Reliable LC/UV Analysis of Nitrite and Nitrate Using the Arc™ Premier System With an Atlantis™ Premier BEH™ C₁₈ AX Column

Nitrite and nitrate are considered as potential precursors in the formation of nitrosamine impurities, carcinogenic compounds that can cause cancer. Herein, we present a reverse phase method developed for the analysis of nitrite and nitrate using an Arc Premier System with UV detection and an Atlantis Premier BEH C₁₈ AX Column. The method is based on direct injection, eliminating the need for a derivatization step prior to analysis. The limits of quantitation (LOQ) for nitrite and nitrate were found to be 0.025 µg/mL and 0.075 µg/mL, respectively. The method demonstrated excellent repeatability, precision, and linearity and was found to be suitable for the determination of nitrite and nitrate in the polyvinylpyrrolidone (PVP) excipient with accurate quantitative performance with 91–100% recovery. 

Benefits

• Successful retention of the nitrite and nitrate ions under reverse phase conditions using an Atlantis Premier BEH C₁₈ AX Column
• Fast and easy screening method by direct injection, eliminating the need for a complex sample derivatization step prior to analysis

Nitrite and nitrate are naturally occurring nitrogen compounds in soil, water, air, and plants.¹ They are also used in food preservation, some pharmaceutical drugs, and in the production of explosives.¹ Recent publications have indicated that nitrite and nitrate are considered precursors or nitrosating agents in the formation of N-nitrosamine compounds, potential human carcinogens.^2–6 Nitrosamines are generally formed by the reaction of secondary or tertiary amines in the pharmaceutical active pharmaceutical ingredients (APIs) with the residual nitrite during the drug product manufacturing process. Nitrate is also recognized as nitrosating agent as it can be reduced to nitrite under certain conditions.⁴ Formulation ingredients, including excipients, can contain nitrosating precursors, which may contribute to nitrosamine contamination in drug products.⁴

The most widely utilized methods for nitrate and nitrite determination involve the use of ion chromatography (IC) with conductivity detection, liquid chromatography (LC) with UV detection, and the Griess test.^4–6 While effective, these methods allow the determination of a single analyte, nitrite, or nitrate, and often require different sample preparation procedures. The Griess test is specific for nitrite and cannot be used to measure nitrate content.⁴ Considering the possible conversion of nitrate to nitrite upon reduction, there is a desire to measure both compounds.

Herein, a single liquid chromatography (LC) method with UV detection is described for the analysis of both nitrite and nitrate, based on direct injection. This reverse phase method offers easy and fast screening for nitrite and nitrate in polyvinylpyrrolidone excipient.

High purity LC/MS grade acetonitrile, ammonium formate, and formic acid were purchased from Fisher Chemicals. Sodium nitrate (NaNO₃) and sodium nitrite (NaNO₂) analytical grade quality were purchased from Sigma. Polyvinylpyrrolidone (PVP) K 15 was obtained from Sigma.

Standard Solutions

Nitrite and nitrate stock solutions were prepared by dissolving sodium nitrite and sodium nitrate salts in water at 5.0 mg/mL. Stock solutions were diluted with water to make a mixture standard solution containing nitrite and nitrate at 0.1 mg/mL concentration. Calibration standards were prepared by diluting the mixture standard solution with water. 

Polyvinylpyrrolidone (PVP) Sample Solutions

Test sample solutions were prepared in water at 1 mg/mL and dissolved by sonication for 20 minutes. After extraction, sample solutions were centrifuged for ten minutes at 3000 rpm and filtered through a PVDF 0.2 µm syringe filter (p/n: WAT200806) prior to analysis.

Data acquisition and analysis performed using Empower Software. Summary reports generated using the report templates are available in the Empower project.

Various column chemistries, organic solvents, mobile phase additives, and pH were explored during the method development to ensure chromatographic retention and separation of nitrite and nitrate ions. The Atlantis Premier BEH C₁₈ AX Column successfully retained and separated nitrite and nitrate under reverse-phase conditions (Figure 1A). The replicate injections of the 0.1 µg/mL standard demonstrated excellent repeatability of the Arc Premier System for nitrite and nitrate analysis with relative standard deviation (RSD) for peak areas of 0.94 and 3.06%, respectively (Figure 1B). 

Limits of Detection and Quantitation (LOD and LOQ)

The minimum concentration levels at which nitrite and nitrate can be reliably detected and quantified using the UV method were determined using the signal-to-noise (S/N) criteria of 3:1 and 10:1, respectively. Calculation of S/N ratio was performed using Empower software by comparing the measured signal for nitrite and nitrate in the standard solution to the blank solution. Data from ten replicate injections of standard solutions was used to establish and assess performance of the method at the LOD and LOQ levels.

Representative chromatograms showing the analysis of nitrite and nitrate LOD and LOQ solutions are shown in Figure 2. The method performance at the LOD and LOQ levels is shown in Figure 3. The LOD and LOQ for nitrite was found to be 0.01 and 0.025 µg/mL, respectively. The LOD and LOQ for nitrate was at 0.05 and 0.075 µg/mL, respectively. The data from ten replicate injections at the LOQ levels demonstrated excellent repeatability of the method, with RSD of peak areas at 4.30% for nitrite and 5.52% for nitrate (Figure 3).

Linearity and Concentration Range

The calibration plot for nitrite and nitrate was constructed in the range of 0.025 to 5 µg/mL and 0.05 to 5 µg/mL, respectively. The calibration curve of the peak areas versus concentration produced a linear relationship with a correlation coefficient (R²) ≥0.999 (Figure 4). 

Polyvinylpyrrolidone Analysis

Polyvinylpyrrolidone, also known as povidone or PVP, is a synthetic polymer used in the pharmaceutical industry as a binder in tablet and capsules manufacturing, a film former for ophthalmic solutions, to aid in flavoring liquids and chewable tablets, and as an adhesive for transdermal systems.⁷

The PVP test samples, prepared at 1 mg/mL in water, were analyzed using the developed method. Representative chromatograms of the PVP sample and the PVP sample spiked with nitrite and nitrate are shown in Figure 5. The method effectively separated the highly concentrated PVP peak from the nitrite and nitrate. In the PVP test sample, no nitrite was detected, but residual levels of nitrate were found, at a concentration of 0.04 µg/mL.

Method Accuracy

The accuracy of the method was assessed by determining recovery of the nitrite and nitrate spiked into PVP samples at three levels including 0.05, 0.1, and 0.5 µg/mL. For nitrate recovery, calculations were corrected for the amount already present in the sample. Recoveries for nitrite and nitrate (n=6 per each level) ranged from 93 to 100% and 91 to 100% (Figure 6), respectively, within the acceptance criteria range of 90–110%.

A single LC/UV with direct injection method was developed for the analysis of both nitrite and nitrate, utilizing the Arc Premier System and an Atlantis Premier BEH C₁₈ AX Column. The method demonstrated excellent performance at the LOQ levels of 0.025 µg/mL and 0.075 µg/mL for nitrite and nitrate respectively. The method has been demonstrated as suitable for the analysis of polyvinylpyrrolidone excipient, with recoveries of nitrite and nitrate between 91 to 100%. The LC-UV method offered a quick and easy solution for screening nitrite and nitrate in the PVP K15 excipient or raw materials.

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