Fast, Simple, Novel and Economical Method for Ultra Trace Determination of Nine Nitrosamines in Sausage by Gas Chromatography-Flameionization Detec- tor and Ascorbic Acid and Sodium Nitrite Effect on Nitrosamine Formation

A simple, fast and low-cost method have been applied for the determination of nine nitrosamines in sausage. This method is based on a liquid-liquid extraction, which examinations were performed by gas chromatography-flame ionization detector (GC-FID) and high-performance liquid chromatography diode array detection (HPLC-DAD) by post-column photolysis and derivatization was used for confirmation and validation of the results. Initially, partitioning of samples was carried out in a water-hexane mixture and then the organic phase was separated and evaporated to dryness, substituted by ethanol and was injected. The detection limit was 0.4 μg Kg-1 and the average relative standard deviation (RSD %) variations for repeated experiments was 6.3%. The extraction recovery variation for the spiked samples was 63-97%. The method was stable and consistent and successfully applied for nitrosamines determination in sausage.


Introduction
The presence of a large amount of free water in meat has been caused on one hand it has high nutritional value; on the other hand, meat is prone to microbial growth and decay. Diverse meat products, such as Sausage, with different flavors, and more lifetime are produced from meat treatment with suitable ingredients. Since antique times, the use of salt for keeping safe of meat revealed that the nitrate existence in salt, as a contaminant, had a meaningful function in the restraint of microorganism growth. The nitrate is converted to nitrite and improves the curing of meat. Nowadays, to prevent the formation of Clostridium botulinum bacterium, nitrite and nitrate salts are commonly added in curing mixtures in order to cause to slow down lipid oxidation and give to treated meats their distinctive taste, color, and odor. There is, however, a lot of controversy over the safety of meat products [1].
Nitrite, in the process of meat manufacturing, is one of the most important and common additives that it is caused desirable texture and color, delay in lipid oxidation, and particularly preventing from the formation of clostridium botulinum [2,3].
In foodstuffs, nitrosamines are produced by the reaction of nitrogen oxide and amines. In acidic medium, hydronitrogen oxide (H2NO2+) is formed from nitrite in food. The source of nitrite is the reduction of nitrate fertilizer or added as a preservative [4]. The reaction of hydro nitrogen oxide with another molecule of nitrite form nitrogen anhydride after dehydration. Nitroso group from nitrogen anhydride reacts with amines in food to form nitrosamines. Stable nitrosamine is formed by secondary amines, while nitrosamines produced from primary amine is destroyed quickly; it is also known that nitrosamine formation from tertiary amine is difficult [4].
But nitrite reaction with amines and amino acids and as a result, N-nitrosamines formation is an important concern.
Cured meat products, tobacco, rubber products, cosmetics, other consumer products, and environmental matrices are a source of nitrosamines [5]. Nitrosamines are carcinogenic, mutagenic and teratogenic. Due to this cause, meat improving by the addition of these nitrosating agents is completely regulated and monitored in some countries. Moreover, some compounds such as ascorbic acid are added to several foods during the processing because these have an inhibitory role in the nitrosation reaction.
The substantial reason is the reduction of nitrite by ascorbic acid and following that changing the reactions of nitrite. The sever-al groups have been investigated mechanisms of the reactions of ascorbic acid with nitrite [6]. The N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) have been classified as most carcinogenic and N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP) and N nitrosopyrrolidine (NPYR) as possibly carcinogenic to humans by International Agency for Research on Cancer (IARC) [7].
The chemical structure of nitrosamines is as R1N(-R2)-N=O, that is, a nitroso group bonded to an amine as following.
Distribution of seven nitrosamines has been studied in different foodstuffs such as agricultural products, seafood, milk, and milk products, meat and meat products, processed meats, oils, butter and margarine, soybean paste, soy sauce, seasoning, sauce, and alcoholic beverages [4].
The various methods for nitrosamine extraction and extract clean-up in the complicated food matrices have been applied, including supercritical fluid extraction,18 solvent extraction, [19] solid-phase extraction, [20] and solid-phase micro-extraction (SPME) [21].
The purpose of the present work was development and validation of an accurate and easy method for the determination of nine volatile N-nitrosamines in sausage (fast food) using gas chromatography-flame ionization detector and ascorbic acid measurement by high-performance liquid chromatography-diode array detector. In addition, in this article, sodium nitrite and ascorbic acid effect on nitrosamine formation have been investigated.
To the best of our knowledge, this article describes the first determination of nine nitrosamines by GC-FID and then method validation by HPLC-DAD with post-column photolysis and derivatization with a low detection limit for the stable and consistent determination of nitrosamines in foodstuffs.

Chemicals and materials
Nitrosamines mix, certified reference material, was

Working standards
Ascorbic acid: A 1000 mg L -1 stock standard was prepared by dissolving 10mg ascorbic acid in 10 mL ultrapure water and used for preparing working standards.
Nitrosamines: A secondary storage solution with a concentration of 1.0 mg L -1 was prepared by diluting nitrosamines mix the standard solution. Then, by sequential dilution of the secondary storage solution in methanol, working standards with concentrations of 1, 2, 5, 10 and 20 µgL-1 were obtained. The outlet of the T-shaped connection connected to the detector.

High-performance liquid chromatography
All tubes and connections were as short as possible in order to prevent band broadening. Configuration and operation details had been presented in ISO.31

Gas chromatography
Samples, for nitrosamines, were determined using GC,

Optimization of the nitrosamines extraction parameters
The extraction efficiency depends on various parameters such as type and volume of extraction solvent, nitrosamines solvent and salt addition. The peak area was utilized for the evaluation of the above-mentioned parameters. All measurements were carried out three times and their mean was considered.

Nitrosamines solvent
Dissolving and diluting of nitrosamines standard solutions were performed in different solvents such as methanol, ethanol, acetone, pentane, hexane, and dichloromethane. The highest signal and the best peaks obtained by using ethanol as solvent.

Salt addition effect
Generally, the increase of the ionic strength can cause a decrease in the solubility of the analytes in the sample solution and enhance extraction efficiency. To evaluate the possibility of the salting-out effect, the extraction efficiency was studied with adding sodium sulfate ranging from 0.5% to 5%. Due to the salting-out effect, the peak area increased as the amount of Na 2 SO 4 increased from 0.5% to 2% owing to very high solution ionic strength and from 2.5% to 5% had negligible changes. By increasing the ionic strength, increasing the extraction recoveries was observed. Based on the experimental results, salt was added in the next experiments.  Table 3.

Accuracy Precision
Results of three replicates of the nitrosamines solutions at low, medium and high levels (1.0, 5.0 and 10.0 µg L−1) during three days were used for evaluation of the method precision indices such as repeatability (intra-day) and intermediate (inter-day) and reproducibility (Intra/inter-day) [32,33]. The average of repeatability relative standard deviation (RSD), intermediate RSD and reproducibility RSD were 6.3%, 4.2%, and 7.5%, respectively.

Stability
The variation in bias over time is stability. A stable measurement process is in statistical control with respect to time.
Stability is the total change in the measurements obtained with a measurement system on the same or similar quality control sample when a single parameter is examined over a long time period.
Based on the obtained results, drift was %10.

Consistency
The degree of repeatability variation over time is consis-

Real sample
A homogeneous sausage sample was prepared by Elka company contained 120 mg kg -1 sodium nitrite and 240 mg kg -1 ascorbic acid. Determinations were carried out on this batch and ascorbic acid and sodium nitrite effect on nitrosamine formation were examined by the use of this sample.
Ascorbic acid, sodium nitrite, and nitrosamines were determined in raw, freeze and fried sausage in various time intervals, each seven days once, during 2 months.

Ascorbic acid determination
About 5 g sample was transferred to a beaker. Extraction was carried out by a 20 ml phosphoric acid (1.7%). The mixture was transferred to a 50 ml flask and diluted to volume by extraction solvent. Then the mixture was centrifuged and filtered by filter syringe and injected to HPLC. Potassium dihydrogen phosphate, pH = 2.9, was used as a mobile phase.

Nitrite determination
A test portion with hot water was extracted, the proteins were precipitated and filtered. By the addition of sulphanilamide and N-1-naphthyl ethylenediamine dihydrochloride to the filtrate, in the presence of nitrite, red color was developed and photometric measurement was carried out at a wavelength of 538 nm.

Nitrosamines
Extraction and preconcentration procedure: Sample was mixed by grinding in a mixer and homogenized. Then 50g ± 0.01g sample was transferred to a round-bottom flask and 100 ml water was added. The mixture was mixed and shaked for 30 min by shaker and then filtered in a 100 ml flask. 2 g sodium sulfate was added and was mixed until was dissolved. After that, 10 ml hexane was transferred and shaked completely. Afterward top phase (organic layer) was transferred to a rotary evaporator flask and the solvent was evaporated entirely. Flask was washed with 1 ml ethanol and was transferred to a vial for GC injection.  The proposed method, relatively, simple, sensitive, fast and low-cost with short analysis time, good repeatability, reproducibility, and recovery for the nitrosamines determination in sausage relative to the previously suggested methods. Therefore this method could readily be used for routine nitrosamines analysis.

Conclusions
In this work, GC-FID was used for the development of a new accurate, novel and inexpensive method for the determination of nitrosamines in sausage. The efficiency was improved by changing the type and volume of extraction solvent, nitrosamine solvent, and salt addition effect. In this regard, nitrosamines detection at 0.4μg Kg −1 levels becomes possible. Therefore, this method could be utilized for quantifying ultra-trace residues of nitrosamines in sausage with good accuracy.
The quantity of N-nitrosamines in raw and fried sausage considered and the trend of nitrosamines concentration at the presence of sodium nitrite and ascorbic acid monitored. The

Method
Quantitation limit (µg  nitrosamines (NDMA, NDEA, and NDBA)amount in the fried sausage was higher than raw sausage and there was a significant difference between them (P < 0.05) and during the time, nitrosamines quantity increased both in raw and fried sausage. The other nitrosamines(NMEA, NDPA, NDPhA, NPIP, NPYR, NMOR) were negligible or were not found in sausage samples. Nitrite and ascorbic acid levels in raw and fried sausage samples decreased during the time and the rate of reduction was more significant in fried ones. Reduction in ascorbic acid amount was more significant than a reduction in nitrite amount, that this fact shows some of the ascorbic acids is used for preventing nitrite participation in nitrosamine formation.
Therefore, amines react with nitrites that exist in sausage and nitrosamines formation occurs by thermal processes.
All trends have been presented in figure 1.
In this research, the amount of nitrite, ascorbic acid, and nine nitrosamines was measured in sausage samples during the time. The concentration range of the nine nitrosamines in the monitored samples was from 2.6 to 17.3 μg kg -1 .