Journal of Biotechnology and Biosafetyjobb.co.in/docs/vol3_issue1/all.pdfMd. Serajul Islam, Md....

Journal of Biotechnology and Biosafety

Volume 3 Issue 1 January/February 2015

An International, Open Access, Peer reviewed,

Bi-Monthly Journal

Editorial

Editor-in-Chief

Chethana G S [email protected]

[email protected]

www.jobb.co.in

Advisory Board

Dr. S.M. Gopinath, Phd HOD, Dept of Biotechnology, Acharya Institute of Technology, Bangalore, INDIA

Dr. Vedamurthy A.B. Phd

Professor, P.G. Department of Studies in Biotechnology and Microbiology, Karnatak

University, Dharwad, India

Dr. Hari Venkatesh K Rajaraman MD(Ay), PGDHM Manager, R&D, Sri Sri Ayurveda Trust, Bangalore, INDIA

R. Rajamani, M.Sc.,M.Phil.,B.Ed. Co-Principle Investigator, SSIAR, Bangalore, INDIA

Dr. Pravina Koteshwar, MBBS, MD Director, Academic Programs, ICRI, India

Editorial Board

Dr. Pushpinder Kaur, Phd Research Associate, CSIR-Institute of Microbial Technology Sector,

Chandigarh, INDIA

Dr. Kavita Sharma, Phd Senior Scientist, Research and Development, Pharmacology Division,

Sigma Test and Research Centre, New Delhi, INDIA

Dr. Kasim Sakran Abass, Phd Associate Professor, College of Nursing,

University of Kirkuk, Kirkuk, IRAQ

Index – JOBB, Volume 3, Issue 1 - January/February 2015

Animal Biotechnology

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 Md. Ashraful Alam, Saleh Ahmed, Akter Mst. Yeasmin, Talukdar Muhammad Waliullah,

Md. Serajul Islam

161-170


UTILIZATION OF SLAUGHTER HOUSE AND KITCHEN BY-PRODUCTS AS PROTEIN SOURCE IN

BROILER DIET

Md. Jahangir Alam, Talukdar Muhammad Waliullah, Md. Saiful Islam, Zannatul Ferdaushi 171-182


A TECHNIQUE TO QUALIFY IN PROFICIENCY TEST OF CHLORAMPHENICOL RESIDUE IN PRAWN

MATRIX UNDER METHOD DEVELOPMENT, VALIDATION, QUANTITATIVE ANALYSIS BY LC-MS/MS

AND PT PARTICIPATION APPROACH. Md. Ashraful Alam, Akter Mst. Yeasmin, Talukdar Muhammad Waliullah, Saleh Ahmed,

Md. Serajul Islam, Md. Manik Mia, Md. Anwar Parvez, Nitta Ranjan Biswas, Sayed Arif Azad

183-190

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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REFERENCE

Allen, J. L., &Meinertz, J. R. (1991) Post-column reaction for simultaneous analysis of chromatic and leuco forms of malachite green and crystal violet by highperformance liquid chromatography with photometric detection. Journal of Chromatography A, 536, 217–222.

Andersen W.C., Turnipseed S.B., Karbiwnyk C.M., Lee R.H., Clark S.B., Rowe W.D., et al.(2009) Multiresidue method for the triphenylmethane dyes in fish: Malachite green, crystal (gentian) violet, and brilliant green. AnalyticaChimicaActa. 637:279-289. C rossRef. MedlineWeb of Science

Angelis I.D., Albo A.G., Nebbia C., Stammati A., Zampaglioni F.(2003) Dacasto M. Cytotoxic effects of malachite green in two human cell lines. Toxicological Letters 144: 58. Search Google Scholar .

Commission Decision. Amending Decision 2002/657/EC as regards the setting of minimum required performance limits (MRPLs) for certain residues in food of animal origin. Official Journal of the European Union. 6:38-39 (2003).

Dowling, G., Mulder, P. P. J., Duffy, C., Regan, L., & Smyth, M. R.(2007) Confirmatory analysis of malachite green, leucomalachite green, crystal violet and leucocrystal violet in salmon by liquid chromatography–tandem mass spectrometry. AnalyticaChimicaActa, 586, 411–419.

European Communities. Implementing Council Directive 96/ 23/EC concerning the performance of analytical methods and the interpretation of results. Commission Decision 2002/657/ EC; 2002. Off J EuropComm, No. L221/8

Lee K.C., Wu J.L., CaiZ.W.(2006)Determination of malachite green and leucomalachite green in edible goldfish muscle by liquid chromatography–ion trap mass spectrometry. Journal of Chromatography B. 843:247-251.

Littlefield, N. A., Blackwell, B. N., Hewitt, C. C., & Gaylor, D. W.(1985) Chronic toxicity and carcinogenicity studies of gentian violet in mice. Fundamental and Applied Toxicology, 5, 902–912. National standard GB/T 19857–2005of PR China.

Li Y.H., Tao Y., Qi X.L., Qiao Y.W., Deng A.P.(2008) Development of a group selective molecularly imprinted polymers based solid phase extraction of malachite green from fish water and fish feed samples. Analytica Chimica Acta. 624:317-325.Cross Ref. MedlineWeb of Science.

Mitrowska K., Posyniak A., Zmudzki J.(2005) Determination of malachite green and leucomalachite green in carp muscle by liquid chromatography with visible and fluorescence detection. Journal of Chromatography A. 1089:187-192. Cross Ref Medline Web of Science.

Mittelstaedt R.A., Mei N., Webb P.J., Shaddock J.G., Dobrovolsky V.N., McGarrity L.J., (2004). Genotoxicity of malachite green and leucomalachite green in female Big Blue B6C3F1 mice. Mutatation Research: Fundamental and Molecular Mechanisms of Mutagenesis. 561: 127-138.

Plakas, S. M.; Doerge, D. R.; Turnipseed, S. B. Disposition and Metabolism of Malachite Green and Other Therapeutic Dyes in Fish. InXe nobiotics in Fish; Smith, D. J., Gingerich, W. H., Beconi-Barker, M. G., Eds.; Plenum Press: New York City, 1999; p. 149-166. Rong-Chun Chen, Kuen-Jou Wei, Ter-Min Wang, Yu-Man Yu, Ju-Ying Li, Shu-Hui Lee, Wei-Hsien Wang, Tyh-Jeng Ren, Chung-Wei Tsai (2 0 1 3). Simultaneous quantification of antibiotic dyes in aquatic products and feeds by liquid chromatography-tandem mass spectrometry, journal of food and drug analysis. 21: 339-346


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Šafarˇík, I., &Šafarˇíková, M.(2002) Detection of low concentrations of malachite green and crystal violet in water. Water Research, 36, 196–200.

Srivastava, S., Sinha, R., & Roy, D.(2004) Toxicological effects of malachite green. Aquatic Toxicology, 66, 319–329.

Sigma Aldrich Part number 677663, http://www.sigmaaldrich.com/catalog/product/fluka/34182

Tao Ding, Jingzhong, Bin Wu, Hulian Shen, Fei Liu and Kefei Wang, LC-MS/MS Determination of Malachite Green and Leucomalachite Green in fish products. Application Note: 385, Thermo Fisher Scientific. http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/ Application%20&%20Technical%20Notes/Chromatography%20Columns%20and%20Supplies/HPLC%20Columns/HPLC%20Columns%20(5um)/AN385_62296_TSQ_Food (1).pdf

Tsai, C. H., Lin, J. D., & Lin, C. H.(2007) Optimization of the separation of malachite green in water by capillary electrophoresis Raman spectroscopy (CE-RS) based on the stacking and sweeping modes. Talanta, 72, 368–372.

Yuan J.T., Liao L.F., Xiao X.L., He B.(2009)Analysis of malachite green and crystal violet in fish with bilinear model. Food Chemistry. 113:1377-1383.CrossRef. Web of Science

Zhang X., Zheng Y., Fried L.E., Du Y., Montana S.J., Sohn A., et al.(2011) Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes. Free Radical Biology & Medicine. 50:811-820. Cross Ref. MedlineWeb of Science.

Zhu, K., Wang, P., Lin, Y., Xiao, S., & Mei, S.(2007) Simultaneous determination of residues of malachite green, crystal violet and their leuco metabolites in aquatic products by liquid chromatography–tandem mass spectrometry. Chinese Journal of Chromatography, 25, 66–69.

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


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Research art i c l e UTILIZATION OF SLAUGHTER HOUSE AND KITCHEN BY-

PRODUCTS AS PROTEIN SOURCE IN BROILER DIET __________________________________________________________________

1,2*Md. Jahangir Alam, 1Talukdar Muhammad Waliullah, 2Md. Saiful Islam, 3Zannatul Ferdaushi ____________________________________ 1Molecular Cell Biology Laboratory, Bioscience Department, GSST, Shizuoka University, Shizuoka 422-8529, Japan 2Dept. of Animal Production & Management, Faculty of Animal Science & Veterinary Medicine, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh 3Dept. of Obstetrics & Gynaecology, Combined Military Hospital, Dhaka Cantonment, Dhaka-1206, Bangladesh *Corresponding author: [email protected], [email protected],

ABSTRACT: A total of 90 day-old straight run broiler chicks were fed ad libitum up to 28 days on a control diet with 5-6% commercial protein concentrate (CPC) and on 2 test diets in which 5-6% slaughter house and kitchen by-product (SHB); and 2.5-3% CPC and 2.5-3% SHB to assess whether it is feasible to substitute costly CPC by SHB. The chemical composition of SHB was 97.45, 69.77, 3430, 0.83, 8.41 and 10.21 for Dry matter %, Crude protein%, Metabolic energy (Kcal/kg), Crude fiber%, Ether extract% and Ash%, respectively. The test diets did not affect significantly (p>0.05) on feed intake, live weight, FCE and survivability of broilers. Dressing percentage (DP) and dressed meat yields at 4th week with different treatments were not differed (p>0.05). Total cost and profit (Taka/kg live broiler) were 105.07 and 14.93 for T1, 101.94 and 18.06 for T2, and 103.52 and 16.48 for T3. The test diets effect significantly (P<0.01) on feed cost, production cost and profitability compared to that of the control. Complete or partial replacement of CPC by SHB reduced feed and production cost due to low cost of SHB, therefore, increased the profitability in raising broilers without hampering body weight gain. It can be concluded that SHB or equal mixture of by product and CPC may be economic and efficient at broiler ration in substitute of CPC.

KEY WORDS: Broiler, Chicken by-product, Kitchen waste, Protein concentrate, Slaughter house by-product _______________________________________________________________________________________

INTRODUCTION: Chicken is among the major economically important livestock in the Bangladesh. The demand for broiler meat is on the rise throughout the world, especially in developing countries due to increased human population, income growth and great need for lean

meat (Sanon et al., 2008). High prices and shortage of feed ingredients are the main constraints involved in animal production. The cost of feed ingredients is increasing at an alarming rate and currently accounts for about 60-65% of the total cost of broiler production, with protein comprising about 13% of the total feed cost (Singh, 1990 and Banerjee, 1992).


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The poultry industry is gradually increasing in Bangladesh where imported costly protein concentrates are used in formulating poultry diet that increased production cost. Indeed, soybean meal, fishmeal and other protein concentrates are becoming very costly. However, a certain amount of protein must be added to the diet to satisfy essential amino acid requirements (Scott et al., 1976). Currently poultry nutritionists are looking for cheaper unconventional energy and protein sources to formulate least cost ration. Various types of unconventional feeds (e.g. Marine Wastes, Frog Wastes, Shrimp Wastes, Rumen Ingesta, Kitchen Wastes, Slaughter house Wastes, Industrial Wastes and Tannery Wastes etc.) are available in Bangladesh, most of which are regarded as wastes and research has been going on incorporating it in diets for poultry (Rahman and Reza, 1983; Islam et al., 1994). Increase world populations and high demands for animal products in the recent years have given impetus to animal husbandry as well as animal products processing throughout the world. These developments in turn, have culminated into generation of different kinds of animal by products in quantities. Different wastes are used in the diet of poultry in some developed and developing countries, but no work has been done using slaughter house and kitchen waste (SHB) in poultry diet in Bangladesh. Imported protein sources like fish meal (65 Taka /kg), soybean meal (50 Taka/kg), Seasame oil cake (40 Taka/kg) and Meat–bone meal (MBM-commercial protein concentrate-60 Taka/kg) were very expensive which enhance the production cost. Whereas the cost of slaughterhouse and kitchen waste (collection, processing and storing cost) was maximum 5.0 Taka/kg. SHB composed of excess fat, fascia and flesh (Subcutaneous tissue), unnecessary portions of the hides and skins (Trimmings), blood and bone, teeth or any other byproduct materials from slaughterhouse and kitchen. Scarcity of animal protein of feed may be fill up by alternate and unconventional protein source from SHB These materials have been found to contain a large amount of protein (Rao, 2000); therefore, they have the potential for use as an inexpensive alternative to currently available protein for the addition to livestock feed. SHB and kitchen wastes are not officially or scientifically used in the diet of poultry throughout the world, but it has also been shown that many Bangladeshi farmers currently use these SHB and kitchen wastes as livestock feed, even though the feeds

are not prepared in a scientific fashion. Moreover, no studies have been conducted to evaluate the use of SHB and kitchen wastes in the diet of broiler in Bangladesh. Therefore, this study was undertaken to evaluate the prepared feed quality, their effects on growth performance of broiler and feasibility of using the SHB and kitchen wastes in the diet of broiler. MATERIALS AND METHODS

The experiment was conducted in the Department of Animal Production & Management, Sher-e-Bangla Agricultural University, Dhaka to develop an efficient method to process different by-products and wastes so that they can be used to as an efficient replacement of conventional animal feed ingredients especially for protein. The whole research was completed within 1 year of period (March/2012 to June/2013). The experiment was conducted for survey and collection of by-products and wastes from different animal slaughterhouse, chicken processing area and residential areas for kitchen waste. Processing and preparation of animal feed from by-product & waste was done in the Laboratory of Animal Production and Management at Sher-e-Bangla Agricultural University, Dhaka. Analysis of prepared feed was performed at Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka. Utilization of prepared feed as livestock (broiler) protein source was conducted in the poultry farm at Sher-e-Bangla Agricultural University campus on trial basis. Survey and collection of slaughter house & kitchen wastes A survey was conducted for slaughter house by-product at Postagola, Mohammadpur, Cantonment Kachukhet and Hazaribagh of Bangladesh estimates that rumen content, blood, bone, horn/hooves, soft tissue (ovary, gallbladder, urinary bladder, eye ball etc.), hides & skins trimmings, fleshings etc. nearly 100% was thrown here and there. Kitchen waste (skin, fat, intestine, digesta, leg, feather etc.) was surveyed and collected from Cantonment Kachukhet market, Taltola market, Town Hall, Krishi market of Dhaka city. Usually they through all of these wastes near the market or road side garbage which is making problem for both of passer and nearest living people as well as market comers.


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Preparation of protein concentrate (Rao, 2000)

Sample collection: Soft tissue, rumen content, blood, bone, horn/hooves, hides & skins trimmings, fleshings, excess fat etc. and Kitchen waste (skin, fat, intestine, digesta, leg, feather etc.) were collected from different places. Cleaning: All the collected byproduct and wastes were properly washed 2-3 times with fresh and clean water to remove dust and foreign materials. Cutting and mixing: All of the cleaned byproduct and wastes were cut in small pieces and mixed properly. Boiling: Mixed wastes were boiled with same volume of water at 1000C for4-5 hours. Drying: The sample was sun and oven dried properly. Grinding: The dried mixture was ground properly by grinding machine. Packing: Prepared protein concentrate from wastes was packed in the gunny bag for preservation.

Chemical analysis of prepared protein concentrate Prepared protein concentrate and commercial protein concentrate were analyzed in the BLRI, Savar, Dhaka for the determination of DM, CP, CF, EE, ash, ME, Ca and P (AOAC, 1990).

Preparation of the experimental rooms The experimental rooms were properly washed and cleaned by using running water and disinfected with anticeptic phenyl solution followed by iosan. The room was properly dried and divided into 9 separate pens of equal size (4ftX3ft) using wire net and bamboo according to treatment and replications. A total of 90 unsexed day-old BV-500 chicks were purchased from CP Bangladesh Ltd. and they were divided into 3 treatment groups having 3 replications of 10 chicks in each (Table 1).

Table 1: The layout of the experiment showing number of chicks allocated in each replication and treatment group.

Number of birds each replication Treatment group R1 R2 R3

Total number of birds

T1 10 10 10 30 T2 10 10 10 30 T3 10 10 10 30

Grand total 30 30 30 90 Where, T1= Control diet (5-6% CPC); T2= Diet in which 5-6% prepared protein concentrate from SHB; T3= Diet in which 2.5-3% SHB and 2.5-3% CPC; CPC= Commercial protein concentrate; SHB= Protein prepared from slaughterhouse and kitchen wastes.

Preparation of experimental diets (Nabil et al., 2009) The amount of feed ingredients required were ground, weighed and thoroughly mixed. Vitamin-mineral premix and coccidiostat were also mixed properly with the feed. Diets for different treatment groups were prepared separately. Each of the three experimental diets were divided into three equal parts and stored for seven days in separate nine (3X3=9) gunny bags, according to treatments and replications. The procedure was followed for both starter and grower diets. The composition of experimental ration is presented in Table 2.

Experimental broiler management Each pen was allotted for 10 birds with a measurement of 4ftX3ft. Therefore, floor space each bird was 1.2 sq. ft. Fresh and dried rice husk was used as litter at a

depth of about 5cm. One round shaped feeder and one round shaped waterer with a capacity of 2.5 liters were provided in each pen. Waterers were thoroughly washed and cleaned every day. The feeders and waters were placed in such way that the birds were able to eat and drink conveniently. Initially, the birds were exposed to a continuous lighting for 23 hours and 30 minutes. During night electric bulb were used to provide necessary light. The birds were placed randomly in 9 pens. The experimental birds were brooded under 200 Watt electric bulbs by using steel sheet as chick guard. The birds were provided with a temperature 35℃ at first week and decreased gradually at the rate of 2.75℃ per week up to 4 weeks of age. During first week, the experimental feed was supplied on newspaper and round waterer was used for supplying drinking water (Sarkar et al., 2008)


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Table 2: Composition of broiler starter and grower diets Diets (Broiler starter) Diets (Broiler grower) Ingredients (%)

T1 T2 T3 T1 T2 T3 Maize 51.35 51.35 51.35 56 56 56 Soybean meal 31 31 31 26 26 26 CPC 5.25 ---- 2.63 6.12 -------- 3.06 SHB ------- 5.25 2.63 ------ 6.12 3.06 Lime stone 1.2 1.2 1.2 1.2 1.2 1.2 Rice polish 7 7 7 6 6 6 Soybean oil 2.5 2.5 2.5 3 3 3 DCP 0.4 0.4 0.4 0.4 0.4 0.4 Lysine 0.2 0.2 0.2 0.2 0.2 0.2 DL 0.18 0.18 0.18 0.16 0.16 0.16 GS 0.25 0.25 0.25 0.25 0.25 0.25 Common salt 0.3 0.3 0.3 0.3 0.3 0.3 NaHCO3 0.01 0.01 0.01 0.01 0.01 0.01 Choline chloride 0.05 0.05 0.05 0.05 0.05 0.05 Toxin binder 0.2 0.2 0.2 0.2 0.2 0.2 Enzyme 0.05 0.05 0.05 0.05 0.05 0.05 Coccidiostat 0.05 0.05 0.05 0.05 0.05 0.05 Antibiotic 0.01 0.01 0.01 0.01 0.01 0.01 Total 100 100 100 100 100 100 Calculated composition: CP% 22.74 22.88 22.71 21.40 21.36 21.31 ME (Kcal/kg) 3070 3092 3095 3173 3190 3175 Ca (%) 1.19 1.10 1.05 1.16 0.99 1.10 Av. P (%) 0.59 0.57 0.43 0.59 0.53 0.50

• Vitamin-mineral premix was mixed @ 2.50g/kg mixed feed. Where, T1= Control diet (5-6% CPC), T2= Diet in which 5-6% SHB and T3= Diet in which 2.5-3% SHB and 2.5-3% CPC; CPC= Commercial protein concentrate; SHB= Protein prepared from slaughterhouse and kitchen wastes.

Methods of feeding: Three experimental diets of broiler starter and finisher were formulated with locally available feed ingredients. Starter diet was fed from 1 to 14 days and finisher diet from 15 to 28 days of age. Feed was supplied ad libitum twice daily throughout the experimental period. Fresh water was made available at all time. Immunization, medication & Sanitation: All birds were vaccinated against Ranikhet, Gumboro and infectious Bronchitis as per recommended schedule at 1, 15 and 19th day of bird age. Bleaching powder, Rhodivit and coccidiostat were used to prevent E. Coli, vitamin deficiency and Coccidiosis, respectively. Feeders and waterers were cleaned and dried daily in the morning and afternoon before use. Strict hygienic measures and sanitation programmes of the experimental house were taken throughout the experimental period. Methods of broiler processing At the end of the experiment, one male and one female broiler from each replication were selected, live weight taken and slaughtered for processing. Feed and water were withdrawn 12 hours prior to slaughter to facilitate proper bleeding. The slaughtered birds were final processed by removing the skin, head, shank, viscera, oil gland, kidneys, heart and liver. The gall bladder was removed from the liver and the pericardial sac and arteries were excised from the heart. The gizzard was removed by cutting and it was split open with knife, emptied, washed and the inner lining was removed by hand.


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The data were calculated and recorded as follows: i) Live weight: The selected birds were

individually weighed in the balance and measurement record taken.

ii) Dressing percentage: DP was calculated using dressed weight was divided by live weight and 100 multiplied that value.

Record keeping during experiment Feed intake: Daily feed intake was recorded and calculated from the total feed supplied to the birds by deducting total feed left-over in each replication and expressed as weekly basis. Live weight: Initial and weekly body weight was recorded for every replication in each treatment. Mortality: Mortality was recorded in each treatment. Survivability was calculated on the basis of mortality record. Temperature and relative humidity: Temperature and relative humidity of the experimental house and pens during the experimental period were recorded three times a day (6.00, 14.00 and 22.00) with the help of digital Thermo-hygrometer. Calculating system: All data were collected and calculated in the following way:

i) Live weight: Average live weights of broilers were determined at day-old and at the end of each week up to 4 week in each replication.

ii) FCR: Feed conversion ratio or efficiency was calculated by using the following formula: FCR= Feed intake (g)/Live weight gain (g)

iii) Production cost: Production cost was estimated by considering expenses for chicks, feeds, labour, vaccine, medicine, litter and miscellaneous cost. All costs were calculated on the basis of market price during the time of experimental period.

Statistical analysis The results are presented as the means and the standard deviation of the means (Means±SD). Data were statistically analyzed by one-way analysis of variance (ANOVA) using the COMPARE MEANS procedure (SPSS 7.5, 1999 software for windows, SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

Survey regarding slaughterhouse & kitchen wastes The survey result showed that slaughter house by-product from different slaughter houses nearly 100% were thrown here and there, which are fully useless and spoilage in time which is similar with the previous opinion of Alam (2001). In the other hand, Kitchen and chicken processing units wastes were produced a huge amount. It was observed from their opinion that usually they through all of these wastes near the market or roadside garbage, which is making problem for both of residents or passer as well as market people. Some of the data found from DLS (1997) that approximately 31,450 MT Soft tissue (intestine, stomach, ovary, gallbladder, urinary bladder etc.), 61,240 MT Rumen content, 20,000 MT Blood, 23,325 MT Bone, 1,865 MT Horn/Hooves, 10,000 MT Hides & Skins trimmings and 15,000 MT Fleshings produced per year in wet basis.

Preparation of protein concentrates from by-products The by-products and wastes from slaughter house and chicken are putresible, but the processed waste to protein concentrate were as like as commercial protein concentrate with good quality and it can be preserved for a long time. Protein concentrate from tannery wastes described by Blazej et al (1969) was helpful for the preparation of waste protein concentrate in the present experiment. Processing of fleshing meal is partially similar to the preparation of waste protein concentrate described by Rao (2000).

Chemical composition The chemical composition of wastes protein concentrate is shown in Table 3. The chemical composition of wastes protein concentrate on DM basis contains more CP than that of commercial (Jasoprot). The crude protein content in Jasoprot is 60% where as in wastes protein concentrate was 69.77. The composition of wastes protein concentrate is close to the values reported by Raju et al (1997) and Huque et al (1992). They reported that sun dried flesh wastes contain 69.8 and 68.8% CP, respectively. Ullah (2002) reported that chemical composition of only kitchen wastes was 25.0%, 22.52%, 2.18%, 7.55%, 11.18% and 56.57% for DM, CP, CF, EE, ASH and NFE, respectively. Here the kitchen wastes nutritive value was low due to mixture of different vegetable wastes.


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Table 3: Chemical composition of prepared waste and commercial protein concentrate Composition Sample name

DM% CP% ME kcal/kg CF% EE% Ash% Wastes protein concentrate (SHB) 97.45 69.77 3430 0.83 8.41 10.21 Commercial protein concentrate (Jasoprot) (CPC) 93.0 60.0 3230 4.0 10.0 8.20

Jasoprot is a commercial brand name of CPC

Performance of broilers The performances of broilers are presented in Table 4. The results of the present research work are stated under the following subheadings to evaluate the effects of diets formulated by replacing commercial protein concentrate (CPC) with slaughterhouse and kitchen by-products (SHB).

Feed consumption: The weekly average feed intakes of broilers in different groups are presented in Table 4. It was observed that different feed intake among different treatment groups were insignificant (p>0.05). Feed intake was increased gradually with their age. The results are almost similar with the findings of Gaweck et al (1981), Stojanovic et al (1984) and Koh et al (1998).

Live weight: Day old chick and the weekly average live weight of broilers in different treatment groups are presented in Table 4. The initial live weights of broilers were almost similar in all dietary treatment groups. It was observed from the results that the weekly average live weights of broilers were increased gradually from 1st to 4th week of age. At the end of every week there were no differences among the different dietary treatment groups. The findings obtained in the present study agree with the result of Gill et al (1992) and Huque et al (1992). It is also evident from the above findings that the body weights of the broiler are not significantly influenced by the replacement of CPC with SHB as in dietary treatment groups.

Table 4: First, second, third and fourth week broiler’s average BW (g), AFI (g), FCR, Av. daily FI (g/d), ADG (g/d) and mortality (%) 0-7day

Treatment Initial BW (1d) Av. BW (g) AFI (g) FCR Av. Daily

FI (g/d) ADG (g/d)

Mortality%

T1 40.59± 0.27

121.25± 9.97

166.66± 3.21

1.37± 0.13

23.81± 0.50

16.91± 1.43 0

T2 40.47± 0.09 125.34± 5.22

165.00± 4.00

1.32± 0.04

23.57± 0.57

17.91± 0.75 0

T3 40.51± 0.11 119.80± 5.06

166.00± 4.35

1.39± 0.06

23.71± 0.62

17.11± 0.72 0

Level of significance NS NS NS NS NS NS 0-14d

Treatment Av. BW (g) AFI (g) FCR Av. Daily FI (g/d) ADG(g/d) Mortality

% T1 355.41±3.97 501.77±4.96 1.42±0.01 35.92±0.26 25.39±0.28 0 T2 363.19±9.21 502.40±5.55 1.38±0.05 35.76±0.17 25.94±0.66 0 T3 358.49±7.46 503.97±5.11 1.41±0.02 35.76±0.19 25.61±0.53 0 Level of significance NS NS NS NS NS NS

0-21d T1 721.41±6.56 1269.93±15.43 1.76±0.03 60.47±0.73 34.35±0.31 0 T2 726.27±7.89 1263.60±16.02 1.74±0.03 60.171±0.76 34.67±0.10 0 T3 724.49±5.90 1270.30±15.04 1.75±0.03 60.49±0.72 34.50±0.28 0


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Level of significance NS NS NS NS NS NS

0-28d (Final) T1 1142.10±10.79 2136.93±18.22 1.87±0.04 76.32±0.45 40.78±0.71 0 T2 1142.19±11.09 2130.93±15.95 1.87±0.01 76.10±0.39 40.79±0.40 0 T3 1139.34±12.53 2136.30±20.14 1.87±0.01 76.30±0.72 40.69±0.18 0 Level of significance NS NS NS NS NS NS

Values indicate Mean±SD and NS means non-significant. Where, T1= Control diet (5-6% CPC), T2= Diet in which 5-6% SHB and T3= Diet in which 2.5-3% SHB and 2.5-3% CPC; 10 birds in each treatment.

Feed conversion: The weekly average feed conversion efficiency or ratio (FCR) among different dietary treatment groups is shown in Table 4. No significant differences were observed among the dietary treatment

groups of T1, T2 and T3. The results are partially close with Delic et al (1982) and Tikhonovskaya and Snitsar (1992).

Survivability: The weekly average survivability of broilers during the experimental period in different dietary treatment groups is shown in Table 4. It was observed that survivability percentages of the broilers were 100% for the dietary treatment groups of T1, T2 and T3 at the entire end of weeks. The present study showed that survivability was not affected by different dietary treatment groups. The results are close with the findings of Tikhonovskaya and Snitsar (1992).

Meat yield and necropsy of the dietary treatments: The data presented in Table 5 showed that live weight and dressing percentage were not significantly different among the dietary treatment groups. The necropsy report indicated that there was no remarkable abnormal lesion found in all of the dietary treatment groups. This result is partially similar with the findings of Gill et al (1992) and Tikhonovskaya and Snitsar (1992).

Table 5: Meat yield and necropsy of the dietary treatments at 29th day of age

Treatments Parameters T1 T2 T3

Level of significance

Body Weight (gm) 1142.10±10.79 1142.19±11.09 1139.34±12.53 NS Dressed wt. (gm) 746.59±7.22 746.88±8.19 744.56±7.98 NS Dressing percentage 65.37±1.31 65.39±0.78 65.35±0.92 NS Footpad Lesion (0-3) 0±0 0±0 0±0 NS Breast Blister N N N - Crooked Keel N N N - Black Tongue N N N - Mouth Lesions N N N - Tracheitis Y:N=1:2 N N - Femoral Head Necrosis N N N - Tibial Dyschondroplasia (0-4) 0±0 0±0 0±0 NS Rickets N N N - Synovitis N N N - Fatty Liver Y:N=1:2 N N - Active Gumboro N N N - Bursa Size (1-4) 1±0 1±0 1±0 NS Airsacs (0-4) 0±0 0±0 0±0 NS Retained Yolk N N N - Ascites Y:N=1:2 N N - Litter Eater N N N -

Necropsy of broiler


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Gizzard Erosions (0-4) 0±0 0±0 0±0 NS Proventriculitis N N N - Tapeworms N N N - Roundworms N N N - Enteritis (0-3) 0±0 0±0 0±0 NS Mucoid Enteritis N N N - Watery Enteritis N N N - Feed Passage N N N - E. Acervulina Gross (0-4) 0±0 0±0 0±0 NS E. Maxima Gross (0-4) 0±0 0±0 0±0 NS E. Tenella Gross (0-4) 0.33±0.11 0±0 0.33±0.11 - Kidney (0-2) 0±0 0±0 0±0 NS

Values indicate Mean±SD and NS means non-significant. Where, T1= Control diet (5-6% CPC), T2= Diet in which 5-6% SHB from wastes and T3= Diet in which 2.5-3% SHB and 2.5-3% CPC; N= No (not found), Y= Yes (found). Cost of production and profitability: Analysis of cost factors shown in Table 6, 7, 8 and 9 indicated that the total cost of control diet for starter and grower was 39.32 and 38.82 taka/kg mixed feed; replaced by-product diet for starter and grower was 36.43 and 35.45 taka/kg mixed feed; partially replaced by-product diet for starter and grower was 37.88 and 37.16 taka/kg mixed feed, respectively. Significant (p<0.01) differences were observed among the different dietary treatment groups for mixed feed cost. The feed cost was highest with the dietary treatment group T1 (control), intermediate with the dietary treatment group T3 (partially replaced with SHB to CPC) and lowest with the dietary treatment group T2 (replaced with SHB to CPC). However, treatments of T2 and T3 (CPC fully or partially replaced by SHB) incurred

comparatively lower cost per kg of broiler production than that of the dietary treatment group T1 (commercial). This might be due to the lower production cost of SHB against the CPC. Profit per kg live broiler was found to be higher (p<0.01) on T2 and T3 than that of T1 (Table 9). The results shown in Table 9 indicate that the feed costs varied among different dietary treatments because of the price of feed ingredients. Incorporating SHB in broiler diets as a substitute of CPC can reduce feed cost. Results (Table 9) also indicated that profit significantly (p<0.01) effected by feed cost and total cost of production. These results are in agreement with the findings of Kushak et al (1990) and Tikhonovskaya and Snitsar (1992) who found less cost per kg live weight gain when CPC was replaced by by-products.

Table 6: Feed cost of per kg mixed feed for control diet (T1) in broiler starter and grower

Starter Grower Ingredients

% Per kg unit Rate (Taka)

Cost (Taka/Kg) % Per kg

unit Rate

(Taka) Cost

(Taka/Kg) Corn 51.35 0.5135 26 13.351 56 0.56 26 14.56 Soyabean meal 31 0.31 50 15.5 26 0.26 50 13 CPC 5.25 0.0525 60 3.15 6.12 0.0612 60 3.672 Limestone 1.2 0.012 15 0.18 1.2 0.012 15 0.18 Rice polish 7 0.07 25 1.75 6 0.06 25 1.5 Soy Oil 2.5 0.025 120 3 3 0.03 120 3.6 DCP 0.4 0.004 60 0.24 0.4 0.004 60 0.24 Lysine 0.2 0.002 255 0.51 0.2 0.002 255 0.51 DL 0.18 0.0018 410 0.738 0.16 0.0016 410 0.656 GS 0.25 0.0025 120 0.3 0.25 0.0025 120 0.3 Salt 0.3 0.003 25 0.075 0.3 0.003 25 0.075 NaHCO3 0.01 0.0001 60 0.006 0.01 0.0001 60 0.006


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Choline chloride 0.05 0.0005 60 0.03 0.05 0.0005 60 0.03 Toxin binder 0.2 0.002 110 0.22 0.2 0.002 110 0.22 Enzyme 0.05 0.0005 300 0.15 0.05 0.0005 300 0.15 Coccidiostat 0.05 0.0005 150 0.075 0.05 0.0005 150 0.075 Antibiotic 0.01 0.0001 410 0.041 0.01 0.0001 410 0.041 Total 100 1 39.316 100 1 38.815 Where, T1= Control diet (5-6% CPC), T2= Diet in which 5-6% SHB and T3= Diet in which 2.5-3% SHB and 2.5-3% CPC. Taka= Bangladeshi currency ($1=80 taka).

Table 7: Feed cost of per kg mixed feed for by-product diet (T2) in broiler starter and grower

Starter Grower Ingredients % Per kg unit Rate (Taka) Cost

(Taka/Kg) % Per kg

unit Rate (Taka) Cost (Taka/Kg)

Corn 51.35 0.5135 26 13.351 56 0.56 26 14.56 Soyabean meal 31 0.31 50 15.5 26 0.26 50 13 SBH 5.25 0.0525 5 0.2625 6.12 0.0612 5 0.306 Limestone 1.2 0.012 15 0.18 1.2 0.012 15 0.18 Rice polish 7 0.07 25 1.75 6 0.06 25 1.5 Soy Oil 2.5 0.025 120 3 3 0.03 120 3.6 DCP 0.4 0.004 60 0.24 0.4 0.004 60 0.24 Lysine 0.2 0.002 255 0.51 0.2 0.002 255 0.51 DL 0.18 0.0018 410 0.738 0.16 0.0016 410 0.656 GS 0.25 0.0025 120 0.3 0.25 0.0025 120 0.3 Salt 0.3 0.003 25 0.075 0.3 0.003 25 0.075 NaHCO3 0.01 0.0001 60 0.006 0.01 0.0001 60 0.006 Choline chloride 0.05 0.0005 60 0.03 0.05 0.0005 60 0.03 Toxin binder 0.2 0.002 110 0.22 0.2 0.002 110 0.22 Enzyme 0.05 0.0005 300 0.15 0.05 0.0005 300 0.15 Coccidiostat 0.05 0.0005 150 0.075 0.05 0.0005 150 0.075 Antibiotic 0.01 0.0001 410 0.041 0.01 0.0001 410 0.041 Total 100 1 36.428 100 1 35.449

Where, T1= Control diet (5-6% CPC), T2= Diet in which 5-6% SHB and T3= Diet in which 2.5-3% SHB and 2.5-3% CPC. Taka= Bangladeshi currency ($1=80 taka). Table 8:Feed cost of per kg mixed feed for both commercial and by-product diet (T3) in broiler starter and grower

Starter Grower Ingredients

% Per kg unit Rate (Taka)

Cost (Taka/Kg) % Per kg unit Rate (Taka) Cost

(Taka/Kg) Corn 51.35 0.5135 26 13.351 56 0.56 26 14.56 Soyabean meal 31 0.31 50 15.5 26 0.26 50 13 CPC 2.63 0.0263 60 1.578 3.06 0.031 60 1.86 SHB 2.63 0.0263 5 0.132 3.06 0.031 5 0.155 Limestone 1.2 0.012 15 0.18 1.2 0.012 15 0.18 Rice polish 7 0.07 25 1.75 6 0.06 25 1.5 Soy Oil 2.5 0.025 120 3 3 0.03 120 3.6 DCP 0.4 0.004 60 0.24 0.4 0.004 60 0.24 Lysine 0.2 0.002 255 0.51 0.2 0.002 255 0.51 DL 0.18 0.0018 410 0.738 0.16 0.0016 410 0.656


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GS 0.25 0.0025 120 0.3 0.25 0.0025 120 0.3 Salt 0.3 0.003 25 0.075 0.3 0.003 25 0.075 NaHCO3 0.01 0.0001 60 0.006 0.01 0.0001 60 0.006 Choline chloride 0.05 0.0005 60 0.03 0.05 0.0005 60 0.03 Toxin binder 0.2 0.002 110 0.22 0.2 0.002 110 0.22 Enzyme 0.05 0.0005 300 0.15 0.05 0.0005 300 0.15 Coccidiostat 0.05 0.0005 150 0.075 0.05 0.0005 150 0.075 Antibiotic 0.01 0.0001 410 0.041 0.01 0.0001 410 0.041 Total 100 1 37.875 100 1 37.158 Where, T1= Control diet (5-6% CPC), T2= Diet in which 5-6% SHB and T3= Diet in which 2.5-3% SHB and 2.5-3% CPC. Taka= Bangladeshi currency ($1=80 taka).

Table 9: Total cost of production and profit per kg broiler fed different diets at 28 days of age

Treatments Parameters T1 T2 T3

Level of significance

Live weight (g/bird) 1142.10±10.79 1142.19±11.09 1139.34±12.53 - Feed cost (Tk/kg live bird) 39.07±1.0 35.94±1.0 37.52±1.0 ** Chick cost (Tk/kg live bird) 50.0±2.0 50.0±2.0 50.0±2.0 - Other cost (Tk. Live bird) 16.0±1.0 16.0±1.0 16.0±1.0 - Total cost (Tk/kg live bird) 105.07±4.0 101.94±4.0 103.52±4.0 ** Market price/kg live bird 120.0±10.0 120.0±10.0 120.0±10.0 - Profit (Tk/kg live bird) 14.93±6.0 18.06±6.0 16.48±6.0 **

Figures in row with dissimilar superscripts differ significantly; NS, Non-significant; *, p<0.05; ** and p<0.01. Where, T1= Control diet (5-6% CPC), T2= Diet in which 5-6% SHB and T3= Diet in which 2.5-3% SHB and 2.5-3% CPC. Taka= Bangladeshi currency ($1=80 taka). Ψ Chick cost was calculated after adjusted for mortality ¶ Other costs included vaccine, medicine, labour, transport, litter, electricity, water etc. ¥ Detail of total cost.

CONCLUSION:

Considering the effects of slaughterhouse byproduct and kitchen wastes (SHB) on growth and meat yield, it is observed that replacement of SHB to the diets did not affect adversely. Moreover, feed cost was reduced and therefore, the production cost was expected to be lower with the diets containing cheaper unconventional SHB as substitute of commercial protein concentrate (CPC). Complete or partial replacement of CPC by SHB reduced feed and production cost due to low cost of SHB, therefore, increased the profitability in raising broilers without hampering body weight gain. It is expected that the present findings will help the livestock raisers to formulate economic and efficient balance rations. However, more studies are needed for

the utilization of SHB as other livestock feeds rather than broiler. ACKNOWLEDGMENT: We would like to extend humble gratitude to the Sher-e-Bangla Agricultural University Research System (SAURES) and University Grants Commission (UGC) of Bangladesh for the financial support of 2012-2013. We also express our sincere thanks to all officials and staffs of the laboratory for their support in works.


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REFERENCES: Alam, M. J. 2001. Control of environmental pollution by utilizing leather industry waste. Bangladesh J. Environ. Sci. 7:11-20.

AOAC. 1990. Official Methods of Analysis. 14th ed., Association of Official Analytical Chemists. Washington DC, USA.

Banerjee, G. C. 1992. In poultry. 3rd ed. Oxford and IBH publishing Co. Pvt. Ltd. New Delhi, Bombay, Calcutta, India.

Blazej, A., Galatik, A. and Minarik, A. 1969. Zpusob izolace bilkovinove hmoty zodpadnich kozeluzskych

louzicich lazni (The method of protein mass isolation from tanning lime liquors). CPS 137077.

Delic, I., Cuperlovic, M., Vucurevic, N., Stojanonic, S., Rede, R., Vukie, M. and Beljanski, V. 1982. Skin and meat meal as a source of protein in diets for fattening chicken. Poultry abstracts, 36 (1/2): 55-69.

DLS. 1997. Department of Livestock Services, Govt. of Bangladesh, Farm gate, Dhaka, Bangladesh. Yearly compiled report.

Gaweck, K. Lipinska, H. and Rutkowski, A. 1981. Meal from residues of the tanning and meat industries in feeds for fattening chickens. Roczniki Naukowa Zootechniki 8 (1): 185-192.

Gill, S. A., Chowdhury, S. M. and Hossain, Z. 1992. Comparative study on the effects of protein from meat processing for broiler ration. Int. J. Agric. Sci. 28 (6): 26-27.

Huque, K.S., Chowdhury, S.A., Hossain, J. and Kamal, A.H.M. 1992. Use of leather shavings as an alternative to fishmeal for cattle and poultry in Bangladesh. Bangladesh J. Livest. Res. BLRI, Savar, Dhaka, pp.37-52.

Islam, M. A., M. D. Hossain, S. M. Bulbul and M. A. R. Howlider. 1994. Unconventional feeds for broilers. Indian Vet. J. 71: 775-780.

Koh, K., Sakurai, K. Hirako, Y. and Karasawa, Y. 1998. Digestibility and nitrogen retention of chickens fed on limed split diets. Anim. Sci. Tech. 69 (1): 62-64.

Kushak, R.I., Travid, I.L., Basova, N.A., Yukhno, E.N., Filipchenkova, L.P., Isidorov, G.E. and Val’dman, A.R. 1990. Effectiveness of different doses of fish protein concentrate in feeding of chickens. Dolady Vsesoyuzhoi Ordena Lenina I ordena Trudovogo Krasnogo Znameni Akademii Sel’ skhozyaistvenny auk in. V.I. Lenina. No. 6:51-54.

Miroslaw, F. and Krzysztof, S. 1986. Preparation and properties of protein concentration from broiler chicken heads. J. Sci. Food Agric. 37: 445-454.

Nabil F. Abd El-Hakim, Mohsen S. Hussein and Hassaein A. Abdel-Halim. 2009. Effect of partial replacement of soybean meal protein with dehydrated alfalfa meal (Medicago sativa L.) on growth performance and feed utilization of male Nile tilapia (Oreochromis niloticus L.) fingerlings reared in tanks. Egypt J. Aquat. Biol. & Fish., 13 (2): 35 - 52 (ISSN 1110 –1131)

Rahman, M. A. and A. Reza. 1983. Study on the effect of unconventional sources of protein and energy for poultry. M.Sc. Thesis. Dept. of Anim. Nutr., Bangladesh Agricultural University, Mymensingh, Bangladesh.

Raju, A.A., Rose, C. and Rao, N.M. 1997. Enzymatic hydrolysis of tannery fleshings using chicken intestine protease. Anim. Feed Sci. Tech. 66 (1-4):139-147.

Rao, D.V.R.P. 2000. Solid waste management. A project of UNIDO. Prakash Feed Mills. Personal communication. Kancheepuram, India. pp. 3-18.

Sanon, H.O., Kabore-Zoungrana, C. and Ledin, I. 2008. Growth and carcass characteristics of male Sahelian goats fed leaves or pods of Pterocarpus lucens or Acacia Senegal. Livest. Sci. 117: 192–202.

Sarkar, P. K., Chowdhury, S. D., Kabir, M. H. and Sarker, P. K. 2008. Comparative study on the productivity and profitability of commercial broiler, cockerel of a layer strain and cross-bred (RIR ♂ × FAYOUMI ♀) CHICKS. Bang. J. Anim. Sci. 37(2) : 89 – 98. (ISSN 0003-3588)

Scott, M. L., Neshem, M. C. and Young, R. J. 1976. Nutrition of the Chicken. Chapter eight. 2nd ed. Published by: M. L. Scott and Associates, Ithaca, New York, USA.


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Singh, R. A. 1990. In: Poultry production. 3rd ed. Kalyany publishers, New Delhi, India.

SPSS. 1999. 7.5 SPSS software for windows, SPSS Inc., Chicago, IL, USA.

Stojanovic, S., Ristic, M. and Stosavlijvic, R. 1984. Nutrition value of hydrolyzes feather meal and hydrolyzed feather and blood meal with addition of lysine concentrate. Stocarstvo (Yugoslavia). 38 (11): 429-434.

Tikhonovskaya, N.D. and Snitsar, A.I. 1992. Use of fishmeal with protein concentrates in the diet for broiler chickens. World’s Poult. Assoc., Netherlands. pp. 620-621.

Ullah, K. 2002. Use of kitchen waste as animal feed. Bangladesh Chemical, Scientific & Industrial Research (BCSIR), Dhaka, Bangladesh. Report on Science Laboratory Hand Book.

Citation of this article: Md. Jahangir Alam, Talukdar Muhammad Waliullah, Md. Saiful Islam, Zannatul Ferdaushi (2015). UTILIZATION OF SLAUGHTER HOUSE

AND KITCHEN BY-PRODUCTS AS PROTEIN SOURCE IN BROILER DIET. Journal of Biotechnology and Biosafety. 3(1):171-182



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Research art i c l e A TECHNIQUE TO QUALIFY IN PROFICIENCY TEST OF CHLORAMPHENICOL RESIDUE IN PRAWN MATRIX UNDER METHOD DEVELOPMENT, VALIDATION, QUANTITATIVE ANALYSIS BY LC-MS/MS AND PT PARTICIPATION APPROACH.

________________________________ 1*Md. Ashraful Alam, 2Akter Mst. Yeasmin, 2Talukdar Muhammad Waliullah, 1Saleh Ahmed, 1Md. Serajul Islam, 1Md. Manik Mia, 1Md. Anwar Parvez, 1Nitta Ranjan Biswas, 1Sayed Arif Azad ________________________________

1. 1FIQC Laboratory, Department of Fisheries, Matshya Bhaban, Ramna, Dhaka-1000, Bangladesh.

2. 2Molecular and Cell Biology Laboratory, Bioscience Department, GSST, Shizuoka University, 836-Oya, Suruga-ku, Shizuoka ╤ 422-8529, Japan. *Corresponding author Email: [email protected]

ABSTRACT Together with the use of validated methods, proficiency testing is an essential element of laboratory quality assurance system, which met the world-class quality. A method was developed and validated for the analysis of chloramphenicol (CAP) in prawn matrix by Liquid Chromatography Mass Spectrometry (LC-MS/MS). Using this method the laboratory participated in proficiency test and qualified successfully. The LC separation was achieved by AQUITY UPLC BEH C18 Column (2.1x50mm; 1.7µm) with gradient elution using a mobile phase of acetonitrile and water. Mass spectral acquisition was done using electrospray ionization in the negative ion mode applying multiple reactions monitoring (MRM) of two diagnostic transition reactions for Chloramphenicol (CAP) m/z 321.17→151.98 and m/z 321.17→257.043 and for d5_CAP m/z 326.17→156.98. Shrimp samples were finally extracted with ethylacetate and evaporated to dryness, followed by a clean-up step using the liquid–liquid extraction with acetonitrile / hexane (1:1 v/v) mixture and reconstituted with 10% acetonitrile in water. The method validation was carried out according to the criteria of Commission decision 2002/657/EC. The calibration curve showed a good linearity in the concentration range from 0.05 to 1.0 ng/g with the correlation coefficient above 0.9992. The decision limit (CCα) and detection capability (CCβ) was 0.045 and 0.080ng/g respectively. The mean recoveries were in the range of 88–109%. Finally the laboratory participated in the FAPAS proficiency test program 02230 (March-April, 2014) as a laboratory ID no 018 and obtained Z-score 0.9 where qualifying Z-score is -2 ≤ Z ≥2 and occasional qualifying Z-score is 2 ˂ Z ˃ 3.

KEY WORDS: LC-MS/MS, Proficiency Test (PT), Prawn, Cloramphenicol, Validation

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INTRODUCTION

Proficiency Testing (PT) Proficiency testing comprises an inter laboratory system for the regular testing of the accuracy that the participant laboratories can achieve. In its usual form, the organisers of the scheme distribute portions of a homogeneous material to each the participants, who analyse the material under typical conditions and report the result to the organisers. The organisers compile the results and inform the participants of the outcome, usually in the form of a score relating to the accuracy of the result (Michael T., 2005). Proficiency Testing and Accreditation Accreditation agencies require analytical laboratories to participate in an appropriate PT scheme where one is available, and demonstrate a system for handling the outcome. This is only one of many requirements of accreditation. Materials distributed PT Scheme The materials distributed are as close as possible to the materials being regularly analysed, so that the results of the scheme represent the capability of the laboratories working under routine conditions.

Purpose of Proficiency Testing The primary purpose of proficiency testing is to help laboratories detect and cure any unacceptably large inaccuracy in their reported results. In other words, it is designed as a self-help system to tell the participants whether they need to modify their procedures. Proficiency tests are not ideally designed for any other purpose, although their results can be used, with due regard to their limitations, and combined with other information, for certain other purposes (Michael T., 2005). Chloramphenicol (CAP) is a broad-spectrum antibiotic drug, which interferes with protein synthesis of many gram-negative and gram-positive bacteria (Austin S. et al., 1967). The wide range antibiotic CAP has toxic effects on humans (Allen E H., 1985). Miscellaneous toxic effects are due to the dichloride carbon alpha to the carbonyl group (Figure1); this carbon readily undergoes substitution with nucleophiles such as those found on proteins (B. R. Baker 1967). Because of many serious side-effects (e.g. fatal aplastic anemia, grey syndrome, severe bone marrow depression) (Joan-Ramon Laporte et al., 1998 and www.drugs.com), use of CAP to treat food-producing animals was banned by the European Community (Off. J. Eur. Commun.1994) and they established MRPL (minimum required performance limit) 0.3µg/kg (Commission Decision 2003/181/EC).

Figure1: Structural formula of chloramphenicol (Pubchem. C. D., CID 5959)


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Thus, a sensitive and reliable method for the determination of CAP at residual levels is very important and qualification in PT assures the reliable test method. The confirmation of suspect positive samples must be carried out by mass spectrometry (MS) coupled to an adequate chromatographic separation. In our study we used Ultra Performance LC (UPLC) and tandem quadruple MS/MS (TQD). The PT sample was analyzed and measured under the calibration curve using contamination free prawn. Method Validation was done according the criteria described in EU decision 2002/657/EC using same matrix.

MATERIAL AND METHODS

Negative Shrimp collection: Sample was collected from deep sea and checked as blank i.e., free from chloramphenicol contamination.

PT Sample Collection: FAPAS proficiency test program 02230 (March-April, 2014) and allocated laboratory number 18.

Equipment Calibration Micropipette: eppendorf, analytical balance (4 dp, Shimadzu AUY 220) and volumetric flaskswere calibrated by national metrology laboratory (NML) of BSTI, Bangladesh. Apparatus and Chemicals: Micro pipette: eppendorf, analytical balance (4 dp, Shimadzu AUY 220), volumetric flask: certified and type A, Syringe filter: 13 mm PTFE 0.2 µm filter (Waters, USA); CAP and d5-CAP standards, assay >99%, Sigma Fluka, Germani; ethylacetate& n-Hexane (HPLC grade), acetonitrile and methanol (HPLC and MS grade), Sigma Aldrich, Germani; NaCl: RANKEM, S0160; Phosphate buffered saline(PBS); Purified Water (deionized) from barnstedapparatus maintaining water quality in case of resistivity18.2 Ω.

Standard Preparation:

1. CAP Mother Stock (2439µg/ml) After verification of calibrated balance chloramphenicol standard was weight in a 5ml volumetric flask (certified

and calibrated) and dissolved in methanol. Standard weight was calculated considering purity of standard.

2. Intermediate CAP Standard (10µg/mL): 4µL of stock standard (2439µg/mL) was taken in 10mL amber volumetric flask and diluted with methanol and store in a refrigerator at 40c.

3. Working Standard of CAP (10ng/mL) and CAP_D5 (30ng/mL) standard: Working standard was prepared by diluting intermediate at concentration 10ng/mL for chloramphenicol(CAP) and that of 30ng/mL for chloramphenicol _D5 (Internal standard)

Samples: Shrimp samples were pasted with blender after removing shell and eight samples [reagent blank, matrix blank, matrix with internal standard (IS) blank and quality control (QC)] were taken as quality samples and another seven were taken to make calibration curve (0.05 to 1ppb). All these samples and proficiency Testing (PT) samples (three replications) were weighed out (3.00 ± 0.01g) into 50mL screw capped polypropylene centrifuge tube separately.

Extraction Procedure: (Storey J. et al., 2003)

100µl of D5-CAP solution (30ng/mL) was added to each of matrix_IS, calibration curve, QC and analytes for equivalent concentration of 1.00ppb and required volume of CAP standard (10ng/mL) was added to the standard curve tubes to make equivalent conc. 0.05 to 1ppb and that of QC samples 0.3 ppb and allowed to stand for 10 min. Then 4mL PBS solution, 1mL of 16% NaCl solution and 4mL of acetonitrile were added. The samples were vortexed for 1 min and centrifuged at 4500 rpm for 20 mins. Supernatant was transferred to 50mL centrifuge tube and 10mL deionized water and 10mL hexane were added and shaked gently for 30 sec and again centrifuged at 4500 rpm for 20 mins. After that the upper n-hexane layer was discarded and 8 ml of ethyl acetate was added and mixed by vortex for 1 min and finally centrifuged at 4500 rpm for 20 mins. Ultimately upper layer (ethylacetate) was transferred to glass tubes and taken to dryness at 550C under N2 gas. Residue was reconstituted in 1mL of 50 % methanol and transferred to vial by filtering with syringe filter (0.2µm) for analysis with UPLC/MS/MS.


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UPLC-MS/MS Analysis:

Instrmennt Calibration Software: Masslynx v4.1 LC Identity: ACQUITY UPLC®

Waters, USA Column:AQUITY UPLC BEH C18, 1.7µm, 2.1x50 mm (Waters, USA), Solvent A: Water, Solvent B: Acetonitrile, Injection Volume (µL): 10, Column temp: 350C, Sample Temp: 100C LC Separation Method: Gradient MS Method Parameters

Instrument Identity: ACQ_TQD#QBB933, Waters, USA.

Capillary (V): 3.2, Cone (V): 3, Extractor (V): 3, RF Lens: 0.20, Source Temp.: 1200C, Dessolvation Temp: 2900C, Cone Gas Flow(L/Hr): 50, Desolvation Gas Flow (L/Hr): 900, Collision Gas Flow (L/Hr): 0.10, LM1 Resolution: 13, HM1 Resolution: 13, Ion Energy1: 0.80, MS mode entrance: 50, exit: 50. MSMS mode: entrance: 1, exit: 1, LM2 Resolution: 14, HM2 Resolution: 15, Ion Energy2: 1.0, Ion mode: ESI (-Ve), Run time (Min): 5, Function type: MRM (multiple reaction monitoring).

Calculation

Ion Ratio, R = Peak area of primary ion (PAPI) Peak area of secondary ion (PASI) Response factor (RF) RF = PAPI of interested substance x ISC Peak area of internal standard ion CAP concentration, X = (RF-b)/a Where, ISC= Internal Standard Concentration a=Slope of calibration curve, b= intercept of calibration curve.

RESULTS

MS–MS detection The detector (TQD MS-MS) was firstly operated in negative ESI–MS mode to select characteristic ions as the precursors of CAP (m/z 321.17) and its deuterated internal standard CAP_d5 (m/z 326.17). Both CAP and CAP_d5 were then analyzed by ESI– MS–MS in a negative ionization product ion scan mode by selecting m/z 321.17 and m/z 326.17 ions as the precursor ion, respectively. The collision-induced dissociation (CID) experiments of these ions, giving rise to m/z 151.98 and m/z 257.043 ions for CAP and m/z 156.98 for CAP_d5 (Figure 3). The selected

transitions for CAP and CAP_d5 for the optimal MS–MS conditions are given in Table 1. The developed gradient method with acetonitrilel and water is shown the Table 2. There were no peak in solvent blank and matrix blank and a single peak in case of matrix_IS. PT samples were tested in that condition and their three replications provided results 0.39, 0.40 and 0.42. In PT participation we reported 0.42 that secured Z score 0.9 where qualifying score is -2 ≤ Z ≥2 (Figure 2)


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Figure 2: Assigned Value and Z-score showing the position of this laboratory (18) in PT program (FAPAS PT report 02230, 2014).

Figure 3: Transitions of CAP and CAP_d5 showing chromatogram


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Table 1: Transitions monitored for CAP and CAP_d5 determination and optimal MS–MS conditions

SL No Parent Daughter Dwell Cone (V) Collision (eV) Compound 1 321.17 151.98 0.05 34 32 CAP 2 321.17 257.043 0.05 34 16 CAP 3 326.17 156.98 0.05 28 24 CAP_d5

Table 2: Gradient Table for chromatographic separation

SL No Time (min) Flow Rate (mL) % A % B 1 Initial 0.300 95 5 2 0.5 0.300 95 5 3 1.20 0.300 5 95 4 2.50 0.300 5 95 5 3.50 0.300 95 5 6 4.00 0.300 95 5

Table 3: Performance data of the UPLC–MS–MS method for the analysis of CAP in spiked samples

Fortification level ng/g Parameter 0.15 0.30 0.45 0.60

Overall mean ± SD 0.14 ± 0.02 0.29 ± 0.02 0.46 ± 0.03 0.59± 0.06 Trueness± SD (%) 93.33 ± 10 96.67 ± 10 97.82 ± 12 98.33 ± 13 Precision (RSD%) 15.3 10.2 13.2 10.1

Analytical performance

Method validation was carried out according to criteria described in Decision 2002/657/EC. The parameters taken into account were: response linearity, decision limit (CCα), detection capability (CCβ), reliability and accuracy. The calibration curve showed a good linearity in the conc. range from 0.05 to 1.0 ng/g with the correlation coefficient above 0.9985. The decision limit (CCα) and the detection capability (CCβ) were 0.045ng/g and 0.08ng/g, respectively. The trueness was expressed in terms of recovery rates. The results are presented in Table 3.


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DISCUSSION

The precursor and daughter ions obtained in the result have a good agreement with previous findings (Hao-Yu Shen, et al., 2005), which indicates the compound was identified accurately. The gradient method for compound separation was developed by acetonitrile and water, which provided very sharp peak. The decision limit (CCα) and the detection capability (CCβ) for CAP were 0.045ng/g and 0.08ng/g, respectively which are significantly below the MRPL of 0.3ng/g and demonstrates that the method

was sufficiently fit for the purpose. The qualification in proficiency testing system organized by FAPAS March-April 2014, noticed that result and analysis done by the system is world-wide qualified and equivalent. AKNOWLEDGEMENT

We would like to provide humble gratitude to the officials and stuffs of Fish Inspection and Quality Control Laboratory. We express our sincere thanks to all associates of this work.

REFERENCE

Allen E H. (1985) Review of chromatographic methods for chloramphenicol residues in milk, eggs, and tissues from food-producing animals. J Assoc off Anal Chem., 68(5):990-9.

Austin S. Weisberger, M.D. (1967)IInhibition of Protein Synthesis by Chloramphenicol, Annual Review of Medicine,18: 483-494

B. R. Baker (1967), Design of Active-Site-Directed Irreversible Enzyme Inhibitors. The Organic Chemistry of the Enzymatic Active-Site, John Wiley and Sons, New York., 159: 3819.1091-a Chloramphenicol Injection Side Effects. Drug.com. http://www.drugs.com /sfx/ chloramphenicol-injection-side-effects.html

Commission Regulation (EC) 1430/94 of 22 June 1994, Off. J. Eur. Commun., L156/6.

European Commission SANNCO/2004/2726-rev 4December 2008, health and consumer protection Directorate-General, Guideline for Implementation of dicision 2002/657/EC.

FAPAS PT report 02230 (2014), Chloramphenicol in prawns, the food and environmental research agency, Sannd Hutton York, YO41 1LZ, UK. www.fapas.com.

Hao-Yu Shen, Hai-Liang Jiang.( 2005) Screening, determination and confirmation of chloramphenicol in seafood, meat and honey using ELISA, HPLC–UVD, GC–ECD, GC–MS–EI–SIM and GCMS–NCI–SIM methods.AnalyticaChimicaActa.535.1–2: 33–41

Joan-Ramon Laporte, Xavier Vidal, Elena Ballarín, and Luisa Ibáñez (1998), Possible association between ocular chloramphenicol and aplasticanaemia the absolute risk is very low. Br J ClinPharmacol,46(2): 181–184.

Joe Storey, Al Pfenning, Sherri Turnipseed, Gene Nandrea, Rebecca Lee, Cathy Burns, Mark Madson (2003), determination of chloramphenicol residues in shrimp and crab tissues by electrospray triple quadrupole LC/MS/MS, Laboratory Information Bulletin (LIB) No. 4306, 19(6)

Michael Thompson, AMC technical brief background paper; analytical method committee, ISSN 1757- 5958, AMCTB 18A January 2005

Pubchem Chemistry database: http://pubchem.ncbi.nlm.nih.gov/compound/chloramphenicol#section=Top, compound summary for CID 5959


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2003/181/EC: Commission Decision of 13 March 2003 amending Decision 2002/657/EC as regards the setting of minimum required performance limits (MRPLs) for

certain residues in food of animal origin, Official J., L71/17.

Citation of this article: Md. Ashraful Alam, Akter Mst. Yeasmin, Talukdar Muhammad Waliullah, Saleh Ahmed, Md. Serajul Islam, Md. Manik Mia, Md. Anwar Parvez, Nitta Ranjan Biswas, Sayed Arif Azad (2015). A TECHNIQUE TO QUALIFY IN PROFICIENCY TEST OF CHLORAMPHENICOL RESIDUE IN PRAWN MATRIX UNDER METHOD DEVELOPMENT, VALIDATION, QUANTITATIVE ANALYSIS BY LC-MS/MS AND PT PARTICIPATION APPROACH. Journal of biotechnology and biosafety. 3(1): 183-190.


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