Analytical method development and dermal absorption of cosmetic preservatives, 2-dodecylisoquinolinium bromide (DB) and hexetidine using Franz diffusion cells.

다국어 초록 (Multilingual Abstract)

2-Dodecylisoquinolinium bromide (DB) and hexetidine are antimicrobial preservatives used in cosmetic products under regulatory concentration limits: DB is allowed up to 0.05% in leave-on cosmetics in Korea, while hexetidine is permitted up to 0.1% in rinse-off products in Korea, and European Union. Because dermal absorption data for both ingredients in cosmetic matrices have been limited, in vitro dermal absorption studies were performed in compliance with the guideline according to the Ministry of Food and Drug Safety of Korea. Prior to the dermal absorption experiments, LC–MS/MS methods using multiple reaction monitoring (MRM) were developed and validated to quantify each analyte in key study matrices, including swabs, stratum corneum (SC), skin (dermis + epidermis), and receptor fluid (RF). The DB method showed well-fitted linearity (r² = 0.9904–0.9995), accuracy (91.8–113.7%), and precision (0.6–8.5%), and the hexetidine method demonstrated excellent performance with linearity (r² = 0.9944–0.9999), accuracy (80.9–108.3%), and precision (0.6–16.1%) in accordance with validation guidelines. Dermal absorption was evaluated using Franz diffusion cells with integrity-verified Micro-pig skin. Cream formulations containing 0.038% or 0.0127% of DB were applied at 10 μL/cm². After 24 hr, the skin surface was wiped with swabs. SC was collected by tape stripping and the remaining skin was sectioned into eight pieces. RF samples were collected at 0, 1, 2, 4, 8, 12, and 24 hr. Total dermal absorption of DB was 0.08 ± 0.05 μg/cm² (2.18 ± 1.06%) for the 0.038% cream and 0.04 ± 0.02 μg/cm² (3.05 ± 1.79%) for the 0.0127% cream, with no detectable DB in RF for either formulation. However, the overall recovery (47.25 ± 25.11%) for the 0.0127% DB cream. did not meet acceptable range for mass balance/recovery (e.g., 85–115%), indicating that the low-dose DB results should be interpreted cautiously. In contrast, the recovery of the 0.038% cream was 97.79 ± 9.84%, which met the acceptance criteria for recovery. For hexetidine, 0.1% and 0.025% gel shampoo (rinse-off) formulations were applied at the same dose (10 μL/cm²) for 30 min, followed by a wash-out step to simulate shampoo-use conditions. Total dermal absorption of hexetidine was 0.008 ± 0.006 μg/cm² (0.07 ± 0.06%) for the 0.1% formulation, and 0.00 ± 0.00 μg/cm² (0.00 ± 0.00%) for the 0.025% formulation. Total recovery was 86.59 ± 13.69% for the 0.1% formulation, and 97.21 ± 2.03% for the 0.025% formulation, and both values met the acceptance criteria for recovery. Collectively, these findings provide substance- and formulation-specific dermal absorption data to support exposure assessment of the preservatives when DB and hexetidine are used as cosmetic ingredients.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• II. Materials and Methods 10
• 1. Chemicals and reagents 10
• 2. Analytical methods 12
• 2.1 Equipment 12
• 2.2 LC conditions 12
• 2.2.1 2-Dodecylisoquinolinium bromide (DB) 12
• 2.2.2 Hexetidine 12
• 2.3 Mass spectrometric conditions 13
• 2.3.1 2-Dodecylisoquinolinium bromide (DB) 13
• 2.3.2 Hexetidine 13
• 2.4 Preparation of calibration standards and QC samples 17
• 2.4.1 2-Dodecylisoquinolinium bromide (DB) 17
• 2.4.2 Hexetidine 17
• 2.5 Analytical method validation 19
• 2.5.1 Selectivity and linearity 19
• 2.5.2 Accuracy and precision 20
• 2.5.3 LLOQ and acceptance criteria 21
• 3. In vitro dermal absorption 21
• 3.1 Selection and preparation of test formulations 21
• 3.2 Skin preparation 24
• 3.3 Receptor fluid selection 24
• 3.4 Micro-pig skin integrity and dosing volume 24
• 3.4.1 In vitro dermal absorption experiment of DB 25
• 3.4.2 In vitro dermal absorption experiment of hexetidine 25
• 4. Statistical analysis 28
• Ⅲ. Results 29
• 1. Validation of analytical methodology 29
• 1.1 Selectivity 29
• 1.2 Linearity 36
• 1.3 Lower limits of quantification (LLOQ) 38
• 1.4 Accuracy & Precision 38
• 2. In vitro dermal absorption 42
• 2.1 Skin integrity 42
• 2.2 In vitro dermal absorption of DB 44
• 2.3 In vitro dermal absorption of hexetidine 44
• Ⅳ. Discussion 49
• V. Conclusion 56
• References 57
• Abstract (in Korean) 63