Figure 7: Flowchart of the QuEChERS AOAC sample preparation procedure. Adapted from reference 7.
QuEChERS (quick, easy, cheap, effective, rugged and safe) is a sample preparation technique that was originally developed
for the extraction of pesticides from fruits and vegetables (4). It is a relatively simpler sample preparation procedure with
two steps: a salting out partitioning extraction involving water and acetonitrile with high concentrations of salts such as
sodium chloride, magnesium sulphate and buffering agents, and a dispersive-SPE step in which an aliquot from the first step
is treated with various sorbents to remove matrix compounds that could interfere with subsequent LC–MS, LC–MS-MS, GC–MS or
GC–MS-MS analysis. The technique has proven to be widely applicable at trace levels for hundreds of pesticides in a variety
of matrices. Standard protocols are available that make it a generic sample preparation procedure.
Figure 8: Overlay HPLC–fluorescence chromatograms of a PAH-spiked fish extract. The black portion of the chromatogram used
260-nm and 352-nm excitation and emission wavelengths, respectively, the red portion used 260-nm and 420-nm wavelengths, and
the blue portion used 260-nm and 440-nm wavelengths. For acenaphthylene, UV detection at 230-nm was used. Column: 50 mm ×
4.6 mm, 1.8-µm dp Agilent Zorbax Eclipse PAH C18; flow rate: 0.8 mL/min; temperature: 18 °C; injection volume: 5 µL; mobile-phase
A: deionized water; mobile-phase B: acetonitrile; gradient: 60% B for 1.5 min, 60–90% B in 6.5 min, 90–100% B in 6 min. Peaks:
1 = naphthalene (20 ng/g), 2 = acenaphthylene (20 ng/g), 3 = acenaphthene (10 ng/g), 4 = fluorene (10 ng/g), 5 = phenanthrene
(10 ng/g), 6 = anthracene (10 ng/g), 7 = fluoranthene (10 ng/g), 8 = pyrene (10 ng/g), 9 = 1,2-benzanthracene (5 ng/g), 10
= chrysene (10 ng/g), 11 = benzo[e]pyrene (5 ng/g), 12 = benz[e]acenapthylene (5 ng/g), 13 = benzo[k]fluoranthene (5 ng/g),
14 = dibenz[a,h]anthracene (5 ng/g), 15 = benzo[ghi]perylene (5 ng/g), 16 = indeno[1,2,3-cd]pyrene (5 ng/g).
Recently, QuEChERS extraction has expanded well beyond the pesticide laboratory and has been used for many matrices ranging
from antibiotics in meat and poultry, veterinary drugs in animal feed, and environmental contaminants in soil. In this example,
using the protocol in Figure 7, QuEChERS was used for the extraction of polycyclic aromatic hydrocarbons (PAHs) in fish. The
PAHs are a large group of organic compounds included in the European Union and the United States Environmental Protection
Agency priority pollutant list because of their mutagenic and carcinogenic properties. In the marine environment, PAHs are
bioavailable to marine species via the food chain, as waterborne compounds and contaminated sediments. This application shows
that tandem MS detection techniques are not necessarily required for just enough sample preparation. Most of the PAHs are highly fluorescent and thus, as shown in Figure 8, reversedphase HPLC was combined
with fluorescence detection to determine 16 of these compounds at a spiking level of less than 10 ng/g (5). QuEChERS extraction
provided excellent recoveries with %RSDs below 2.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC