A Sensitive Multiresidue Method for the Determination of Pesticides in Marijuana by Liquid Chromatographytandem Mass Spectrometr

Home , Chlorantraniliprole

Journal of Chromatography A, 1603 (2019) 231–239

Contents lists available at ScienceDirect

Journal of Chromatography A

j ournal homepage: www.elsevier.com/locate/chroma

A sensitive multiresidue method for the determination of pesticides

in marijuana by liquid chromatography–tandem mass spectrometry

a,b a a,∗

Daniela Daniel , Fernando Silva Lopes , Claudimir Lucio do Lago

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, Butantã, CEP 05508-000, São Paulo, Brazil

b ◦

Agilent Technologies Brasil Ltda, Av. Marcos Penteado de Ulhoa Rodrigues, 939 - 6 andar Castelo Branco Ofﬁce Park, Torre Jacarandá, CEP 06460-040,

Barueri, Brazil

a r a

t i c l e i n f o b s t r a c t

Article history: A multiresidue method based on QuEChERS extraction followed liquid chromatography coupled with

Received 23 April 2019

tandem mass spectrometry was developed and validated for the determination of 42 pesticides in mar-

Received in revised form 27 June 2019

ijuana. Less than 6 min is required for detection of all species. By using original QuEChERS, the sample

Accepted 3 July 2019 −1

preparation is also fast and simple. In the range from 1.0 to 50 ␮g kg , the coefﬁcients of determination

Available online 4 July 2019 2

(r ) were greater than 0.980, and relative standard deviations for replicate injections were lower than

−1

4.6%. The limit of detection (LOD) and limit of quantiﬁcation (LOQ) were lower than 0.32 ␮g kg and

Keywords: −1

1.07 ␮g kg , respectively. Precision and accuracy were veriﬁed through recovery of spiked samples at

Pesticides −1

␮

Marijuana three distinct levels of concentration (1.0, 5.0, and 50.0 g kg ) in ﬁve replicates. Recovery values ranged

QuEChERS from 82 to 119% with RSD lower than 6%. The method was applied to the detection of pesticide residues

Tandem mass spectrometry in six marijuana samples seized by the Police of Rio de Janeiro, Brazil, where imidacloprid, metazachlor,

buprofezin, and metalaxyl were found in four of them.

1. Introduction addition, pesticides are to a greater or lesser degree harmful to

humans [5,6]. The toxic effects of the pesticides in cannabis can

Non-medical cannabis, known as marijuana, is a psychoactive cause extra health problems for marijuana users with immuno-

drug commonly used for recreational purposes and is the most con- logical problems, liver diseases, or any other health issue [7].

sumed illicit substance in the world [1]. Despite of the global trend Furthermore, the risk of possible formation of more toxic products

toward marijuana legalization for medical or recreational uses, the than the pesticides themselves during the heating of the material

legality of marijuana varies from country to country [1–3]. Although when smoked should be considered [8]. Therefore, serious health

there is a lack of direct ofﬁcial information about the illegal global issues can be expected for chronic marijuana users who most likely

marijuana market, it is noticeable by indirect indicators, such as the inhale substantial amounts of pesticides. Although there is some

number of seizures, that this has become greater every year. The regulation and control for the use of pesticides in countries where

2018 UNODC (United Nations Ofﬁce on Drugs and Crimes) World cannabis has medicinal or recreational use liberated [9,10], the fact

Drug Report estimates a production of 4682 tons of cannabis per is that there is no control on the use of pesticides on illegal cannabis

year and a population of 192.2 million consumers worldwide [1]. crops and their levels may be underestimated.

As in any other crop, this growing supply of cannabis cannot The ﬁrst report in the scientiﬁc literature about the presence of

be dissociated from the use of pesticides to control disease and pesticide residues in marijuana occurred in 1978 [11], but because

increase productivity. The use of pesticides in any kind of crop is it is an illegal market few information is still available. The ﬁrst

a widespread practice and the same occurs for cannabis, even for studies about pesticides in marijuana dealt primarily only with

indoors plantation [4]. the presence of paraquat, which was analyzed by reversed-phase

In this regard, pesticides proﬁle can be used to track down the paired-ion high performance liquid chromatography [12,13].

illegal market, similarly to the adulterants proﬁle for cocaine. In Gradually the number of pesticides as well as the analytical tech-

niques used for their detection in marijuana has been updated.

Gas and liquid chromatographies coupled to mass spectrometry

∗ besides capillary electrophoresis with UV detection are some of the

Corresponding author.

E-mail address: [email protected] (C.L. do Lago).

https://doi.org/10.1016/j.chroma.2019.07.006

232 D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239

Table 1

Retention time (tR) and MS/MS acquisition parameters used for the identiﬁcation and quantiﬁcation of pesticides in marijuana by LC–MS/MS.

a b c d

Compound # tR (min) Q1 (m/z) Q3 (m/z) CE (V) FE (V) 152.2* 12

Aminocarb 1 0.751 209.1 105

137.2 24 160.1* 16

Carbendazim 2 0.775 192.1 105

132.1 32 125.0* 10

Methamidophos 3 0.825 142.0 85

94.0 10 95.0* 20 Acephate 4 0.825 184 70

49.0 24 211.1* 8 Thiamethoxam 5 0.961 292.0 85

181.1 20 106.0* 0

Methomyl 6 0.994 163.1 50

88.0 4 208.9* 12 Imidacloprid 7 1.095 256.0 80

175.0 12 126.0* 12

Acetamiprid 8 1.104 223.1 80

56.1 27 198.8* 0 Dimethoate 9 1.119 230.0 70

125.0 16 199.0* 4

Flonicamid 10 1.119 230.1 110

125.0 16 144.1* 16

Spiroxamine 11 1.243 298.3 100

100.1 32 116.2* 0

Aldicarb 12 1.414 208.1 70

89.1 12 95.0* 16 Flutriafol 13 1.747 302.1 90

70.1 56 145.2* 4 Carbaryl 14 1.944 202.1 65

127.1 28 220.1* 10 Metalaxyl 15 2.089 280.2 95

45.1 36 106.9* 20

Pyrimethanil 16 2.224 200.1 120

82.0 25 210.1* 4

Metazachlor 17 2.425 278.1 70

134.2 15 452.9* 8

Chlorantraniliprole 18 2.512 483.9 105

285.9 16 301.1* 20 Dimethomorph(E) 19 2.552 388.1 145

165.1 32 169.0* 32

Fludioxonil 20 2.766 247.0 95

126.0 32 302.2* 36 Spirotetramat 21 2.770 374.2 120

216.1 36 91.1* 36

Cyprodinil 22 2.901 226.1 140

76.9 50 372.1* 8

Azoxystrobin 23 2.986 404.1 110

344.1 24 307.1* 16

Boscalid 24 3.149 343.0 145

272.1 32 97.1* 20

Fenhexamid 25 3.165 302.1 130

55.1 40 124.9* 40

Tebuconazole 26 3.239 308.1 100

70.0 47 198.2* 4

Bifenazate 27 3.298 301.1 95

170.1 16 159.0* 20

Hexaconazole 28 3.408 314.1 95

70.1 30 126.9* 5

Malathion 29 3.447 331.0 80

99.0 10 198.0* 20

Chlorpyriphos 30 3.588 349.9 100

197.8 20 330.0* 12

Fipronil 31 3.754 435.0 70

250.0 28 337.0* 10

Difenconazole 32 3.861 406.1 120

251.0 20 205.0* 12

Cyhexatin 33 3.879 369.2 72

81.2 28 158.0* 15

Hexaﬂumuron 34 4.137 461.0 120

141.0 45 163.1* 20

Pyraclostrobin 35 4.137 388.1 95

193.8 8 169.1* 32 Diazinon 36 4.249 305.1 105

97.0 40 201.1* 5

Buprofezin 37 4.401 306.2 105

116.1 10 186.0* 12

Triﬂoxystrobin 38 4.446 409.1 110

D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239 233

Table 1 (Continued)

a b c d

Compound # tR (min) Q1 (m/z) Q3 (m/z) CE (V) FE (V)

145.0 52 196.9* 36

Quinoxyfen 39 4.647 308.0 115

161.9 45 252.1* 8 Benfuracarb 40 4.795 411.2 95

162.1 40 227.9* 8

Hexythiazox 41 5.070 353.1 90

168.1 24 366.2* 12 Fenpyroximate(E) 42 5.137 422.2 135

138.1 32

Precursor ion (Q1).

Fragment ions (Q3). c

Collision energy. d

Fragmentor energy.

More intensity MRM transition used for quantiﬁcation purposes.

Fig. 1. Distribution of recoveries (A) and matrix effect (B) for the targeted pesticides in the two evaluated methods.

techniques employed in the detection of pesticides in marijuana literature between original QuEChERS and its buffered versions for

[4,14–17]. the determination of pesticides in marijuana samples.

Several methods of extraction were used for the determina- Herein, an extremely sensitive and reliable analytical method

tion of different classes of pesticides in vegetal matrices, such based on original QuEChERS and liquid chromatography–tandem

as solid-liquid extraction (SLE) and solid phase extraction (SPE) mass spectrometry for the determination of 42 pesticides in mar-

[18]. Extraction using the QuEChERS (Quick, Easy, Cheap, Effective, ijuana samples is introduced – nine of them are reported for the

Rugged and Safe) method introduced by Anastassiades et al. [19] ﬁrst time.

has been shown to be the most effective and efﬁcient in extracting

pesticides residues in a wide variety of matrices, including mar-

ijuana [14,17]. The QuEChERS method has become popular every 2. Material and methods

day because of its successful ability to extract polar compounds and

to be easily adapted to improve recoveries of pH-dependent com- 2.1. Chemicals, materials, and standards

pounds, for example, in many complex matrices [20,21]. However,

although buffered versions of QuEChERS improve the results for Acetonitrile (J.T. Mallinckrodt Baker Inc., Phillipsburg, NJ, USA),

pesticides affected by pH [22], there is no comparative study in the LC-MS grade, was used throughout the work to prepare mobile

phase, standard solutions and samples. A Certiﬁed Reference Mate-

234 D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239

rial (CRM) of a LC/MS Pesticide Comprehensive Test Mix (ULTRA sodium acetate and 6.0 g of anhydrous magnesium sulfate were

Scientiﬁc’s ISO 9001, Rhode Island, USA) composed by aminocarb, added. The tube was capped tightly, shaken in vortex again for 1 min

carbendazim (azole), methamidophos, acephate, thiamethoxam, and centrifuged at 5000 rpm for 5 min. The QuEChERS clean-up step

methomyl, imidacloprid, acetamiprid, aldicarb, azoxystrobin, ben- and all other subsequent steps were the same described before.

furacarb, bifenazate (D 2341), boscalid (nicobifen), buprofezin,

carbaryl, chlorantraniliprole, chlorpyriphos, cyhexatin, cyprodinil,

2.3. Instrumental

diazinon (dimpylate), difenconazole, dimethoate, dimethomorph

(E), fenhexamid, fenpyroximate (E), fipronil, flonicamid, flu-

An Agilent UHPLC 1290 Inﬁnity II chromatographic system –

dioxonil, ﬂutriafol, hexaconazole, hexaﬂumuron, hexythiazox,

composed of binary pump, automatic injector, column thermostat,

malathion, metalaxyl, metazachlor, pyraclostrobin, pyrimethanil,

and sampler – coupled to a triple quadrupole mass spectrometer

quinoxyfen, spirotetramat, spiroxamine, tebuconazole, and tri-

−1 LC–MS/MS 6470 (Agilent Technologies, CA, USA) was used to deter-

ﬂoxystrobin (100.0 ± 0.2 ␮g mL each) in acetonitrile was

mine pesticide residues, using AJS (Agilent Jet Stream) ion source

obtained from Agilent Technologies (Santa Clara, CA, USA) and

◦ in the positive mode. LC separation was performed on a ZORBAX

stored at −15 C. A standard stock solution of the pesticides were

× ␮

−1 RRHD Eclipse Plus C18 column (2.1 mm 150 mm, 1.8 m) (Agilent

prepared at the concentration of 1000 ␮g L each in acetonitrile

◦ Technologies, CA, USA) column and the column oven temperature

and stored at −15 C, while standard solutions were prepared daily ◦

was set at 40 C. The mobile phase consisted of (A) 0.1% formic

by appropriate dilution of different aliquots of the stock solution

acid in water and (B) acetonitrile and the following elution pro-

with acetonitrile.

gram was used: started at 50% B where it was held for 0.5 min

All aqueous solutions were prepared with 18 M·cm deionized

and linearly increasing to 95% B for 4 min, after that 95% B was

water (Milli-Q Direct, Millipore, Molsheim, France) and formic acid

maintained for 2 min, being the initial ratio resumed and maintain-

(> 99%) and acetic acid (> 96%), both bought from Supelco (Belle-

ing this condition for 1 min for column reconditioning. The elution

fonte, PA, USA). The QuEChERS kits used for the extraction and

conditions were optimized using a mobile phase ﬂow of 0.3 mL

cleanup of marijuana samples were obtained from Agilent Tech- −1

min . The injection volume was 1 ␮L and the oven temperature

nologies (CA, USA). The used instruments also included a centrifuge ◦

was 40 C. MassHunter software (Agilent Technologies, CA, USA)

Hettich Mikro 220 (Beverly, MA, USA) and a Shimadzu AUW220D

was used for data acquisition and processing. The mass spectrom-

scale with resolution of 0.01 mg (Kyoto, JP). One of the marijuana ◦

eter parameters used were: gas temperature 300 C, gas ﬂow 10 L

samples, in which none of the pesticides were found, was used as −1 −1

min , nebulizer 20 psi, sheath gas ﬂow 10 L min , sheath gas tem-

blank matrix. ◦

perature 300 C, capillary voltage 4.0 kV (positive mode), and nozzle

0.5 kV. Tandem MS detection was performed using the dynamic

2.2. Sample preparation

Multiple Reaction Monitoring (MRM) mode by time segment, with

2 transitions being monitored for each analyte, in order to obtain at

Six sample of marijuana were provided by the Service of Foren-

least 4 identiﬁcation points. The most intense transition was used

sic Chemistry of the Carlos Éboli Institute of Criminalistics (Rio de

for quantiﬁcation and the other ones were used as qualifying ions.

Janeiro, Brazil). The method validation – including recovery and

Table 1 lists the monitored ions, the MS/MS acquisition parame-

calibration studies – was performed with the same samples, which

ters used for the identiﬁcation and quantiﬁcation of the pesticides

were checked for pesticide residues. All samples were manually

residues in seized marijuana, as well as the retention time of each grinded.

target analyte.

2.2.1. Original QuEChERS

A 1.0 g aliquot of dry homogenized sample was placed into 50- 3. Results and discussion

mL polypropylene (PP) disposable centrifuge tube. Then, a ceramic

homogenizer and 10.0 mL of Milli-Q water were added, and the 3.1. Comparison of the QuEChERS methods

sample was left in contact for 10 min. Afterwards, 10.0 mL of ace-

tonitrile was added and vigorously shaken in vortex for 1 min. Two well-known sample preparation methods were evaluated

For the QuEChERS extraction step, 1.0 g of sodium chloride and for two ﬁgures of merit: recovery and matrix effect. One of the

4.0 g of anhydrous magnesium sulfate were added. The tube was methods is the original QuEChERS method and the other one is the

capped tightly, shaken in vortex again for 1 min and centrifuged at buffered AOAC 2007.01 QuEChERS [19,20], which was used in all

5000 rpm for 5 min. The QuEChERS clean-up step was performed by the previous works about the determination of pesticide residues in

placing a 1.0 mL aliquot of the supernatant from the previous step marijuana by HPLC-MS [14,17,21]. For instance, Perez-Parada et al.,

into a 2-mL PP tube containing 25.0 mg of PSA and 150 mg of MgSO4. evaluated three different buffered QuEChERS extractions (acetate

The tube was capped, shaken in vortex for 1 min and centrifuged at buffered, citrate buffered, and a modiﬁed citrate buffered QuECh-

5000 rpm for 5 min. After that, a 0.5 mL aliquot of the supernatant ERS) for the determination of pesticides in marijuana [21]. Although

was diluted with mobile phase 1:1 (v/v), ﬁltered through a 0.2 ␮m 61 pesticides were tested, recovery (7–120% range) and accuracy

PTFE membrane, and analyzed. For the blank spiked samples, dif- (< 20%) for buffered citrate medium were reported for only 46 ana-

ferent amounts of pesticide mix were added to the blank marijuana lytes.

sample before addition of water and left in contact for 10 min. After The best approach for compensating the matrix effect is the

that the same protocol was followed. use of isotopically labeled internal standards, but it is not prac-

ticable in multi-residue analysis. A more appropriate approach is

2.2.2. Acetate buffered QuEChERS to use matrix-matched calibration curves [23,24]. Therefore, mar-

This sample preparation was performed according to AOAC- ijuana samples were previously analyzed to verify the absence of

−1

2007.1 [20]. A 1.5 g aliquot of dry homogenized sample was placed the studied pesticides and then they were fortiﬁed at 50 ␮g kg

into 50-mL polypropylene (PP) disposable centrifuge tube. Then, for the tests. Matrix effects were evaluated by comparison of the

a ceramic homogenizer and 15.0 mL of Milli-Q water were added peak areas of the fortiﬁed samples with those ones from standard

and the sample was left in contact for 10 min. Afterwards 15.0 mL of solutions of same concentration.

glacial acetic acid in acetonitrile was added and vigorously shaken Recoveries and matrix effects are shown in Fig. 1. AOAC 2007.01

in vortex for 1 min. For the QuEChERS extraction step, 1.5 g of QuEChERS showed a more spread proﬁle of recoveries than the

D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239 235

original QuEChERS method, which was centered around 100% 3.2.1. Speciﬁcity

recoveries. An enhancement effect due to the matrix was observed The speciﬁcity was evaluated through the analyses of diverse

for metalaxyl, ﬂudioxonil, quinoxyfen, and ﬁpronil in both sam- marijuana samples, which were compared to blank samples spiked

−1

ple treatments, while the bifenazate presented the enhancement with the mix of pesticide standards at 10 ␮g kg . The method

effect only using the original QuEChERS method. Signal suppression was considered speciﬁc because no signiﬁcant interference was

– which is mainly caused by the competition between the analytes detected at migration time, molecular ion and its two fragments, as

and the co-extractives during the electrospray ionization [25,26] – well as in the ratio between relative abundance of fragment ions.

was observed for all other pesticides. The number of analytes sub- All 42 pesticides determined can be differentiated from the other

ject to a severe suppression (matrix effect > 50%) is smaller for the compounds present in the samples as demonstrated in Fig. 2.

unbuffered method.

The pH of the extract in original version was 6.5 while the pH

of the AOAC version was 5.0, which suggests that a greater amount

3.2.2. Linearity

of co-extractives is obtained at lower pH. Therefore, the original

The linearity of the matrix-matched calibration curves was

QuEChERS method was the method selected for validation.

studied at seven different concentration levels ranging from 1 to

−1

50 ␮g kg in ﬁve replicates each level. The calibration curves were

obtained by plotting the peak area ratios against to the concen-

3.2. Original QuEChERS method validation trations. The developed method proved to be homoscedastic and

the pesticide concentrations were calculated from the simple linear

The validation of the method was performed by evaluating regression equation established by the least squares method. The

selectivity, linearity, limit of detection (LOD), limit of quantiﬁcation determination coefﬁcients (r ) obtained from the analytical curves

(LOQ), intra/inter-assay precision, accuracy, recovery, and stability for all 42 pesticides were> 0.996. The parameters are summarized

of the analytes [27,28]. in Table 2.

Table 2

Analytical Figures of Merit.

Proposed method Other methods Pesticide

2 −1 −1 −1 a −1 b −1

y = a + bx r LOD (␮g kg ) LOQ (␮g kg ) GRL (␮g kg ) LCL (␮g kg ) LOQ (␮g kg )

Aminocarb y = 4374x + 11178 0.998 0.07 0.24 ND ND ND

Carbendazim y = 2152.5x + 41938 0.990 0.10 0.33 2000 ND 200

Methamidophos y = 693.1x + 688.3 0.999 0.12 0.39 1000 ND 50

Acephate y = 679.8x + 891.5 0.998 0.15 0.49 100 20 200

Thiamethoxam y = 609.8x + 871.2 0.996 0.07 0.22 5000 10 200

Methomyl y = 2761.7x + 3475.8 0.998 0.03 0.11 1000 50 200

Imidacloprid y = 395.3x + 5812.2 0.992 0.10 0.33 5000 20 50

Acetamiprid y = 1765.8x + 6857.1 0.993 0.25 0.85 3000 50 200

Dimethoate y = 1596.8x + 2701.2 0.997 0.09 0.31 500 10 200

Flonicamid y = 314.9x + 1201.3 0.993 0.18 0.61 ND 25 ND

Spiroxamine y = 20213x - 3856.6 0.999 0.04 0.14 ND ND 200

Aldicarb y = 31.9x + 44.8 0.995 0.18 0.60 500 500 50

Flutriafol y = 3826.9x + 6235.3 0.997 0.12 0.42 ND ND ND

Carbaryl y = 2878.2x + 26261 0.998 0.06 0.20 500 ND 200

Metalaxyl y = 17077x + 18372 0.999 0.02 0.07 2000 25 10

Pyrimethanil y = 3152.5x + 2484.8 0.999 0.10 0.33 ND 20 10

Metazachlor y = 15095x + 25167 0.998 0.05 0.17 ND ND 200

Chlorantraniliprole y = 1544.9x + 679.2 0.993 0.26 0.86 10000 ND ND

Dimethomorph(E) y = 3872.9x + 7303 0.997 0.03 0.11 2000 ND ND

Fludioxonil y = 53.3x + 59.8 0.998 0.08 0.28 ND 20 ND

Spirotetramat y = 4825.1x + 731.2 1.000 0.03 0.08 ND 20 ND

Cyprodinil y = 3730.7x + 893.1 1.000 0.05 0.17 ND 10 ND

Azoxystrobin y = 18493x + 16409 0.999 0.02 0.07 ND 10 200

Boscalid y = 469.4x + 1331.6 0.996 0.05 0.15 ND 10 200

Fenhexamid y = 478.7x + 454.3 0.997 0.03 0.10 ND ND ND

Tebuconazole y = 3450x - 1337.2 1.000 0.09 0.31 ND 10 50

Bifenazate y = 3574.1x - 1230.6 0.999 0.24 0.81 ND 10 ND

Hexaconazole y = 3660.3x + 9901.4 0.998 0.12 0.42 ND ND 10

Malathion y = 1060.6x + 19.5 0.999 0.04 0.12 500 20 10

Chlorpyriphos y = 69.13x + 56.6 0.980 0.32 1.07 500 ND 200

Fipronil y = 755.5x + 990.1 0.995 0.22 0.72 ND 60 ND

Difenconazole y = 2420x + 4532.6 0.997 0.04 0.13 ND ND ND

Cyhexatin y = 508.1x + 15.4 1.000 0.04 0.14 ND ND ND

Hexaﬂumuron y = 85.7x + 12.8 0.992 0.07 0.23 ND ND ND

Pyraclostrobin y = 6169.3x + 4458.1 0.999 0.02 0.06 ND 20 200

Diazinon y = 11787x - 1315.7 0.999 0.15 0.51 100 500 50

Buprofezin y = 7159.1x + 8917.7 0.998 0.04 0.12 ND 10 ND

Triﬂoxystrobin y = 2848.8x + 2238.4 0.999 0.04 0.15 ND 10 10

Quinoxyfen y = 1638.1x + 1021 0.999 0.05 0.18 ND ND ND

Benfuracarb y = 1465.9x + 238.3 0.995 0.21 0.69 ND ND ND

Hexythiazox y = 99.6x + 64.9 0.998 0.09 0.31 ND ND 200

Fenpyroximate(E) y = 3566.5x + 1956.2 1.000 0.04 0.12 ND 20 ND

Lowest calibrated level (LCL) values obtained by Moulins et al. [17].

LOQ values obtained by Pérez-Parada et al. [21].

ND stands for “not deﬁned”.

236 D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239

Table 3

Precision and accuracy expressed as inter- and intra-day relative standard deviation (RSD) for the proposed LC–MS/MS method at different concentration levels.

Intra-day precision (RSD, %) Inter-day precision (RSD, %) Compound

−1 −1 −1 −1 −1 −1

1 ␮g kg 10 ␮g kg 50 ␮g kg 1 ␮g kg 10 ␮g kg 50 ␮g kg

Aminocarb 4.3 2.6 3.3 3.8 1.2 2.9

Carbendazim 2.0 0.8 2.2 4.8 2.7 4.8

Methamidophos 1.2 2.0 1.7 5.1 3.9 3.3

Acephate 3.8 2.6 1.2 4.3 2.0 3.4

Thiamethoxam 3.6 4.3 1.6 5.3 1.1 3.2

Methomyl 2.0 1.5 0.8 4.9 2.5 1.4

Imidacloprid 1.3 3.9 1.4 3.3 2.8 2.0

Acetamiprid 3.8 3.1 1.0 5.5 3.2 2.0

Dimethoate 3.8 2.3 1.5 4.3 3.4 1.5

Flonicamid 2.0 4.6 2.0 2.7 4.1 3.2

Spiroxamine 0.5 0.4 2.6 2.6 0.6 0.6

Aldicarb 5.9 0.8 3.4 5.1 4.4 5.4

Flutriafol 1.2 0.8 0.9 3.2 1.2 0.8

Carbaryl 1.4 1.1 0.2 4.7 1.2 1.7

Metalaxyl 2.6 0.4 0.3 1.9 0.8 0.7

Pyrimethanil 2.6 1.5 1.0 2.7 1.3 0.5

Metazachlor 0.6 0.6 0.3 1.7 0.8 1.2

Chlorantraniliprole 1.4 2.0 1.5 5.2 2.4 1.3

Dimethomorph(E) 3.2 1.0 0.7 4.6 1.9 0.9

Fludioxonil 2.4 1.8 0.4 2.7 4.3 2.4

Spirotetramat 1.2 0.5 0.8 1.2 1.7 1.3

Cyprodinil 2.3 0.3 1.9 5.1 1.8 2.5

Azoxystrobin 3.5 3.4 4.0 2.1 0.7 1.2

Boscalid 3.7 3.6 1.8 2.1 3.3 1.8

Fenhexamid 1.0 0.5 3.1 2.7 5.5 2.8

Tebuconazole 3.1 1.6 0.4 4.5 1.4 1.4

Bifenazate 1.7 4.1 0.4 4.5 2.7 1.2

Hexaconazole 4.5 1.3 2.9 3.1 1.5 1.5

Malathion 5.6 2.4 2.8 6.2 4.8 4.5

Chlorpyriphos 4.8 0.6 0.8 1.5 4.7 5.3

Fipronil 4.3 1.8 2.3 1.6 1.5 1.6

Difenconazole 1.9 5.4 2.1 4.1 1.6 0.8

Cyhexatin 4.5 0.5 0.6 3.6 3.2 1.6

Hexaﬂumuron 3.3 5.4 2.6 3.8 4.8 4.5

Pyraclostrobin 0.9 0.6 2.6 2.6 2.3 2.3

Diazinon 0.6 1.6 1.4 4.2 0.8 1.1

Buprofezin 2.4 1.7 3.0 2.6 0.6 1.2

Triﬂoxystrobin 3.9 4.7 1.6 3.7 5.4 1.9

Quinoxyfen 3.1 1.2 2.7 1.9 1.8 0.9

Benfuracarb 5.4 3.6 3.9 3.8 1.5 0.1

Hexythiazox 2.1 3.0 1.4 3.4 4.4 2.8

Fenpyroximate(E) 4.3 2.6 3.3 3.1 1.6 1.1

3.2.3. Limit of detection and limit of quantiﬁcation ent, they were also compared. In this case, the LOQ values of the

The limit of detection (LOD) and limit of quantiﬁcation (LOQ) present method are between 12 and 980 times smaller. Schneider

were determined considering the corresponding concentration to et al. [14] tested 50 seized illegal cannabis plants for 160 differ-

produce a signal 3 and 10 times the baseline noise, in a close region ent pesticides, but the ﬁgures of merit were reported only for the

to the migration time of each pesticide, respectively. The LODs seven pesticides that were detected in the samples. The smallest

−1

ranged from 0.02 up to 0.32 ␮g kg . LOD was obtained for hexythiazox, imidacloprid, and tebuconazole.

−1

␮

Although there are no maximum residue limits (MRLs) for mar- However, the LOD of 1 g kg is 10 times greater than the corre-

ijuana used for recreational purposes yet, we can compare the sponding LOD of the present work. Therefore, the present method

obtained results with the guidance residues levels (GRLs) estab- improves the limits in at least one order of magnitude when com-

lished for tobacco by CORESTA (Cooperative Center for Scientiﬁc pared to previous works for multiresidue analysis of pesticides in

Research on Tobacco) [29]. From this perspective, the results cannabis.

presented in Table 2 show that the proposed method allows quan-

tifying all pesticides in many orders of magnitude below their

corresponding GRLs.

For the sake of comparison, the results obtained by two other 3.2.4. Precision and accuracy

groups are also reported in Table 2. Pérez-Parada et al. [21] studied Precision and accuracy – expressed in terms of relative standard

three different methods based on LC MS/MS for a total of 61 pesti- deviation (RSD) – were determined by analyzing ﬁve replicates of

cides. The results of the best-performance method (method C) for marijuana samples spiked with a known amount of pesticide mix

−1

pesticides that are common to both works are reported in Table 2. at three different levels (1, 10 and 50 ␮g kg ). Intra-day precision

The LOQ values obtained in the present work are between 24 and (repeatability) was determined through the analysis in triplicate,

3333 times smaller than the corresponding ones. Moulins et al. [17] conducted on the same day, while the same replicates were ana-

also developed three methods for a set of 39 pesticides. Instead of lyzed in three different days for the determination of the inter-assay

LOQ, the reported value in this case is the lowest calibrated level precision. In both cases, the precision expressed as RSD (relative

(LCL), which is obtained when the peak height is 5 times greater standard deviation) was lower than 6.2% for all pesticides, as shown

than the noise. Although the deﬁnitions of LCL and LOQ are differ- in Table 3.

D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239 237

Table 4

Recoveries at three concentration levels for pesticides in marijuana by the proposed LC–MS/MS method (n = 5).

Spiked concentration levels Compound

−1 −1 −1

1 ␮g kg 10 ␮g kg 50 ␮g kg

Rec (%) RSD (%) Rec (%) RSD (%) Rec (%) RSD (%)

Aminocarb 113.5 0.9 92.3 1.0 104.6 2.6

Carbendazim 109.4 0.5 109.1 0.6 119.4 2.2

Methamidophos 112.8 3.7 94.1 2.0 99.5 1.7

Acephate 116.7 2.1 87.3 2.2 105.5 1.2

Thiamethoxam 112.1 3.0 95.8 3.2 105.2 1,5

Methomyl 101.4 2.5 99.2 1.2 102.5 0.8

Imidacloprid 91.5 4.7 89.1 3.7 101.2 1.4

Acetamiprid 101.6 1.9 90.0 2.8 99.9 1.0

Dimethoate 98.8 2.5 91.9 2.2 100.5 1.8

Flonicamid 96.0 2.3 100.9 4.3 100.3 2.1

Spiroxamine 95.6 1.4 101.8 1.3 94.6 0.3

Aldicarb 102.0 2.8 96.5 6.5 108.7 4,4

Flutriafol 103.2 1.9 95.3 1.8 100.6 1.9

Carbaryl 107.8 1.6 96.8 2,1 98.6 1.2

Metalaxyl 108.7 2.0 94.2 1.4 100.0 1.3

Pyrimethanil 117.3 1.0 90.4 1.3 102.7 1.0

Metazachlor 101.8 1.4 94.9 1.3 100.6 1.0

Chlorantraniliprole 104.4 0.6 90.1 1.8 102.0 1.4

Dimethomorph(E) 105.4 4.4 97.8 0.9 102.3 1.7

Fludioxonil 104.6 0.3 96.8 2.1 98.0 2.4

Spirotetramat 108.2 2.0 96.7 0.7 97.4 0.8

Cyprodinil 100.2 3.5 92.5 1.3 101.3 1.9

Azoxystrobin 91.6 0.9 111.3 0.7 102.6 0.9

Boscalid 98.7 1.4 91.5 3.0 104.7 1.7

Fenhexamid 117.0 2.0 118.9 1.5 90.0 3.1

Tebuconazole 95.7 1.3 91.3 1.6 103.0 1.4

Bifenazate 86.9 2.6 95.7 1.5 100,5 1.6

Hexaconazole 107.6 3.7 94.8 4.5 102.8 1.6

Malathion 103.8 1.7 89.9 3.1 102.2 2.8

Chlorpyriphos 98.7 5.6 96.4 3.8 113.2 5.1

Fipronil 100.0 5.8 103.7 1.7 96.2 1.9

Difenconazole 102.3 4.3 89.9 1.6 102.7 1.7

Cyhexatin 103.4 0.6 100.0 1.7 103.7 2.1

Hexaﬂumuron 102.2 6.1 99.2 4.2 100.9 1.3

Pyraclostrobin 101.3 5.3 101.8 5.4 103.1 5.6

Diazinon 103.2 3.9 100.1 1.6 100.0 3.6

Buprofezin 100.2 1.6 90.1 1.6 103.5 2.4

Triﬂoxystrobin 98.3 4.2 100.0 1.7 81.6 2.9

Quinoxyfen 103.9 5.8 98.5 1.8 102.0 1.6

Benfuracarb 94.2 4.1 91.9 0.9 106.7 2.6

Hexythiazox 100.3 6.1 98.8 6.7 107.5 5.9

Fenpyroximate(E) 99.0 2.7 105.4 2.4 99.2 3.7

Table 5

−1

Pesticides in marijuana samples. Concentration expressed in ␮g kg .

Sample

Pesticide

S1 S2 S3 S4 S5 S6

Imidacloprid n.d. n.d. n.d. 35.4 20.4 63.4

Metazachor n.d. n.d. n.d. n.d. 22.6 12.4

Buprofezin n.d. 6.6 n.d. n.d. n.d. n.d.

Metalaxyl n.d. 6.4 n.d. n.d. 23.0 3.2

a non-detected.

3.2.5. Recovery and stability 3.3. Real sample analysis

Recovery experiments were carried out in ﬁve replicates at three

−1

different levels of concentration (1, 10 and 50 ␮g kg ) by adding The validated method was applied to the simultaneous deter-

known volumes of pesticide standard mix to blank marijuana sam- mination of 42 pesticide residues in 6 marijuana samples provided

ples. The recovery was calculated by comparing the average area by the Rio de Janeiro Department of Technical and Scientiﬁc Police.

response of extracted samples (spiked before extraction) to that Imidacloprid, metazachlor, buprofezin, and metalaxyl were found

spiked after extraction. The achieved recoveries (Table 4) were in 4 samples (Table 5) at concentration levels ranging from 3.2 to

−1

␮

between 81.6–119.4 % with RSD smaller than 6.7% for all species. 63.4 g kg – all of them at levels lower than the respective GRLs

Concerning analyte stability, there was no signiﬁcant decline established by CORESTA. The samples with concentration above the

(smaller than 1%) in the pesticide signal from marijuana spiked dynamic range were diluted, re-analyzed and the concentration

samples stored without contact with air, light, and kept under was corrected by corresponding dilution factor. Fig. 3 shows the

refrigeration for one week. normalized MRM chromatogram of a marijuana sample with pos-

itive result for imidacloprid, metalaxyl, and metazachlor. Thanks

238 D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239

to the low LOQ values achieved with the developed method, it was

possible to quantify metalaxyl, for example, in three analyzed sam-

−1

ples – in two of them at concentrations lower than 10 ␮g kg .

Using previous methods described in the literature [17,21], this

pesticide would have been unnoticed in the samples.

4. Conclusion

The results demonstrated that the original QuEChERS sample

preparation combined with HPLC–MS/MS is a powerful tool for

the quantitative determination of the 42 pesticides in marijuana

−1

samples with limits of detection lower than 0.32 ␮g kg , which

is smaller than the result found in the literature [14,17,21] and

several orders of magnitude lower than the GRLs values estab-

lished for the tobacco industry by CORESTA [29]. In addition, 9

pesticides, among the 42 pesticides that were evaluated in this

work, have never been studied in marijuana before: aminocarb,

metazachlor, chlorantraniliprole, dimethomorph (E), fenhexamid,

cyhexatin, hexaﬂumuron, quinoxyfen and benfuracarb. From this

group, metazachlor was found in two analyzed samples. The sam-

ple preparation step used to require most of the time of an analyst

during the determination. The use of QuEChERS, however, makes

simpler this task. All the 42 pesticides were eluted in less than

6 min. The present study showed that pesticide contamination in

marijuana occurs frequently: 67% of the analyzed samples were

contaminated with residues of imidacloprid, metazachlor, bupro-

fezin, or metalaxyl.

Funding

This work was supported by FAPESP, Brazil (grant 2012/06642-1

and 2017/13137-5). C.L.L. thanks CNPq, Brazil (researcher fellow-

ship 304415/2013-8).

Fig. 2. Normalized dynamic MRM chromatogram of blank marijuana sample spiked

with 10 ␮g/kg of pesticide mix standard. The elution order is the same shown in

Table 1. Ethical approval

This article does not contain any studies with human partici-

pants or animals performed by any of the authors.

Informed consent

Informed consent is not applicable in this study.

CRediT authorship contribution statement

Daniela Daniel: Conceptualization, Methodology, Investigation,

Validation, Formal analysis, Writing - original draft. Fernando

Silva Lopes: Investigation, Formal analysis, Writing - original

draft. Claudimir Lucio do Lago: Funding acquisition, Supervision,

Methodology, Writing - review & editing.

Acknowledgement

The authors wish to thank the seized marijuana samples pro-

vide by Service of Forensic Chemistry of the Carlos Éboli Institute

of Criminalistics (Rio de Janeiro, Brazil).

References

[1] UNODC, World Drug Report, in, United Nations Ofﬁce on Drugs and Crime,

2018 https://www.unodc.org/wdr2018/.

[2] D. Stone, Cannabis, pesticides and conﬂicting laws: the dilemma for legalized

States and implications for public health, Regul. Toxicol. Pharmacol. 69 (2014)

284–288.

Fig. 3. Normalized MRM chromatogram of marijuana sample 6, with positive result

[3] T. Decorte, G.R. Potter, The globalisation of cannabis cultivation: a growing

for presence of imidacloprid, metalaxyl and metazachlor.

challenge, Int. J. Drug Policy 26 (2015) 221–225.

D. Daniel et al. / J. Chromatogr. A 1603 (2019) 231–239 239

[4] E. Cuypers, W. Vanhove, J. Gotink, A. Bonneure, P. Van Damme, J. Tytgat, The [19] M. Anastassiades, S.J. Lehotay, D. Stajnbaher, F.J. Schenck, Fast and easy

use of pesticides in Belgian illicit indoor cannabis plantations, Forensic Sci. multiresidue method employing acetonitrile extraction/partitioning and

¨ ¨

Int. 277 (2017) 59–65. dispersive solid-phase extractionfor the determination of pesticide residues

[5] A.F. Hernandez, T. Parron, A.M. Tsatsakis, M. Requena, R. Alarcon, O. in produce, J. AOAC Int. 86 (2003) 412–431.

Lopez-Guarnido, Toxic effects of pesticide mixtures at a molecular level: their [20] S.J. Lehotay, Determination of pesticide residues in foods by acetonitrile

relevance to human health, Toxicology 307 (2013) 136–145. extraction and partitioning with magnesium sulfate: collaborative study, J.

[6] C.K. Winter, Pesticide tolerances and their relevance as safety standards, AOAC Int. 90 (2007) 485–520.

Regul. Toxicol. Pharmacol. 15 (1992) 137–150. [21] A. Pérez-Parada, B. Alonso, C. Rodríguez, N. Besil, V. Cesio, L. Diana, A.

[7] J.M. McPartland, P.L. Pruitt, Medical marijuana and its use by the Burgueno,˜ P. Bazzurro, A. Bojorge, N. Gerez, H. Heinzen, Evaluation of Three

immunocompromised, Altern. Ther. Health Med. 3 (1997) 39–45. Multiresidue Methods for the Determination of Pesticides in Marijuana

[8] M.B.W. Lorenz, F. Korte, Thermolysis of pesticide residues during tobacco (Cannabis sativa L.) with Liquid Chromatography-Tandem Mass

smoking, Chemosphere 16 (1987) 521–522. Spectrometry, Chromatographia 79 (2016) 1069–1083.

[9] K. Islam, R. Chand, D. Han, Y.-S. Kim, Microchip capillary electrophoresis [22] S.J. Lehotay, K. Mastovska, A.R. Lightﬁeld, Use of buffering and other means to

based electroanalysis of triazine herbicides, Bull. Environ. Contam. Toxicol. 94 improve results of problematic pesticides in a fast and easy method for

(2015) 41–45. residue analysis of fruits and vegetables, J. AOAC Int. 88 (2005) 615–629.

[10] T. Subritzky, S. Lenton, S. Pettigrew, Legal cannabis industry adopting [23] J. Zrostlikova, J. Hajslova, J. Poustka, P. Begany, Alternative calibration

strategies of the tobacco industry, Drug Alcohol Rev. 35 (2016) 511–513. approaches to compensate the effect of co-extracted matrix components in

[11] C.E. Turner, M.A. Elsohly, F.P. Cheng, L.M. Torres, Marijuana and paraquat, liquid chromatography-electrospray ionisation tandem mass spectrometry

JAMA 240 (1978), 1857-1857. analysis of pesticide residues in plant materials, J. Chromatogr. A 973 (2002)

[12] J.A. Liddle, L.L. Needham, Z.J. Rollen, B.R. Roark, D.D. Bayse, Characterization of 13–26.

the contamination of marijuana with paraquat, Bull. Environ. Contam. [24] B. Kmellar, L. Abranko, P. Fodor, S.J. Lehotay, Routine approach to qualitatively

Toxicol. 24 (1980) 49–53. screening 300 pesticides and quantiﬁcation of those frequently detected in

[13] L. Needham, D. Paschal, Z.J. Rollen, J. Liddle, D. Bayse, Determination of fruit and vegetables using liquid chromatography tandem mass spectrometry

paraquat in marijuana by reversed-phase paired-ion high performance liquid (LC-MS/MS), Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk

chromatography, J. Chromatogr. Sci. 17 (1979) 87–90. Assess. 27 (2010) 1415–1430.

[14] S. Schneider, R. Bebing, C. Dauberschmidt, Detection of pesticides in seized [25] C.G. Enke, A predictive model for matrix and analyte effects in electrospray

illegal cannabis plants, Anal. Methods 6 (2014) 515–520. ionization of singly-charged ionic analytes, Anal. Chem. 69 (1997) 4885–4893.

[15] R. Lanaro, J.L. Costa, S.O. Cazenave, L.A. Zanolli-Filho, M.F. Tavares, A.A. Chasin, [26] D. Remane, M.R. Meyer, D.K. Wissenbach, H.H. Maurer, Ion suppression and

Determination of herbicides paraquat, glyphosate, and enhancement effects of co-eluting analytes in multi-analyte approaches:

aminomethylphosphonic acid in marijuana samples by capillary systematic investigation using ultra-high-performance liquid

electrophoresis, J. Forensic Sci. 60 (Suppl 1) (2015) S241–247. chromatography/mass spectrometry with atmospheric-pressure chemical

[16] N. Sullivan, S. Elzinga, J.C. Raber, Determination of pesticide residues in ionization or electrospray ionization, Rapid Commun. Mass Spectrom. 24

cannabis smoke, J. Toxicol. 2013 (2013), 378168. (2010) 3103–3108.

[17] J.R. Moulins, M. Blais, K. Montsion, J. Tully, W. Mohan, M. Gagnon, T. [27] H. Evard, A. Kruve, I. Leito, Tutorial on estimating the limit of detection using

McRitchie, K. Kwong, N. Snider, D.R. Blais, Multiresidue method of analysis of LC-MS analysis, part I: theoretical review, Anal. Chim. Acta 942 (2016) 23–39.

pesticides in medical Cannabis, J. AOAC Int. 101 (2018) 1948–1960. [28] H. Evard, A. Kruve, I. Leito, Tutorial on estimating the limit of detection using

[18] D.A. Lambropoulou, T.A. Albanis, Methods of sample preparation for LC-MS analysis, part II: practical aspects, Anal. Chim. Acta 942 (2016) 40–49.

determination of pesticide residues in food matrices by [29] CORESTA, The concept and implementation of agrochemcial guidance residue

chromatography–mass spectrometry-based techniques: a review, Anal. levels Guide, vol. 1, 2016.

Bioanal. Chem. 389 (2007) 1663–1683.