Journal of Pharmaceutical and Biomedical Analysis 49 (2009) 1287–1291 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Short communication Isolation and characterization of a potential process related impurity of phenazopyridine HCl by preparative HPLC followed by MS–MS and 2D-NMR spectroscopy R. Nageswara Rao ∗ , Pawan K. Maurya, A. Narasa Raju Analytical Chemistry Division, Discovery Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India article info Article history: Received 12 November 2008 Received in revised form 26 February 2009 Accepted 3 March 2009 Available online 20 March 2009 Keywords: Phenazopyridine HCl Impurity profile Semi-preparative HPLC ESI-MS/MS 2D-NMR abstract During the process development of phenazopyridine HCl bulk drug, a potential impurity was detected in the routine impurity profiles by HPLC. Using MS–MS and multidimensional NMR techniques, the trace level impurity was unambiguously identified to be 3-phenyl-5-phenylazo-pyridine-2,6-diamine after its isolation from phenazopyridine HCl by semi-preparative HPLC. The formation of the impurity was discussed. To our knowledge, it is a novel impurity not reported elsewhere. © 2009 Published by Elsevier B.V. 1. Introduction The impurity profile of a drug substance is critical to its safety assessment and its manufacturing process. As per the guidelines of United States Pharmacopeia, the impurities that exceed 0.1% in a drug must be identified prior to clinical trials. Because the impuri- ties are usually process related, they are most probably structurally similar to the synthesized target drugs. High performance liquid chromatography in combination with multistage mass spectrome- try (HPLC/MS n ) is extremely useful for its capability to afford both molecular masses and structural information. Phenazopyridine hydrochloride (PPH) has been used for long time in conjunction with antibacterial agents for the treatment of urinary-tract infections [1–3]. It exerts an analgesic effect on the mucosa of the urinary tract and is used to provide symptomatic relief of pain in conditions such as cystitis and urethritis [4–6]. It is absorbed from the gastrointestinal tract and is excreted mainly from the urine [7]. During its production in large scale, an unknown impurity was detected by HPLC in all batches reducing the quality of the bulk drug. A variety of analytical techniques including amperometry in a flowing stream at the glassy carbon electrode [8], UV spec- ∗ Corresponding author. Tel.: +91 040 27193193; fax: +91 040 27160387. E-mail addresses: rnrao@iict.res.in, rnrao55@yahoo.com (R. Nageswara Rao). trophotometry [9], and colorimetry [10,11] were used to assay PPH. Quantitation of PPH was carried out by spectrophotometric [12–14], HPLC [15], polarographic [16] and gravimetric [17] methods. The US pharmacopeia [18] adopts a spectrophotometric method for assay of PPH. Simultaneous determination of PPH and sulfonamides were also reported [19–21]. Iqbal et al. [22] have studied photo degra- dation of PPH and characterized its major degradation products. Sabry [23] has studied forced degradation of PPH by HPLC and spec- trofluorimetric analyses. However, to the best of our knowledge, the impurity profiles of PPH particularly process related were not reported in the literature. The present manuscript describes a comprehensive investiga- tion on isolation and characterization of a major process related impurity of PPH by semi-preparative HPLC followed by ESI-MS–MS, 1 H NMR and 2D-NMR spectroscopy. The possible mechanism of its formation was proposed. 2. Experimental 2.1. Materials and reagents All the reagents were of analytical-reagent grade unless stated otherwise. Glass-distilled and de-ionized water (Nanopure, Barn- sted, USA), HPLC-grade acetonitrile and methanol (S.D. Fine Chem., Mumbai, India) were used. The investigated samples of PPH bulk drug materials, crude samples and impurities were synthesized in the laboratory. 0731-7085/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.jpba.2009.03.004