Nitric oxide (NO) is a cell-signaling agent in physiologic processes including vasodilation, inhibition of clotting, and inflammation. A sensitive chromatography-mass spectrometry method was developed to measure two of its metabolites, nitrite$\left({{\mathrm{NO}}^{-}}_2\right)$and nitrat$\left({\mathrm{NO}}_3\right)$,in biological fluids. Internal standards,${\mathbf{NO}}^{\mathbf{-}}{\mathbf{15}}_2$and${}^{\mathbf{15}}{\mathbf{NO}}^{\mathbf{-}}\mathbf{3}$, were added to the fluid at concentrations of 80.0 and, respectively.Natural${}^{\mathbf{14}}{{\mathbf{NO}}^{\mathbf{-}}}_{\mathbf{2}}$and${}^{\mathbf{14}}{{\mathbf{NO}}^{\mathbf{-}}}_{\mathbf{3}}$plus the internal standards were then converted to volatile derivatives:

Because biological fluids are so complex, the derivatives were first isolated by high-performance liquid chromatography. For quantitative analysis, liquid chromatography peaks corresponding to the two products were injected into a gas chromatograph, ionized by negative ion chemical ionization (giving major peaks for${{\mathbf{NO}}^{\mathbf{-}}}_{\mathbf{2}}$and${{\mathbf{NO}}_{\mathbf{3}}}^{\mathbf{-}}$and the products measured by selected ion monitoring. Results are shown in the figure on the next page. If the internal standards undergo the same reactions and same separations at the same rate as the${}^{\mathbf{14}}\mathbf{N}$analytes, then the concentrations of analytes are simply$\mathbf{\left[}\mathbf{14}{{\mathbf{NO}}_{\mathbf{x}}}^{\mathbf{-}}\mathbf{\right]}\mathbf{=}\mathbf{\left[}\mathbf{15}\mathbf{N}{{\mathbf{O}}_{\mathbf{x}}}^{\mathbf{-}}\mathbf{\right]}\mathbf{(}\mathbf{R}\mathbf{-}{\mathbf{R}}_{\mathbf{blank}}\mathbf{)}$

where R is the measured peak area ratio$(m/z46/47\mathrm{for}\mathrm{nitrite}\mathrm{and}m/z62/63\mathrm{for}\mathrm{nitrite})$for nitrite andfor nitrate) and${\mathbf{R}}_{\mathbf{blank}}$is the measured ratio of peak areas in a blank prepared from the same buffers and reagents with no added nitrite or nitrate. The ratios of peak areas areand. The ratios for the blank were$\mathbf{m}\mathbf{/}\mathbf{z}\mathbf{46}\mathbf{/}\mathbf{47}\mathbf{=}\mathbf{0}\mathbf{.}\mathbf{062}$and$\mathbf{m}\mathbf{/}\mathbf{z}\mathbf{62}\mathbf{/}\mathbf{63}\mathbf{=}\mathbf{0}\mathbf{.}\mathbf{058}$. Find the concentrations of nitrite and nitrate in the urine.

Step by step solution

01

Definition of Nitrate and nitrite

Nitrate [NO3-] and nitrite [NO2-] are ions that occur naturally in the nitrogen cycle. Inorganic fertilisers contain nitrate, while sodium nitrite is employed as a food preservative, particularly in cured meats.

02

Determine the concentrations of nitrite and nitrate in the urine.

Nitrate and Nitrite concentrations in urine must be estimated.

Calculate the nitrate and nitrite concentrations in urine

Blanks ratio =0.040 and 0.058, respectively.

The formula below can be used to compute the concentration of the analytes.

$\left[{}^{14}N{O_x}^{-}\right]=\left[{}^{15}N{O^{-}}_x\right]\left(R-R_{blank}\right)$

For Nitrite:

$$\begin{gathered} \left[{}^{14}\mathrm{N}{{\mathrm{O}}_2}^{-}\right]=\left[{}^{15}\mathrm{N}{{\mathrm{O}}^{-}}_{\mathrm{x}}\right]\left(\mathrm{R}-{\mathrm{R}}_{\mathrm{blank}}\right) \\ \left[{}^{14}\mathrm{N}{{\mathrm{O}}_2}^{-}\right]=\left[80.0\mathrm{\mu M}\right]\left(0.062-0.040\right) \\ \left[{}^{14}\mathrm{N}{{\mathrm{O}}_2}^{-}\right]=1.8\mathrm{\mu M} \end{gathered}$$

Nitrite concentration is a measurement of how much nitrite is present in$=1.8\mathrm{\mu M}$

For Nitrate:

$$\begin{gathered} \left[{}^{14}\mathrm{N}{{\mathrm{O}}_3}^{-}\right]=\left[{}^{15}\mathrm{N}{{\mathrm{O}}^{-}}_3\right]\left(\mathrm{R}-{\mathrm{R}}_{\mathrm{blank}}\right) \\ \left[{}^{14}\mathrm{N}{{\mathrm{O}}_3}^{-}\right]=\left[800.0\mathrm{\mu M}\right]\left(0.538-0.058\right) \\ \left[{}^{14}\mathrm{N}{{\mathrm{O}}_2}^{-}\right]=384\mathrm{\mu M} \end{gathered}$$

The amount of nitrate in the body$=384\mathrm{\mu M}$

Nitrate and Nitrite concentrations in urine were measured $384\mathrm{\mu M}$and $1.8\mu \mathrm{M}$respectively

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