MALDI-TOF imaging analysis of benzalkonium chloride penetration in ex vivo human skin.

Benzalkonium chloride (BZK), alkyldimethylbenzlamonium chloride, is a cationic surfactant that is used as an antiseptic. BZK is classified as a quaternary ammonium compound composed of molecules of several alkyl chains of differing lengths, that dictate its effectiveness towards different microbes. As a result, BZK has become one of the most used preservatives in antibacterial solutions. Despite its widespread use, it is not clear whether BZK penetrates human skin. To answer this question, BZK treated skin was analyzed using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry imaging. Solutions containing BZK and differing excipients, including citric acid, caprylyl glycol, and vitamin E, were applied ex vivo to excised human skin using Franz diffusion cells. Treated skin was embedded in gelatin and sectioned prior to MALDI-TOF imaging. BZK penetrates through the epidermis and into the dermis, and the penetration depth was significantly altered by pH and additives in tested solutions

Determined thickness of the epidermis of each treatment group regardless of the skin origin.

Determined thickness of the epidermis of each treatment group regardless of the skin origin.</p

Collected mass list of 0.123% BZK.

Mass list collected of 0.123% BZK solution spotted with 40 mg/mL CHCA mixed with acetone. Rows highlighted in green correspond to confirmed BZK ions. Rows highlighted in yellow correspond to matrix ions matching those observed in matrix scans. (XLSX)</p

Graph of depths of single BZK ions into dermis layer of skin.

(A) Bar chart of average depth of ion m/z 304 into the dermal layer. (B) Bar chart of average depth of ion m/z 332 into the dermal layer. One-way ANOVA was performed to determine significance where P<0.05 *, P<0.01 **, P< 0.001 ***, and P<0.0001 ****.</p

Epidermal thicknesses of each specific skin location based on treatment group.

Epidermal thicknesses of each specific skin location based on treatment group.</p

Imaging of gelatin compared to negative and positive control.

Ion heat maps of gelatin without skin, negative control, and positive control. (A-C) Selected ions of m/z 172.06 ±0.43, (D-F) ion m/z 212.04 ± 0.53, (G-I) ion m/z 335.14 ± 0.84, (J-L) and ion m/z 379.13 ±0.95 are all CHCA matrix ions and are represented with purple. (M-O) Ions heat maps of ion m/z 304.30 ±0.76 (BZK C12) in red. (P-R) Ion heat maps m/z 332.33 ± 0.83 (BZK C14) in blue. (TIF)</p

Pearson’s correlation coefficient values.

Recorded Pearson’s correlation coefficient values for each skin section MALDI imaging analysis based on comparing its m/z 304 ion heat map to its m/z 332 ion heat map. (XLSX)</p

Comparison of freezing media and gelatin negative controls.

Negative control skin embedded in PolyFreeze freezing medium showing (A) ion m/z 304, (B) ion m/z 332, and (C) overlay of both ions. (D) Mass spectra of selected raster spot in epidermis of negative control prepared in PolyFreeze. (E) Mass spectra of selected raster spot in dermis of negative control prepared in PolyFreeze. Negative control embedded in gelatin showing the presence of ions of interest (F) ion m/z 304, (G) ion m/z 332, and (H) the overlay of both within the region of interest. (I) Mass spectra of selected raster spot in epidermis of negative control embedded in gelatin. (J) Mass spectra of selected raster spot in dermis of negative control embedded in gelatin. All ion heat maps are pulled with background images still present. Selected raster spot mass spectra are noted on ion heat maps with labeled yellow circle. (TIF)</p