Determination of Subsidiary Colors in D&C Red No. 36 (Pigment Red 4) by HPLC and UHPLC
Adrian Weisz1, Marianita Perez-Gonzalez1, Rosemary Hall1
1U.S. Food and Drug Administration, Office of Cosmetics and Colors, Office of the Chief Scientist, College Park, MD 20740, USA.
Background:
Batches of Pigment Red 4 (PR4) submitted to the U.S. Food and Drug Administration (FDA) for certification as D&C Red No. 36 (R36) must comply with specifications listed in the U.S. Code of Federal Regulations (CFR). Currently, a tedious thin-layer chromatography (TLC) method is used to enforce limiting specifications for four subsidiary colors: Pigment Orange 5 (PO5, CAS 3468-63-1) and Yellow-1-Naphthol (Y1N, CAS 36265-89-1), each ≤0.5%, and Pigment Red 1 (PR1, CAS 6410-10-2) and Pigment Red 6 (PR6, CAS 6410-13-5), each ≤0.3%.
Objective:
To develop improved analytical methods for determination of the subsidiary colors in samples from batches submitted for certification as R36.
Methods:
High-performance liquid chromatography (HPLC) and ultrahigh-performance liquid chromatography (UHPLC) were the techniques used for development of the new methods. Reference materials for PO5, PR1, and PR6 were purchased, and a standard for Y1N was synthesized, with its identity confirmed by HRMS and NMR. Calibration curves were prepared for quantification of the subsidiary colors by each method.
Results:
Both newly developed methods enabled baseline separation of the four subsidiary colors and their identification based on defined retention times, elution sequence, and UV-Vis spectra. Linearity was demonstrated for both methods, with R2 values >0.999 for each of the analytes. Ranges of other validation data included: LOD, 0.006-0.018%; LOQ, 0.007-0.05%; recoveries, 85.5 ± 7.4% - 101.8 ± 2.0%. The new methods were implemented to quantify the analytes in 24 surveyed batches of R36. They yielded identical or nearly identical values for the analyzed subsidiary colors. Obtained levels in all but two samples were below CFR limits.
Conclusions:
For routine batch-certification analyses of subsidiary colors in R36, either the HPLC or UHPLC method developed in this study can replace the outdated TLC procedure.
Highlights:
The R36 subsidiary colors PO5, Y1N, PR1, and PR6 can be accurately quantified using the newly developed HPLC and UHPLC methods.
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