OASIS HLB cartridge (3 mL × 60 mg) was selected. The wash and elution solvents and volumes were optimized during the method development stage for the cartridge with the best retention capacity. Prior to loading of sample

Fig. 3. MRM spectra of amicarthiazol (200 ␮g L−1 .) at different constituents (Left: 0.05% (v v−1 ) (top), 0.1% (v v−1 ) (middle)), and 0.2% (v v−1 ) (bottom) formic acid; Right: 0.05% (v v−1 ) (top), 0.1% (v v−1 ) (middle) and 0.2% (v v−1 ) (bottom) ammonia water.

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solution, the cartridge was equilibrated using 3 mL 20% (v v−1 ) of methanol. 10 mL of the standard (1000 ␮g L−1 , diluted with 20% (v v−1 ) methanol/water) was transferred to the top of the cartridge. The cartridge was washed with 2 mL 50% (v v−1 ) of methanol/water, then followed by eluting with 5 mL 100% of methanol. The eluent was dried under a stream of nitrogen on a water bath at 45 ◦ C. The residue was re-dissolved by 2 mL methanol, then filtered through a 0.22 ␮m one-off filter membrane and undergone for UPLC-MS/MS analysis. 2.7. Validation procedure Validation was performed according to the National Standard of the People’s Republic of China (GB/T 5009.1-2003) [19], agricultural industry standard of the People’s Republic of China (NY/T 788-2004) [20], and OECD guidance document on pesticide residue analytical methods (ENV/JM/MONO (2007) 17) [21]. The analytical figures of merit determined were the limit of detection (LOD), limit of quantification (LOQ), linearity, matrix effect and precision. The LOD and LOQ were defined as the concentrations that will give signal/noise ratios of 3 and 10, respectively. Linearity was studied using matrix-matched calibrations by analyzing five working standards (between 5 and 1000 ␮g L−1 ) in triplicate. The matrix effect in the present work was calculated by comparing the slope of the standards curve in solvent with matrix-matched standards curve. Mean recoveries and precision were determined by analyzing spiked samples in quintuplicate at three spiking levels. The intra-day and inter-day precision was defined in terms of the relative standard deviation (RSD). The intra-day precision was determined within one day by analyzing 5 replicates soils samples spiked with the amicarthiazol at 100 ␮g L−1 . The inter-day precision was determined on 10 separate days using 10 replicated soils samples spiked at the above concentration.

cartridges, retention may be expected based on the polarity of the analyte. Additional retention due to the presence of ionizable functional groups may play an important role when cartridges are used. C18, C18-N and Oasis HLB cartridges were selected due to their reversed-phase capabilities; Strata-X-C, Cleanert COOH, and HyperSep Retain-CX cartridges were selected due to their cationexchange capabilities, which retained an amino group very well. Initial testing of a variety of SPE cartridges showed that the OASIS HLB cartridge had the best retention capacity of the amicarthiazol (Fig. 4A). while the HLB have a special polar-hook for enhanced capture of polar analyte and excellent wettability, the retention capacity could be improved when aqueous standard solution containing 20% (v v−1 ) methanol/water was transferred to the top of the cartridge. To study the retention of a compound on the various cationexchange sorbents, the cartridges should be used in their ideal equilibrium conditions. As a matter of fact, it was difficut to optimize the ideal condition. Commonly, amicarthiazol, which contains an amino group, was expected to retained very well on the cation-exchange cartridge. However, the retention capacity of Cleanert COOH (weak cation-exchange) was very poor, and StrataX-C (mix strong cation-exchange), HyperSep Retain-CX (strong cation-exchange) cartridges yielded a high break-through recoveries (28.2% and 41.8%, respectively). The reason might be that the acidity of the eluent was not strongly enough for these three cationexchange cartridges retenting the analyte. As Xu [22] showed that

3. Results and discussion 3.1. Optimization of UPLC-MS/MS Standard solution of 100 ␮g L−1 was prepared in methanol/ water (50:50, v v−1 ) for MS optimization by infusion experiments. The cone voltage and collision energy were optimized by constant infusion of amicarthiazol at a rate of 7 ␮L min−1 . The precursor ion selected in the first quadrupole was submitted to cone voltage, which was varied from 0 to 100 V to find the maximum response for amicarthiazol. The precursor ion obtained was [M + H]+ at m/z 235 when the cone voltage was 30 V; Collision-induced dissociation (Fig. 2) produced dominant product ions at m/z 115 and 141, and the optimum collision energies were 30 eV, 28 eV, respectively. The MRM mode was selected for this investigation. The signal intensity in LC-MS can be strongly influenced by the mobile phase composition. In order to select a LC eluent composition that would provide an overall optimum response for MS detection, six different constituents (only added into water phase) were tested: 0.05% (v v−1 ), 0.1% (v v−1 ) and 0.2% (v v−1 ) ammonia water, 0.05% (v v−1 ), 0.1% (v v−1 ) and 0.2% (v v−1 ) formic acid. The results (Fig. 3) indicated that the addition of 0.1% formic acid and 0.1% ammonia water in water phase both gave the satisfied signal response in positive ESI mode, but the latter gave a very poor peak shape, even a split peak for amicarthiazol. So the final choice of mobile phase was methanol/water containing 0.1% formic acid. 3.2. Optimization of the SPE clean-up protocol The first procedure of the SPE development focused on finding an appropriate sorbent which retained the amicarthiazol. A rapid sorbent screening of 6 different sorbents ranging from hydrophobic to polar and ion-exchange were performed. On most

Fig. 4. Optimization of sample clean up using SPE. A: Break-through recoveries of analyte using various SPE cartridges (1000 ␮g L−1 , n = 2); B: Elution profile of analyte for HLB cartridge (1000 ␮g L−1 , n = 3); C: Elution curve of analyte for HLB cartridge (1000 ␮g L−1 , n = 3).

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Fig. 5. MRM spectra of amicarthiazol in different water samples (left column: blank sample, middle column: matrix-matched standards (50 ␮g L−1 ); right column: spiked water samples (10 ␮g L−1 ); The type of water sample from top to bottom: tap water, lake water, river water, pond water and spring water).

amicarthiazol has a better stability in an alkaline or a weak alkaline medium, while there was not such stability in acidic medium. The eluent with strong acidity hence was not applied at the present study. The organic solvent strength of the eluent was optimized by using 2 mL per time of mixture (methanol/water) with an increment interval of 10% methanol from 20% to 100% (all v v−1 ). Result (Fig. 4B) showed that no break-through was observed when the crude extract (10 mL) was applied to the SPE cartridge. Amicarthiazol was almost all retained on the cartridge when methanol was less than 50%, then it was gradually eluted form 0 to 100% when methanol was increased in eluent. So, prior to elution with 100% methanol, a washing step of 50% (v v−1 ) methanol/water (2 mL) was set to move interferents. To estimate the amount of

remaining amicarthiazol on the cartridge a series elution steps with 2 mL methanol per time, collected individually, were incorporated (Fig. 4C). In the first elution step, the recovery was somewhat lower (total of 35%), the second elution resulted in a total recovery of more than 90%. Around 3% of amicarthiazol was eluted in the last three steps, so an elution step of 5 mL 100% methanol was incorporated in the final method. 3.3. Validation 3.3.1. Accuracy Recoveries and repeatability experiments were established in order to evaluate the methods’ trueness and precision, respectively. Five types of water samples (lake water, river water, pond water,

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Table 3 The recoveries and precisions for the different amicarthiazol-spiked samples (n = 5). Matrix matched standard curve equation

R2

Matrix effecta

Linear range (␮g L−1 )

S/N ratiob

LODc (␮g L−1 )

LOQc (␮g L−1 )

8.4 5.2 1.1

y = 3111.8x + 3020.5

1

0.69

5–1000

41.5

0.36

1.20

75.5 86.7 85.2

9.4 7.4 4.9

y = 4405x + 34424

0.9994

0.98

5–1000

61

0.25

0.82

10 100 1000

78.5 83.0 92.6

10.2 7.2 5.4

y = 2269x + 2313.1

0.9992

0.505

5–1000

53.8

0.28

0.93

Red soil

10 100 1000

97.1 93.4 93.6

1.4 5.7 2.9

y = 2703.2x + 4445.7

0.9999

0.602

5–1000

46.8

0.32

1.07

Powder soil

10 100 1000

74.4 84.8 87.3

10.0 6.7 4.2

y = 3764.9x + 39512

0.9973

0.84

5–1000

35.3

0.425

1.42

Tap water

10 100 1000

92.9 83.8 102.5

11.5 7.26 1.7

y = 4577.1x + 51830

0.997

1.02

5–1000

150.3

0.099

0.332

Lake water

10 100 1000

81.3 81 98.1

3.7 6.2 4.9

y = 4652.7x + 39392

0.9996

1.04

5–1000

205.6

0.073

0.243

Spring water

10 100 1000

80.1 75.45 94.78

5.45 1.83 9.0

y = 2681x + 11651

0.9989

0.597

5–1000

74

0.203

0.676

River water

10 100 1000

81.79 89.02 94.3

3.8 8.4 1.7

y = 2439.4x + 27170

0.9975

0.543

5–1000

57.3

0.262

0.873

Pond water

10 100 1000

72.14 83.85 109.9

5.2 5.1 6.0

y = 2575.1x + 18251

0.995

0.573

5–1000

64.7

0.232

0.772

Recovery (%)

Sample name

Spiked level (␮g kg−1 )

Black soil

10 100 1000

92.0 90.1 89.4

Paddy soil

10 100 1000

Moisture soil

a b c

RSD (%)

The matrix effect was defined as the ratios of the slopes from standard curves in matrixes and in solvent. S/N ratios were calculated at the matrix matched standard of 5 ␮g L−1 . The LODs and LOQs were calculated on the basis of a peak − peak signal-to-noise (S/N) value that was S/N = 3 and 10 at the matrix matched standard of 5 ␮g L−1 .

spring water and tap water) and soil samples (black soil, paddy soil, moisture soil, powder soil and red soil) were used as blank sample for spike and recovery experiments. Table 3 showed the recoveries of amicarthiazol in soils and water matrixes. As could be seen, the mean recoveries were 74.4–97.1%, 72.1–109.9% for soil and water, respectively. Figs. 5 and 6 displayed the typical chromatograms obtained for different control samples and spiked samples at the concentration level of 10 ␮g kg−1 (or L−1 ). Although a peak appeared at retention time of the standard in the blank profile, but it could be obviously found that the S/N of the peak was less than 3 for almost all water and soil samples (except lake water, S/N around 4), which could be ignored. For lake water, the S/N was more than 200 at 5 ␮g/L (lowest limit of linearity) of matrix-matched standard. So, even if the peak (S/N around 4) in the blank profile meant the analyte, it could not disturb real sample analysis. Hu et al. [2] reported a residual analysis method of amicarthiazol in tobacco by HPLC coupled with SPE extraction. The lowest fortified level was 1 ␮g g−1 for root, leaf and stem samples but the LOD was not given. The retention time of the analyte was around 7 min. The residues of amicarthiazol in capsicum field and capsicum were analyzed by WANG et al. [6]. The lowest fortified concentration was 0.225 mg kg−1 for both capsicum field and capsicum samples. The LOD was 0.0035 mg kg−1 . The retention time of the analyte was around 3.5 min. Compared to the lowest fortified level and LOD, the present study gave a higher sensitivity (nearly 20–100 folds and 8–48 folds for lowest fortified level and LOD, respectively) than that of the previous reports. Additionally, the retention time of the established assay was shorten than the that of the previous

assays, which could promote the efficiency of amicarthiazol residue analysis. 3.3.2. Intra-day and inter-day precision The intra-day precision (n = 5) at 100 ␮g kg−1 spiked concentration was 4.7%, and the inter-day precision over 10 days (n = 10) was 15.9%. Result showed good reproducibility and precision of the developed method. 3.3.3. Linearity and matrix effect Two calibration curves, named “standard” and “matrixmatched”, were constructed. Standard solutions were prepared by diluting appropriate volumes of the working standard solution within the range of 5–1000 ␮g L−1 (five concentration levels). Matrix-matched calibration standards were constructed by redissolved of matrix extracts of control samples in working standards directly. Good linearities were achieved in all cases with correlation coefficients better than 0.99. A significant drawback in the MS analysis performed with electrospray (ESI) as ionization technique is the appearance of matrix effect (ME). This occurs due to the high sensitivity of ESI source to different components present in the matrix, which can lead to signal suppression or enhancement, thereby leading to false quantitative results [23]. The matrix effects in the present work were calculated by comparing the slope of the standards curve in solvent (A) with matrix-matched standards curve (B). The ME value (expressed in %) calculated as: ME (%) = B/A × 100. ME value >100 indicates enhancement whereas those