Journal of Biotechnology and Biosafety …jobb.co.in/docs/vol3_issue1/paper1.pdfJournal of Biotechnology and Biosafety! Volume3,!Issue1,January2February2015,1612170! ISSN232220406

Journal of Biotechnology and Biosafety Volume 3, Issue 1, January-‐February 2015,161-‐170

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www.jobb.co.in International, Peer reviewed, Open access, Bimonthly Online Journal

Journal of Biotechnology and Biosafety Volume 2, Issue 5, September-October, 2014, 131-140

ISSN 2322-0406 Journal of Biotechnology and Biosafety


Research article CYTOTOXIC EFFECT OF CYPERMETHRIN AND ITS SYNERGIST PBO ON ALPHITOBIUS DIAPERINUS (PANZER) (COLEOPTERA: TENEBRIONIDAE)

FOR BIOLOGICAL SECURITY OF STORED GRAINS AND CEREALS ______________________________________ Akter Mst Yeasmin1, Talukdar Muhammad Waliullah1*, Md. Ashraful Alam2, ASM Shafiqur Rahman3

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1Molecular and Cell Biology Laboratory, Bioscience Department, GSST, Shizuoka University. 836-Oya, Suruga-ku, Shizuoka ╤ 422-8529, Japan. 2FIQC Laboratory, Department of Fisheries, Matshya Bhaban, Ramna, Dhaka-1000, Bangladesh. 3Department of Zoology, University of Rajshahi, Rajshahi 6205, Bangladesh.

* Correspondence author E-mail: [email protected]

ABSTRACT To investigate the co-toxicity and co-efficient activity of Cypermethrin (Cythrin 20EC), a pyrethroid and Pyperonyl butoxide (PBO) against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae). Repellency test was done by the residual film assay technique. Statistically the dose mortality relationship was expressed as a median lethal dose (LD50) by the probit analysis. The regression lines and isoboles were drawn using the Fig-P (Biosoft) package. The Co-efficient values showed that all ratios of cypermethrin and piperonyl butoxide offered synergistic action to both larvae and adult. We observed that the toxicity of the cypermethrin was decreased as the ratio (amount) of PBO was increased. The individual LD50 value of cypermethrin for adult is 0.1235µgcm-2. But in the mixture, the share of cypermethrin are 0.0080, 0.0058, 0.0018 and 0.0015µgcm-2 at ratios of 1:1, 1:3, 1:5;1:10 when PBO causes reduction of dose level of 93.52%, 95.30%, 98.54% and 98.78% respectively. In case of larvae the individual LD50 value of cypermethrin is 0.0476µgcm-2. But in the mixture, the share of cypermethrin are 0.0055, 0.0046, 0.0022 and 0.0013µgcm-

2 at ratios of 1:1, 1:3, 1:5;1:10 when PBO causes reduction of dose level of 76.89%, 61.34%, 72.26% and 69.74% respectively. The study suggests that the mortality rate of lesser meal worm is increase with the increase of insecticide dose. The LD50 values of the insecticides are inversely related to the toxicity of the insecticides i.e. higher the LD50 value lower the toxicity of the insecticide.

Key words: Pyrethroid, Neuromuscular transmission, Acetone, Darkling beetle, Integration of pest management, Residual film assay. ____________________________________________________________________________________

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Research art i c l e QUANTITATIVE DETERMINATION, VALIDATION AND CONFIRMATORY ANALYSIS OF MALACHITE GREEN, LEUCOMALACHITE GREEN, CRYSTAL VIOLET AND LEUCOCRYSTAL VIOLET IN FISH AND SHRIMP MATRIX BY LIQUID CHROMATOGRAPHY–ELECTROSPRAY IONISATION–TANDEM MASS SPECTROMETRY ________________________________________ 1*Md. Ashraful Alam, 1Saleh Ahmed, 2Akter Mst. Yeasmin, 2Talukdar Muhammad Waliullah, 1Md. Serajul Islam ________________________________________

1FIQC Laboratory, Department of Fisheries, Matshya Bhaban, Ramna, Dhaka- 1000, Bangladesh. 2Molecular and Cell Biology Laboratory, Bioscience Department, GSST, Shizuoka University, 836-Oya, Suruga-ku, Shizuoka ╤ 422-8529, Japan. Corresponding author Email: [email protected]

ABSTRACT A confirmatory method has been developed to analyse malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in Rui fish (Labeo rohita) and Bagda shrimp (Penaeus monodon). Samples were extracted with dichloromethane by liquid-liquid extraction process and reconstituted with 80% of acetonitrile with water. Aliquots of the extracts were analysed by LC-MS/MS with electrospray ionization in positive mode using multiple reaction monitoring. The method was validated in fish and shrimp matrices, according to the criteria defined in Commission Decision 2002/657/EC. In case of fish and shrimp sample the decision limit (CCα) was in the range of 0.75-0.92µg/kg and detection capability (CCβ) was in the range of 1.28-1.57µg/kg. Fortifying samples (n=7) in three separate assays, showing the accuracy between 93% and 108%. The precision of the method, expressed as RSD values for the within-laboratory reproducibility, at the six levels of fortification (0.5, 1, 1.5, 2.0, 3.0 and 4.0µg/kg), was less than 11%. The ability to simultaneously quantify residues of MG, LMG, CV and LCV and to confirm the chemical structure of a marker residue by using LC-MS/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of these dyes in aquaculture.

Keywords: MG, LMG, CV, LCV, Fish, Shrimp ____________________________________________________________________________________


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INTRODUCTION Accurate monitoring of chemical residue levels in food and agriculture products is essential to assure the safety of the food supply and manage global health risks. Malachite green (MG) and crystal violet (CV) are effective and inexpensive fungicide used in aquaculture, particularly in Asian countries. Because of their disinfection and sterilization properties, they are widely used in aquatic products throughout the world (Li Y.H. et. al,. 2008 and Yuan J.T. et al., 2009). MG has been widely used as a topical fungicide and antiprotozoal agent in fish farming throughout the world for several decades. MG and CV are readily absorbed by fish and reduced to the corresponding metabolites, MG to leuco form Leucomalachite green (LMG), CV to leuco form Leucocrystal violet (LCV) which are the majority of prevalent residues present in fish tissues (Andersen W.C et al., 2009), as shown in Figure 1. It has been found that dyes of this family (like rosaniline) can induce hepatic and renal tumors in mice and reproductive abnormalities in fish, and the dyes have been linked to increased risk of human bladder cancer

(Mittelstaedt R.A. et al., 2004 and Zhang X. et al.,2011). MG is highly cytotoxic to mammalian cells and also acts as a liver tumor-enhancing agent (Angelis I.D.et al.,2003). MG, CV and their metabolites were reported to cause human carcinogenesis and mutagenesis (Littlefield, N. A. et al., 1985 and Srivastava, S. et al., 2004).

For this reason, the European Commission requires methods that can determine MG and LMG residues in the meat of aquaculture products. In addition, the Commission has established a minimum required performance limit (MRPL) of 2µg/kg for the sum of MG and LMG (Commission Decision EU/EC 2003). The US Food and Drug Administration explicitly banned the use of MG in fish farming in 1991 due to its suspected carcinogenic properties. However, due to their low cost and high efficacy, these harmful dyes are still used and will probably continue to be used in the aquaculture in some parts of the world. Therefore, it is very important to develop sensitive detection methods for the simultaneous determination of MG and CV and their metabolites in foodstuffs such as fish, shrimp samples.

Figure 1: Structures and conversion of MG and LMG; CV and LCV (Rong-Chun Chen et. al., 2013 and Plakas, S. M. et al., 1999)


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Figure 2: Structure of Leucomalachite Green d5 (Sigma Aldrich, P/N 677663)

Several methods have been proposed for the simultaneous determination of MG and CV and their metabolites, such as liquid chromatography–tandem mass spectrometry (Dowling, G. et al., 2007 and Zhu, K. et al., 2007), liquid chromatography–visible spectrophotometry (Allen, J. L. et al., 1991), capillary electrophoresis–Raman spectroscopy (Tsai, C. H. et al., 2007) and magnetic solid phase extraction–spectrometry (Šafarˇík, I. et al., 2002). Mitrowska K. et al., 2005 extracted MG and LMG from carp sample with acetonitrile–acetate buffer mixture followed by portioning with dichloromethane, cleanup on a SCX solid-phase extraction (SPE) cartridge and detection by tandem visible absorbance and fluorescence detectors connected inline without any post column procedure. Lee K.C. et al., 2006, reported that 16mL of acetonitrile containing 250mg ascorbic acid and 0.8% perchloric acid were used for extraction from edible goldfish muscle, followed by partitioning with dichloromethane and cleaning up with a Strata-x 33µm polymeric cartridge and detection with ion trap mass spectrometry. These proposed methods often require complicated operations of pretreatment, which prompt us to develop some alternative methods with simple pretreatments for the simultaneous determination of MG, CV and their metabolites. In this study, a method for simultaneously determining four dyes in aquatic products has been established without using any SPE cartridge. The method was validated in fish and shrimp matrices, according to the criteria defined in Commission Decision 2002/657/EC.

MATERIALS AND METHODOLOGY

Negative Sample Collection

Negative fish samples were cultured and collected in a standard condition without adding any dye contamination. Shrimp samples were collected from deep sea.

Equipments and Apparatus

Auto pipette 50-200µL, 200-1000µL, Model: eppendorf- 4450418; Test tubes, Model: IWAKI TE32 pyrex, Asahi, Indonesia; Analytical Balance (4 decimal points) Model: Shimadzu Auy 220; Centrifuge, Model: Nuve, NF 1200; Centrifuge tubes (griner B532); Nitrogen evaporator (Organomation Associates Jnc.); Microvials and caps (Waters); 13mm PTFE 0.2µm filter (Waters EDGE, USA); Column: Acquity UPLC, C18 1.7µm, 2.1 x 50mm, Waters Corp. USA; Polypropylene centrifuge tubes (50mL) (Griner, B 532); separation funnel; Volumetric flasks, 10mL (Schoot Duran); Volumetric flasks, 100mL; Vortex mixer, Model: Barnstead Thermolyne.

Chemicals and reagents

Standard: Malachite green (Fluka), Crystal violet (Fluka), Leucomalachite green (Fluka), Leucocryastal violet (Fluka), Leucomalachite green d5 (Fluka), Hydroxylamine hydrochloride, P- tolune sulfonic acid, Acetonitrile HPLC grade, Acetonitrile MS grade, Dichloromethane, Formic acid, Amonium acetate buffer

Preparation General Solutions (Tao Ding et al., Ap. N. 385)

Mobile phase: Solvent A: 0.1% formic acid in water, Solvent B: 0.1% formic acid in CAN; Hydroxyl amine hydrochloride solution (0.25 g/L): 255mg of hydroxylamine hydrochloride was taken in 1000mL volumetric flask and volume was made up to the mark


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with deionized water; p-tolune sulfonic acid (0.05 mol/L): 4.9g of p-tolune sulfonic acid (C7H8O3S.H2O, M=190.25, assay=98%) was taken in a 500mL volumetric flask and volume was made up to mark with deionized water; ammonium acetate (0.1 mol/L): 3.933g of ammonium acetate (CH3COONH4, M=77.08 and assay=98%) was taken in a 500mL volumetric flask and volume was made up to mark with deionized water.

Preparation Standard Solution: (Tao Ding et al., Ap. N. 385)

Stock solution was prepared by considering actual weight and purity of dry standard powder.

a. MG, LMG, CV, LCV Stock solutions (~1mg/mL): 14.6mg MG oxalate, 10mg LMG, 12.2mg CV chloride and 10mg LCV was added into separate 10 ml amber vials and dissolved in acetonitrile. These solutions were stored in refrigerator (2 – 8 °C) for 6 months.

b. Combined solution (10.0µg/mL): ~100 µL (depending on the exact concentration) of each stock standard solution was taken into a 10.0 ml amber volumetric flask and made up-to mark with acetonitrile. This solution was stored in refrigerator (2 – 8 °C) for 1 month.

c. Combined Working Solution (100ng/mL) and External Standard Solution (5.0ng/mL): Combined working solution and external standard solution was prepared separately from combined solution by diluting with solvent in amber vial freshly.

d. LMG d5 Stock Solution (~1mg/mL): 10mg LMG was added into 10mL amber vial and dissolved in acetonitrile.

e. LMG d5 Working Solution (250ng/mL): LMG d5 working solution was prepared from LMG d5 stock solution by diluting with solvent in amber vial freshly.

Preparation of matrix based calibration curve: combined working standard solution (100ng/mL) was spiked in 5.00g negative shrimp/fish matrix at volume of 25, 50, 100, 150, 250, 500µL to get equivalent concentrations 0.5, 1.0, 2.0, 3.0, 5.0 and 10ppb respectively and internal standard LMG d5 (250ng/mL) was spiked in each sample at the volume of 100µL to get concentration 5.0ppb.

Extraction Procedure (Tao Ding et al., Ap. N. 385)

5g of blended sample was taken in polypropylene centrifuge tubes (50mL) and 1mL of 0.25g/L hydroxylamine hydrochloride, 1mL of 0.05 mol/L p-tolunesulfonic acids, 2 mL of 0.1mol/L NH4-HAc buffer (pH 4.5) and 20mL of acetonitrile were added. Then homogenize for 2 minutes and centrifuge at 3000 rpm for 3 minutes. Then the supernatant was collected into a 250mL separation funnel. To the acetonitrile crude extract add 30mL of dichloromethane (DCM) and 35mL deionized water and shake for 2 minute and collect the DCM (Lower portion). Then evaporate the combined DCM solvent to dryness and reconstitute in 3mL of 80% ACN and passed through 0.2µm syringe filter. LMG d5 (Figure 2) was spiked at the initial stage of extraction procedure to compensate for any analyte loss.

Quality Control Measures Solvent blank, reagent blank, matrix blank and positive control samples were used each analytical batch as an internal quality control measures.

UP LC-MS-MS Analysis

Instrumentation: UPLC-MS-MS i. Liquid Chromatograph – Acquity UPLC, Waters Corp. USA ii. Analytical Column – Acquity UPLC, C18 1.7µm, 2.1 x 50mm, Waters Corp. USA iii. Mass Spectrometer – Acquity TQD, Waters Corp. USA Inlet parameters Pump A1: 0.1% FA in water, Pump B1: o.1% FA in acetonitrile, Stop Time (min): 5, Injection Volume (µL): 10.00, Column Temp: 350C, LC Separation Method: Gradient. MS Method Parameters Function: MRM (multiple reaction monitoring) of 9 channels, Solvent Delay (min): 0.00, Inter Channel Delay (Sec): 0.02, Span (Daltons): 0.00, Start time (Min): 0.00, End Time (Min): 5.00, Repeats: 1, Dwell(s) time (Sec): 0.05


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Tune Parameters Source (ESI+) Capillary (kV): 2, cone (V): 30, extractor (V): 3, RF(V):0.1, source temp(0C): 115, desolvation temp (0C): 375, desolvation gas flow (L/hr):800, cone gas flow (L/hr): 50, collision gas flow(mL/Min): 0.10

Analyser LM Resolution 1:11, HM resolution 1:12, Ion energy 1:1.0, Entrance voltage: 50, Exit voltage: 50, LM resolution 2:11, HM resolution 2:12, Ion energy 2:1.0, Multiplier voltage: 650, Collision gas: Argon @ 3.5 x 10-3 mbar

CALCULATION

Ion Ratio (R) = Peak area of Primary ion Peak area of Secondary ion

Response factor (RF) RF = PAPI x Internal standard concentration Peak area of internal standard ion

PAPI=Peak area of Primary ion (of interested substance) ISC=Internal standard concentration Concentration (X)

X = RF-b a Where; X = concentration of interested substance that found in sample (ppb) RF = Response factor of ion product a = Slope from the calibration curve b = intercept of calibration curve

Confirmation criteria:

Dyes were considered as positively identified in the samples when the peak area ratio of the various transitions was within the tolerance set by Commission Decision 2002/657/EC. In addition, the relative retention time of the analyte must be equal to that of the calibration standard to within ± 2.5%.

RESULT AND DISCUSSION

The LC/MS/MS method was developed to provide confirmatory data for the analysis of fish and shrimp samples for MG, LMG, CV, LCV, and LMG d5 whose structures are shown in Figure1& 2. For a method to be deemed confirmatory, one parent ion and two daughter ions must be monitored. This yielded four identification points, which provided a suitable confirmatory method in accordance with 2002/657/EC: 2002. The MS/MS

fragmentation conditions were investigated and collision energies were optimized for each individual compound. Product ion spectra resulting from collision-induced dissociation were examined and suitable ions selected for multiple reaction monitoring (MRM) schemes (Table 1). The precursor and daughter ions obtained in the result have good agreement with previous findings (Dowling, G. et al., 2007), which indicates compounds were identified accurately. UPLC columns conditions were studied in order to optimize the chromatographic separation in terms of resolution and overall analysis time due to the different properties of compounds and a gradient separation was established (Table 2) which provided nice resolution and good chromatograms (Figure 3). There were no significant peaks in solvent blank, reagent blank, matrix blank that noticed that experiment was done in contamination free condition for respective compounds.


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Table 1: Ion monitored and MS/MS fragmentation conditions

Comp. Name Mass Prnt (m/z) Dau (m/z) Cone(V) Coll(eV) RT (min) 164.998 8 62 2.18 Malachite green (MG) 328.1 329.132 208.048 8 34 2.19 239.082 50 32 2.43 Leucomalachite green (LMG) 330.2 331.168 316.087 50 20 2.43 165.062 14 80 2.22 Crystal violet (CV) 371.2 372.17 340.188 14 54 2.21 238.209 52 26 2.23 Leucocrystal violet (LCV) 373.2 374.232 359.039 52 20 2.22

LMG d5 335.2 336.168 321.09 45 22 2.42

Table 2: Gradient table

Time Pump A/ Buffer solution Pump B/ Acetonitrile Flow (mL/min) Curve 0 95 5 0.250 6 0.50 95 5 0.250 6 1.20 95 5 0.250 6 1.50 5 95 0.250 6 2.30 5 95 0.250 6 2.35 95 5 0.250 6 3.50 95 5 0.250 6 5.00 95 5 0.250 11


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Figure 3: Chromatogram of MG, LMG, CV, LCV and LMG d5 with their daughter ions.

Table 3: Decision Limits & Detection capability (CCα & CCβ)

Shrimp Fish Analyte CCα(µg/kg) CCβ (µg/kg) CCα (µg/kg) CCβ (µg/kg)

MG 0.86 1.46 0.84, 1.44 LMG 0.88 1.5 0.87 1.48 CV 0.92 1.57 0.89 1.52 LCV 0.75 1.28 0.77 1.30


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Figure 4: A comparative representation of CCα and CCβ in shrimp and fish matrices.

Validation of the method was carried out in accordance with the Decision 2002/657/EC, which establishes criteria and procedures for the validation of methods. The following parameters were determined: decision limit (CCα), detection capability (CCβ), linearity, accuracy, precision, selectivity, specificity and matrix effect. The decision limits (CCα) and Detection capabilities (CCβ) are shown in the Table3 and Figure4. CCα values for all compounds were less than MRPL, which indicates the method was fitted for the purpose. This study was used for all over the year for dyes analysis. The findings demonstrate the method is robust and suitable for routine quality control operations to detect simultaneously MG, CV and its metabolite (LMG and LCV) in fish and shrimp. CONCLUSION A relatively stable, fast, and selective LC-MS/MS method for the simultaneous determination and confirmation of MG, LMG, CV and LCV fish and shrimp muscles was

developed without using any SPE cartridge.There are few published confirmatory methods for the simultaneous determination of MG, LMG, CV and LCV in fish and shrimp muscles that are validated according to the Commission Decision 2002/657/EC. This study shows that the required sensitivities for MG and LMG were obtained and met the MRPLs (Minimum Required Performance Limits) of 2mg/kg. Although there is no MRPL set for CV and LCV the method is sensitive for CV and LCV also. The method performed very well in terms of accuracy and stability (over 2 years, n =2000). The results of this study were satisfactory for the development of a rugged analytical method. AKNOWLEDGEMENT We would like to provide humble gratitude to the laboratory in-charge of FIQC laboratory, director of Fish Inspection and Quality Control and DG of the department of Fisheries. We express our sincere thanks to all officials and staffs of the laboratory for their support in works.

MG LMG CV LCV

0 0.2 0.4 0.6 0.8 1

1.2 1.4 1.6

CCα(µg

/kg)

CCβ(µg

/kg)

CCα(µg

/kg)

CCβ(µg

/kg)

Shrimp Fish

MG

LMG

CV

LCV


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Citation of this article: Md. Ashraful Alam, Saleh Ahmed, Akter Mst. Yeasmin, Talukdar Muhammad Waliullah, Md. Serajul Islam (2015). QUANTITATIVE DETERMINATION, VALIDATION AND CONFIRMATORY ANALYSIS OF MALACHITE GREEN, LEUCOMALACHITE GREEN, CRYSTAL VIOLET AND LEUCOCRYSTAL VIOLET IN FISH AND SHRIMP MATRIX BY LIQUID CHROMATOGRAPHY–ELECTROSPRAY IONISATION–TANDEM MASS SPECTROMETRY. Journal of Biotechnology and Biossafety. 3(1): 161-170

Source of Support: Nil Conflict of Interest: None Declared