Ion chromatographic method for simultaneous determination of nitrate and nitrite in human saliva

doi:10.1016/S0378-4347(00)00264-4

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 744, Issue 2, 21 July 2000, Pages 433-437

https://doi.org/10.1016/S0378-4347(00)00264-4 Get rights and content

Abstract

A simple, rapid, accurate and sensitive method is proposed for the simultaneous determination of nitrite and nitrate in human saliva. Nitrite and nitrate present in the human saliva were determined after 10- to 100-fold dilution with ion chromatography (IC) using suppressed conductivity detection. Recoveries of nitrite and nitrate were found to be ranged between 95% and 101%. The method was linear (r²=0.9991) over the concentration working range. The detection limits were found to be 15.0 μg/l and 33.5 μg/l, for nitrite and nitrate, respectively. Ions that are present in human saliva and several other ions that are suspected to affect nitrite and nitrate determination were checked. It was found that most of the ions did not cause any interference in the determination. The method allows simultaneous determination of nitrite and nitrate in human saliva.

Introduction

Extensive interest has been attained in determining nitrite and nitrate in biological materials. Nitrite and nitrate are generally regarded as hazardous compounds, because they are highly toxic to humans and especially to infants, which usually results in methemoglobinemia and possibly death [1], [2]. Nitrite has the possibility to react with secondary amines and amides and form various carcinogenic N-nitroso compounds [3].

Salivary nitrite is produced due to microbial reduction of nitrate in the oral cavity. Therefore, ingested nitrite through foods and salivary nitrite can react in vivo with various amines and amides in the food in the acidic environment of human stomach or other parts of the body to form N-nitroso compounds [3]. This reason suggests the need for accurate control and monitoring of nitrite and nitrate level in human saliva, Many researchers have studied the effect of various nitrate-rich diets on salivary nitrite levels and ultimately on vivo N-nitrosation [3].

Several methods have been reported for the determination of nitrite and nitrate in foods and biological materials [4], [5], [6], [7], [8], these methods include high-performance liquid chromatography (HPLC) or flow injection with chemiluminescence detection [4], gas chromatography (GC) [5], [6], HPLC using a micellar mobile phase [7], HPLC by reversed-phase ion pair liquid chromatography [8] and spectrophotometric methods [9], [10], [11]. Where a spectrophotometric method is widely used, it is based on a diazotization of various aromatic amines with nitrite in acidic medium and on a subsequent coupling of the diazonium ions with N-(1-naphthyl)ethylenediamine [9], 8 quinoline [10], or resorcinol [11]. In general, spectrophotometric methods are subject to various interferences and lack of specificity. GC methods in general need a derivatization reaction and then partitioning to the organic layer prior to analysis, which is a complicated process. In liquid chromatography, nitrite is liable to be oxidized in the acidic medium during the process [12].

In the present communication we describe a selective and sensitive analytical method in which nitrite and nitrate are determined directly from human saliva using ion chromatography (IC) using an Ion Pac AS12A column, with suppressed conductivity detection. The method allows simultaneous determination of nitrite and nitrate in human saliva.

Section snippets

Instrumentation

A Dionex (Sunnyvale, CA, USA) a suppressed conductivity detection ASR-II, Auto suppression-II, recycle mode and LC25 enclosure system, was used. Chromatographic data were analyzed using a C-R6A chromatopac chart. The Ion Pac AS12A (200×4 mm) column was obtained from Dionex.

Materials

Human saliva samples were collected from different volunteers. Sodium nitrite and sodium nitrate were obtained from (Wako, Japan). Sodium carbonate and sodium hydrogencarbonate were also obtained from (Wako).

Ion chromatography

Nitrite and

Results and discussion

Fig. 1 shows the chromatogram of the standards nitrate and nitrite. Chromatograms of nitrite and nitrate for saliva samples obtained from different volunteers are shown in Fig. 2a and b. The chromatograms of of the spiked saliva with nitrite and nitrate are shown in Fig. 2c and d. The simultaneous determination of nitrite and nitrate in saliva by the Ion Pac AS12A column was performed with suppressed conductivity detection. The dissolved solution of solid saliva is centrifuged and was directly

Conclusion

The proposed analytical method for determining nitrate and nitrite in human saliva is simple, rapid and accurate. The anion-exchange column in conjunction with suppressed conductivity detection was adopted to provide to a selective determination of nitrate and nitrite from human saliva without interference from the matrix components present in the saliva. The saliva sample needs only centrifugation to remove the insoluble matter and no sample preparation or clean-up process is necessary. The

Acknowledgements

The authors are grateful to Japan Society for the promotion of Science (JSPS), Japan, for the award of a postdoctoral fellowship (P98448) and financial support. This work is partly supported by the Grant-in-Aids (10558085, 10650797) from The Ministry of Education, Science, Sports and Culture, Japan.

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Short communicationIon chromatographic method for simultaneous determination of nitrate and nitrite in human saliva

Abstract

Introduction

Section snippets

Instrumentation

Materials

Ion chromatography

Results and discussion

Conclusion

Acknowledgements

Toxicol. Appl. Pharmacol.

J. Chromatogr.

J. Chromatogr.

Anal. Chim. Acta

Talanta

Short communication
Ion chromatographic method for simultaneous determination of nitrate and nitrite in human saliva