Optimized UHPLC–derivatization method for low-level nitrite detection in pharmaceutical excipients

Abstract

This work presents a method optimization strategy for determining nitrite ions in pharmaceutical excipients. After derivatization, nitrite ions were analyzed using ultra-high-pressure liquid chromatography with UV detection. The article details how to achieve a sensitivity of 1 ng/mL (0.1 ppm) in pharmaceutical excipients while maintaining an analysis time of less than one minute. The results of nitrite measurements in several excipients obtained using the limit test to validate the analytical method are presented. The article also explains how to further reduce the detection limit while maintaining the analysis time. The advantage of the presented method is that it offers simple sample preparation and reproducible recovery above 90%. The article also shows what problems can be caused by incorrect sample preparation and excipient storage.

Highlights

Griess-Ilosvay reaction is suitable for the low nitrite determination in a UHPLC.
For efficient analysis, the UHPLC system must be optimized.
Nitrite content can be analyzed in 1 min.
The role of solvent effect is important in large-volume sample dosing.
Gradient elution in (U)HPLC is not always beneficial.

Introduction

Nitrite ions (NO₂⁻) in pharmaceutical excipients are a critical concern as they act as precursors for forming carcinogenic N-nitrosamines, which can contaminate drug products and pose cancer risks, prompting strict regulatory guidance (FDA, EMA) requiring risk assessments, testing, and mitigation strategies, including supplier controls and formulation changes, to reduce these ppm-level impurities [1], [2], [3]. The nitrite ion problem in excipients stems from its role as a nitrosating agent, reacting with amino groups in active pharmaceuticals ingredient (APIs) under acidic conditions to N-nitrosamines [4], [5].

Since excipients can be used for different drug products (DP), the nitrite content must always be determined individually, after a risk assessment. In most cases, it is sufficient to know whether the nitrite ion concentration is below or above a limit level (e.g. 1 ppm), there is no need to determine the exact amount, therefore, in accordance with the ICH Q2(R2) guideline [6], performing a limit test is sufficient during method validation. However, it should be kept in mind that an excipient may have multiple limit levels, depending on the DP into which it is incorporated. Therefore, it is advisable to supplement limit test validation with a linearity test. In the modern determination of nitrites within pharmaceutical excipients, the use of advanced separation techniques is indispensable, as each method offers distinct applications, advantages, and inherent limitations.

Ion chromatography (IC) is a highly selective and sensitive technique for nitrite analysis, especially in complex matrices. In IC, anions are separated on an ion-exchanger column and detected, typically by conductivity [7] or ultraviolet (UV) absorption [8]. Its advantage is that it allows direct determination and small (ppm level) concentrations can be detected, but for some reasons it is less widespread in the pharmaceutical industry, at least compared to high performance liquid chromatography (HPLC). The lower measurement limit of IC separation can be reduced further with post-column derivatization [9] or ion chromatography coupled with mass spectrometry (IC-MS) [10]. However, these procedures are too complicated for routine analytical measurements.

A variety of chromatographic techniques are available for the determination of nitrites following their conversion through derivatization. One possibility is cyclamate derivatization, where cyclamate reacts with nitrite under acidic conditions. The cyclohexene formed can be easily measured by head space gas chromatography (HS-GC) using flame ionization (FID) or mass spectrometry (MS) detection [11]. This technique allows for nitrite determination at ppb levels, but its limitations include that the sample matrix can influence derivatization, and the analysis time, due to HS sample preparation, is longer than liquid chromatographic methods.

DAN derivatization [12] followed by fluorescence (FL) or MS detection [13] coupled with (U)HPLC can also achieve ppb-level nitrite determination, but these detector types are not as widespread. In the case of such low concentrations, in addition to the appropriate purity of the reagents, care must be taken to minimize the time the materials used are in contact with air, because the binding of nitrogen oxides present may interfere with the measurement.

The most widely used technique for nitrite determination is the Griess-Ilosvay reaction [14]. In this method, nitrite reacts with sulfanilic acid under acidic conditions to form a diazonium salt, which then couples with N-(1-naphthyl)ethylenediamine to produce a pink azo dye. The intensity of the color, measured spectrophotometrically at around 540 nm, is directly proportional to the nitrite concentration. This method is simple, sensitive, and suitable for routine analysis, but may be affected by interfering with substances that also react with the reagents or cause sample coloration. The derivative, however, can be analyzed by HPLC, thus ensuring selectivity from interfering factors. The analysis time can be significantly reduced if modern UHPLC technique is used instead of conventional HPLC [15].

The detection limits of various analytical methods have been reported extensively [16], [17], [18]; however, details regarding method optimization, precise sample preparation, and strategies to enhance the detection limit are often lacking, even though such knowledge is critically important for analysts.

In this work, the optimization of nitrite determination after Griess-Ilosvay derivatization is described, from the details of sample preparation to the optimal setup of the fast UHPLC system where the analysis time is only 1 min.

Continue reading here

Róbert Kormány, Optimized UHPLC–derivatization method for low-level nitrite detection in pharmaceutical excipients, Journal of Pharmaceutical and Biomedical Analysis, Volume 276, 2026, 117472, ISSN 0731-7085, https://doi.org/10.1016/j.jpba.2026.117472.