Quantitative proton magnetic resonance determination of N,N-dimethylformamide in one intermediate of the Quimi-Hib vaccine Raine Garrido Arteaga, a * Felix Cardoso San Jorge, a María del C. Rodríguez Montero, a Violeta Fernández Santana, a† Vicente Vérez Bencomo a and Herman Vélez Castro a Quimi-Hib is a conjugate vaccine against Haemophilus influenza type b (Hib) where the Hib antigen is the only one produced by chemical synthesis. NMR has become the alternative of choice for the identity of intermediates during the chemical synthesis of Hib antigen. We explore a rapid quantitative proton magnetic resonance (qHNMR) assay for the determination of N,N-dimethylformamide (DMF) as a residual in one of the critical intermediates. The proposed assay has been shown to be accurate, precise for intermediate precision conditions (relative standard deviation <3% for spectrometer-to-spectrometer variations), specific (no detected interferences), and rugged (percentage difference <3% for day-to-day and spectrometer-to- spectrometer variations). The quantitative NMR assay can replace the common chromatographic methods for monitoring the DMF contents in one crucial step of the synthetic scheme. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: NMR; proton; quantitative analysis; Hib; vaccine Introduction Haemophilus influenzae type b (Hib) is a Gram-negative bacterium that causes meningitis, pneumonia, and otitis media, mainly in children under 4 years old. The active pharmaceutical ingredient (API) of Quimi-Hib vaccine is composed of Hib antigen obtained by chemical synthesis and further conjugated to tetanus tox- oid. [1] NMR is one of the most widely used techniques in the eval- uation of active ingredients and intermediates of high added value. [2] In the last decade, the application of quantitative proton magnetic resonance (qHNMR) had an increasing impact on the pharmaceutical industry. The qHNMR method involves proton NMR experiments with several modified parameters in order to obtain spectra with quantifiable signals (99.9% recovery of equilib- rium magnetization). These parameters depend essentially on the relaxation times of nuclei to be evaluated for the analyte and the reference. [3] There are theoretical values for the parameters that yield the best qHNMR spectra. These assays usually take hours depending on the analyte and reference quantities in the sample. However, the parameters can be optimized experimentally, decreasing the delay of the assay. This paper discusses a method of qHNMR developed for the control of N,N-dimethylformamide (DMF) in 5-O-Allyl-1-O-(2,5-di-O-benzyl-b-D-ribofuranosyl)-2,3,4- tri-O-benzyl-D-ribitol (compound 1) that represents one critical intermediate of the synthesis scheme of Hib antigen [4] (Fig. 1). Materials and Methods Sample preparation The sample of compound 1 was supplied by the Quality Department of the Center for Genetic Engineer and Biotechnology. Seven deuterated chloroform stock solutions were prepared with 1.7 mg ml 1 tert-butanol (t-BuOH) as internal reference for qHNMR, 0.2 mg ml  1 TMS, and different quantities of DMF [7.5 mg ml 1 (A), 8.3 mg ml 1 (B), 10.2 mg ml 1 (C), 11.5 mg ml 1 (D), 12.8 mg ml 1 (E), 14.2 mg ml 1 (F), and 15.5 mg ml 1 (G)] using 5 ml (0.5%) volumet- ric flasks. Three sets of samples were prepared by dissolving 10 mg of compound 1 in 0.6 ml of each of the above-mentioned stock solutions. All reagents and solvents were of spectroscopic quality. Instrument setting NMR analyses were carried out on two Bruker Avance DPX 250 instruments operating at 250.13 MHz. The main spectrometer (A) operates with a 5-mm quadruple nuclei probe ( 1 H, 13 C, 31 P, and 19 F), whereas the second spectrometer (B) operates with a 5-mm 1 H and 13 C dual probe, both at a temperature of 27  C. The inversion–recovery experiment was run with a list of 26 * Correspondence to: Raine Garrido Arteaga, Center of Biomolecular Chemistry, 200 Street and 21 Ave., Atabey, Playa, La Habana, Cuba 11600. E-mail: raine. garrido@cqb.cu † In memory of Dr Violeta Fernández Santana, who passed away on 20 November 2011. a Center of Biomolecular Chemistry, 200 Street and 21 Ave. Atabey, Playa, La Habana, Cuba, 11600 Abbreviations used: Hib, Haemophilus influenzae, type b; API, active pharma- ceutical ingredient; qHNMR, quantitative proton magnetic resonance; DMF, N, N-dimethylformamide; t-BuOH, tert-butanol; QNP, quadruple nuclei probe; ICH, International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; RSD%, relative standard deviation Magn. Reson. Chem. (2012) Copyright © 2012 John Wiley & Sons, Ltd. Research Article Received: 15 March 2012 Revised: 15 May 2012 Accepted: 18 May 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/mrc.3831