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Journal of Chromatography A, 1135 (2006) 135–141

Aflatoxin M1 determination in cheese by liquid

chromatography–tandem mass spectrometry
Chiara Cavaliere, Patrizia Foglia, Chiara Guarino, Francesca Marzioni,
Manuela Nazzari, Roberto Samperi, Aldo Laganà ∗
Department of Chemistry, “La Sapienza” University, Box no. 34 - Rome 62, Piazzale Aldo Moro 5, 00185 Rome, Italy
Received 1 March 2006; received in revised form 19 July 2006; accepted 21 July 2006
Available online 23 October 2006

A new method for determining aflatoxin M1 (AFM1) in cheese by liquid chromatography–tandem mass spectrometry has been developed.
Two methodologies were compared for sample extraction. The first one involves sample extraction with dichloromethane for hard, aged cheese
or acetone for fresh cheese and includes a preliminary matrix solid-phase dispersion–extraction step before solid-phase extraction (SPE) clean-up
by a Carbograph-4 cartridge. The second method uses a water/methanol solution (90:10, v/v) extraction at 150 ◦ C before clean-up. The average
recoveries of AFM1 from samples spiked at levels of 0.25–0.45 ␮g/kg, were 81–92% and the precision (RSD) ranged from 3 to 7% with the first
method, whilst the average recoveries were 79–84%, and RSD ranged from 7 to 15% for the second method. Due to different matrix effect, the
quantification limits were 0.019–0.025 ␮g/kg in the first case and 0.048–0.143 ␮g/kg in the second one, depending on cheese typology.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Aflatoxin M1; Cheese; Matrix solid-phase dispersion; Hot water extraction; Liquid chromatography; Tandem mass spectrometry

1. Introduction animal to animal, from day-to-day, and from one milking to the
next [13,14].
Aflatoxin M1 (AFM1) is a hydroxylated metabolite of the In order to reduce aflatoxicosis risk, most of the developed
potential carcinogen aflatoxin B1 (AFB1), and may be found in countries have regulated the maximum permissible levels of
milk of lactating animals which have ingested feedstuffs con- AFM1 in milk. The limits are highly variable, depending on
taminated by AFB1 [1–6]. Both AFB1 and AFM1 can cause the degree of development and economic involvement of the
human disease specially primary liver cancer involving genetic countries in setting regulatory limits [15].
mutations P53 gene in DNA [2,7,8], DNA damage, chromoso- In milk, AFM1 maximum allowable level was set to
mal anomalies and cell transformation in mammalian cells in 0.05 ␮g/kg by European Union [16] and 0.5 ␮g/kg by U.S. Food
vitro, in insects, lower eukaryotes and bacteria [9]. AFM1 has and Drug Administration [17], while in baby-food products this
shown an acute toxicity and carcinogenicity comparable to that level cannot be greater than 0.025 ␮g/kg [18].
of the parent molecule, therefore it is now classified by IARC The potential hazardous human exposure to AFM1 via con-
as group 1 human carcinogen [10]. sumption of milk products has been demonstrated [19–21].
It has been reported that approximately 0.3–6.2% of the afla- AFM1 is relatively stable in raw and processed milk prod-
toxin B1 initially present in animal feeds appear as AFM1 in ucts, and is slightly affected by pasteurization or processing
milk and a linear relationship has been found between intake of into cheese [22,23]. In addition, the protein fraction of milk, in
AFB1 in contaminated feed and the AFM1 content of milk in particular casein, binds AFM1 [24], thus, if raw milk contains
cows [11,12], but this transmission rate was shown to vary from AFM1, cheese made from such milk will also contain AFM1
[22,25,26]. Even if the presence of AFM1 in cheese seems to
be very variable [27], it has been observed that AFM1 concen-
∗ Corresponding author. Tel.: +39 06 49913679; fax: +39 06 490631. trations was 2.5–3.3 times higher in soft cheeses and 3.9–5.8
E-mail address: (A. Laganà). times higher in hard cheeses than in the milk from which the

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
136 C. Cavaliere et al. / J. Chromatogr. A 1135 (2006) 135–141

cheeses were made [28]. These values can be ascribed to the The aim of this work was to develop and evaluate a
condensation of the raw material occurring during cheese man- LC–MS/MS method for determining AFM1 in sample of Grana
ufacture [15]. Examination of different types of cheese showed or Parmigiano cheeses (typical hard, long maturing cheeses)
high stability of AFM1 during maturation and storage [29,30]. and mozzarella (a typical fresh cheese) at concentration below
The population can therefore be indirectly exposed to aflatoxins the provisional adopted limits. Two sample pretreatment proce-
not only by the consumption of milk but also of milk products dures that do not make use of immunosorbent cartridge were
such as cheese [31,32]. Hence, the detection and determina- compared.
tion of AFM1 in dairy products, particularly in cheese (where
there is an AFM1 enrichment factor), is of increasing interest 2. Experimental
For cheese, Switzerland and Turkey have introduced a legal 2.1. Reagents and chemicals
limit for AFM1 to 0.25 ␮g/kg [1,34,35]. At the present time
European Union has not imposed a maximum tolerance level for Crystalline standards of AFM1 and AFG1 (from Aspergillus
aflatoxin in cheese, even if Italian Minister of Health have posed Flavus) were purchased from Sigma–Aldrich (St. Luis, MO,
a provisional limit to 0.45 ␮g/kg for AFM1 in hard cheeses [36] USA). The standard stock solution title was controlled by com-
(eg. Grana padano and Parmigiano reggiano type). This limit paring its mass spectrometer response with a calibrated solu-
is higher than Swiss one, but it is provisional and it is only for tion supplied by M. Miraglia, a partner involved in the project
matured hard cheese that, because of loss of water, at the same AFLARID. AFG1 was used as internal standard (I.S.). Individ-
initial milk contamination, reaches higher toxin concentration ual stock solutions were prepared in acetonitrile at 50 ␮g/mL
at final process. level for AFM1 and at 1000 ␮g/mL for I.S., stored at −20 ◦ C
Immunological methods such as enzyme-linked immunosor- and kept in the dark at room temperature before use. Work-
bent assay (ELISA) are commonly used for screening purpose ing standard solutions were prepared by suitable dilution of
[15,23,26,27,37]. ELISA is not fully reliable due to cross- stocks. These solutions were kept at 4 ◦ C and renewed weekly.
reaction interferences, especially at concentrations just lower All organic solvents were HPLC grade from Carlo Erba (Milan,
than 0.05 ␮g/kg [38]. To allow an effective control of the contam- Italy) and were used as received. Concentrated ammonia, formic
ination of cow milk and dairy products by AFM1, very sensitive and acetic acids were RPE grade from Carlo Erba. Ultra-pure
and reliable analytical methods have been developed. Many of water was produced from distilled water by a Milli-Q system
them are based on solvent extraction followed by partition with (Millipore Corporation, Billerica, MA, USA).
hexane or heptane to eliminate fat share [15,23,27,37,39–41].
Clean-up is then obtained by solid-phase extraction (SPE) or 2.2. Safety considerations
immunoaffinity chromatography in combination with reversed-
phase HPLC and fluorescence detection with or without deriva- Aflatoxins are carcinogenic compounds. Consequently, solu-
tization [25,32,33,41–44]. tions and extracts should be handled with extreme care. Gloves
There are still very few published methods for AFM1 analy- and other protective clothing were worn as safety precaution
sis in complex food matrices like cheese and only one utilized during the handling of the compounds. Crystalline aflatoxin
LC–MS as identification and quantification technique [40]; how- standards were handled in a glove box. Glassware used for stan-
ever, the limit of quantification (LOQ) achieved for AFM1 is dard or sample was soaked in 3% aqueous sodium hypochlorite
higher (0.6 ␮g/kg) than the proposed limit. LC–MS can pro- to destroy AFM1 residue before cleaning and re-use.
vide decisive advantages in performing identification as well as Aflatoxins are subject to light degradation. Analytical work
determination of analytes at trace levels. However, the coupling was protected from daylight and aflatoxin standard solutions
of both techniques is really efficacious if a suitable combi- kept in amber vials.
nation of sample preparation, chromatographic conditions and
interface is selected [45]. The matrix complexity can give rise 2.3. Samples
to an unpredictable matrix effect so, although very selective,
also LC–MS/MS may require time and labour intensive sample Two different kinds of cheese were chosen: one is hard,
preparation steps. cooked and long matured cheese, with a water content of about
Matrix solid-phase dispersion (MSPD) is an extraction tech- 30–32%, protein 34% and fats 29%, the other is a fresh cheese
nique suitable for extracting selected analytes from liquid or soft which medium composition is water 59%, protein 19% and
solid samples [46,47]. lipidic content 20%. Several Protected Designation of Origin
The use of hot water as an effective extractant for solid sam- (P.O.D.), whole Italian hard cheeses (Parmigiano reggiano and
ples was proposed by Hawthorne et al. [48]. The polarity of water Grana padano) [50] and different brand of mozzarella were ran-
decreases as the temperature is increased. This means that selec- domly purchased from local retail markets and drug stores in
tive extraction of polar and medium polar compounds can be Rome. Sampling for hard cheeses was done by the aid of the
performed by suitably adjusting the water temperature. Recently, retailer. A whole cheese (about 40 kg) was split lengthwise and
hot water extraction (HWE) has been proposed as an alternative about 500 g piece was cut through the shape diameter, whereas
extraction procedure for residue determination in food, in which for mozzarella a whole cheese (200–400 g) was taken. The sam-
sand as dispersing solid was employed [49]. ples were purred in plastic bags (for food use) and transferred
C. Cavaliere et al. / J. Chromatogr. A 1135 (2006) 135–141 137

to the laboratory in an ice box. In the laboratory, cheeses were a homogeneous dry powder was obtained. Finally, the matrix
stored in a refrigerator (4 ± 2 ◦ C). All samples were analyzed dispersed sample was packed into a 6 mL polypropylene car-
within a week. tridge pre-filled with 250 mg of the same C18 material, and a
From these “global samples” were obtained “laboratory sam- frit was placed above the packing material. The MSPD packed
ples” of about 200 and 50 g for hard cheese and mozzarella, column was placed onto a vacuum manifold and the analyte was
respectively. Hard cheese sample was finely grated, whereas recovered by passing through the cartridge, at a flow rate of
mozzarella was cut in small pieces and homogenized. There- about 1–2 mL/min, a methanol/water mixture (80:20, v/v). Ten
after sub-samples of about 1 g were taken for analysis. milliliter of eluate were collected in a graduate tube.

2.4. Sample preparation 2.4.3. Procedure II: HWE

One gram of cheese was taken. Cheese was placed in a porce-
2.4.1. Apparatus and materials lain mortar with 4 g of sand and blended with the pestle, until
A Polytron homogenizer was purchased from Kinematica homogeneous and dry material was obtained. This material was
(Luzern, CH). The design of homemade apparatus used for packed into the extraction cell, taking care to tap the tube to avoid
HWE was very similar to that shown in a previous paper [51]. loose packing of the particles. Any void space remaining after
Briefly, it consisted of an LC pump to supply solvent through packing the solid material was filled with sand. Stainless steel
1/16-in. O.D. (1/30-in. I.D.) stainless steel tubing (including a frits were located above and below the packing. The tube was
2-m preheating coil) to the extraction cell. Both the preheating put into the oven and filled with a mixture of water/methanol
coil and the extraction cell were placed inside a GC oven. A (90:10, v/v) at room temperature till the first drop go out. At
10 cm × 8.3 mm-I.D. stainless steel column matched with stain- this point, the output needle valve was closed and the oven was
less steel frits (2 ␮m pore size) was used as extraction cell. heated at 150 ◦ C for 5 min. The valve was opened, keeping a
Sand (Crystobalite, 40–200 mesh size) was supplied by Fluka 10 atm pressure, and the analyte was eluted from the cell by the
AG (Fluka Chemie GmbH, Buchs, Switzerland). C18 (Discov- water/methanol solution at 1 mL/min flow rate, collecting 20 mL
ery DSC-18), polypropylene tubes (6 mL), polyethylene frits in a calibrate glass tube.
and the vacuum manifold were from Supelco (Bellefonte, PA,
USA). Carbograph-4 was purchased by LARA (Rome, Italy); 2.4.4. Clean-up
Carbograph-4 is a graphitized carbon black (GCB) with a surface The extracts coming from procedure I or procedure II
area of 210 m2 /g and particle size range of 120–400 mesh, sim- were cleaned-up by a Carbograph-4 SPE cartridge, as reported
ilar to Carboprep 200 (Restek, USA) and Envicarb X (Supelco, in a our previously published paper [45], while 5 mL of
USA). PTFE syringe filters (0.2 ␮m; 13 mm diameter) were from methanol containing 2% acetic acid were used to wash the
Alltech (Deerfield, IL, USA). cartridge. AFM1 was eluted from Carbograph-4 with 10 ml of
Sand cartridges were prepared by filling 6 mL tubes with dichloromethane/methanol/acetic acid (88:10:2, v/v/v), before
about 3 g of the solid placed between two frits. C18 and evaporation, the eluate was spiked with 25 ␮L of a I.S. solution
Carbograph-4 cartridges were filled with 250 mg of the adsor- 0.04 ␮g/␮L. After evaporation, the residue was reconstituted
bents. C18 was washed immediately prior to use with 2 mL of with 500 ␮L of starting mobile phase for LC and the obtained
methanol, while before processing samples, the Carbograph-4 solution was forced through a PTFE syringe filter. A 20 ␮L
cartridges were attached to a vacuum manifold apparatus and aliquot of the final solution was analyzed by LC/ESI-MS/MS.
washed sequentially with 5 mL of dichloromethane/methanol/
acetic acid (88:10:2, v/v/v), 5 mL of methanol and 10 mL of 2.5. Recovery experiments
Milli-Q water.
Before analysis, samples were checked to evaluate their con-
2.4.2. Procedure I: solvent extraction/MSPD tamination level applying procedure I. Only samples aflatoxin-
One gram of hard cheese was placed into a 50 mL poly- free or at low contamination level were selected. Recovery stud-
carbonate tube followed by adding 10 mL of dichloromethane, ies were therefore carried out on three samples of mozzarella
homogenized twice for 10 s using a Polytron homogenizer. The cheese (two analyte-free and one at 0.029 ␮g/kg), two Parmi-
homogenize was then placed on the top of a sand cartridge posi- giano reggiano samples (one analyte-free and one contaminated
tioned in the vacuum manifold and filtered. The vacuum was at 0.104 ␮g/kg) and one Grana padano sample (analyte-free).
adjusted to the maximum and the extract was collected into a One gram of sample was placed in a flat amber glass vessel and
3 cm I.D. round-bottomed glass tube. The extraction vessel was artificially fortified with variable volumes of AFM1 working
washed with 10 mL of the extracting solvent, and this washing standard solution diluted in 1 mL of acetone, taking care to uni-
was also passed through the sand cartridge and collected in the formly spread it on the sample. An intimate contact between the
tube. The same procedure was applied to mozzarella cheese, analytes and the sample was obtained by mixing with a spatula
with the exception of solvent used for extraction, consisting in for some minutes. The samples were allowed to air drying at
acetone. 25 ◦ C in a ventilate oven, to eliminate the organic solvent. The
The filtrate was evaporated near to dryness in a bath set a spiked samples were then treated following both extraction pro-
50 ◦ C under a gentle stream of nitrogen. One gram of C18 was cedure described above, and analysed. Analysis of spiked and
added to the oily residue and mixed with a small spatula until unspiked samples was conducted in duplicate.
138 C. Cavaliere et al. / J. Chromatogr. A 1135 (2006) 135–141

2.6. Analysis by LC–MS/MS milk consisting in a protein precipitation step with acetone, dilu-
tion of the acetonic extract with water and clean-up by GCB [45].
Liquid chromatography was performed by using Perkin- We tried to use the same scheme for cheese but it was an useless
Elmer series 200 micropumps (Norwalk, CT, USA) including attempt. Dilution of acetonic extracts with water resulted in a
a vacuum solvent degassing unit, and coupled with a Perkin- foggy aqueous samples that plugged the SPE cartridges. This
Elmer autosampler equipped with a 20 ␮L loop. The purified effect was likely due to the co-extracted lipidic fraction more
samples were chromatographed on an Alltima C18 column abundant in cheese, especially hard, aged cheese, than in milk. A
(150 mm × 1 mm I.D., 5 ␮m particle size) from Alltech (Deer- defatting step was necessary between extraction and Carbograph
field, IL, USA) with a Securityguard ODS, 4 mm × 2.1 mm clean-up. The most widely used defatting method is partition of
I.D. precolumn supplied by Phenomenex (Torrance, CA, USA). fats by liquid–liquid extraction (LLE) [15,27,23,37,39–41], but
Analysis was performed as reported in our previous work [45]. several were shortcomings of this method. The extensive use of
An Applied Biosystems/MDS Sciex Q TRAP linear ion trap glassware may result in cumulative loss by adsorption on glass
mass spectrometer (Concord, Ontario, Canada), coupled with of hydrophobic analytes. LLE requires the use of relatively large
a TurboIonSpray (TISP) was used. Applied Biosystems/MDS amounts of highly purified solvents that are expensive as well
Sciex Analyst software version 1.3.2 was employed for data as flammable and toxic. Vigorous shaking of solvent and liq-
acquisition and processing. Mass calibrations and resolution uid matrix may create serious emulsion problems, owing to the
adjustments on the resolving quadrupoles were automatically presence in the sample of natural surfactants. Emulsions can
performed as elsewhere described [45]. be eliminated only by additional time-consuming operations.
Ionization and mass spectrometric conditions were optimized MSPD can overcome these drawbacks because the analytical
by infusing at a flow rate of 5 ␮L/min AFM1 and AFG1, protocol is drastically simplified and shortened, the possibility
10 ng/␮L standard solution. TISP interface was operated in the of emulsion formation is eliminated and solvent consumption is
positive ionization mode. Multiple reaction monitoring (MRM) substantially reduced.
mode of operation was used for identification and quantification As an alternative, we tested a more selective extraction step
of the compounds. The [M + H]+ ions of the aflatoxins (m/z 329 based on the versatility of water as extracting solvent when the
for both compounds) were used as parent ions. The daughter temperature is properly adjusted.
ions detected were: m/z 273 and 259 for AFM1 and m/z 311 and Therefore, we developed and evaluated two different sam-
243 for AFG1. The tuning parameters selected for detection of ple preparation procedures: in the first one the analyte extrac-
two compounds have been reported in a previous paper [45]. tion was performed by homogenization with an organic solvent
(dichloromethane or acetone); this extraction is very effective
2.7. Quantitation and statistical evaluation but not selective, therefore needs extensive clean-up. The sec-
ond developed protocol was based on a more selective extraction,
Linearity was evaluated as reported in our published work carried out with a hot water/methanol solution 90:10 (v/v), fol-
[45], and a linear range between quantification limit and 70 ng lowed by a simpler purification step.
(R2 = 0.995) was found. AFM1 was quantified using exter-
nal calibration, matrix matched, or standard addition proce- 3.1.1. Procedure I: solvent
dures (see “Section 3” paragraph). For the external calibra- extraction/MSPD/Carbograph-4 clean-up
tion, standard solutions were made at five concentration levels Initial extraction with acetone gave the best recovery
(0.05–10 pg/␮L). Matrix matched calibration was also made for a watery fresh cheese such as “mozzarella”, whereas
at the same five concentration levels (0.05–10 pg/␮L) by for- dichloromethane was a better extracting solvent for the hard
tifying extracts obtained from an analyte-free sample. Standard cheese type tested. This fact can be easily explained considering
additions procedure was done at three levels of spiking after the relative content of water and fats.
evaluating the original contamination level. The combined ion One gram of C18 material was a sufficient quantity to obtain
current profiles for both fragment ions were extracted from the a dry matrix dispersion from the oily residue obtained after sol-
LC/MRM dataset. The peak area versus injected amount chart vent removing, amenable for packing an extraction cartridge.
was obtained by measuring the resulting peak area and relating The preloaded packing was necessary for optimal defatting, that
this area to I.S. one. Statistical comparisons were performed by was reached also by selective elution using an appropriate vol-
ANOVA (p = 0.05). ume of methanol/water mixture (80/20, v/v). This eluate can be
diluted with water and passed through the Carbograph-4 clean-
3. Results and discussion up cartridge.

3.1. Extraction and clean-up 3.1.2. Procedure II: HWE/Carbograph-4 clean-up

As reported in many published papers [48,51,52], the influ-
As reported in the introduction, one of our goals was to obtain, ence of extraction parameters versus the analyte recovery was
from cheese samples, extracts amenable for LC–MS/MS anal- studied. All parameters were evaluated with triplicate experi-
ysis, with a simple, time effective procedure, without using the ments by comparing a hard cheese sample aflatoxin-free spiked
expensive immunosorbent cartridges. In a very recent paper we before and after extraction with AFM1 at 0.45 ␮g/kg. Briefly,
devised a sample preparation method for determining AFM1 in initial extraction experiments were performed by using pure
C. Cavaliere et al. / J. Chromatogr. A 1135 (2006) 135–141 139

Table 1
Matrix effect obtained using the two sample preparation procedures (n = 4)
Procedure I: solvent extraction/MSPD Procedure II: HWE
Mozzarella cheese Hard cheese Mozzarella cheese Hard cheese

Relative peak areaa (RSD) Relative peak area (RSD)

AFM1 0.97 (5%) 0.84 (9%) 0.81 (7%) 0.52 (11%)

AFG1 1.02 (6%) 0.89 (7%) 0.80 (8%) 0.61 (9%)
a Peak area of the analyte injected from a cheese extract relative to that of the analyte injected from a standard solution.

water heated at 120 ◦ C as extractant and the recovery of AFM1 tification of two compounds was performed by comparing their
was 52 ± 19%. Increasing the extraction temperature to 150 ◦ C absolute peak areas to those obtained injecting a standard solu-
improved recovery to 68 ± 16% and using an extraction tem- tion. The results are reported in Table 1. As can be seen the
perature of 180 ◦ C did not improved significantly AFM1 recov- procedure II exhibits a more prominent matrix effect, espe-
ery, this was likely due to some thermal decomposition [51]. cially analyzing hard cheese, this demonstrate the effectiveness
Therefore, to enhance the extraction efficiency without thermal of MSPD step included in procedure I.
degradation, 10% of methanol was added to water operating
at 150 ◦ C: this modification was enough to increase recovery 3.2.2. Recovery and precision
to 85 ± 12%. Doubling methanol percentage, AFM1 amounts Recovery experiments were performed on artificially forti-
removed from cheese sample did not significantly increase, but fied cheese samples, at two contamination level: 0.25 ␮g/kg
the extract appeared to contain a greater amount of matrix com- and 0.45 ␮g/kg which were, respectively, AFM1 Switzerland
ponents compared with one obtained with 10% of methanol. and Turkish legal limit and provisional limit in hard cheeses of
Extraction with hot liquids can be performed in static mode, Italian Minister of Health. Recoveries were calculated by com-
dynamic mode, or a combination of them [52]. Using 20 mL of a parison with samples fortified after clean-up, excluding in this
water/methanol solution (90:10, v/v) at 150 ◦ C, we investigated way matrix effect. Results of these experiments are summarized
the effect of the static extraction period duration on analyte- in Table 2. Recoveries for method I ranged between 81 and
extracted amounts, varying the time from 0 to 10 min. When the 92% with relative standard deviation not larger than 7%, while
static extraction step was not performed, lower recoveries were method II seems to provide a little lower performance, although
obtained and more failed extraction happened, due to clogging none of data were significantly different at p = 0.05.
of the extraction cell. Static extraction time exceeding 5 min
did not yield higher efficiency. Thus, the extraction was per- 3.2.3. Quantification limit
formed, as reported in the experimental section, using 20 mL Instrumental LOQ was estimated from the LC–MS MRM
of a water/methanol solution (90:10, v/v) at 150 ◦ C and with a chromatogram resulting from analysis of AFM1 standard solu-
static extraction time of 5 min. This extract was amenable for tion at 0.1 pg/␮L concentration (2 pg injected). The sum of
Carbograph-4 clean-up after dilution with water. the ion currents of both precursor-fragment ion transitions
was extracted from dataset, the resulting trace was two-times
3.2. Method comparison smoothed by applying the mean smoothing method. LOQ was
then calculated on the basis of a minimal accepted value of
3.2.1. Matrix effect signal-to-noise ratio (S/N) of 10 and its value was about 0.6 pg.
Matrix effect, for both procedures, was evaluated by com- Method quantification limits (MQLs) for both procedures were
paring analyte-free samples spiked, with a solution containing calculated as reported for LOQ but analyzing real contaminated
AFG1 and AFM1, after clean-up with a reference solution. Prac- samples, at a concentration close to 10 times signal to noise, and
tically, two Mozzarella cheese samples and two hard cheese correcting the obtained values for calculated procedure recov-
samples (one Parmigiano reggiano and one Grana padano) ery. Data are listed in Table 3. In the same table, in order to well
were extracted as reported under two experimental procedures. compare the increase of MQLs as regards LOQ, due to recovery
Before evaporation, the SPE-eluates were spiked with AFM1 and matrix effect, we also reported LOQ as concentration rather
and AFG1, respectively, at 0.45 ␮g/kg and 1 ␮g/kg level. Quan- than as injected amount.

Table 2
Recoveries and precision (n = 6)
Procedure I: solvent extraction/MSPD Procedure II: HWE

0.25 ␮g/kg 0.45 ␮g/kg 0.25 ␮g/kg 0.45 ␮g/kg

R% (RSD) R% (RSD) R% (RSD) R% (RSD)

Mozzarella cheese 92 (5%) 90 (3%) 79 (7%) 82 (8%)

Hard cheese 83 (6%) 81 (7%) 84 (11%) 80 (15%)
140 C. Cavaliere et al. / J. Chromatogr. A 1135 (2006) 135–141

As can be seen, although both procedures have MQLs below Table 3

the lower current legal limits, the first procedure gave better Comparison among method quantification limit using the two sample prepara-
tion procedures
MQLs than the second one. This is due to an enhanced purifica-
tion that minimizes matrix effect and chemical noise. This could Procedure I: solvent Procedure II:
be important when, fixed a legal limit, there is the needing to a extraction/MSPD HWE
confirmatory method having well defined performance criteria. LOQa MQLb (␮g/kg) MQL (␮g/kg)
For example, a recent decision of European Union [53] estab- (␮g/kg)
lishes that for undoubted compound identification, an S/N > 3 Mozzarella cheese 0.019 0.048
should be achieved, at a maximum tolerable level, for the worst 0.015
Hard cheese 0.025 0.143
transition. In Fig. 1 are reported the extracted ion current pro- a Instrumental limit of quantification (S/N = 10).
b Method quantification limit (S/N = 10).

files for the second most intense transition of AFM1 relative to a

Parmigiano reggiano sample analyzed after procedure I (upper)
and II (lower). The contaminant level found was 0.34 ␮g/kg and
S/N ratios were, respectively, 140 and 17.

3.3. Real sample quantification

Some cheeses purchased from Italian retail markets were ana-

lyzed. For mozzarella extracts prepared with the procedure I,
for which no matrix effect was evidenced, the external cali-
bration was applied. In a first attempt, for all other samples,
matrix matched calibration was used. Analyzing some hard
cheeses aged more than 20 months, very different values with
the two procedures were found. This was due to the quantifi-
cation method adopted: for hard cheeses matrix effect can vary
with aging, and this outcome is more evident for the procedure
II. In this case only the standard addition calibration gives right
values and, as reported in Table 4, a good accordance with the
two extraction procedures was obtained.

Table 4
Aflatoxin M1 levels in real sample
Sample AFM1 (␮g/kg)

Procedure I: solvent Procedure II:

extraction/MSPD HWE

Mozzarella nqa nqb

Mozzarella 0.029a nqb
Mozzarella nqa nqb
Mozzarella 0.071a 0.077b
Grana padano (16 months ageing) nqb nqc
Parmigiano reggiano (18 months nqb nqc
Grana padano (20 months ageing) 0.340b 0.332c
Grana padano (20 months ageing) 0.749b 0.763c
Parmigiano reggiano (24 months 0.178b 0.176c
Parmigiano reggiano (24 months 0.081b nqc
Grana padano (24 months ageing) 0.222b 0.229c
Parmigiano reggiano (36 months 0.334b 0.344c
Parmigiano reggiano (36 months 0.104b nqc

Fig. 1. Chromatograms showing the extracted ion current profiles for the second nq: not quantified.
most intense transition (329 → 259) of Aflatoxin M1 (0.34 ␮g/kg level) relative a Quantified using external calibration.

to a Parmigiano reggiano sample analyzed after sample preparation I (upper) b Quantified using matrix matched calibration.

and II (lower). c Quantified using standard addition procedures.

C. Cavaliere et al. / J. Chromatogr. A 1135 (2006) 135–141 141

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