HYALURONIC ACID ANALYSIS IN COSMETIC PRODUCTS

Quality control in cosmetics and perfumery products, as well as product development and R&D work, require various analyses such as essences, preservatives, hyaluronic acid and SPF (sun protection factor) testing to meet regulatory requirements. These analyses require different analytical techniques such as HPLC, GC, GCMS, LCMSMS, UV-VISNIR. Hyaluronic acid is an acidic mucopolysaccharide with alternating bonds of D-N-acetylglucosamine and D-gluconic acid, distributed throughout the body in tissues such as skin, tendons, muscles, cartilage, blood vessels and brain, with a molecular weight exceeding 1,000,000. Because hyaluronic acid occurs naturally in living organisms, it has high biocompatibility and is used in applications such as surgical procedures. Since hyaluronic acid progressively degrades in vivo, there is increasing demand for higher molecular weight hyaluronic acid with longer duration of action. Hyaluronic acid also has strong moisturizing effects and is therefore used as a cosmetic ingredient. Pharmaceutical formulation impurities of hyaluronic acid and production stage fragments are monitored by LCMSMS ESI. This article presents examples of the use of Size Exclusion Chromatography (SEC) in the analysis of hyaluronic acid

SHIMADZU Prominence Size Exclusion Chromatography (SEC) System

Analysis of Standard Samples

Figure 1 shows the structural formula of hyaluronic acid. When Size Exclusion Chromatography is used for the analysis of macromolecules containing ionic dissociation groups, these ionic groups within the molecule repel each other causing the molecule to expand. These ionic interactions can be suppressed by adding a salt to the mobile phase. In particular, where a hydrophilic molecule such as polysaccharides is concerned, the presence of any type of ionic group will frequently result in model deformation and inconsistent measurement results. In this example, sodium sulfate was used as the salt. The analytical conditions are shown in Table 1.

Figure 1. Structural Formula of Hyaluronic Acid

Figure 2. Chromatogram of Sodium Hyaluronate Obtained from Rooster Comb

Table 1 Analytical Conditions

Figure 3. Chromatogram of Sodium Hyaluronate from Human Umbilical Cord.

Figure 4. Chromatogram of Non-Alcoholic Beverage

Figure 2 shows an analysis example of sodium hyaluronate obtained from rooster comb. The sample was dissolved to 0.1% concentration in 50 mM aqueous sodium sulfate solution used as mobile phase. Figure 3 shows an analysis example of sodium hyaluronate derived from human umbilical cord. Similar to Figure 2, the sample was dissolved to 0.1% concentration in 50 mM aqueous sodium sulfate solution.

Analysis of Cosmetic Products and Non-Alcoholic Beverages

We analyzed several cosmetic products and non-alcoholic beverages that claim to contain hyaluronic acid. The analytical conditions are shown in Table 1. Chromatograms of a non-alcoholic beverage are shown in Figure 4, and cosmetic product A and cosmetic product B are shown in Figures 5 and 6, respectively. Cosmetic A was analyzed after 10-fold dilution in 50 mM aqueous sodium sulfate solution used as mobile phase. The non-alcoholic beverage and cosmetic B were analyzed without dilution, but after filtration through a 0.45 µm membrane filter. The peaks (pik) between 11 and 17 minutes are assumed to be hyaluronic acid. Ozan Halisçelik Senior Chemist Service Manager Assistant Ant Teknik Cihazlar      

Figure 5. Chromatogram of Cosmetic Product A

Figure 6. Chromatogram of Cosmetic Product B.