Assay for ppm Levels of Azide in Drug Substances

Nov 01, 2012

The removal of azide as a potential impurity from a drug substance may be critical to its safety profile, such that quantitation of this impurity becomes an important control parameter. This paper describes a simple and practical assay for azide using chemical derivatization and high performance liquid chromatography. The method is shown to be suitable for the intended purpose on three example test materials. Potential issues for wider applications are discussed.

In the manufacturing of pharmaceutical drug substances in which azides are used as reagents or when they are generated somehow in the synthesis, it may be necessary to demonstrate that these impurities are sufficiently removed to levels below an appropriate safety threshold. The threshold would depend on the target dose and route of administration. Sodium azide is an example of an azide for which the environmental exposure limits have been reasonably well characterized (1). Testing for azides can be performed on the final drug substance, but may also be performed at critical control points in a synthesis such as with related compounds like starting materials and intermediates.

Figure 1: PFBB– azide derivatization reaction
This article describes the use of pentafluorobenzyl bromide (PFBB) as a derivatization agent to form the pentafluorobenzyl-azide (PFB-Az) derivative, based on a known approach for synthesizing azides (2,3) as diagrammed in Figure 1.

Relative to azide, the derivative can be easily detected by UV absorbance detection and is sufficiently nonpolar for retention on conventional reversed-phase high performance liquid chromatography (HPLC) analytical columns. Thus, there is potential to manage this reaction for application to parts-per-million analysis of azide as an impurity in drug substances and related compounds.

The literature describes the assay of azide in different modes, such as using ion-based HPLC (4–6), derivatization-based gas chromatography (GC) (7,8), and derivatization-based HPLC (9,10). The reagent PFBB has been used in conjunction with GC analysis (7,8), and other derivatization agents have been used in conjunction with HPLC analysis (9,10).

This article covers the assay of free azide, based on the use of sodium azide as the standard and spiking agent in method development. The work described herein is on three drug substance–related compounds exhibiting a range of polarity and complexity. Because of their proprietary nature, the three materials of interest will be designated as compounds A, B, and C, where A is the most polar with the smallest molecular size, and C is the least polar with the largest molecular size.



Sodium azide, pentafluorobenzylbromide (PFBB), dimethyl sulfoxide (DMSO), trifluoroacetic acid, and acetonitrile were obtained from Sigma-Aldrich. Distilled deionized water was obtained through an in-house purification system.

Azide Standard and Test Sample Preparation

Azide standards were prepared using sodium azide in DMSO, in which target parts-per-million concentrations were set relative to free azide concentration. Test samples of compounds A, B, and C were prepared at a 10-mg/mL target concentration in DMSO.

Derivatization Conditions

A PFBB stock solution was first prepared by transferring 100 μL of PFBB to a 5-mL volumetric flask, then the substance was diluted to flask volume with DMSO, and the contents were mixed well. When 100 μL of this stock was added to a 5-mL volume of a standard or test sample solution, taking into account density and concentrations, the PFBB was present at 10-fold molar equivalents in excess over 1000 ppm free azide. A derivatized blank was prepared in a similar fashion in which 100 μL of the PFBB stock solution was added to 5 mL of DMSO. The derivatization had no quenching procedure, but it was shown that the reaction, whether for standards or test samples, required ~4–6 h to reach completion, and that the solutions exhibited no significant changes in HPLC profiles after at least 16 h in ambient conditions.

Chromatographic Analysis Conditions