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Confirmation of Aflatoxin in Fish and Shrimp by Lc/ms-ms (zevo Tqd)

Mohammed Anwar Parvez
Technologist
Quality Control Laboratory, Dhaka, Department of Fisheries, Bangladesh

Md. Barkatul Alam
Technical Manager
Quality Control Laboratory, Dhaka, Department of Fisheries, Bangladesh

Md Manik Mia Technologist
Quality Assurance Manager
Quality Control Laboratory, Dhaka, Department of Fisheries, Bangladesh

Abstract/ Executive Summary:

Mycotoxins are agricultural contaminants of fungal origin occurring at all latitudes worldwide and being characterized by acute and chronic effects on human health and animal wellness, depending on the species sensitivity. Various types of crops like maize, wheat, soybeans etc are used as raw materials for preparing feed of fish and shrimp. They are particularly susceptible to infection by Aspergillus following prolonged exposure to a high humidity environment. For this reason, the fish and shrimp samples should be tested for identifying and quantifying mycotoxin. The major mycotoxins of food concern are aflatoxin (B₁, B₂, G₁, G₂) for its toxicity. This paper focus on the confirmation of aflatoxin in fish and shrimp by developing method and validated it by LC/MS-MS (ZEVO TQD) which is important for ensuring the safety of fishery product for human consumption. The monitored MRM transitions for B1, B2, G1, G2 were m/z 313→ 57 and m/z 313→71, m/z 318→88 and m/z 318→256, m/z 328.75→242.99 and m/z 328.75→199.9, m/z 330.34→88 and m/z 330.34→106 respectively. Limit of decision (CCα) for B₁, B₂, G₁, G₂ were 0.59 μg/kg, 0.70 μg/kg, 0.68 μg/kg, 0.83 μg/kg respectively and detection capability (CCβ) for B₁, B₂, G₁, G₂ were 1.01μg/kg, 1.19 μg/kg, 1.15 μg/kg, 1.40 μg/kg respectively.

Keywords: Aflatoxin, CCα, CCβ, LC-MS, MS, Shrimp, Fish

Location: In Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Shrimp, Fish

Objectives:

To confirmation of aflatoxin in fish and shrimp by LC/MS-MS (ZEVO TQD).

Methodology:

Standards and reagents:
Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2, M1 standard ware purchased from Sigma-Aldrich (st. Louis, MO). MS grade formic acid, methanol and acetonitrile were also from Sigma-Aldrich (st. Louis, MO). Besides these HPLC grade chemicals (Ethyl acetate, Acetonitrile, Methanol), Appropriate dispersive sorbent (Z-Sep+) and Deionized water were used.
Sample preparation procedure:
i. Take (1 ± 0.05) g of blend sample in to 50 ml centrifuge tube.
ii. Add 100 ?l from 50 ppb M1
iii. Add 9 ml of ACN
iv. Shake 10 minutes and centrifuge at 8000 rpm for 15 minutes
v. Take 8 ml of supernatant
vi. Add 90 μl formic acid (MS Grade)
vii. Add 500 mg Appropriate dispersive sorbent (Z-Sep+)
viii. Vortex for 5 minutes and centrifuge at 8000 rpm for 15 minutes
ix. Take 7 ml of supernatant in a glass tube
x. Evaporate to Dryness under nitrogen gas Pressure
xi. Reconstitute by 1 ml of 0.1% Formic acid With 10% Acetonitrile
xii. Pass the sample through a 0.45 μm syringe filter and collect in a vial for subsequent LC-MS/MS analysis.

Chromatography conditions:
LC/MS-MS Module: Acquity UPLC ZEVO TQD (Waters, USA)
Column: Acquity UPLC BEH C18 1.7 m, 2.1x100 mm column, Waters, made in Ireland.
Column Temperature: Ambient.

MS condition:

Mass Spectrometer: ZEVO TQD (Waters, USA)-

Source (ESP+) Capillary (kv) : 4

Cone (v) : 42 Extractor (v) :

3 RF Lens : 0.2

Source Temp (ºc) : 135

Dessolvation Temp (ºc) : 400

Desolvation gas flow (L/h) : 900

Cone gas flow (L/h) : 50

Analyser

LM Resolution 1 : 9.7

HM Resolution 1 : 15

Ion Energy 1 : 0.2

Entrance voltage : 50

Exit voltage : 50

LM Resolution 2 : 10.8

HM Resolution 2 : 14.9

Ion Energy 2 : 0.7

Multiplier voltage : 650
Collision gas : Argon @ 3.5 x 10-3 mbar
Span (Daltons) : 0.00
Dwell time (Sec) : 0.030
Inter Channel Delay (Sec) : 0.02
Inter-Scan Delay (Sec) : 0.02.

Source of Information: Asian Australas. J. Food Saf. Secur. 2020, 4 (1), 14-21; ISSN 2523-1073 (Print) 2523-2983(Online)

Article Link (Online Journal): www.ebupress.com/journal/aajfss

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The method was developed and validated as per guideline and commission decision 2002/657/EC. The recovery, linearity and other parameters explains that the developed method is good enough for the confirmatory analysis of Aflatoxin (B1, B2, G1, G2) by LC/MS-MS in fish & shrimp matrix.

Knowledge Management: Journal