Screening freshness of seafood by measuring trimethylamine (TMA) levels using helium-plasma ionization mass spectrometry (HePI-MS)
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Trimethylamine (TMA) is a marker used for monitoring the quality of seafood because it is the primary component of the “fishy” odor.
The levels of TMA in seafood samples were directly measured by helium-plasma ionization mass spectrometry (HePI-MS). Each sample was directly exposed to the HePI source, and the intensity of the m/z 60 signal for protonated TMA was monitored by a selected-ion-recording (SIR) protocol. Using a set of TMA-spiked water standards, the TMA levels in seafood samples were quantified.
The signal intensity of the m/z 60 ion from shrimp samples maintained at room temperature for 2 days can be attenuated to baseline levels by adding lime juice. The amounts of TMA in samples of salmon and shrimp recovered from some sushi preparations, and in squid samples, were found to be 0.24 μg, 0.16 μg, and 17.2 μg per gram, respectively.
HePI-MS is an efficient technique to screen and monitor the TMA content and assess the quality of seafood.
KeywordsTrimethylamine (TMA) Helium-plasma ionization HePI Ambient mass spectrometry Seafood quality Fish odor Screening freshness of seafood
Helium-plasma ionization mass spectrometry
Fresh seafood is highly perishable and therefore requires utmost care during processing, transportation, and storage in order to prevent decomposition. Approximately 200 million metric tons of seafood is directly consumed by people globally per annum as reported by the UN Food and Agricultural Organization. Despite the high perishability, many connoisseurs prefer consuming seafood preparations raw or only lightly preserved. As a result, the market has recently seen an increasing demand for fresh seafood. Thus, in order to meet consumer demands and comply with legislative regulations, a quality assessment performed on the product before it is offered to the consumers is of paramount importance. In seafood, biogenic amines are formed upon storage thorough enzymatic and microbial action on amino-acids. Among the biogenic amines, trimethylamine (TMA) is the primary component that imparts the fishy odor (Bedia Erim 2013). Thus, TMA is commonly used as a marker to qualitatively and semi-quantitatively detect the spoilage of fish (Oetjen and Karl 1999; Pedrosa-Menabrito and Regenstein 1990; Timm and Jørgensen 2002). TMA is produced by the oxidation of choline by bacteria in marine animals by TMA-lyase. TMA also accumulates by the reduction of trimethylamine N-oxide (TMAO) by the enzyme TMAO reductase in the tissues of decaying marine animals. TMA is toxic to humans: it is oxidized in the liver to form TMAO (Seibel and Walsh 2002), which has been recognized as an agent that causes cardiovascular disease (Landfald et al. 2017).
Several sensory- and instrument-based techniques are available to monitor the quality of seafood. Most of these methods rely on the detection of TMA, which is one of the main compounds responsible for the malodor of poor-quality seafood (Oetjen and Karl 1999; Timm and Jørgensen 2002). The correlation between extracted TMA and the age and quality of seafood has been well-demonstrated (Malle et al. 1996; Malle and Tao 1987; Oetjen and Karl 1999; Romero-González et al. 2012; Timm and Jørgensen 2002).
More elaborate instrumental methods have evolved through the years. At present, gas chromatography (Namieśnik et al. 2003; Shim and Baek 2012), solid-phase micro-extraction (Chan et al. 2016; Shim and Baek 2012), or solvent extraction (daCosta et al. 1990; Oetjen and Karl 1999), ion mobility (Bota and Harrington 2006; Cheng et al. 2017), nuclear magnetic resonance spectroscopy (Podadera et al. 2005), ion chromatography (Erupe et al. 2010; Li et al. 2009), capillary electrophoresis (Li and Lee 2007; Timm and Jørgensen 2002), high-resolution rotational terahertz (THz) spectroscopy (Hindle et al. 2018), and high-performance liquid chromatography methods (Cháfer-Pericás et al. 2004; Hyötyläinen et al. 2001; Romero-González et al. 2012) are the most widely adopted techniques to measure the amounts of primary and secondary amines in various matrices. The major advantages of these chromatographic techniques are higher sensitivity, specificity, and ability to determine several substances simultaneously.
A drawback of many traditional analytical techniques employed to determine the quality of seafood is the time-consuming and laborious TMA extraction step, and the difficulty in handling low-molecular-mass amines due to their high water solubility and volatility. The technique we have developed—based on ambient-pressure helium-plasma ionization mass spectrometry (HePI-MS)—does not require the TMA extraction step or chromatographic separation.
In this study, we employed ambient-pressure HePI-MS (Yang and Attygalle 2011) to screen the freshness of seafood, because the technique affords a direct measurement of TMA levels in samples, without the need to resort to chemical extraction, or any other prior sample preparation. HePI is a versatile ambient-ionization MS technique, applicable to the analysis of a wide variety of samples, both organic and inorganic. It has been applied, for example, to the analysis of energetic materials (Yang et al. 2012), pharmaceutical preparations (Attygalle et al. 2014a, 2014b, Xia et al. 2016), halobenzenes (Attygalle et al. 2014a, 2014b; Gangam et al. 2015), phenolics and quinones (Hassan et al. 2017), inorganic nitrates (Pavlov and Attygalle 2013), and inorganic mercury compounds (Weerasinghe et al. 2014). A major advantage of HePI is that it is highly portable and adaptable: any mass spectrometer with an electrospray ion source can be transformed into an ambient HePI instrument with ease, and no extensive hardware modifications are necessary (see Experimental Section). In addition, unlike other helium-mediated sources such as Direct Analysis in Real Time (DART) and Flowing Atmospheric-Pressure Afterglow (FAPA), HePI is extremely economical in its helium consumption. Another important feature of HePI is that if a sample is sufficiently volatile or can be volatilized, it can be detected without any significant interference from the sample matrix. In this study, we investigated the capabilities of HePI to measure TMA levels in several seafood samples from various specimens of fish at different time points of storage at room temperature (0 to 96 h). Additionally, the reduction of the amount of free TMA by the addition of lime juice, similar to the action of many other common acidic seafood condiments (e.g., lemon juice and tartar sauce), was demonstrated.
Materials and sample preparation
High-purity helium (99.999%, Airgas, Radnor, PA) was used for all experiments. Trimethylamine was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Concentrated hydrochloric acid and NaHCO3 were purchased from Fisher Scientific (Hampton, NH). Lime juice (ReaLime® 100%) and samples of seafood [cod, char, salmon, and squid (mantle only, not the tentacles)], displayed on ice, were purchased from a local store (Wegmans, Woodbridge, NJ) and transported to the laboratory on a bed of ice. Similarly, samples of shrimp- and salmon-sushi, and fresh squid samples were purchased from the same store, and a sample of fresh shrimp was obtained from another local store (99 Ranch Market, Jersey City, NJ). From each seafood, eighteen representative samples (25 mm × 3 mm x 3 mm; 0.8 g each) were separated and placed in Eppendorf tubes (2.0 mL), which were kept at room temperature with lids tightly closed. For shrimp, the representative samples were cut from the region immediately below the head, and for the sushi samples, samples were prepared only from the meat portion.
Mass spectrometric analysis
Prior to analysis, each Eppendorf tube containing a sample was opened and attached to the inner side of the ion-source glass cover. Mass spectra were acquired (m/z 25–200) at a rate of 2 scans per second, and the results were processed using MassLynx 4.0 software (Waters Corp., Milford, MA, USA). The cone voltage was held at 15 V; the source and desolvation gas temperatures were both maintained at 110 °C. After each analysis, the ion source was cleaned thoroughly by wiping it with a cotton swab soaked in methanol, and a background check was made to ensure that the m/z 60 signal intensity had returned back to the background level before a new sample was introduced.
Three samples of each seafood (cod, char, salmon, shrimp, and squid), maintained inside Eppendorf tubes at room temperature, were analyzed at 0, 6, 24, 48, 72, and 96 h. Each tube containing a sample was opened and placed immediately in the HePI source. The tube neck was positioned at a pre-determined fixed point at 1.0 cm distance from both the entrance-cone orifice and the HePI plasma flame. The amount of TMA emanating from each sample was recorded for 0.5 min by a selected-ion-recording (SIR) experiment monitoring the abundances of the m/z 60 and 61 ions (dwell time 0.1 s; inter-scan delay 0.1 s). Each sample was analyzed in duplicate by the SIR procedure. Then, the average m/z 60 peak intensity and that of m/z 61 were calculated for each 0.5 min acquisition period.
Trimethylamine standard curve
The concentration of the commercial TMA solution was determined to be 3.5 M, by titrating it against a 0.12 M standard HCl solution, using methyl orange as the indicator. The HCl solution was standardized using NaHCO3 as a primary standard.
A stock solution of TMA (1300 μg/mL) was prepared from the 3.5M TMA solution and diluted quantitatively to make a series of standards (650, 325, 162.5, 81.3, 40.6, 20.3, 10.2, 5.1, 2.5, and 1.3 μg/mL). Aliquots of each standard (200 μL) were transferred to Eppendorf tubes, and the intensity of the m/z 60 ion generated from the headspace of each sample was monitored by an SIR experiment conducted under HePI-MS conditions. A standard curve was generated by plotting the intensity of the m/z 60 ion peak in six replicates, against the amount of TMA present in each sample.
Estimating the freshness of seafood samples by the amount of TMA detected
Samples from shrimp- and salmon-sushi, as well as squid were analyzed in duplicate, immediately after they were brought to the laboratory, by an SIR experiment. The peak intensity at m/z 60 was monitored for 0.5 min for each sample (dwell time 0.1 s). Then, the average m/z 60 peak intensity was calculated for the total acquisition period of 0.5 min.
Effect of lime juice on TMA levels
After recoding positive-ion spectra (m/z 25–200) for 0.7 min, a 0.8-g sample of shrimp which had been kept at room temperature for 48 h was placed in the ion source. The volatiles emanating from the sample were monitored by recording a chronogram. After recording spectra for 1.2 min, a 500-μL aliquot of lime juice was added, using a pipette, to the shrimp sample, and spectra were acquired for another 1 min.
Results and discussion
TMA amounts found in some sushi and squid samples
Average amount (μg/g)
Squid sample (0.8 g)
A piece of salmon recovered from a sushi sample (0.8 g)
A piece of shrimp recovered from a sushi sample (0.8 g)
A major advantage of ambient-ionization mass spectrometric methods of analysis is specifically the fact that signals for analytes of interest can be elicited even from very complex samples without any measurable matrix interference—TMA is a gas. Once it emanates from a sample, it can be ionized and detected very efficiently because unlike in electrospray ionization, it does not have to be desorbed from a solution. In the samples used in this study, the matrix consists mostly of fat and protein. Unlike in electrospray or MALDI techniques, fats and proteins do not undergo ionization directly by HePI, and therefore do not interfere with TMA signals.
Masking of TMA odor with lime juice
The described method can be used to rapidly screen the quality of seafood in a high-throughput manner, due to the simplified sample preparation procedure, which does not involve the solvent extraction of the analyte. The amount of TMA present can be determined semi-quantitatively. Herein, we have demonstrated that the reducing or completely eliminating the malodor associated with decaying seafood in seafood samples, by treating seafood with lime juice or vinegar, is due to reducing the amount of free TMA.
The authors thank Ramu Errabelli, Sihang Xu, and Zhaoyu Zheng for helpful discussions.
IH and TO carried out the experiments and collected, analyzed, and interpreted the experimental results. JP and ABA supervised the work and interpreted the experimental results. All authors contributed to the manuscript drafts, and read and approved the final manuscript.
This work has not been financially supported.
The authors declare that they have no competing interests.
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