Food Chemistry 147 (2014) 287–294

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Micro-solid phase extraction with liquid chromatography–tandem mass

spectrometry for the determination of aflatoxins in coffee and malt
beverage
Wejdan Shakir Khayoon a, Bahruddin Saad a,⇑, Baharuddin Salleh b, Normaliza Hj Abdul Manaf c,
Aishah A. Latiff c
a
School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
b
School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
c
Doping Control Centre, Universiti Sains Malaysia, 11800 Penang, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: A single step extraction-cleanup procedure using porous membrane-protected micro-solid phase extrac-
Received 19 July 2012 tion (l-SPE) in conjunction with liquid chromatography–tandem mass spectrometry for the extraction
Received in revised form 15 February 2013 and determination of aflatoxins (AFs) B1, B2, G1 and G2 from food was successfully developed. After the
Accepted 8 September 2013
extraction, AFs were desorbed from the l-SPE device by ultrasonication using acetonitrile. The optimum
Available online 19 September 2013
extraction conditions were: sorbent material, C8; sorbent mass, 20 mg; extraction time, 90 min; stirring
speed, 1000 rpm; sample volume, 10 mL; desorption solvent, acetonitrile; solvent volume, 350 lL and
Keywords:
ultrasonication period, 25 min without salt addition. Under the optimum conditions, enrichment factor
Aflatoxins
Micro-solid phase extraction
of 11, 9, 9 and 10 for AFG2, AFG1, AFB2 and AFB1, respectively were achieved. Good linearity and correla-
Liquid chromatography–tandem mass tion coefficient was obtained over the concentration range of 0.4–50 ng g1 (r2 0.9988–0.9999). Good
spectrometry recoveries for AFs ranging from 86.0–109% were obtained. The method was applied to 40 samples involv-
Mycotoxins ing malt beverage (19) and canned coffee (21). No AFs were detected in the selected samples.
Food Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Mycotoxins are toxic low molecular weight metabolites (MW
700) produced by fungi species, such as Aspergillus (A.), Fusarium,
and Penicillum that grow on a variety of agricultural commodities
in humid and temperate conditions. Mycotoxins came to the fore-
front in 1960 when an outbreak of X-disease caused death to
100,000 turkey poultries. The reason behind the death was later
attributed to the consumption of groundnuts that was infected
by Aspergillus flavus and contaminated by aflatoxins (AFs) (Beltran,
Ibanz, Sancho, & Hernandez, 2009; Turner, Subrahmanyam, & Pilet-
sky, 2009). Although more than 300 mycotoxins are known, only
approximately 20 mycotoxins are expected to occur frequently in
food and animal feedstuffs. Among all mycotoxins, AFs are the
most toxic, followed by ochratoxin A (OTA) (Beltrán et al., 2011).
Amongst the 20 AFs which have been identified, only aflatoxins
B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) were found to con-
taminate agricultural commodities in tropical areas (Lutfullah &
Hussain, 2012). The carcinogenic and toxic effects of AFs led the
International Agency for Research on Cancer (IARC) to classify
⇑ Corresponding author. Tel.: +60 4 6534047; fax: +60 4 6574857.
E-mail address: bahrud@usm.my (B. Saad).
AFB1 as carcinogen (Group 1) whereas other AFs and OTA have
been classified as possible carcinogen (Group 2B) (IARC., 2002).
Wheat, sorghum, Brazil nuts, almonds, walnuts, pecans, dried
fruits, legumes, coffee, peppers, potatoes, rice, copra, filberts, beer,
medicinal herbs, meat products of animal feed, milk and milk
products are reported to be infected by Aspergillus and contami-
nated with AFs (Ventura et al., 2004). The Commission Regulation
(EU) has set 2.0 ng g1 for AFB1 and 4.0 ng g1 for total aflatoxins in
the groundnuts, oil seeds, dried fruits, and processed products in-
tended for direct human consumption (European Commission,
2010a,b). Different approaches have been reported for the determi-
nation of AFs in food and animal feed e.g., capillary electrophoresis
(Peña, Alcaraz, Arce, Ríos, & Valcárcel, 2002) thin layer chromatog-
raphy (Stroka, Otterdijk, & Anklam, 2000), high performance liquid
chromatography (HPLC) (Khayoon, Saad, Lee, & Salleh, 2012) and
enzyme-linked immunosorbent assay (Li et al., 2009).
Before the analysis, sample preparation including extraction,
clean-up and further pre-concentration are important. Qualitative
and quantitative analysis is further complicated by the heteroge-
neous distribution of mycotoxins in agricultural commodities.
Therefore a simple analytical method which minimises the use of
solvents and less labour intensive is needed. HPLC with fluores-
cence detection, employing post-column derivatization has
become the most common technique for the determination of

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.09.049
288 W.S. Khayoon et al. / Food Chemistry 147 (2014) 287–294
AFs in food and feed (Alcaide-Molina, Ruiz-Jiménez, Mata-
Granados, & Luque de Castro, 2009; Quinto, Spadaccino, Palermo,
& Centonze, 2009).
Liquid–liquid extraction (LLE) and solid-phase extraction (SPE)
are commonly used to extract AFs from food matrices. More re-
cently, microextraction techniques have been developed to over-
come some of the limitations of these conventional methods.
Solid phase microextraction (SPME) (Quinto et al., 2009) and dis-
persive liquid–liquid microextraction (DLLME) were developed
for the extraction of AFs from food (Campone, Piccinelli, Celano,
& Rastrelli, 2011). The SPME technique is simple, solvent-less, en-
ables the extraction of polar and non-polar compounds, and com-
bines sampling and extraction into one step. The limitations of the
SPME technique are the fragility of the fibre and batch to batch var-
iation of fibre coatings (Ridgway, Lalljie, & Smith, 2007). Although
the DLLME technique involves short extraction time, simple to
operate, but it is difficult to be automated (Mahugo-Santana,
Sosa-Ferrera, Torres-Padrón, & Santana-Rodríguez, 2011).
Recently micro-solid phase extraction (l-SPE) has been devel-
oped to reduce solvent consumption, sorbents usage and sample
handling (Ahmadi, Shahsavari, & Rahimi-Nasrabadi, 2008). l-SPE
is an interesting alternative to the multistep SPE method for the
preconcentration of analytes in complex samples. It is suitable
for the extraction of analytes in complex matrices as sample
clean-up and the extraction step is carried out simultaneously.
To prepare the l-SPE device, sorbent materials such as C18
(Basheer, Chong, Hii, & Lee, 2007), multiwalled carbon nanotube
(Basheer, Alnedhary, Rao, Valliyaveettil, & Lee, 2006), hybrid or-
ganic–inorganic silica monolith (Zheng, Ruan, & Feng, 2009),
graphite fibre (Xu & Lee, 2008) or molecularly imprinted polymers
(Lee, Saad, Khayoon, & Salleh, 2012) were used.
l-SPE involves the use of a sorbent that is trapped in a porous
membrane sheet. Due to the porosity of the membrane, the analyte
is able to diffuse freely and extracted by the sorbent. While the
extraction is performed, the sample is stirred and the tumbling
of the l-SPE device facilitated mass transfer. After the extraction,
desorption is carried out by ultrasonification where the extraction
device immersed in a suitable organic solvent. l-SPE technique
was applied for the analysis of acidic drugs (Basheer et al., 2007),
pesticides (Ahmadi et al., 2008), per-O-methylated mono and
disaccharides (Ciucanu, Swallow, & Căpritßă, 2004) estrogens (Kan-
imozhi et al., 2011) and OTA (Lee et al., 2012). The main objective
of this work was to develop a l-SPE technique that can be applied
for the analysis of AFs in food. After the l-SPE step, LC-MS/MS was
used for the determination of AFs. The effects of extraction condi-
tions and desorption conditions will be evaluated.
2. Experimental
2.1. Chemicals and reagent
AFs standards and ammonium acetate were purchased from Sig-
ma–Aldrich (St. Louis, MO, USA). Methanol, acetonitrile (HPLC
grade) and acetone were purchased from Fisher Scientific (Lough-
borough, Leicestershire, UK). Ultrapure water (resistivity,
18.2 MX cm1) was produced by a Milli-Q system (Millipore, USA),
and was used throughout for the preparation of solutions. Flat-sheet
Accural porous polypropylene membrane with 0.2-lm pore size was
purchased from Membrana (Wuppertal, Germany). C8, C18 and C30
sorbents were obtained from Supelco (Bellefonte, PA, USA).
2.2. Instrumental and chromatographic conditions
An Agilent 6410 Triple Quadrupole UPLC-MS/MS (Agilent Tech-
nologies, Waldbronn, Germany) equipped with Agilent LC 1200
series auto sampler, binary pump, and thermostatted column com-
partment, operated by Mass Hunter software version B.01.04 was
used. The injection volume was 10 lL. Under the electrospray ion-
isation (ESI+) mode, the mass spectrometer parameters were: cap-
illary voltage of 4000 V; gas temperature and flow, 350 °C and
10.5 L min1, respectively. Nitrogen gas (99.99% purity) was used
for both drying and collisions. The separation was carried out on
Eclipse Plus C18 column (150 mm  2.1 mm I.D., 3.5 lm particle
size) that was purchased from Agilent (Waldbronn, Germany). A
gradient elution of ammonium acetate (10 mM) and methanol
(100%) as mobile phases A and B, respectively. was used. A seg-
mented gradient of 20% to 70% B in 9 min followed by ramping
of B to 100% in 9.5 min and was held constantly until 10.5 min then
B was reduced gradually to 20% for 8.1 min. The flow rate was
0.3 mL min1 and the column temperature was 40 °C.
2.3. Preparation of standard solutions
Stock solutions of AFs (1000 ng g1) standards were prepared in
acetonitrile (ACN) and stored at 4 °C. Working solutions (0.5–
50 ng g1) were prepared in ACN.
2.4. Safety considerations
Due to the potential toxicity and carcinogenic nature of AFs,
safety precautions were taken into consideration during the han-
dling by wearing gloves, safety glasses and disposable face mask.
AFs standards and working solutions were protected from light.
Decontamination of possible AFs from glasswares, vials and tubes
were effected by submerging all these glasswares in sodium hypo-
chlorite solution (5.25%) for 72 h before washing and re-using.
2.5. Food samples
Canned coffee (21) and malt beverage (19) samples were pur-
chased from local supermarkets and stored at ambient
temperature.
2.6. Preparation of l-SPE device
The l-SPE device consists of the sorbent material that was en-
closed within a polypropylene membrane envelope. The longer
edge of a rectangular polypropylene membrane was folded over
a width of 0.75 cm and the edge of the flap was heat sealed to
the main sheet. The fold-over section was then trimmed off from
the main membrane sheet and cut (2 cm intervals) into individual
rectangular pieces (2 cm  1.5 cm). One of the two open ends of
each pieces was then heat-sealed using an electrical sealer. A glass
Pasteur pipette was used to introduce sorbent (20 mg) via the
remaining open end of envelope that was later heat-sealed to se-
cure the content. Before use, each device was cleaned by ultrason-
ication in ultrapure water (2 min) and ACN (2 min). It was then
stored in ACN till use.
2.7. l-SPE procedure
Food sample (i.e., malt and coffee) was degassed using ultra-
sound. A 10 mL of food sample that was spiked with AFs (2 ng g1)
was added to a sample vial (25 mL) containing magnetic stirring
bar (15 mm  5 mm). Next, the l-SPE device was conditioned by
ultrasonication in ultrapure water (2 min), followed by in acetoni-
trile (2 min) and then dried with lint-free tissue. A l-SPE device
was placed in a 10 mL sample and that was stirred at 1000 rpm
(Fig. 1). After the extraction (90 min), the device was removed,
rinsed in water, dried with lint-free tissue and placed in a 750 lL
desorption vial. 350 lL of acetonitrile was added and AFs were
 W.S. Khayoon et al. / Food Chemistry 147 (2014) 287–294
desorbed by ultrasonication for 25 min. After desorption, the l-SPE
device was removed from the desorption vial and the extract was
dried using gentle stream of nitrogen gas. The residue was recon-
stituted in 100 lL of MeOH: water (10:90, v/v) and injected di-
rectly to the LC–MS/MS unit.
3. Results and discussion
3.1. Optimisation of chromatographic conditions
Table 1 shows the LC–MS/MS conditions, precursor, product
ions, linearity and correlation coefficients for AFs under the
adopted chromatographic conditions. The precursor ions (m/z)
were 313.1, 315.1, 329.1 and 331.0 for AFB1, AFB2, AFG1 and
AFG2, respectively.
3.2. Optimisation of l-SPE conditions
Optimisation studies were carried out on uncontaminated malt
beverage sample that was spiked with 2 ng g1 AFs. This level was
chosen as it is the mean AFs level permitted as stipulated by the
European Union. The extraction of analytes using the l-SPE tech-
nique is similar to SPME as the extraction is an equilibrium-driven
procedure. Moreover, the extraction efficiency (EE) is dependent
on the partitioning of analyte to sorbent (Basheer et al., 2007).
Thus, detail extraction conditions including the effects of type of
sorbent (material and mass), extraction conditions (stirring speed,
exposure time, salt addition and sample volume) and desorption
conditions (desorption time, solvent type and volume) were
investigated.
3.2.1. Effect of sorbent type and mass
Different commercially-available sorbent materials were inves-
tigated, i.e., C30, C8 and C18. C8 gave the highest extraction effi-
ciency for all the AFs (Fig. 2A). The C8 sorbent contains silica
surface that is covered with linear octyl chain which prefer to
interact with the moderately polar analytes. Moreover, the C8
material exhibits interaction with the AFs via van der Waals forces.
It is interesting to note that the shorter chain length sorbent is able
to absorbe AFs better than the longer chain length. The effect of C8
mass (5–30 mg) on the extraction of AFs was studied. As expected,
as the mass increased, higher extraction efficiency was found.
However, when more than 20 mg was used, no additional enhance-
ment was observed (Fig. 2B). Therefore, 20 mg of C8 was selected
for the subsequent experiments.
Fig. 1. Schematic diagram of experimental set-up and SEM image of l-SPE sorbent (C8).
289
3.2.2. Effect of exposure time
The amount of analyte extracted is dependent on the rate of its
mass transfer from the aqueous phase to the sorbent in the l-SPE
device (Basheer et al., 2008). Therefore, extraction time is another
important factor to be considered. To study the effect of extraction
time on the extraction efficiency, various extraction times (15–
90 min) were studied. The extraction efficiencies increased rapidly
by increasing the extraction up to 90 min (Fig. 2C). Therefore,
90 min extraction was selected for the subsequent experiments.
3.2.3. Effect of stirring speed
Stirring or sonicating of the aqueous solution helps to accelerate
the extraction. Stirring permits the continuous exposure of the
extraction surface to fresh aqueous sample and enhances mass
transfer in the sorbent device, leading to induced convection in
the membrane phase (Tahmasebi, Yamini, & Saleh, 2009). The ef-
fect of stirring speeds (400–1200 rpm) on the extraction efficiency
was examined (Fig. 2D). Highest extraction efficiency was obtained
when stirred at 1000 rpm. However, it was found that high stirring
speeds generate problems such as production of air bubbles on the
surface of the membrane device leading poorer precision. Thus,
1000 rpm was selected for the rest of the studies.
3.2.4. Effect of salt addition
Addition of salt decreases the solubility of analytes in the sam-
ple solution by increasing the ionic strength (salting out effect) and
enhances their partitioning into the solid sorbent. The effect of salt
was investigated by adding 0, 2.5, 5 and 10% (w/v) sodium chloride,
respectively to 10 mL of sample that was spiked with 2 ng g1 AFs.
Aflatoxins are freely soluble in moderately polar solvent and dis-
solve in water to the extent of 10–20 mg L1. The addition of so-
dium chloride was expected to enhance the extraction efficiency
of l-SPE since analyte was dissolved in acetonitrile and then mixed
with the aqueous sample. Addition of salt did not result in an in-
crease in extraction efficiency and the mean reduction in peak
areas for the AFs was 32%. Moreover, the addition of large amounts
of salt to the aqueous phase increases its viscosity. Thus, the veloc-
ity of the mass transfer processes of the analytes from the aqueous
matrix to the sorbent phase is diminished and, consequently, the
time required to reach equilibrium increases (Castells, Santos, &
Galceran, 2003). Hence, no salt was added in further experiments.
3.2.5. Effect of sample volume
Sample volume is particularly important in determining the
loading capacity of the l-SPE device. This is critical when working
at the microscale, due to the very small amount of the sorbent
290 W.S. Khayoon et al. / Food Chemistry 147 (2014) 287–294

Table 1
Analytical characteristics of the l-SPE method.

AFs ttR (min) Precursor ion Product ion Collision energy (V) Linear range Regression equation r2 LOD (ng g1) LOQ (ng g1)
(m/z) (m/z) (ng g1)
AFG2 7.1 331 [M + H]+ 313 (Q) 25 0.63–50m 536.95x + 11557 0.9988 0.20 0.63
285 (q) 28 1.94–50c 265.71x  199.24 0.9996 0.58 1.94
245 (q) 30
AFG1 7.6 329 [M + H]+ 311 (q) 20 1.11–50m 1162.7x  181.6 0.9997 0.33 1.11
283 (q) 25 2.52–30c 131.82x  57.72 0.9991 0.76 2.52
243 (Q) 27
AFB2 8.1 315 [M + H]+ 287 (Q) 25 0.44–50m 843.24x  3.6739 0.9999 0.13 0.44
259 (q) 30 0.98–50c 424.69x  357.23 0.9991 0.30 0.98
243 (q) 42
AFB1 8.5 313 [M + H]+ 285 (q) 22 0.40–50m 1700.8x + 601.23 0.9999 0.12 0.40
269 (q) 32 0.76–50c 700.51x  455.06 0.9997 0.23 0.76
241 (Q) 40

tR, retention time; fragmentation energy, 170 V, data obtained from malt beverage (m) and coffee samples (c).

Fig. 2. (A) Effect of l-SPE sorbent type on the extraction efficiency. Extraction conditions: extraction time, 60 min; extraction speed, 1000 rpm; desorption time, 20 min;
350 lL of ACN as a desorption solvent; 10 mL of sample spiked with 2 ng g1 AFs; 20 mg each sorbent; without salt addition. (B) Effect of l-SPE sorbent mass on the
extraction efficiency. Extraction conditions: extraction time, 60 min; extraction speed, 1000 rpm; desorption time, 20 min; 350 lL of ACN as a desorption solvent and 10 mL
of sample spiked with 2 ng g1 AFs; without salt addition. (C) Effect of exposure time on the extraction efficiency of AFs. Extraction conditions: 20 mg of C8 sorbent;
extraction speed, 1000 rpm; desorption time, 20 min; 350 lL of ACN as a desorption solvent; 10 mL of sample spiked with 2 ng g1AFs; without salt addition. (D) Effect of
stirring speed on the extraction efficiency of AFs. Extraction conditions: 20 mg of C8 sorbent, extraction time 90 min, desorption time 20 min; 350 lL of ACN as a desorption
solvent; 10 mL of sample spiked with 2 ng g1 AFs; without salt addition.

material used to extract AFs (Basheer et al., 2006). The effect of
sample volume (5–50 mL) on the extraction efficiency was studied.
Increasing in sample volume increases the extraction efficiency.
This is due to the increase of analyte enrichment with increasing
sample volume whereas a limit of this enrichment is reached when
the desorption sites are fully saturated with the analytes. Higher
extraction efficiency was obtained when 10 mL was used. The
observation could be due to the insufficient length of the extrac-
tion time needed to achieve extraction equilibrium. Generally in
microextraction, with increasing sample size, the equilibrium time
also increases. Since our extraction time was long (total 115 min;
90 min for extraction and 25 min for desorption), we did not fur-
ther optimise the extraction time. The optimum sample volume se-
lected was 10 mL.
3.2.6. Effect of desorption solvent, volume and time
Selection of desorption solvent is mainly based on the solubility
of the analyte. Thus, methanol (100%), methanol:water (90%), ace-
tonitirile (100%), acetonitrile:water (90%), methanol:acetonitrile
(50%) and acetone (100%) were studied. Highest desorption was
obtained when 100% of acetonitrile was used. Different volumes
(300–500 lL) of 100% acetonitrile were examined. Small volumes
(less than 300 lL) were not considered as was not sufficient to im-
merse l-SPE device, whereas higher volume caused a decrease in
peak area. 350 lL of 100% ACN was found to be sufficient to com-
pletely desorb the analyte. Therefore, 350 lL of acetonitrile was
chosen as the optimum desorption volume.
The effect of desorption (ultrasonication) time (5–30 min) was
performed with 350 lL of the desorption solvent (100% ACN).
 W.S. Khayoon et al. / Food Chemistry 147 (2014) 287–294 291

25 min desorption time is sufficient. Above 25 min, decrease in
desorption efficiency was observed. This is probably due to the
re-adsorption of analytes by the sorbent materials. After the first
desorption, the l-SPE device was further desorbed in ACN for a
second time to test for possible carryover effects. No analytes were
detected after the second desorption step. This indicates that the
l-SPE device could be reused by rinsing the used l-SPE device in
water followed by ultrasonication with ACN. There is no carryover
under the above conditions. However, it was found that the l-SPE
devices can be reusable for about 30 extractions.
3.2.7. Adopted extraction conditions
The adopted extraction conditions are: sorbent, 20 mg of C8;
stirring speed, 1000 rpm; exposure time, 90 min; sample volume,
10 mL; without salt addition; desorption solvent, 350 lL of ACN;
ultrasonication period, 25 min.
Enrichment factor (EF) is defined as the ratio of analyte concen-
tration in the final extract to that in the original water sample:
EF ¼ Cs =Cw
where Cs and Cw are the analyte concentration in the final extract
and in the original sample solution, respectively. Extraction is usu-
ally quantified as the extraction efficiency (EE%) which is the frac-
tion of analyte removed by the acceptor from the original sample
solution and was computed as:
EE ¼ ns =nw ¼ ðCs Vs Þ=ðCw Vw Þ ¼ EFðVs =Vw Þ
Where ns and nw are the analyte mass in the final extract and in the
original sample solution, Vs and Vw the volume of the concentrated
extract and the original sample solution, respectively. The EF and EE
for C8, C18 and C30 sorbents were compared under the optimum
extraction conditions (Table 2). The results show that the highest
EF and EE were obtained were C8 was used since the C8 had strong
interaction with AFs. The EF (and EE) values are higher than the
results obtained by using carbon nanotubes for the extraction of
2-nitrophenol, 2,6-dichloroaniline and naphthalene, ranged from
3–7 (14–27) (Sae-Khow & Mitra, 2009).
Table 2
Enrichment factor (EF) and extraction efficiency (EE) of AFs under the optimum
conditions.
AFs C8 C18
EF EE EF EE
AFG2 11 39 5 18
AFG1 9 32 4 15
AFB2 9 32 6 22
AFB1 10 35 5 16
3.3. Analytical characteristics
3.3.1. Linearity, detection and quantification limits
Calibration curves were obtained under the optimised condi-
tions using malt beverage and coffee sample that were originally
free from AFs and were spiked with different levels of AFs. To as-
sess the possible effects of endogenous components in the food
items, matrix-matched calibration was used for the quantification
and validation. Linear regression analysis was performed on the
peak area (y) versus concentration (x) in ng g1 for AFs. Each point
of the calibration curve corresponded to the mean value obtained
from five measurements. Good linearity were obtained for each
analyte (r2 0.9988–0.9999) over the concentration range of 0.4–
50 ng g1 for all AFs in malt beverage sample and 0.76–50 ng g1
for AFG2, AFB2 and AFB1 whereas 2.52–30 ng g1 for AFG1 in coffee
samples (Table 1). The shorter linear range for coffee and the AFG1
in coffee indicates that there is slight effect of the matrix on the
detector response for these samples. The limit of detection (LOD)
was estimated at signal-to-noise ratio of 3, whereas the LOQ was
ð1Þ estimated at signal-to-noise ratio of 10. The results obtained from
malt and beverage samples are summarised in Table 1. The LOQs
are lower than the maximum levels for AFs established by the
European Union, suggesting the suitability of the developed meth-
od for determining trace amounts of AFs in the selected food
samples.
3.3.2. Precision, accuracy and trueness
ð2Þ
Precision of the method is expressed by the repeatability and
reproducibility data. Intra-day repeatability was obtained at four
concentration levels of each AFs that were spiked to the malt and
coffee samples. Six replicate measurements for each level was con-
ducted. Inter-day reproducibility data was obtained by spiking to
the food extract with four concentration levels of AFs and all sam-
ples were analysed on three different days. Acceptable precision as
reflected from the relative standard deviations (RSDs) values for
the intra-day (0.92–7.20%) and inter-day (0.92–6.11%) (Table 3)
was obtained.
The trueness of the method was established from the percent
recoveries of spiked amounts of AFB1, AFB2, AFG1 and AFG2 to dif-
ferent categories of food items. Thus, four levels of AFs were spiked
to malt and coffee. Five replicates were studied at each concentra-
tion. The results showed that the recovery values ranges from
86.0% to 109% (Table 3).
C30
EF EE
3.4. Comparison with previous methods
3 9
3 9 The developed l-SPE method was compared with the other re-
4 15
3 11
ported methods (Table 4). It is clear that the proposed method pro-
vide better recoveries that the other reported methods. The good

Table 3
Intra-day, inter-day and recoveries values of spiked malt and coffee samples.

Sample Amount spik (ng g1) Intra-day repeatability, (%RSD, Inter-day reproducibility, (%RSD, Recovery (%) ± RSD (%) (n = 5)
n = 5) n = 15)
AFG2 AFG1 AFB2 AFB1 AFG2 AFG1 AFB2 AFB1 AFG2 AFG1 AFB2 AFB1
M alt 0.5 3.68 2.91 2.62 1.38 3.00 2.49 3.10 1.86 96.50 ± 1.46 98.46 ± 0.44 100.9 ± 2.80 95.41 ± 0.82
5 1.10 1.23 1.05 1.90 1.94 1.98 1.71 1.89 96.20 ± 3.15 97.23 ± 1.82 96.87 ± 2.70 99.05 ± 2.00
20 1.15 1.63 1.11 1.31 1.28 2.55 2.32 2.20 100.7 ± 1.10 95.58 ± 1.14 98.94 ± 2.74 100.9 ± 3.49
50 2.46 1.85 1.56 1.42 2.09 2.34 2.38 1.98 99.67 ± 1.25 100.5 ± 0.61 100.0 ± 1.05 99.93 ± 1.15
Coffee 1.0 2.86 5.9 6.50 7.20 4.93 5.24 5.96 6.11 97.60 ± 4.90 93.18 ± 1.56 109.5 ± 3.00 93.38 ± 12.4
5 1.47 3.66 2.69 2.89 5.49 4.54 3.01 2.19 95.33 ± 3.70 91.06 ± 4.59 86.00 ± 1.37 94.43 ± 2.80
20 1.92 1.65 1.11 0.22 2.10 1.99 1.63 2.72 99.90 ± 2.41 99.62 ± 2.91 94.42 ± 1.61 98.58 ± 2.50
30 – – 11.23 – – 3.92 – – – 100.6 ± 2.91 – –
50 0.92 2.22 – 1.57 0.92 – 1.11 1.38 100.5 ± 0.49 – 99.95 ± 0.23 99.46 ± 2.30
292 W.S. Khayoon et al. / Food Chemistry 147 (2014) 287–294

Table 4
Comparison of the developed l-SPE method with the other reported microextraction techniques.

Sample preparation Sample analysed Instrument Linearity LOD LOQ Recovery (%) References
SPME Cereal flour HPLC–FLD 0.06–10 ng g1 0.035–0.2 ng g1 0.1–0.63 ng g1 60–67 (Quinto, Spadaccino, Palermo,
& Centonze, 2009)
SPME Food LC–MS 0.05–2.0 ng mL1 2.1–2.8 pg mol1 20 pg g1 80.8–109.1 (Nonaka, Saito, Hanioka, Narimatsu,
& Kataoka, 2009)
1 1 1
DLLM Cereal HPLC–FLD 0.05–5 lg kg 0.01–0.17 lg kg 0.04–0.57 lg kg 67–92 (Campone, Piccinelli, Celano,
& Rastrelli, 2011)
l-SPE Malt LC–MS/MS 0.5–50 ng g1 0.12–0.33 ng g1 0.4–1.11 ng g1 95.41–100.86 Present study
l-SPE Coffee LC–MS/MS 1.0–50 ng g1 0.23–0.76 ng g1 0.76–2.52 ng g1 86.00–109.5 Present study

FLD, fluorescence; PDA, photodiode array.

Fig. 3. (A) Total ion chromatograms of malt beverage sample spiked with AFs (1.0 ng g1) (i) before l-SPE, and (ii) after l-SPE. (B) Total ion chromatograms of coffee beverage
sample spiked with AFs (50.0 ng g1) (i) before l-SPE, and (ii) after l-SPE.
 W.S. Khayoon et al. / Food Chemistry 147 (2014) 287–294 293

Fig. 3 (continued)

recoveries obtained by the developed l-SPE method are mainly 4. Conclusion

attributed to the good enrichment and extraction that was
achieved (Nonaka, Saito, Hanioka, Narimatsu, & Kataoka, 2009). An innovative l-SPE method for the determination of AFs is for
the first time reported. The porosity of the polypropylene mem-
brane of the l-SPE device is effective in eliminating interferences
from the sample materials without further clean-up. The method
3.5. Analysis of real samples
is remarkably simpler than the official method, clean-up using
immunoaffinity column is not required. The developed method
The developed method was used to analyse AFs in malt bever-
uses only 20 mg of sorbent, whereas the SPE technique necessi-
age and coffee samples. A total of 40 samples including 19 samples
tates about 100 mg of sorbent. It was also noted that only 350 lL
of malt beverages and 21 samples of coffee were analysed under
of eluting solvent was required. When used with the sensitive
the optimum conditions. It was found that no AFs were detected
LC–MS/MS, derivatization is also not required, thus overcoming
in the analysed food samples. Fig. 3A and B show total ion chro-
some of the drawbacks that accompany the derivatization step
matogram of malt beverage and coffee samples spiked with AFs be-
(e.g., corrosion effects of TFA and the hazardous impacts of its han-
fore and after applying the l-SPE technique. From the values of the
dling). With suitable sorbents, the l-SPE technique can be ex-
peak areas, it is clear that there is a significant enrichment of the
tended to the analysis of other mycotoxins.
analytes after undergoing the l-SPE treatment.
294 W.S. Khayoon et al. / Food Chemistry 147 (2014) 287–294
Acknowledgements
This work was financially supported by Universiti Sains Malay-
sia (USM) Postgraduate Research Grant Scheme (USM-RU-PRGS),
1001/PKIMIA/842071 and Research Fellowship program.
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