Identification of nitrite treated tuna fish meat via the determination of nitrous oxide by head space-gas chromatography/mass spectrometry

Tuna fish meat is an expensive and highly perishable sea food. Fresh meat has a bright red colour which soon turns into an unsightly brown during storage. To prolong the aspect of freshness, the red colour is stabilised or even enhanced e.g. with carbon monoxide or nitric oxide, the product of a nitrite / ascorbic acid treatment, which bind as a ligand to myoglobin. These procedures are illegal. Here we present a method for identifying tuna meat samples, which have undergone fraudulent wet salting with nitrite. The method uses headspace-gas chromatography/mass spectrometry (GC/MS) for the determination of nitrous oxide, which is formed as the final product of the two-step reduction nitrite (added agent) to nitric oxide (ligand) to nitrous oxide (target compound). Complex bound nitric oxide is set free with sulfuric acid, which also promotes the reduction to nitrous oxide. The method was validated using 15N labelled nitrite as well as treated and untreated reference fish samples. A survey of 13 samples taken from the Swiss market in 2019 showed that 45 % of all samples were illegally treated with nitrite.

As Tuna fish species have been overfished on the one hand, and their meat being a sought-after but highly perishable food stuff on the other hand, the seafood industry has felt itself constrained to undertake efforts in increasing the shelf life of this commodity. Being a bright red in its fresh state, but turning into an unsightly brown during storage, attempts have also been made in extending or even enhancing the attractive aspect of fresh meat. The red colour of meat in its natural state is due to myoglobin in its reduced form (Mb-Fe +II -O 2 ). Being susceptible to autoxidation, the resulting metmyoglobin (Mb-Fe +III -O 2 ) is brown in colour. One way of stabilising the red colour is by gassing with carbon monoxide (CO), which binds as a ligand to myoglobin (Mb-Fe +II -CO). A second way consists in applying the anion nitrite (NO 2 -) in combination with the reducing agent ascorbic acid to form nitric oxide (NO), which forms a complex with both myoglobin (Mb-Fe +II -NO) and metmyoglobin (Mb-Fe +III -NO) 1 . Whereas the colour hue of the CO complex is described as cherry red, that of the NO-complexes are described as pink red. Neither procedure is authorised for fresh fish 2 . However, with these possibilities at hand, abuse for simulating the fresh aspect of tuna fish meat is a tempting option 3,4 , all the more so regarding the currently insufficient tools for fraud detection: Whereas, for CO, analytical methods are at hand to support law enforcement 5,6 , attempts to detect nitrite treated meat, either by searching for nitrite residues or traces of nitrosamines, have not been reported as being successful yet 7 . Here we report on our method for the detection of nitrite treatment (brining), which originates from the method for CO determination and determines nitrous oxide (N 2 O) as the reduction product of nitric oxide (NO), stemming from the complex Mb-Fe +II -NO ( Figure 1). This approach allows the determination of CO and N 2 O even within the same GC/MS run.

Samples and sample preparation
Tuna samples (frozen loins, tuna saku, loose and pre-prepared packages) were taken from retailers and importers in Basel and stored at -18°C until sample preparation. An aliquot of 15 g was weighed into a 70 mL centrifuge tube and 15 g of crushed ice as well as 15 mL of water were added. The mixture was homogenised at 7,000-9,000 rpm with a polytron homogeniser (Kinematica AG, Switzerland) and centrifuged for 5 min at 3,500 rpm. An aliquot of the resulting supernatant (5 mL) was transferred into a 20 mL headspace vial. After adding 5 µL of n-octanol and 2 mL of sulphuric acid (20 % in deionised water) the vial was immediately sealed and gently shaken by hand for 30 s. Certified NO-gas 100 ppmv (122.6 ng/mL) and N 2 O-gas 100 ppmv in N 2 (180.6 ng/mL) served as standards for a one-point calibration: A headspace vial was filled with 5 mL of deionised water and 2 mL of sulphuric acid (20 %), the cap only loosely fitted and the reference gas introduced with a needle through the septum. The cap was then sealed tight.

Amendments from Version 1
Implementing reviewer comments we now indicate, that the method also allows for the simultaneous determination of CO and N 2 O in the same GC run. We further specified the type of calibration used (one-point) and the limit of detection for N 2 O.

REVISED
Headspace -gas chromatography/mass spectrometry (GC/MS) The sealed vials were placed into the incubation unit of the CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland) for 60 min at 25°C and analysed with a Trace 1310 GC /

Brining experiments
For brining experiments, 15 g of nine untreated fish samples (certified as 100 % tuna from the Maldives, the Philippines, China, Vietnam) were each cut into pieces of about 1 cm and treated with 20 mL of brine solution (10% NaCl and 0.4% NaNO 2 in deionised water) 8 at room temperature for 24 hours. After removing the solution, the now reddish looking tuna samples along with eight untreated control samples were each bathed twice in 15 mL of antioxidant solution (3.0 % ascorbic acid and sodium ascorbate in deionised water) for one hour at room temperature and subsequently washed twice with antioxidant solution. Thereafter, samples were prepared as described above and analysed by headspace-GC/MS. Verification experiment: The whole procedure was also done with three tuna samples using Na 15 NO 2 for the brine solution instead.

Method development
In contrast to carbon monoxide, where the agent used for treatment, the ligand responsible for colour stabilisation and the target compound for sample screening is one and the same molecule, nitrite treatment identification has to consider the two-step reduction from nitrite to nitric oxide to nitrous oxide. This required changing the original GC temperature programme of the CO method: Instead of an isothermic run at 40°C, oven temperature was ramped up from 70°C to 220°C.
Both carbon monoxide and nitric oxide are bound in the sample as a ligand. Their release is enhanced by adding sulfuric acid. Being a free radical, however, nitric oxide is far more reactive than carbon monoxide: Instead of a sharp symmetrical peak, chromatograms show nitric oxide as a broad hump, barely distinguishable from baseline noise. The direct approach therefore is not feasible. In an acidic environment, however, two NO molecules, upon reduction, react to nitrous oxide (N 2 O), which shows up as a perfect peak in the chromatogram (Figure 2), allowing for the indirect detection of NO. For the purpose of identifying nitrite treatment, a semi quantitative assessment of N 2 O proved to be sufficient, as any clear signal is alone due to a former nitrite treatment.

Brining experiments
Verification of this mechanism was performed using isotope labelled nitrite (Na 15 NO 2 ) for brining ( Figure 1). During subsequent analysis, both the 15 NO hump and 15 N 2 O peak were detected (Figure 2), which is consistent with the postulated reaction pathway. In addition, brining experiments with nine tuna samples using unlabelled NaNO 2 showed sharp N 2 O signals in the range of 1000 to 12000 µg/kg (Figure 3, underlying  data 9 ). In contrast, untreated reference samples, even though they were exposed to antioxidants, were free of nitrous oxide (limit of detection about 30 µg/kg). Therefore, naturally occurring relevant concentrations of N 2 O can be excluded.

Validation with reference samples
The method was validated with seven reference tuna samples of a trustworthy source from 2017 which were stored at -18°C for two years in our lab (see underlying data 10 ). Five of them were declared as untreated and consists of 100% tuna. The remaining two samples were declared as treated with rosemary extract (E392) and/or nitrite and labelled with additives such as salt and antioxidants (E300, E301, E331). The untreated samples were free of N 2 O (< 30 µg/kg) whereas the treated ones showed stable N 2 O signals of 260 and 750 µg/kg. These results not only confirm our findings from the brining experiments, but are also consistent with the corresponding documents of the reference tuna samples.

Market survey
The method was then used on a routine basis in a market survey of 13 samples of raw tuna collected in Basel in 2019 (see underlying data 11 ). Seven samples (54%) were negative. Six samples (46%) were tested positive with N 2 O peaks corresponding to levels of between 90 and 1130 µg/kg. As a consequence, these six samples, all originating from Vietnam, were objected according to law.

Conclusion
With our experiments using labelled Na 15 NO 2 and the comparison of our results with the corresponding documents of the reference tuna samples, we could demonstrate that our method using nitrous oxide as a target compound is suitable for identifying nitrite manipulated tuna. Its use in a market survey also showed, that the method is fit for purpose. In addition, the method was even capable of identifying nitrite treatment in samples stored at -18°C for two years before analysis.

Data availability
As a law enforcement body, we are bound to official secrecy. Considering this restriction, names of products and importers were anonymised.

Grant information
The author(s) declared that no grants were involved in supporting this work. 1.

Are sufficient details provided to allow replication of the method development and its use by others? Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: food and FCM analysis, GC-MS, on-line LC-GC, comprehensive two-dimensional GCxGC I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. © 2019 Busch U. This is an open access peer review report distributed under the terms of the Creative Commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.

Ulrich Busch
Bavarian Health and Food Safety Authority, Oberschleißheim, Germany