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

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

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

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

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

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

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

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

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

Collected spectra of all matrix, excipients, and solutions.

Collected mass spectra of (A) CHCA matrix, (B) sDHB matrix, (C) 0.123% BZK solution, (D) citric acid, (E) 1.0% caprylyl glycol solution, (F) 0.1% vitamin E solution, (G) BZK + citric acid treatment solution, (H) BZK + caprylyl glycol, (I) BZK + vitamin E, (J) Solution 1, and (K) Solution 2. All were collected in reflectron positive mode with 5,000 laser pulses. Mass range was set to m/z 100–600. All solutions were spotted with 40 mg/mL CHCA in acetone as the matrix. Labeled peaks of m/z 172, 212, 335, and 379 were all found to be CHCA matrix peaks. (TIF)</p

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