Extraction of Polycyclic Aromatic Hydrocarbons (PAH) from Fish Using the QuEChERS Approach*

September 1, 2010

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2010, Volume 0, Issue 0

The QuEChERS approach is used for the extraction of 16 PAH compounds from fish with analysis by LC using fluorescence detection.

Don Shelly and Craig A. Perman, UCT, LLC

The QuEChERS approach is used for the extraction of 16 PAH compounds from fish with analysis by LC using fluorescence detection.


Extracts were analyzed using a Shimadzu LC system (Shimadzu Corporation, Kyoto, Japan) equipped with a LC-20AB pump, DGU-20AS degasser, and a fluorescence RF-10AXL (FLD) detector.

1) Sample Preparation

a) Transfer 5 g homogenized fish to a 50 mL centrifuge tube

b) Add 10 mL acetonitrile

c) Shake vigorously

d) Add the contents of UCT ECMSSC50CT-MP pouch containing 4 g MgSO4 & 1 g NaCl to the tube

e) Immediately vortex the mixture for 3 min

f) Centrifuge the tube for 3 min at 3400 rpm

g) The supernatant is ready for clean-up

2) Sample Cleanup

a) Add 3 mL of the supernatant to centrifuge tube UCT ECMPSC1815CT (900 mg MgSO4, 300 mg PSA, and 150 mg endcapped C18)

b) Shake for 1 min

c) Centrifuge for 1 min at 3400 rpm

d) Filter supernatant using 0.20 μm PTFE membrane filter

e) Sample is ready for analysis

3) Analysis Conditions

a) Separation is performed using a C18 column (CC 150/4 Nucleosil 100-5 C18 PAH, 150 × 4.0 mm; 5 μm particle size; Macherey-Nagel, Duren, Germany) or equivalent. Maintained at room temperature.

b) Inject 15 μL

c) Mobile phase: Initial 50% ACN and 50% water

d) Use a linear gradient to 100% ACN over 15 min

e) Flow rate is 0.8 mL/min

f) Hold for 13 min

g) Initial conditions are achieved in 1 min and maintained for 6 min before next run

h) Total run time is 40 min

i) Fluorescence Wavelength Program: Each compound is detected at its optimum excitation/emission pair.

315/260 nm naphthalene, acenaphthene, fluorene

366/260 nm phenanthrene

430/260 nm anthracene, fluoranthene, pyrene,

benz(a)anthracene, chrysene, benzo(b)fluoranthene,

benzo(k)fluoranthene, benzo(a)pyrene, dibenz(a,h)anthracene,

benzo(g,h,i)perylene and dibenzo(a,l)pyrene

505/290 nm (indeno(1,2,3-cd)pyrene

Figure 1: Overlay LC-FLD chromatograms of a matrix-matched standard PAHs mixture (a) containing 16 PAHs (Naph-100.3 μg/L, Ace-100.3 μg/L, Flu-19.8 μg/L, Phe-10.0 μg/L, Ant-10.0 μg/L, Fln-19.7 μg/L, Pyr-9.69 μg/L, B(a)A-9.84 μg/L, Chry-9.91 μg/L, B(b)Ft-20.1 μg/L, B(k)Ft-9.98 μg/L, B(a)P-10.0 μg/L, DB(a,l)P-20.0 μg/L, DB(a,h)A-20.0 μg/L, B(g,h,i)P-19.9 μg/L and InP-10.0 μg/L) and (b) a blank horse mackerel extract.


This QuEChERS approach allows efficient extraction of PAHs from multiple homogenized fish samples in 20 min or less while providing a clean extract. Sample clean-up prevents instrument downtime.

*Adapted from: Ramalhosa, Maria Joao, et. al., "Analysis of polycyclic aromatic hydrocarbons in fish: evaluation of a quick, easy, cheap, effective, rugged, and safe extraction method," J. Sep. Sci., 32, 3529–3538 (2009).


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