An improved mass spectrometry-based measurement of NO metabolites in biological fluids

doi:10.1016/j.freeradbiomed.2012.12.002

Free Radical Biology and Medicine

Volume 56, March 2013, Pages 1-8

https://doi.org/10.1016/j.freeradbiomed.2012.12.002 Get rights and content

Abstract

Assessment of NO metabolism in vivo relies on the accurate measurement of its metabolites nitrite (NO₂⁻), nitrate (NO₃⁻), and nitrosothiols (RSNOs) in biological fluids. We report a sensitive method to simultaneously determine NO₂⁻ and NO₃⁻ in biological matrixes. Tetraoctylammonium was used to catalyze the complete conversion of NO₂⁻ and NO₃⁻ to stable pentafluorobenzyl (PFB) derivatives directly from aqueous acetone medium before gas chromatography and negative-ion chemical ionization mass spectrometry (GC/NICI/MS). This catalyst dramatically improved the yield of PFB derivatives for NO₂⁻ (4.5 times) and NO₃⁻ (55 times) compared to noncatalyzed derivatization methods. Analysis was performed using ¹⁵N-labeled internal standards by selected-ion monitoring at m/z 46 for fragment NO₂⁻ and m/z 47 for its isotope analogue, ¹⁵NO₂⁻, and m/z 62 for NO₃⁻ and m/z 63 for ¹⁵NO₃⁻. This method allowed specific detection of both PFB derivatives over a wide dynamic range with a limit of detection below 4.5 pg for NO₂⁻ and 2.5 pg for NO₃⁻. After the specific conversion of RSNOs by HgCl₂ to NO₂⁻, this GC/NICI/MS analysis was used to measure RSNOs in plasma. A further comparison with the widely used tri-iodide chemiluminescence (I₃⁻-CL) assay indicated that the GC/MS assay validated the lower physiological RSNO and nitrite levels reported using I₃⁻-CL detection compared with values obtained using UV–photolysis methods. Plasma levels of RSNOs determined by GC/MS and I₃⁻-CL were well correlated (r = 0.8). The improved GC/MS method was successfully used to determine the changes in plasma, urinary, and salivary NO₂⁻ and NO₃⁻ as well as plasma RSNOs in humans after either a low-NO₃⁻ or a high-NO₃⁻ meal.

Graphical abstract

Highlights

► The study of NO biology requires accurate assessment of its metabolites in vivo. ► We developed an improved GC/MS method for measurements of NO₂⁻, NO₃⁻, and RSNOs. ► The results are in close agreement with those obtained using the tri-iodide assay. ► The methods were tested on NO changes in humans after a low-NO₃⁻ or high-NO₃⁻ meal. ► A technical advance was achieved in a challenging area of NO metabolism.

Section snippets

Chemicals and standards

Sodium [¹⁵N]nitrite, sodium [¹⁵N]nitrate (>98% ¹⁵N), sulfanilamide (SFA), N-ethylmaleimide (NEM), 2,3,4,5,6-pentafluorobenzyl bromide (PFB-Br), tetraoctylammonium bromide (TOA-Br), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetate (DTPA), albumin, cysteine, potassium iodide (KI), iodine (I₂), isooctane, pyridine, and toluene were purchased from Sigma–Aldrich (St. Louis, MO, USA). Mercuric chloride (HgCl₂; Sigma) is very toxic if swallowed or upon extended exposure to the

Chromatographic separation and mass spectrometric characteristics

The chromatographic separation and the mass spectra of volatile PFB–NO₂ and PFB–NO₃ derivatives are shown in Fig. 1. PFB derivatization allowed baseline separation of NO₂⁻ and NO₃⁻ with m/z 46 for fragment NO₂⁻ and m/z 47 for its isotope analogue, ¹⁵NO₂⁻, and m/z 62 for NO₃⁻ and m/z 63 for ¹⁵NO₃⁻ (Fig. 1, top). The full-scan MS spectrum indicates that the two peaks at m/z 46 and m/z 62 correspond to the loss of PFB radical from the molecular ions of PFB–NO₂ and PFB–NO₃, respectively (Fig. 1,

Discussion

The analysis of NO₂⁻ and NO₃⁻ in biological samples is associated with analytical difficulties. A significant advance in this field was achieved by the simultaneous measurement of both NO₂⁻ and NO₃⁻ as their PFB–NO₂ and PFB–ONO₂ derivatives using GC/MS [13]. This avoids the need for chemical reduction conversion of NO₃⁻ to NO₂⁻ required by previous assays. However, the formation of PFB–NO₃ was incomplete and was found to proceed considerably more slowly compared to PFB–NO₂, making the

Conclusions

This work demonstrates an improved GC/MS method for the simultaneous analysis of NO₂⁻ and NO₃⁻ in biological fluids. NO₂⁻ and NO₃⁻ anions were completely converted into PFB derivatives via a convenient TOA-catalyzed reaction and were specifically detected by GC/MS. The further combination of the specific conversion of RSNOs by HgCl₂ to NO₂⁻ and GC/MS analysis made it adaptable to determining RSNOs at physiological plasma concentration.

Acknowledgments

We greatly appreciate the support of a grant from the National Health and Medical Research Council of Australia. Xingbin Yang was supported by the National Natural Science Foundation of China (C31171678). We acknowledge Mr. Claude Backory for nursing assistance.

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Gas chromatography (GC) methods for the determination of inorganic anions and structurally related compounds are reviewed. In their native form, such analytes are polar and non-volatile, therefore they require derivatization before GC analysis. Several chemistries have been employed to convert anions to volatile molecules with applications to a wide set of analytes: nitrite, nitrate, halides, azide, bromate, iodate, borate, carbonate, thiocyanate, cyanide, sulfide, silicates, phosphates, phosphonates, selenite, selenate, arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid have been measured following GC separation.
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Plasma nitrate and nitrite concentrations increased with the HN treatment in comparison to the LN and control treatments (P < 0.001). Plasma carotenoids increased with the HN and LN treatments compared with the control (P < 0.01). HN treatment did not reduce systolic blood pressure [24-h ambulatory—HN: 127.4 ± 1.1 mm Hg; LN: 128.6 ± 1.1 mm Hg; control: 126.2 ± 1.1 mm Hg (P = 0.20); home—HN: 127.4 ± 0.7 mm Hg; LN: 128.7 ± 0.7 mm Hg; control: 128.3 ± 0.7 mm Hg (P = 0.36); clinic—HN: 128.4 ± 1.3 mm Hg; LN: 130.3 ± 1.3 mm Hg; control: 129.8 ± 1.3 mm Hg (P = 0.49)] or diastolic blood pressure compared with LN and control treatments (P > 0.05) after adjustment for pretreatment values, treatment period, and treatment order. Similarly, no differences were observed between treatments for arterial stiffness measures (P > 0.05).
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Original ContributionAn improved mass spectrometry-based measurement of NO metabolites in biological fluids

Abstract

Graphical abstract

Highlights

Section snippets

Chemicals and standards

Chromatographic separation and mass spectrometric characteristics

Discussion

Conclusions

Acknowledgments

J. Am. Coll. Cardiol.

Blood

Blood

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

J. Chromatogr. B

J. Biol. Chem.

Free Radic. Biol. Med.

J. Chromatogr. B

Free Radic. Biol. Med.

J. Am. Coll. Cardiol.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Free Radic. Biol. Med.

Free Radic. Biol. Med

J. Chromatogr. B

J. Chromatogr. B

J. Chromatogr. A

Original Contribution
An improved mass spectrometry-based measurement of NO metabolites in biological fluids