Assay of Parabens via Benchtop NMR: An Alternative to HPLC

Parabens are among the most widely used preservatives in a variety of products, ranging from food to cosmetics and pharmaceuticals.¹ This class of molecules is based on p-hydroxybenzoic acid, with substitutions of the carboxylic acid fragment by ester groups of varying chain lengths (Figure 1). Recently, parabens have been the subject of much study due to research showing that these may act as endocrine-disrupting chemicals (EDCs).² As such, these are expected to be more heavily regulated in coming years, and methods for precisely determining the purity of parabens being introduced in consumer products will be important.

Figure 1. General structure of the parabens discussed herein (methylparaben (R = CH₃); ethylparaben (R = CH₂CH₃); propylparaben (R = CH₂CH₂CH₃))

The current monographs published by The United States Pharmacopeia – The National Formulary (USP–NF) for the parabens discussed in this work describe a method using high-performance liquid chromatography (HPLC) with an ultraviolet (UV) detector for assays of these products.³ The requirements for expensive reference standards, calibration curves, and large amounts of high-purity solvents (typically ≥99.9%) make this approach tedious and costly. Here, we demonstrate the use of benchtop NMR for paraben assays using benzyl benzoate as an internal calibrant for quantitative NMR (qNMR).

For all three parabens discussed herein, sample preparation and data collection could be performed in approximately 5 minutes per sample. To demonstrate the performance of the method under reproducibility and repeatability conditions, three physical samples for each paraben were prepared (~20 mg of both paraben and benzyl benzoate in 0.6 mL of methanol-d₄), and each was analyzed in triplicate at 60 MHz.⁴ In Figure 2, stacked spectra of each analyzed paraben are shown.

Figure 2. Stacked ¹H (60.4 MHz) NMR spectra of methylparaben, ethylparaben, and propylparaben in methanol-d₄. The structures of the parabens and the internal calibrant, benzyl benzoate (top right), are shown and their corresponding signals used for qNMR are labeled. The asterisk represents the residual solvent peak for methanol.

The NMR assay results are presented in Table 1 and compared to the values included in the certificate of analysis (CoA) provided by the supplier. The CoA also confirms that these values were determined according to the official USP–NF monograph method. In all cases, the %diff values are within 0.4% of the reported values, and the RSD values are ≤0.85, as required in the monograph for the HPLC method.

Table 1. Results of ¹H NMR assays of methylparaben, ethylparaben, and propylparaben. The relative standard deviation (RSD) values are included in parentheses. The reported values are those included in the CoA provided by the supplier.

The results obtained herein demonstrate that benchtop NMR technology can perform paraben assays in an efficient, less wasteful, and inexpensive manner when compared to the traditional HPLC approach. The sample preparation and analysis are straightforward, and the data processing is minimal, only requiring that the spectra be phased, corrected for baseline distortions, and integrated, meaning that this process could easily be automated.

If you have any questions about the incorporation of benchtop NMR into your
USP–NF workflows, or about the work presented herein, please don’t hesitate to contact us!

References
(1) Soni, M. G.; Carabin, I. G.; Burdock, G. A. Food Chem. Toxicol. 2005, 43, 985–1015.
(2) Nowak, K.; Ratajczak-Wrona, W.; Górska, M.; Jabłońska, E. Mol. Cell. Endocrinol. 2018, 474, 238–251.
(3) (a) Methylparaben (2020). The United States Pharmacopeia − The National Formulary. Rockville, MD, USA.
     (b) Ethylparaben (2020). The United States Pharmacopeia − The National Formulary. Rockville, MD, USA.
     (c) Propylparaben (2020). The United States Pharmacopeia − The National Formulary. Rockville, MD, USA.
(4) The parabens studied in this work were analyzed as received and without further purification. Protameen Chemicals: Methylparaben, lot: IA2711; Ethylparaben, lot: FA1711; Propylparaben, lot: GK1911.