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

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

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

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

Treated skin single ion heat maps.

Selected ion heat maps of (A) BZK in water, (B) water, (C) Solution 1, (D) Solution 2, (E) BZK + citric acid (pH 4.7), (F) BZK + caprylyl glycol, (G) BZK + vitamin E. The ion m/z 304.30 is shown in red and ion m/z 332.33 in blue. All yellow scale bars represent 2 mm. Skin is oriented with epidermis on the right side. Dotted lines are the selected regions of interest drawn along the edges of the skin.</p

Total ion penetration into human skin.

(A) Average penetration observed of ion m/z 304 into the skin. (B) Average penetration observed of ion m/z 332 into the skin. Both are showing the observed thickness of the epidermis in purple and continued ion depth into the dermis in pink. (TIF)</p

Observed average depth of BZK into the dermal layer of skin.

Observed average depth of BZK into the dermal layer of skin.</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

Commercial solution compositions.

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.</div

Average difference in BZK ion penetration.

Differences between the average penetration depth of ion m/z 304 to ion m/z 332. A positive value indicates ion m/z 304 was observed deeper while a negative value indicated ion m/z 332 penetrated deeper. (XLSX)</p