Pharmacology & Pharmacy, 2013, 4, 611-618
Published Online November 2013 (
Open Access PP
Rapid and Simple Extraction Method for Volatile
N-Nitrosamines in Meat Products
Mohammad Al-Kaseem*, Zaid Al-Assaf, Franswa Karabet
Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria.
Email: *
Received September 21st, 2013; revised October 22nd, 2013; accepted October 29th, 2013
Copyright © 2013 Mohammad Al-Kaseem et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A new methodology for extraction, pre-concentration and analysis of volatile nitrosamines in meat-derived products
was developed and compared with conventional methods (Distillation and two-step solid-phase extraction). The sam-
ples (canned sausages, cured meat, luncheon and smoked meat) were treated with an aqueous sodium hydroxide (NaOH)
by autoclaving at 121˚C for 10 min and extracted by liquid-liquid extraction with dichloromethane, then the nitrosa-
mines were pre-concentrated using activated silica. Then, gas chromatography coupled with flame ionization detector
was used for the separation and determination of the different nitrosamines contained in a real sample and gas chroma-
tography with mass spectrometry detection was used as the confirmation technique. The newly invented autoclaving
method allowed the determination of nitrosamine compounds at trace levels with limit of detection ranged from 0.077
to 0.18 ppb and quantitation limits were from 0.26 to 0.6 ppb for all nitrosamines, and found to be superior to the con-
ventional ones, yielding approximately about 10% - 20% increasing in the recovery compared with the mean recovery
obtained when applying conventional methods.
Keywords: GC-FID; N-Nitrosamines; LLE; Meat; Autoclave
1. Introduction
N-Nitrosamines (NAs), mainly N-nitrosodimethylamine
(NDMA), and N-nitrosodiethylamine (NDEA) are re-
ceiving special attention, because they present high mu-
tagenic and carcinogenic potential that can induce tumors
in variety of organs, including the liver, lung, kidney,
bladder, pancreas, esophagus and tongue depending on
the species, but not in the skin, brain, colon or bone. For
example, N-nitrosodimethylamine at the levels of 20
ppm can induce liver cancer in a human [1]. The Interna-
tional Agency for Research on Cancer (IARC) classified
NDMA and NDEA as most carcinogenic to humans, and
N-nitrosodi-n-butylamine (NDBA), N-nitrosopiperidine
(NPIP) and N-nitrosopyrrolidine (NPYR) as possibly
carcinogenic to humans [2,3]. In the diet, they occur as
unintentional byproducts of food preparation, preserva-
tion and processing [4], although they can also occur in
the environmental tobacco smoke [5], and may be formed
endogenously within the human body [6]. These com-
pounds are formed by the reaction of secondary amines
with nitrosating agents, such as nitrates or nitrites, which
are commonly used in the manufacture of meat products
[4-7]. For this reason, the use of these nitrosating agents
for curing meat is strictly controlled in some countries
[8]. Moreover, some known inhibitors of the nitrosation
reaction, such as ascorbic acid, are used in the processing
of several foods [9]. The determination of nitrosamines
in food and water samples has been carried out by dif-
ferent analytical methods, including colorimetry [10,11],
capillary electro-chromatography [12], micellar electro-
kinetic capillary chromatography [13], gas chromatogra-
phy with flame ionization detection, nitrogen phospho-
rous detection, thermal energy detection, nitrogen chem-
iluminescence detection [14-17], and mass spectrometry
detection [18-20], high-performance liquid chromatog-
raphy with thermal energy analyzer, mass spectrometry
and fluorescence detection [21,22]. The extraction of the
nitrosamine from the complex food matrices and the
cleanup of the extract have been the critical points of the
sample preparation step, and several approaches are do-
cumented in the literatures, including distillation (steam
or vacuum) [23-25], solvent extraction, [26] solid-phase
*Corresponding author.
Rapid and Simple Extraction Method for Volatile N-Nitrosamines in Meat Products
extraction [27], solid-phase micro-extraction (SPME)
[28], and supercritical fluid extraction [29].
This paper describes the development of a simple
method for the extraction of volatile nitrosamines from
meat products using sodium hydroxide to enhance the
stability under autoclaving conditions and comparing the
new method with the conventional procedures (vacuum
distillation and two-step solid-phase extraction with Ex-
trelut and Florisil sorbents).
2. Methods
2.1. Samples
All meat samples (10 samples of each of cured meat,
sausage, luncheon, and smoked meat) were purchased
from supermarkets in Damascus. The samples were
crushed and mix with no addition of the fluid and then
stored in 20˚C before analysis.
2.2. Materials
EPA 521 nitrosamine mix standard was purchased from
Supleco (USA), this solution contained seven analytes at
2000 µg/mL of each: N-nitrosodimethylamine (NDMA),
N-nitrosomethylethylamine (NMEA), N-nitrosodiethyla-
mine (NDEA), N nitrosopyrrolidine (NPYR), N-nitroso-
din-propylamine (NDPA), N-nitrosopiperidine (NPIP)
and N-nitrosodi-n-butylamine (NDBA). For sample pre-
paration, sodium hydroxide, ethanol, Methanol, hexane,
octane and dichloromethane (DCM) and Florisil were
purchased from Sigma-Aldrich (USA). Florisil water
sep-pak cartridges from Waters (Milford, MA, USA).
Extrelut and Activated Silica Gel (silica gel 60 extra pure.
70 - 230 mesh) were obtained from Merck (Germany).
All other chemicals (ascorbic acid, sulfanilic acid, sul-
furic acid, anhydrous sodium sulfate and sodium chlo-
ride) used in this research were of analytical laboratory
2.3. Apparatus
Pyrex tubes (20 ml) with heat-stable Teflon-lined caps
and glass column (30 cm × 1.5 cm) were used. Kuderna-
Danish (KD) concentrator was used for the concentration
of organic solvents. Autoclave (Selecta, Spain, 4001757)
and gas chromatograph equipped with a flame ionization
detector controlled by computer running GC-Solution
software (GC-2014, Shimadzu, Japan) and an optima-
XLB Capillary Columns (0.25 mm i.d. 30 m, 0.25 μm)
from (MACHEREY-NAGEL GmbH & Co) were used.
Agilent gas chromatography 7890A equipped with mass
selective detector MS 5975C controlled by computer
running Agilent Chemstation software (Agilent Tech-
nologies, Madrid, Spain) for confirmation of the results.
Local steam distillation device and glassware were used
for sample preparation.
2.4. Preparation of Nitrosamine Mix Standard
A series of working standard solutions were prepared by
appropriate dilution of the EPA 521 nitrosamine mix
with dichloromethane and stored at 20˚C before use.
From the primary stock solution 2000 µg/mL of each
nitrosamine, a 1/200 dilution was done to get 100 µg/mL
secondary stock solution of each nitrosamine. Sequen-
tially dilute secondary stock solution was performed to
get standards titrating at 4, 10, 50, 100, 500, 1000 µg/L
in by taking the following volumes from secondary stock
solution, 40 µl, 100 µl, 500 µl, 1000 µl, 5 mL, 10 mL in
series of 100 mL volumetric flasks respectively, these
solution kept in the absence of light.
2.5. Analytical Conditions
GC analysis was carried out using GC-2014 gas chro-
matograph equipped with flame ionization. (GC-FID)
(Shimadzo Technologies, Japan). One microliter of the
extracted solution sample was injected into optima-XLB
Capillary Column (30 m × 0.25 mm I.D. × 0.25 film
thickness (df)). For the gas chromatograph separation of
N-nitrosamines, the injection port and detector tempera-
ture were kept at 250˚C and 300˚C. The oven program
was as follows: 40˚C, held for 3 min; ramp to 100˚C at
10˚C/min, held isothermally for 1 min; ramp to 250˚C at
15˚C/min, held isothermally at 250˚C for 2 min. The
sample was injected in splitless mode; using an injection
volume of 1 µl. Nitrogen was used as make-up gas. The
purity of all gases used was greater than 99.999%. The
velocity of the helium carrier was 1 mL/min. GC-MS
were carried out to confirmation of the results using an
optima XLB column (30 m × 0.25 mm I.D. × 0.25 film
thickness (df) column with same conditions used in GC-
FID and the injector and transfer line temperature in GC-
MS were 240˚C and 275˚C, respectively. The ion source
temperature was 240˚C and operated in positive electron
ionization (EI) 70 eV mode.
2.6. Conditions for Autoclave Treatment
In this study, half ml of the N-nitrosamine Mix standard
containing 50 μg/L of each nitrosamine was diluted with
10 mL of 0, 0.5, 1 N, 2 N aqueous sodium hydroxide and
10 mg of ascorbic acid (Inhibitor of endogenous nitrosa-
tion) in 20 mL Pyrex tubes tightly capped and autoclaved
at 121˚C for 5, 10, 30 min. After being allowed to stand
at room temperature, the autoclaved solution was trans-
ferred to 50 mL separatory funnel. The tubes was rinsed
twice with 5 mL of ethanol and then 10 mL of dichloro-
methane, and the rinsing solutions and 10 mL of 10%
aqueous sodium chloride were combined with the origi-
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Rapid and Simple Extraction Method for Volatile N-Nitrosamines in Meat Products 613
nal extract in the separatory funnel. After being shaken,
the dichloromethane layer was collected, and the water
layer was re-extracted with 10 ml of dichloromethane.
The dichloromethane extracts were combined, dried over
anhydrous sodium sulfate and concentrated to approxi-
mately 0.5 mL using KD concentrator and nitrogen gas
flow. The concentrate was loaded onto a silica gel col-
umn (30 cm × 1.5 cm) (equilibrated with dichloro-
methane) and the column was eluted with 10 ml of di-
chloromethane. After the addition of 100 µL of octane
(to prevent exsiccation of the solvent), the elute was
concentrated to 1 mL using KD concentrator and nitro-
gen gas flow and analyzed by GC-FID using 1 µL injec-
tion volume, and confirmatory analyses were performed
on GC-MS.
2.7. Extraction of N-Nitrosamine from Meat
2.7.1. Autocl a ve Treatment
Approximately one gram of meat sample was placed in
the Pyrex tube into which 10 mL of sodium hydroxide 1
N was poured. The tube was capped tightly and auto-
claved at 121˚C for 5, 10 and 30 min; dichloromethane
extract was obtained by the procedure described for the
standard solution in (Section 2.6). The individual extracts
were analyzed by GC-FID, GC-MS as in the case of the
standard solution (Section 2.5).
2.7.2. Steam Distillation
Extraction was done as described by Komarova and Ve-
likanov (2000) [22]. A weighed portion (80 - 100 g) of a
meat reduced in a homogenizer was placed in a 500-mL
round-bottom flask connected with a steam generator and
a condenser. Distilled water (100 - 150 mL depending on
the humidity of the foodstuff) and 0.1 mL of 50 ppb so-
lution of each nitrosamine were added to the foodstuff
and stirred. Next, 10 g of sodium chloride, 10 g of so-
dium or magnesium sulfate, 5 mL of a 2% sulfanilic acid
solution (to inhabit endogenous nitrosation), and 10 mL
of a 1 N sulfuric acid solution were added, and N-ni-
trosamines were steam distilled off collecting 250 mL of
the distillate. The distillate was transferred into a separa-
tory funnel, and N-nitrosamines were extracted with di-
chloromethane five times (with portions of 10 mL each).
Each portion of the extract was passed through a funnel
with a red-ribbon paper filter filled with 5 g of anhydrous
sodium sulfate. The filter was washed with 10 mL of the
extractant. All portions of the extract were combined and
condensate using KD to 1 ml and analyzed using GC-
2.7.3. Two-Step Solid-Phase Extraction
Extraction of nitrosamines was performed using the same
method as Yurchenko and Molder (2007) [31]. A two-
step solid-phase extraction using Extrelut and Florisil
sorbents was used for sample cleaning. Each sample (6.0
± 1.0 g) was minced and mixed with 0.1 mol·L1 NaOH
(6 mL). For the first step, 6 g of Extrelut was placed at
the bottom of the glass column (30 cm × 1.5 cm) and
wetted with 20 mL hexane/dichloromethane 40:60 (v:v).
Then, the sample was eluted with two 20 mL portions of
hexane/dichloromethane solution 40:60 (v: v). the eluate
was collected in a 50 mL concentrator flask and evapo-
rated under a nitrogen stream. During the second step, 1
g of Florisil was placed at the bottom of the Florisil car-
tridge (6.5 cm × 1.3 cm) and wetted with 6 mL di-
chloromethane/methanol 95:5 (v:v). Then the sample
solution was eluted with 6 mL dichloromethane/metha-
nol solution 95:5 (v: v). the solution was evaporated un-
der a nitrogen stream to about 1 mL. The prepared solu-
tion was transferred to the GC auto sampler vial. Extrac-
tions were performed in triplicate [30].
3. Results and Discussion
3.1. Optimization of Conditions for Autoclave
Figure 1 shows a chromatogram of the N-nitrosamines
standard solution obtained by GC-FID. The total recov-
ery of nitrosamine for the autoclave treatment in water
ranged from 61.1% to 98% depending on the heating
period as shown in Table 1. This indicates that N-ni-
trosamines remained almost stable during the autoclave
treatment because of the stability of N-nitrosamines un-
der the alkaline conditions and difficulty to destroy them
under these conditions [31], however, the total recovery
of N-nitrosamines slightly decreased as the NaOH con-
centration increased or the heating period elongated. To
clarify the reason for the decrease in the total N-ni-
trosamine recovery, the volatile nitrosamines in Figure 1
were analyzed and the results are shown in Table 2.
The recoveries of some N-nitrosamines with rather
short retention times such NDMA and NDEA were sat-
isfactory under any conditions. In contrast, the decrease
in the recovery was notable for the peaks with much
longer retention times as the NaOH concentration in-
creased or the heating period became longer. Since
NPYR and NPIP giving longer retention times that, the
decrease in the total N-nitrosamines recovery would re-
sult from the instability under the conditions of high
NaOH concentration and lasted heating at 121˚C. Con-
sequently, it was proposed that samples for N-nitrosa-
mines analysis should be treated with 1 N NaOH for 10
3.2. Efficacy of Autoclave Treatment for
Extraction of N-Nitrosamines from Meat
Table 3 shows the peak areas and total peak areas of N-
nitrosamines for all extraction procedures, the peak areas
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Rapid and Simple Extraction Method for Volatile N-Nitrosamines in Meat Products
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Figure 1. Chromatogram of 50 µg/L of N-nitrosamine mix (GC-FID).
Table 1. Effect of alkali concentration and autoclaving times on total recovery of N-nitrosamine.
Autoclaving time (min)
5 10 30
Alkali concentration
Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)
0 N NaOH (water) 61.1 6.8 76 8.9 62.7 9.24
0.5 N NaOH 85.9 2.7 97.8 4.3 87.9 8.5
1 N NaOH 88.4 2.8 98.1 1.05 88.4 3.1
2 N NaOH 87.2 5.6 87.6 4.6 88.5 6.4
Each value represents mean (S) and relative standard deviation (RSD) in three experiments.
Table 2. Effect of alkali concentration and autoclaving times on recovery of N-nitrosamines.
Autoclaving time (min)
5 min 10 min 15 min 5 min 10 min 15 min 5 min 10 min 15 min
With NaOH 0.5 N
(Recovery %)
With NaOH 1 N
(Recovery %)
With NaOH 2 N
(Recovery %)
NDMA 80.0 89.7 88.9 90.2 105.5 93.7 88.4 80.7 80.6
NMEA 88 89 76 67 89 89 76 73 89
NDEA 85.6 87.8 85.6 80.1 102.3 91.4 88.5 79.3 80.5
NPYR 77.1 79.0 77.1 80.2 99.1 90.2 86.9 75.3 70.3
NDPA 80.6 86.3 76.4 80.3 98.5 93.8 86.4 77.1 83.6
NPIP 73.2 80.2 75.3 86.3 99.3 88.9 83.4 77.3 86.1
NDBA 69.9 73.7 69.3 80.3 92.1 88.3 85.3 82.9 71.6
Each value represent mean of three experiments, N; normality, N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine
(NDEA), N nitrosopyrrolidine (NPYR), N-nitrosodin-propylamine (NDPA), N-nitrosopiperidine (NPIP) and N-nitrosodi-n-butylamine (NDBA).
Rapid and Simple Extraction Method for Volatile N-Nitrosamines in Meat Products 615
Table 3. Determination of N-nitrosamines with and without autoclave treatment, and with conventional methods.
With autoclave treatment Without autoclave treatmentDistillation methodTwo step solid phase extraction
(min) Area Relative ratio* Area RV* Area RV* Area RV*
NDMA 5.617 203348 0.08 103456 0.51 120087 0.59 180323 0.88
NMEA 7.344 287513 0.113 123456 0.43 160000 0.55 247909 0.86
NDEA 8.733 353106 0.139 201345 0.57 98000 0.27 314876 0.89
NPYR 12.104 442116 0.175 286776 0.65 121564 0.27 399887 0.90
NDPA 12.224 355971 0.14 173456 0.49 123546 0.34 291006 0.81
NPIP 12.853 400387 0.158 127999 0.32 259099 0.64 328987 0.82
NDBA 14.681 483347 0.191 254888 0.53 178988 0.37 393000 0.81
total 2525788 1 1271376 1061284 2155988
Relative ratio*: area/total area, RV*: peak area as the percentages of that of the autoclave treatment, NAs: nitrosamines, RT: retention time.
after the conventional treatments were shown as the per-
centages of the peak areas corresponding of autoclave
treatment. All N-nitrosamines tested were detected after
autoclaving. The peaks of N-nitrosamines were sharp and
completely separated, and there is an increasing of the
recovery (expressed as peak area) after autoclave treat-
ment compared with the same sample when extracted
without an autoclaving, or by conventional methods. Ob-
viously, the relative percentage of peak area for each
nitrosamine (Table 3) under autoclaving conditions was
as twice as that of extraction without autoclaving or by
distillation, and it was about 10% - 20% more than that
of the two-step solid-phase extraction.
3.3. Method Validation
The new method was validated to demonstrate that it is
suitable for its intended purpose by the standard proce-
dure to evaluate adequate validation characteristics [30,
32]. Retention times of selected N-nitrosamines was de-
termined by using standard solutions of each nitrosamine,
and the confirmation was performed using GC-MS. Fig-
ure 1 shows the chromatogram obtained by GC-FID of
50 ppb (part per billion) standard solution of each N-
nitrosamines. Figure 2 shows the chromatogram ob-
tained by unspiked meat sample. Table 4 shows the con-
firmation results of seven nitrosamines obtained when
analyzing the same solution and identifying the retention
times using GC-MS. In order to examine the linearity a
series of working standard solutions at the concentrations
of 4, 10, 50, 100, 500, 1000 µg/L of each N-nitrosamine
were prepared and analyzed under the selected chroma-
tographic and flame ionization detector conditions. The
linear equations, linear ranges and correlation coeffi-
cients for the seven nitrosamines are shown in Table 4.
The limits of quantification with a signal-to-noise ratio of
10 for the seven nitrosamines were from 0.26 to 0.6 ppb
with limits of detection with a signal-to-noise ratio of 3
for the seven nitrosamines were from 0.077 to 0.18 ppb.
The recovery and precision experiments were conducted
by spiking different blank samples at three spiking levels
of 1, 5, 10 µg/L, six replicates at each level, the mean
recoveries were from 89% - 105.5% with the relative
standard deviations (RSDs) from 2.8% to 4.4%.
3.4. Sample Results
Meat samples were treated with 1 N NaOH for 10 min in
autoclaved conditions and then analyzed by GC-FID and
the results are shown in Table 5. The results of 40 sam-
ples of different meat products which examined for the
test of the extraction procedure shows the presence of
some volatile nitrosamine in all different products, and
the absence of NDEA, NDPA, and NDBA in all the
sample tested which may related to the presence of these
three nitrosamines in low amounts which may below
under the quantitation limit of our procedure, or the real
absence of these nitrosamine in the sample tested. Statis-
tically when applying (PASW Statistics 18) one way
ANOVA test for statistical comparisons between the
means of sum of seven nitrosamines in meat samples,
there was significant difference (p > 0.05) which may
related to the low number of different samples, or be-
cause that some of tested samples are commonly pre-
served by nitrite salts (sausages and luncheon) which
contributing in nitrosamine formation and the effect of
processing (smoking) in the formation of these carcino-
genic compounds in smoked meat.
4. Conclusion
It was included in this paper that the extent of recovery
of N-nitrosamines from the meat products was highly
dependent on the extraction conditions, and it was con-
cluded that extraction recovery could be increased when
adopting the autoclave conditions and sodium hydroxide
to increase the stability of volatile nitrosamine, and add-
ing ascorbic acid to inhabit endogenous nitrosation which
may occur during extraction We recommend the newly .
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Rapid and Simple Extraction Method for Volatile N-Nitrosamines in Meat Products
Figure 2. Chromatogram of unspiked meat sample.
Table 4. Linear equations, linear ranges and correlation coefficients of seven volatile nitrosamines.
N-nitrosamine RT*
Confirmation RT*
GC-MS Linear equation Linear range
NDMA 5.617 5.617 y = 4161.7x 9274.8 0.6 - 500 0.9997 0.6
NMEA 7.344 7.344 y = 5816.1x 14015
0.43 - 500
NDEA 8.733 8.733 y = 7095.3x 13214
0.33 - 500
NPYR 12.104 12.104 y = 8699.3x 8839.9
0.26 - 500
NDPA 12.224 12.224 y = 6674.6x 3555.2
0.25 - 500
NPIP 12.853 12.853 y = 7992.3x 141000
0.34 - 500
NDBA 14.681 14.681 y = 9805.1x 16361
0.30 - 500
RT*: retention time (minutes), GC-FID: gas chromatography-flame ionization detection, LOQ: limit of quantitation, LOD: limit of detection.
Table 5. Results on nitrosamine results in meat samples.
Mean concentration (n = 3) of N-nitrosamine µg/kg
products No. of samples
Sum of seven N-nitrosamine µg/kg
Sausage 10 nd* nd* nd* nd* 2.78 2.98 4.76
Cured Meat 10 nd* nd* nd* nd* 3.2 nd 3.20
Luncheon 10 nd* nd* nd* nd* 2.60 nd 2.60
Smoked meat 10 nd* nd* nd* nd* 3.70 2.60 6.60
nd*: not detected.
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