THE HONG KONG POLYTECHNIC UNIVERSITY

DEPARTMENT OF APPLIED BIOLOGY AND

CHEMICAL TECHNOLOGY
BSc(Hons) in Food Safety and Technology
ABCT4402
Advanced Analytical Techniques for Food Hazards
Laboratory Manual

Expt. 1: Sample preparation for GC/MS and GC/ECD analysis of

pesticides
1.

Introduction

Many polychlorinated aromatic compounds were introduced in the 1940s as pesticides, of which
lindane, DDT, and DDE are well known examples which have been used for over 30 years. It was
later discovered that most of these compounds are carcinogenic and their use has been banned or
strictly restricted worldwide. However, many of these compounds are not bio-degradable and are
persistently found in the natural environment, such as soil and sediment.These pesticides get into the
food chain (e.g. meat, fish and vegetables) and eventually into humans through food consumption.
Thus, the residual level (at ng/ml or ppb levels) of pesticides in agricultural produce is a major
concern in regard to food safety and health risk in many countries.
Phosphorous-containing pesticides, such as malathion and methamidophos, are bio-degradable and
are therefore short-lived in food and environmental samples. Many incidences of acute
food poisoning are due to contamination of high levels of metamidophos in fresh vegetables.
Apart from screening methods such as enzymatic assays and TLC, GC/MS is routinely used as the
final confirmatory method in pesticide analysis. The GC/MS approach offers many advantages,
including high separation efficiency of capillary columns, high sensitivity and rich mass spectral
data for the identification of residual pesticide.
In pesticide residue analysis, sample preparation is very important prior to GC/MS or GC-ECD
analysis. This work can eliminate as much chemical interferences as possible prior to GC/MS or
GC-ECD analysis whilst, at the same time, reserving most of the pesticides in samples. The
preparatory work is usually tedious and time-consuming and involves a combined use of solvent
extraction and column chromatography. In this experiment, vegetable extracts which have been
cleaned up are provided for GC/MS analysis.

Many organochlorine and organophosphorus-type pesticides can cause toxic effects to

humans. These classes of pesticides can be analyzed by GC/MS and GC/ECD. Experiment 2 and
3 will introduce the use of GC/ECD and GC/MS in pesticide analysis.

2.

Materials and methods

1.
2.

Weigh and blend 10 g of a vegetable sample.

Add 1 ml of surrogate (10 g/ml, internal standard), 20 ml of ethyl acetate and 20 g of
sodium sulfate to the blended vegetable sample. Homogenize the mixture for about 5 min.

3.
4.
5.
6.

Centrifuge the homogenized sample at 2000 rpm for 10 min. Transfer the supernatant
through anhydrous sodium sulfate into a measuring cylinder (record the volume of filtrate
collected).
Transfer the filtrate to a 100-ml round bottle flask and concentrate the sample using a
rotary evaporator.
Add 10 ml of a mixed solution of dichloromethane (DCM)/cyclohexane (1:1), and add 2g
of anhydrous Na2SO4. Centrifuge the sample at 2000 rpm for 10 min.
Filter off the Na2SO4 and collect the filtrate.

Sample cleanup
1.

Gel Permeation Chromatography (GPC)

Set up the GPC according to the following conditions:

GPC is used to separate the pesticides from large molecules (e.g proteins and polysaccharides) in
the sample. Transfer the extract to the column and add 5 ml of DCM to the column. Transfer the
sample to a 100-ml round bottle flask and concentrate it using a rotary evaporator. Add 10 ml of
15% diethyl ether (in petroleum ether) to the sample, and transfer the sample to a SPE column
(rinsed with 5 ml of 15% diethyl ether in petroleum ether).
2.

Solid Phase Extraction (SPE)

The SPE column (Florisil, 500mg), rinsed with 5 ml of 15% diethyl ether in petroleum ether, is
required for sample clean-up. Collect the sample with a round bottle flask and concentrate it
using a rotator evaporator. Add 1 ml of iso-octane and transfer it to a sample bottle. Add 3 ml of
conc. sulfuric acid and shake it. Collect the clear upper layer and transfer it to another bottle
containing copper powder. Allow the sample to stand overnight.
GPC conditions
Column
Mobile phase
Flow rate
Sample loop
vol.
Dump time
Collection
time

Bio-beads S-X3 glass column, 60g, 43

cm or equivalent
Dichloromethane : cyclohexane (1:1)
5 mL/min
5 mL
31 min
20 min

Expt. 2: Analysis of pesticides by GC/ECD

INTRODUCTION
GC/ECD is an indispensable tool in analysis of residual pesticides in food products (e.g.
vegetables and tea leaves). Prior to such analysis, sample preparation is often required to
extract the pesticide analytes out of the food product and keep it in conditions suitable for
GC/ECD analysis. Please also refer to the Introduction of Experiment 1: Sample preparation for
GC/MS and GC/ECD analysis of pesticides.
OBJECTIVE
The objective of this experiment is to demonstrate the working principle and the use of a
computerized GC/ECD system in identification of pesticide residues in food samples.
EQUIPMENT
HP 6890 Series II Gas Chromatography coupled with Electron Capture Detector (ECD)
CHEMICALS
Pesticides: DDT and aldrin
Safety Precautions: pesticides may cause acute and/or chronic toxicity. Sample preparation
should be handled carefully in a fume cupboard.
EXPERIMENTAL
Inject the standards and samples (DDT and aldrin) into the GC/ECD machine.
Instrumental conditions for GC/EDC analysis:
Initial temperature
Initial time

100C
2 minutes

Rate
(C/min)

Final Temp.

Final Time

Level 1

10.0

165

10.0

Level 2

3.0

230

10.0

Level 3

15.0

280

10.0

Questions:
(1)

Determine the concentration of the pesticides in the samples.

(2)

Describe the working principle of electron capture detector (ECD)?

(3)

Why is ECD more preferable than flame ionization detector (FID) in GC analysis of
organochlorine and organophosphorus-type pesticides? Justify your reason(s) by comparing
these two detectors.

(4)

GC/ECD and GC/MS are often used in combination to analyze pesticides in foods. What
is/are the purpose(s) of using GC/MS in addition to GC/ECD?

Expt. 3: Analysis of pesticides by GC/MS

INTRODUCTION
In addition to GC/ECD, GC/MS is often used in analysis of pesticides in food products because
of the ability of mass spectrometric detector (MSD) to identify various analytes according to their
mass values. Prior to such analysis, sample preparation is often required to extract the pesticide
residues from the sample. The details of sample preparation for foods (e.g. vegetables) contaminated
with pesticides are described in Experiment 1: Sample preparation for GC/MS and GC/ECD
analysis of pesticides.
OBJECTIVE
The objective of this experiment is to demonstrate the working principle and the use of
computerized GC/MS in the analysis of pesticides. Computer-aid data processing for GC/MS will
also be demonstrated.
EQUIPMENT
Agilent 7890 Series Gas Chromatography coupled with Mass Spectrometric Detector (MSD)
CHEMICALS
Pesticides: DDT and Aldrin
Safety Precautions: pesticides may cause acute and/or chronic toxicity. Sample preparation
should be handled carefully in a fume cupboard.
PROCEDURE
A.

Optimization of Mass Spectrometric Conditions and Mass Calibration

Before a GC/MS run, the DC and/or RF voltages of the ion source and the quadrupole mass
analyzer have to be adjusted and optimized for the best possible mass resolution and
sensitivity of detection. For a modern GC/MS system, this is carried out automatically by
an 'AUTOTUNE' program which optimizes the various ion source and quadrupole rod
voltages, calibrates the mass axis of the mass spectrometer, and adjusts peak widths. A
reference compound, PFTBA (perfluoro-tributylamine, (C4F9)3N) is used in the optimization
and mass calibration processes by comparing the ion intensities and m/z ratios with a
reference spectrum of PFTBA.

The AUTOTUNE program operates in five steps. First, the system adjusts the peak widths
at m/z = 69 and 219 to 0.6 amu by varying the DC to RF voltage ratio on the mass filter.
Next, the amplitude of 219 is maximized by adjusting the ion source voltage control
parameters and the X-ray shield. While doing this, the peak height of 69 is kept between
16,384 and 32,767 absolute abundance (if possible) by changing the multiplier voltage. The
third part of AUTOTUNE re-calibrates the resolution; this time at m/z = 69 and 502, again
to 6.0 amu peak width. Then the ion optics are adjusted to maximize the amplitude of m/z =
502. A report is then generated indicating the relative peak heights of m/z = 69, 219 and
502 and also the intensities of the isotope peaks at m/z = 70, 220 and 503.

B.

GC/MS Conditions
The GC/MS analysis conditions are pre-programmed into a 'METHOD' file in this
experiment. The procedure is outlined in as follows:
1.

Choose Methods from the menu bar of the MS Top window, followed by LOAD
and EDIT entire method parameters. Choose 'METHOD INFORMATION',
'INSTRUMENT/ACQUISITION' and enter 'METHOD COMMENTS'.

2.

Select Agilent 7890A GC inlets and ATUNE.U tune file by clicking the OK button.

3.

GC Column Temperature Programming Conditions:

Initial temperature
Initial time

80C
1 minute

Rate
(C/min)

Final Temp.

Final Time

Level 1

30.0

180

1.0

Level 2

3.0

205

4.0

Level 3

20.0

240

2.0

Inject the standards and samples (DDT and aldrin) into the GC/MS.
C.

GC/MS Analysis and Data Acquisition

1.

Load and Run the METHOD file in the GC/MS system.

2.

Choose AcquireData from the menu bar of the MS Top window.

- Select AcquireData from the AcquireData menu.

- Fill in the Data File Name (e.g. GROUPXX, where XX stands for the Group
Number). Fill in the Operator Name, Sample Name, and Misc Info fields to
document the injection.
- Click the OK Pushbutton to initiate the run. The Prepare To Inject box will
appear.
- Wait until the Not Ready light on the GC going off.
3.

D.

Inject 1L sample (standard and unknown) into the instrument and press the Start
button on the GC panel.

Post GC/MS Data Analysis

1.

Obtaining a Total Ion Chromatogram (TIC)

- Choose Data Analysis from the menu bar of the MS Top window.
- Select Main Panel from the Data Analysis menu.
- Select Load from the File menu.
- Double-click on the file name in the list for data processing.
The TIC chromatogram would appear on the monitor screen.

2.

Viewing a Mass Spectrum

- Point with the mouse to the X-axis of the chromatographic peak for which
you want to see the mass spectrum.
- Double-click the right mouse button to retrieve the mass spectrum.

3.

Printing Display Graphics

- Select Print from the File menu.
- Choose TIC & Spectrum.
- Click OK to initiate the printing process.

4.

Target

Compound

Analysis

and

Extracted

Ion

Chromatogram

The molecular ions and/or major fragment ions of common pesticides are shown in Table I.
The presence or absence of these common pesticides in the vegetable extracts could
be confirmed by observing the Extracted Ion Chromatograms of targeted m/z values.
In this experiment, you are asked to confirm the presence/absence of four common
pesticides found in vegetables in Hong Kong, e.g. lindane, DDT, malathion and
methamidophos.
- Select Extract Ion Chromatograms from the Chromatogram menu.
- Fill in the masses of the major fragment ions of the 4 target compounds in this
analysis (i.e. m/z = 183, 235, 125 and 94) respectively.
8

- Click OK to view the chromatograms.

If the presence of one pesticide is suspected, test/confirm its presence by viewing the
Extracted Ion Chromatogram of other m/z values of fragment ions. The presence of
at least two extracted ion chromatographic peaks at the same retention time is usually
taken as sufficient evidence that the target compound is present and a full library
search is warranted.
5.

Library Search of Unknown Peaks in the TIC chromatogram (for GC/MS only)
- After loading the mass spectrum from the chromatogram, select Select Library from
the spectrum menu.
- At line 1, press ?(shift + /) to select the available library NBS75K.L, or type in
NBS75K.L in Search Order 1.
- Point with the mouse to the mass spectrum.
- Double-click the right mouse button to initiate the Library Search.

6.

Quantitative Analysis
Quantitative analysis of pesticide residues in the vegetable extracts can be done by
comparing the chromatographic peak areas with those of the internal standards or
standard solutions. The procedures for finding chromatographic peak areas are as
follows:
- LOAD data file, then choose CHROMATOGRAM.
- AUTO INTERGATE the TIC peaks.
- LIST RESULT to display retention times and peak areas. Choose DONE or
PRINT.
- Choose % REPORT or PURITY REPORT to see other formats of data
reporting for quantitative analysis.
- Double click on the top left corner to exit the CHROMATOGRAM mode.
TABLE I. Molecular/Major Fragment Ions of Common Pesticides
Compound

Selected Ions (m/z)

Dieldrin
p-Terphenyl-d14(S)
p,p'-DDE

79, 107, 149

244, 122
318, 176, 246

p,p'-DDT

235, 165, 239

Methamidophos
Malathion

94, 141
125, 173

Questions
(1)

Use the spectral library to identify the pesticides in the vegetable samples.

(2)

Determine the concentrations of the pesticides in the vegetable samples.

(3)

What are the advantages of using GC/MS in the analysis of pesticides in vegetable
samples?

(4)

Give two other applications of GC/MS in food analysis.

10

Expt. 4: Analysis of ions in soft drinks by ion chromatography

Introduction
Ion Chromatography (ion-exchange chromatography) is used for separation of ions (cationic and
anionic species). In this experiment, students are required to find out the retention time and the
amount of various anions, such as F-, Cl-, NO3-, PO43-, SO42-, in soft drinks. For example, the
formulation of Coke contains phosphoric acid while soft drink (e.g. 7-up) contains sodium
benzoate.This technique is very useful for qualitative and quantitative determination of various
ions in soft-drinks.
Objective
The objective of this experiment is to analyze ions in a soft drink by Ion Chromatography
Materials and method
IC system: Metrohm 761D Ion Chrom system with Metrosep A Supp 5 100 column (catlog.
No. 6.1006.510 M)
Solvent: (1) 1.0mM NaHCO3 and 3.2mM Na2CO3, (2) MilliQ water, (3) 0.5% H2SO4
Sample: Coke
Standards: 1000 ppb F-, Cl-, NO3-, PO43-, SO42- and benzoate

Experimental
1. Prepare standard mixtures of F-, Cl-, NO-3, PO43-, SO42- and benzoate anions with
concentrations of 100, 200, 500 and 1000 ppb each.
2. Inject the standards and the sample into the instrument and determine the retention time and
the amount of various anions in the soft drink.

11

Typical chromatogram for a standard mixture of the six anions involved in this experiment

Questions
(1) Describe the working principle of ion chromatography in the determination of anionic
species in soft drinks.
(2) From your observation, estimate the detection limit for each anion species in the soft drink.
(3) Identify common anions in the soft drink you examined and determinate the concentration of
each species.

12

Expt. 5: Aluminium analysis by ICP-OES

1.

Introduction

Jellyfish is one of the common cuisines for Chinese people. To make jellyfish to have good
texture, a mixture of salt and aluminium potassium sulphate (also known as alum) is often used
to process jellyfish. Frequent consumption of these products would lead to memory loss, if a
high amount of aluminium is consumed. In this experiment, students will perform quantitative
analysis of aluminium in selected jellyfish sample using inductively coupled plasma emission
spectroscopy (ICP-OES).
Before analyzing food samples with ICP-OES (or ICP-MS and flame AAS), acid digestion is
often performed to extract the metals from the samples. Concentrated nitric acid (~70% HNO3)
is often used in this task. The sample/HNO3 mixture is heated up on a hot plate. Alternatively,
microwave oven can also be used in acid digestion. This approach can digest samples at a
higher temperature in a shorter time compared to the conventional hot-plate method. The
digested sample is then made up with water to a known volume using a volumetric flask.
2.

Objective

The aim of this experiment is to let students be familiar with the use of ICP-OES for the
determination of aluminum in jellyfish.
3.

Materials and Method

(a)

Jellyfish sample
Reagents: Concentrated nitric acid (HNO3); DDI water (obtained from Milli-Q-Plus ultra
pure water system); standard aluminium stock solution (1000 ppm) should be prepared in
volumetric flask (made up with 1% HNO3 (v/v)) and stored in plastic bottles. The reagent
blank is 1% HNO3 (v/v) in water.

(a)

Equipment
ICP-OES Agilent 710-ES instrument system installed with ICP Expert II version 2.0

(b)

Procedure

Sample preparation
1. Weigh the jellyfish sample (1.5-2 g, in a breaker). Record the weight accurately.
2. Add about 5-6 mL AR grade concentrated nitric acid.
3. Turn on the heater and digest the sample for about 1 h.
13

4. Transfer the digested sample solution quantitatively to a 100-mL volumetric flask and make up
with DDI water.
5. Prepare aluminium standard solutions of the following concentration:
Element
Al

Concentration (ppm)
0.5, 1, 2, 4 and 10

Each student should analyze the sample with the ICP-OES instrument individually
1. Make sure that the liquid argon supply, the cooling system and the instrument are in the
ON status. Ask your lab demonstrator about the machine operation if you have any
questions.
2. Figure

shows

the

PC

operational

interface

of

the

instrument:

3. Check the worksheet and instrument icons for the setting of operation conditions and
instrumental analysis.
4. Check the peristaltic pump program: (i) speed: 0.5 ml/min; (ii) uptake time: 30 s; (iii)
stable time: 30 s.
5. Set up and optimize the ICP-OES, and calibrate the instrument with calibration standards.
The wavelengths (237.312, 308.215, 309.271, 394.401, 396.152 nm) can be selected for
the measurements. Observe the peak shape of the selected wavelengths, and judge
whether 394.401 and 396.152 are better choices than others.
6. Fig 2 shows the ICP-MS operation conditions.
14

7. Fig 3 shows the calibration curve of a selected wavelength

Questions
(1)
(2)

Determine the concentration of Al in the sample.

Suggest three major possible improvements in the ICP-OES analysis.
15

(3)
(4)

Describe the working principle of ICP-OES.

ICP-MS and ICP-OES are widely used in the analysis of metals. Discuss and compare the
advantages and disadvantages of these two techniques.

References
1. Au, A. M., Ko, R., Boo, F. O., Hsu, R., Perez, G. and Yang, Z., Screening Methods for
Drugs and Heavy Metals in Chinese Patent Medicines, Bull. Environ. Contam. Toxicol.
2000, (65), 112-119.
2. Centre for food safety
(http://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_43_04.
html)
Safety precautions:
Students should be aware that the instrument is operated at high temperature and a strong acid
is used in this experiment. Hence, all operations should be performed in a fume cupboard and
personal protection tools, such as safety goggles and laboratory coats should be worn
throughout the experiment.

16

Expt. 6:
Analysis of malachite green by electrospray ionization mass spectrometry
(ESI-MS)
Introduction
Malachite Green (MG) is a fungicide illegally used in aquaculture. On uptake, MG is rapidly
converted to Leucomalachite Green (LMG), which accumulates in fatty fish tissues. Both MG
and LMG have demonstrated putative carcinogenic activity, and have been banned in fishery in
many countries. Analysis of MG and LMG in fish products is an important issue for food safety.
ESI-MS is the method of choice for such measurements. In this experiment, first, MG and LMG
are analyzed by ESI-MS respectively and their masses are measured. Subsequently, the
precursor ions of the two compounds are selected and fragmented through collision induced
dissociation (CID), and the fragment ions generated are recorded to create the MS/MS spectra
for both compounds.

(from http://www.restek.com/graphics/figure_fff_003-1.gif)
Objectives

To analyze MG and LMG by ESI-MS.

To understand how MS/MS works.

Materials and Methods

Sample preparation
Prepare 10 ppm solutions of MG and LMG in 50/50 methanol/water with 0.1% formic acid
respectively.

17

Calibration of instrument
Calibrate the Quattro Ultima triple quadrupole mass spectrometer with sodium iodide before the
experiment.
Full scan measurements
Introduce the prepared samples into the mass spectrometer using a syringe pump. Scan Q3 to
acquire spectra for MG and LMG respectively.
Major acquisition settings include:
Syringe pump flowrate: 5 L/min
Ionization mode: +ESI
Capillary voltage: 3000 V
Cone voltage: 30 V
Source temperature: 80 oC
Desolvation temperature: 150 oC
MS/MS measurements
Introduce the prepared samples into the mass spectrometer using a syringe pump. Select m/z 329
and m/z 331 as the precursor ions for MG and LMG respectively using the Q1. Introduce
collision gas into Q2, in which CID of the selected precursor ions takes place. Scan Q3 to
obtain MS/MS spectra for MG and LMG respectively. Adjust the collision energy for desired
MS/MS spectra.
Questions:
1.

Explain how ions are produced during ESI process.

2.

Briefly describe the concept of selected ion monitoring (SIM) and selected reaction
monitoring (SRM) in mass spectrometry-based quantitative analysis of chemical
compounds. Choose appropriate ions for SIM and SRM for MG and LMG.

18