Journal of Chromatography B, 951–952 (2014) 89–95
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of amitraz and its metabolites in whole blood using solid-phase extraction and liquid chromatography–tandem mass spectrometry Hao Guo, Pan Zhang ∗ , Junwei Wang, Jing Zheng Center of Evidence Identiﬁcation, Chongqing Public Security Bureau, Chongqing 400021, China
a r t i c l e
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Article history: Received 30 October 2013 Received in revised form 20 January 2014 Accepted 21 January 2014 Available online 28 January 2014 Keywords: SPE LC–MS/MS Amitraz Whole blood
a b s t r a c t A method was developed for determination of amitraz and its metabolites, N-[2,4-(dimethylphenyl)N -methylformamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA) in whole blood. The analytes were extracted by solid-phase extraction (SPE) using dichloromethane, acetonitrile and methanol (2:1:1) mixture as elute solution. Analysis was performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the positive ion mode using multiple reaction monitoring (MRM) technique. Collision-induced dissociation (CID) of amitraz at the electrospray source in MS/MS was observed in the analytic conditions. The method was validated in human whole blood spiked at three concentration levels. The low limit of detection (LOD) and the low limit of quantiﬁcation (LOQ) for all the analytes were below 0.5 g/L and 2 g/L, respectively. Recoveries were between 90.2% and 104.5%, Bias and relative standard deviation (RSD) were below 15% (n = 6). The good linear relationships were obtained in certain concentration ranges of amitraz and its metabolites. The results demonstrated the method is exclusive, sensitive and accurate, and can be applied in forensic toxicology. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Amitraz (N -2,4-(dimethylphenyl)-N-[2,4-(dimethylphenyl) imino]-N-methyl-methanimidamide) is a formamidine derivative insecticide and acaricide, which has been widely used in agriculture and horticulture for control of ticks and manage mites in animals [1,2]. Amitraz poisoning of human beings occurs in many countries, especially in China, where it is authorized for numerous applications . In the body, it interacts with the ␣-2 adrenocepter and causes a series of symptoms, such as central nervous, respiratory systems depression, bradycardia, hypotension and convulsions [4,5]. The amitraz undergoes a rapid degradation or/and metabolism in the body yielding N[2,4-(dimethylphenyl)-N -methylformamidine (DMPF) which is also used as insecticide, 2,4-dimethylformamidine (DMF), and end product 2,4-dimethylaniline (DMA). The chemical structures of amitraz and its metabolites are shown in Fig. 1. Subchronic toxicity studies with metabolites showed that their toxicity on molar base is comparable to that of the parent compound [6,7]. The incidence of amitraz ingestion has occurred every year in China. During the past ﬁve years, there were ten more amitraz poisoning
∗ Corresponding author. E-mail address: [email protected]
(P. Zhang). 1570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2014.01.027
cases happened in our territory. Due to the death investigation and forensic application, sensitive and reliable analytic method of amitraz and its metabolites is required. Several techniques, such as gas chromatographic–mass spectrometry (GC–MS) [8–10], liquid chromatographic–mass spectrometry (LC–MS) [11–13], high performance liquid chromatography (HPLC) , have been used to analyze amitraz and its metabolites, but most of publications have described the methods in wines, fruits, and beeswax. Only few reports exist for biological matrices: Chou et al. and Saito et al. reported a GC–MS and LC–MS method for quantitatively measuring amitraz and its metabolites in urine and serum, respectively [10,11]. In forensic science practices, whole blood analysis is essential for hemolyzed blood samples . However, there has been no method reported for determination of amitraz and its metabolites in whole blood to the best of our knowledge, In whole blood, the presence of matrix interference could adversely affect analyte quantiﬁcation and identiﬁcation. Therefore, the extract clean-up method is so critical in toxicological analysis that it could avoid the matrix interference and even more, reduce the detection limits. In general, protein precipitation (PP), liquid–liquid extraction (LLE) and solid phase extraction (SPE) are currently used as pretreatment techniques in toxicological analyses . Although PP and LLE are simple methods, inadequate puriﬁcation of samples may cause contamination to the instrument and problems with reliability of quantitative values. While SPE, which is
H. Guo et al. / J. Chromatogr. B 951–952 (2014) 89–95
Fig. 1. Product ion spectra of a standard solution and proposed fragments structures of (a) amitraz, (b) DMF, (c) DMPF and (d) DMA.
a combination of clean-up and exaction procedure, has high selectivity and the extraction may contain fewer interfering substances than after PP or LLE, the procedure for extraction is relatively laborious and time-consuming. In recent years, SPE has been widely used in forensic analysis to separate the analyte in whole blood .
The present work described and validated a sensitive, reliable and speciﬁc method to determine amitraz and its metabolites in human blood samples. The method involves SPE procedure and liquid chromatography–tandem mass spectrometry (LC–MS/MS) determination. The applicability of the proposed method was
H. Guo et al. / J. Chromatogr. B 951–952 (2014) 89–95
demonstrated in qualiﬁcation and quantiﬁcation of amitraz and its metabolites in forensic toxicology. 2. Experimental 2.1. Chemicals and materials LC–MS grade methanol, acetonitrile, ethyl acetate and dichloromethane (DCM) were purchased from Fisher (Germany). Amitraz, N-[2,4-(dimethylphenyl)-N -methyl-formamidine (DMPF), 2,4dimethylformamidine (DMF) and end product 2,4-dimethylaniline (DMA) were purchased from Sigma–Aldrich (St. Louis, MO, US). Deionized water (>18.2 M cm resistivity) was puriﬁed using a Milli-Q system (Millipore, Molsheim, France). Drug-free human whole blood samples were supplied by Chongqing Blood Centre (Chongqing, China). The poisoning blood samples were provided from a hospital and were obtained from subjects suspected of being poisoned by amitraz. 2.2. Sample preparation Initially, an accurately measured volume of 1.0 mL of the whole blood sample was diluted to a ﬁnal volume of 3.0 mL with deionized water. Solid-phase extraction was performed with the Blod Elut C18 cartridges using a Gilson auto-SPE machine. Before use, the cartridges were conditioned with 3.0 mL methanol and 3.0 mL deionized water. The diluted samples were percolated through the cartridges at a ﬂow rate 5 mL/min. The cartridges were then rinsed with 3.0 mL deionized water and vacuum-dried for 10 min to remove excess water. Finally, the retained compounds were eluted with 2 mL dichloromethane, acetonitrile, methanol mixture (2:1:1) and the elution was collected in a test tube. The eluent was evaporated until near dryness by a gentle nitrogen stream with a water batch at 30 ◦ C. Finally, the residue were re-dissolved with 100 L acetonitrile and ﬁltered with 0.22 m PTFE ﬁlter (Millex FG, Millipore, Milford, MA, USA) for LC–MS/MS analysis. 2.3. LC–MS/MS The LC–MS/MS was equipped with a Shimadzu 20A LC system (Shimadzu, Japan) that included an autosampler. Separation was attained on a Waters (Paris, France) Atlantis dC18 (150 mm × 3.9 mm, 5 m) analytical column, preceded by a security guard cartridge dC18 (10 mm × 3.9 mm, 5 m), using a gradient that started at 10% of methanol (A) and 90% of 0.1% formate acid in water (B) held for 1 min. Then B was decreased linearly to 10% in 2 min and held for 5 min, and back to the initial conditions in 1 min which held in 6 min. The ﬂow rate was 0.5 mL/min, and 20 L of standard solutions or extraction were injected. API 2000 tandem mass spectrometry was applied. The Analyst 1.4.1 software was used for MS/MS analyses. The mass spectrometer was operated in the positive ionization mode for the LC–MS/MS analyses. To optimize the MS parameters, the samples of the Amitraz, DMPF, DMF and DMA (1000 g/L) were constantly added at a ﬂow rate of 10 L/min using a syringe pump (maximal volume 1 mL, Hamilton, Bonaduz, Switzerland) in the infusion mode. The ion spray voltage was set at 4 kV and the source temperature was set at 350 ◦ C. Nitrogen was used as a nebulizing gas (GS 1, 60 psi), turbo spray gas (GS 2, 65 psi) and curtain gas (25 psi). The collision-activated dissociation (CAD) was set to a medium level. The declustering potential (DP) was optimized, while the ion spray voltage, nebulizing and curtain gas conditions were used in default mode. The dwell time was set at 0.1 s, and the MS scan was performed in positive ion modes. The product ion spectrum (MS–MS) was generated at optimized DPs to identify the prominent product ions of the analytes using nitrogen as the collision gas. The collision
energies (CE) for the precursor to product ions transition were optimized by CE ramping via direct infusion. For both analytes, multiple reaction monitoring (MRM) mode was used. For the source-CID studies, solutions of amitraz were continuously infused by means of a syringe pump (Harvard, Quebec, Canada) with a microliter syringe at a ﬂow-rate of 10 L/min while ramping the oriﬁce voltage in 5 V steps from 0 to 200 V. Prior to the oriﬁce-ramping experiments, ring voltage (400 V) was optimized using the protonated 1 molecule of amitraz (M+H, m/z 294). The “breakdown curves” were obtained by plotting the ion abundances of molecular ions and fragment ions against the increasing oriﬁce voltage. 2.4. Stock solution, calibrators and quality control standards Amitraz, DMPF, DMF and DMA were accurately weighed, transferred to volumetric ﬂasks and dissolved in acetonitrile to produce individual stock solutions of 1.0 g/L. These solutions were thoroughly mixed and stored at −20 ◦ C in tightly closed bottles until use. Stand working solutions of amitraz, DMPF, DMF and DMA were diluted with acetonitrile in appropriate concentration from mother liquor. The standard working solutions were spiked into drug-free human whole blood sample to create the ﬁnal amitraz, DMF, DMPF and DMA concentration of 2, 10, 20, 50, 200, 1000 g/L. This sequence of spiked human whole blood solutions was considered to the set of matrix-matched calibration standards and three concentration levels of 10, 50 and 200 g/L of amitraz, DMF, DMPF and DMA in whole blood were considered quality control (QC) samples. 2.5. Method validation The analytic method was validated according to the single laboratory validation approaches. The performance of the method was evaluated considering different validation parameters that include the following parameters. Selectivity and matrix effect. Extracted drug-free blood samples from different persons were analyzed to test the selectivity. The extracts were reconstituted with 100 L of acetonitrile containing a known amount of amitraz, DMPF, DMF and DMA at three concentration levels. To investigate the matrix effect (co-eluting undetected endogenous matrix compounds that may inﬂuence analyte ionization), the reconstituted samples were analyzed and the peak areas of the analytes were compared with that in standard solution at the same concentration. The low limit of detection (LOD), the low limit of quantiﬁcation (LOQ) and Calibration curves. The LOD and LOQ were estimated from extracted blank blood sample, spiked with decreasing concentration of the analytes, where the response of the qualiﬁer ion was equal to 3 and 10 times the response of the blank extract, respectively. Once evaluated, three samples were spiked at the estimated levels and extracted according to the proposed procedure. The LOQ was deﬁned in this study as the lowest calibrator. Calibration curves were analyzed by a 1/x weighted linear regression of standard peak area of duplicates based on six standard spiked levels of concentrations in a range of 2–1000 g/L. The calibration curves (y = ax + b) were constructed by the plots of the peak area of the analytes concentrations (x, g/L) of standards. The analyte concentrations of the unknown samples were determined by interpolation of the calibration curves. Accuracy and precision, recovery. The intra-day accuracy and precision of each sample preparation were determined by the injection of three QC samples (10.0, 500.0, 200.0 g/L) in six replicates at the same day, including the extraction procedures. The inter-day accuracy and precision were also determined in a similar manner on the different days. A comparison was made between the obtained
H. Guo et al. / J. Chromatogr. B 951–952 (2014) 89–95
values and the experimental values. Accuracy and precision were expressed as bias (%) and percentage relative standard deviation (RSD %). The mean value of bias should be within 15% of the actual value, and the precision determined at each concentration level should not exceed 15% RSD. The processing recovery of each analyte was determined by comparing the peak areas obtained from QC samples with the spiked standard calibration curves of the same concentration. 3. Results and discussion 3.1. SPE method development
Table 2 MS parameters of the studied compounds. MWa
When tuned with ﬂow injection analysis (FIA) using single standard solution, protonated molecule [M+H]+ were observed at m/z 294, 163,150 and 122 in Q1 full scan for amitraz and its metabolites, respectively. The major fragments observed in MS–MS spectrum of amitraz were at m/z 163, 132 and 122, while Table 1 Recoveries (in %) obtained in the SPE of amitraz, DMPF, DMF and DMA (200 g/L) with different solvents from whole blood using Bond Elut C18 column.
Amitraz DMPF DMF DMA a
49.8a 84.3 72.1 76.3
73.5 89.4 69.4 44.6
90.9 18.5 10.2 65.3
98.4 19.6 15.6 79.8
89.2 85.5 70.1 80.1
Recoveries (in %). The mixture elution was consisted of DCM: acetonitrile: methanol on the ratio of 2:1:1. b
3.2. Mass spectrometry
Among the different pre-treatment approaches, SPE offers a good compromise between convenience and efﬁciency, and is ideally suited to routine forensic analysis. Direct SPE of the samples with no dilution seems difﬁcult, owing to the existence of protein. The SPE step was optimized in terms of dilution of sample before SPE and elution solvent. To meet the objectives for the monitoring of amitraz and metabolites in blood samples, the performance of Bond Elut C18 and Oasis HLB were compared. The efﬁciency of the SPE sorbents was evaluated by spiking 1 mL of blood with 200 ng of each compounds. The adsorption efﬁciencies of four compounds on C18 column were over 90 percents, while the efﬁciencies of DMF and DMA on HLB column were less than 50 percents. Therefore, Bond Elut C18 has been chosen as the SPE sorbent in the pre-treatments steps. The selection of extracting solvent in sample treatment process with a polarity to match the analyte was beneﬁcial to improve efﬁciency and recovery. Methanol, acetonitrile, ethyl acetate and dichloromethane (DCM) were considered for SPE process. The recoveries obtained in the SPE of amitraz, DMPF, DMF and DMA (200 g/L) with different solvents from whole blood were shown in Table 1. From above table we can see that none of the four solvents provided well recoveries for all the analytes: Low recoveries (