Analysis of 10 nucleotides and related compounds in Litopenaeus vannamei during chilled storage by HPLC-DAD

https://doi.org/10.1016/j.lwt.2015.11.047Get rights and content

Highlights

  • An HPLC-DAD method for analyzing 10 nucleotides and related compounds in L. vannamei.

  • Column, pH and mobile phase were optimized and method was validated.

  • Changes of NRCs in L. vannamei when stored at 4 °C were related to its flavor/freshness.

Abstract

An HPLC-DAD method for the analysis of 10 nucleotides and related compounds (NRCs) in Litopenaeus vannamei was established. Hyperchloric acid was used to precipitate proteins. Samples were separated on an ODS-3 C18 (4.6 mm × 250 mm, 5 μm) column with 0.02 M phosphate buffer (pH = 5.8) as mobile phase A. All 10 NRCs were completely separated within 25 min. Good linearity was seen in the concentration range from 0 to 500 μmol/L with correlation coefficients between 0.9996 and 0.9999. Limit of detection was between 0.02 and 0.10 μg/mL and spike recoveries were between 72.5% and 110%. This method is facile, reliable, and accurate for the analysis of 10 NRCs in shrimp muscle samples. Using this method, the changes of NRCs in L. vannamei were studied during chilled storage at 4 °C. Important correlations between the changes of NRCs and the flavor/freshness of L. vannamei were proposed.

Introduction

The Pacific white shrimp, Litopenaeus vannamei, is native to the Eastern Pacific coast through central Gulf of Mexico. It is one of the most commercially farmed species worldwide, accounting for over 70% of all farmed shrimp. Due to its high protein content, low fat content and delicate texture, L. vannamei is gaining popularity among consumers (Qian et al., 2013, Xie et al., 2012).

Nucleotides and related compounds (NRCs) are a group of compounds composed of a nitrogen-containing unit (purine, pyrimidine, nicotinamide or others) linked to a sugar and a phosphate group. Examples include adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosinic acid (IMP), inosine (HxR), hypoxanthine (Hx), guanosine monophosphate (GMP), adenosine (Ado), adenine (Ad), xanthine (Xt), etc. NRCs are important substances in the muscles of aquatic animals such as shrimp. Close relationship has been found between the flavor and freshness of aquatic foods and NRCs. In terms of flavor, AMP can suppress bitterness and produce pleasant sweetness and saltiness, acting as a good flavor enhancer in aquatic foods. Hx can interact with certain amino acids and peptides resulting in bitterness (Tikk et al., 2006). IMP and GMP, the major flavor enhancer nucleotides in aquatic foodstuff, can be used with monosodium glutamate (MSG), producing stronger flavor enhancing effect than MSG alone. They have been widely used as new flavor enhancers in all types of food (Aristoy and Toldra, 2009, Matoba et al., 1988). As for freshness, it is commonly accepted that the ATP in fish muscle sequentially degrades after death to ADP, AMP, IMP, HxR, and Hx. Hx is subsequently oxidized to Xt (Venugopal, 2002). K value, a freshness indicator, is defined as the ratio of the sum of HxR and Hx to that of ATP, ADP, AMP, IMP, HxR, and Hx. K value has proven to be an important chemical indicator for fish freshness according to freshness studies of horse mackerel, turbot, and yellowfin tuna during chilled storage (Nejib et al., 2005, Santiago et al., 2005, Vanesa et al., 2005). K value has been widely used for the evaluation of fish quality (Kuda et al., 2007, Mendes et al., 2001). Recent studies showed that the degradation pathway of ATP in certain ocean invertebrates was different from that in fish. The ATP degradation pathway in the muscle tissues of clams and scallops was found to follow ATP→ADP→AMP→Ado→HxR→Hx (Arai and Saito, 1961, Hiltz and Dyer, 2011). There was no IMP intermediate in between AMP and HxR. In the muscle tissues of ark clam, mogai, and abalone, this process followed ATP→ADP→AMP→Ado→Ad (Arai and Saito, 1961, Iwamoto et al., 1991, Saito et al., 1958, Yoneda et al., 2002). On the other hand, two ATP degradation pathways were proposed in the muscle tissues of Dungeness crab and kuruma prawn, i.e. ATP→ADP→AMP→IMP→HxR→Hx and ATP→ADP→AMP→Ado→HxR→Hx (Groniger and Brandt, 1969, Shirai and Kikuchi, 2002). Therefore, it remains inconclusive whether K value can be used as a freshness indicator in invertebrates. Regardless, fast and accurate determination of NRCs in L. vannamei can provide preliminary identification of the ATP degradation pathway in postmortem muscles, which is of great significance for the flavor and freshness of aquatic foodstuff.

Current methods for the analysis of NRCs include enzymatic method (Angel & Fermin, 1981), ion exchange chromatography (Gao et al., 2006, Iwamoto et al., 1987), capillary electrophoresis (Kaname et al., 2005, Tsoda, 1983), reverse phase liquid chromatography (Bhatt et al., 2012, Graven et al., 2014, Isabel et al., 2001, Özogul et al., 2000, Wang et al., 2007; ), sensor method (Devi et al., 2013, Devi et al., 2011), etc. Reverse phase HPLC has the advantages of high speed and simplicity, which render it the principal method currently used in biological and food sciences (Jose, Barat, Eduardo, et al., 2008). However, for the analysis of ionic species, ion pairing is usually required. Ion pair reagents are pricy and may contaminate the column, shortening the lifetime of the column. Moreover, many methods have limitations on the type of analytes and some require long analysis time. Particularly, no fast and facile HPLC method is available for the analysis of 10 NRCs in L. vannamei. In this study, a high performance liquid chromatography coupled with diode array detection (HPLC-DAD) method for the analysis of 10 NRCs in L. vannamei was established. Validation of the method was performed and good results were obtained. The concentration change of the 10 target NRCs during chilled storage (4 °C) was analyzed.

Section snippets

Instruments and materials

Live shrimp (L. vannamei) with a body weight of 10–15 g were purchased from Luchaogang Market of Shanghai and put in an oxygenated sampling vessel, which was quickly taken back to the lab. Upon arrival, the shrimp were washed with ice water and blotted dry. They were stored at 4 ± 1 °C before further processing.

HPLC analysis was performed on a Waters Alliance 2695 HPLC system equipped with a Waters 2996 photodiode array (PDA) detector and Empower 2 software package. A Shimadzu Kubota 520

Optimization of chromatographic conditions

Separation of the 10 NRCs was optimized in terms of peak shape, degree of separation, and analysis efficiency. Optimal, column, pH and composition of the mobile phase were selected.

Conclusion

In this study, an HPLC-DAD method for the analysis of 10 NRCs in L. vannamei was developed and evaluated. The target 10 NRCs were ATP, ADP, AMP, IMP, HxR, Hx, GMP, Ado, Ad, and Xt. This new method is faster, more efficient, more sensitive, and has better separation power than current methods. It meets the requirements for precision, repeatability, linearity, limit of detection and spike recovery. This method is effective in determining NRCs and deciphering the ATP degradation pathway in L.

Acknowledgments

This work was supported in part by National “Twelfth Five-Year” Plan for Science & Technology Support (Grant No: 2012BAD29B06), Shanghai Science and Technology Key Project on Agriculture from Shanghai Municipal Agricultural Commission (Grant No: (2013)3rd-4).

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