transdermal delivery 
 of therapeutic agents represents an attractive 
 alternative to oral or parenteral delivery and has witnessed increased 
 clinical application in the past two decades . although oral analgesics 
 are commonly prescribed for the treatment of acute and chronic pain , 
 adverse , potentially fatal effects such as respiratory depression , 
 nausea , and addiction can ensue from systemic drug exposure . clinically effective drug concentrations can 
 be introduced at a peripherally located site of injury or inflammation 
 by topical administration without resulting in high systemic concentrations 
 that may increase the likelihood of untoward side effects . understanding drug penetration and diffusivity through skin , however , 
 poses significant challenges given the structural complexity of this 
 organ and the many disparate mechanisms that transport small molecules 
 across the dermal layers . methods for 
 determining transdermal drug absorption in skin include skin extraction 
 measurements , quantitative autoradiography , and spectroscopic methods ( fluorescence and ftir ) . in this 
 work , we present a desorption ionization mass spectrometric imaging 
 technique for identifying small molecule analytes in skin and for 
 mapping compound distribution through the dermal layers . we suggest 
 that this technique represents a superior approach to measuring the 
 extent of skin permeation of topically applied compounds . many 
 clinically employed analgesics and anesthetics for relieving 
 pain modulate voltage - gated sodium channels ( navs ) , which 
 are a family of integral membrane proteins responsible for the rising 
 phase of action potentials in electrically conducting cells . one such example is lidocaine , a prescription - based 
 injectable and topical pharmaceutical agent . in addition to clinically 
 employed anesthetics , natural neurotoxic alkaloids , including saxitoxin , 
 neosaxitoxin , batrachotoxin , and aconitine , are sodium 
 channel modulators , some of which are currently being investigated 
 for potential usage as pain therapeutics owing to the high affinity 
 and specificity that such compounds demonstrate for navs . little is known about 
 the ability of these compounds to penetrate human skin to produce 
 a topical analgesic effect . mass spectrometry imaging is a label - free 
 method with which the distribution of a multitude of compounds can 
 be mapped in biological tissues with high specificity and offers an 
 efficient , straightforward method for this type of analysis . we have used high mass resolution / mass accuracy desorption 
 electrospray 
 ionization mass spectrometric imaging ( desi - msi ) to investigate the 
 permeation of natural sodium channel blockers and novel synthetic 
 analogues following topical application of these compounds to ex vivo 
 human skin . desi - msi enables two - dimensional mapping of a sample analyte 
 in the ambient environment , without the need for extensive sample 
 preparation . samples are bombarded with microdroplets of acetonitrile 
 that dissolve hundreds of endogenous lipids and metabolites as well 
 as exogenous compounds present in tissue . the splash forms secondary 
 microdroplets that enter a mass spectrometer , providing a detailed 
 chemical map of the distribution of molecules within the sample surface . after desi - msi , the same tissue section can 
 be stained , optically imaged , and compared to selected 2d desi - ms 
 ion images . the compounds investigated 
 in this study include common analgesics such as lidocaine and prilocaine 
 as well as aconitine , an herbal neurotoxin used in chinese medicine , 
 saxitoxin , a sodium channel inhibitor synonymous with paralytic shellfish 
 poisoning , and novel synthetic analogues of both saxitoxin and batrachotoxin . the effect of sunburn injury on the penetration of these sodium channel 
 blocking agents was also examined . using desi - msi , we demonstrate 
 that the compound of interest can be detected with unambiguous determination 
 among the hundreds of other endogenous skin compounds concurrently 
 being analyzed . the spatial distribution of these agents has been 
 mapped and directly compared to the morphology of the skin sections 
 to assess transdermal penetration . human skin 
 samples were obtained from four patients undergoing surgery at stanford 
 medical school . the skin graft was placed in a shallow dish 
 partially filled with synthetic interstitial fluid . confined circular 
 treatment areas were created by delineating the application site with 
 a felt - tip pen and encircling this mark with petroleum jelly . the 
 compounds were directly applied to the treatment area with a pipet , 
 and after 1 , 4 , or 10 h of application , the skin was frozen and cross 
 sectional tissue samples carefully prepared to avoid cross - contamination . positive ion mode desi - msi was performed using an orbitrap for mass 
 analysis at a mass resolution of 60  000 ( see the supporting information for experimental details ) . rich and distinctive molecular profiles were observed from the different 
 layers of skin ( epidermis , dermis , hypodermis ) by high - resolution 
 desi - ms imaging . many of the ions were identified as sodium or potassium 
 adducts of complex glycerophospholipids : glycerophosphocholines ( pc ) , 
 lyso - pc , sphingomyelins , glycerophosphoethanolamines , and glycerophosphoglycerols ; 
 and glycerolipids : triacylglycerols ( tg ) and diacylglycerols ( table s1 ) . ion images showing the distribution 
 of these compounds within the different layers of the skin samples 
 are shown in figure s1 , alongside the optical 
 image of the hematoxylin and eosin ( h&e ) stained tissue sections . pathological evaluation of the h&e stained human skin tissue sections 
 reveals the presence of epidermis ( 3060 m thickness ) , 
 superficial dermis ( 250450 m thickness ) , deep dermis 
 ( 22003000 m thickness ) and hypodermis ( 6001200 
 m thickness ) layers within all the human samples analyzed . topical application of local anesthetics and nav modulators 
 was initially performed using ethanol as the vehicle , the alcohol 
 most commonly used as a permeation enhancer . despite the complexity of the desi mass spectra obtained from the 
 different layers of the human skin , high mass resolution / high mass 
 accuracy measurements allowed for clear mass spectral separation and 
 identification of the compounds of interest . saxitoxin was detected 
 at m / z 282.1303 ( mass error of 2.14 
 ppm ) , corresponding to the protonated molecule with loss of an equivalent 
 of h2o , whereas lidocaine , aconitine , and an analogue of 
 batrachotoxin , btx - a , were observed as 
 protonated molecules at m / z 235.1805 
 ( mass error of 0.09 ppm ) , m / z 646.3220 
 ( mass error of 0.35 ppm ) , and m / z 504.2373 ( mass error of 1.44 ppm ) , respectively . errors in these 
 measurements were calculated from an average of a few mass spectra . figure 1 shows the desi - msi results obtained 
 for human skin tissue sections from the same patient in which saxitoxin , 
 lidocaine , aconitine , and btx - a were topically applied following the 
 protocol outlined above . the optical image of the h&e stained 
 skin sample following desi - msi is also depicted to assist visualization 
 of the outline of the tissue section . as observed in the desi - ms ion 
 image , saxitoxin did not exhibit any penetration in the human skin 
 tissue ( figure 1b ) . the molecule is completely 
 localized to the application site at the top of the tissue section . the low molecular weight of this compound notwithstanding , the inability of saxitoxin to penetrate the epidermis 
 is unsurprising given its charged , hydrophilic nature . by contrast , 
 the desi - ms ion image of lidocaine ( figure 1e ) shows that this molecule readily diffuses throughout the tissue 
 section , penetrating to the deep dermis layer ( as determined by comparison 
 with the h&e stained tissue section ) . similar to lidocaine , desi - ms 
 images of aconitine and btx - a also reflect extensive tissue penetration . both compounds were detected deep within the tissue section , the former 
 reaching the hypodermis of the skin . ion images for samples treated 
 with either aconitine or btx - a reveal a higher relative signal intensity 
 in the top layers of the skin than in the hypodermis ( figure 1h , k , respectively ) . desi - ms 
 ion images for saxitoxin , lidocaine , aconitine , and btx - a using ethanol 
 as vehicle were reproducible in skin sections obtained from the three 
 different donors . overall , the average penetration depth of lidocaine 
 was 1.24 mm ( 5 samples ) , of aconitine was 3.05 mm ( 6 samples ) , of 
 btx - a was 2.00 mm ( 4 samples ) , and no penetration was observed for 
 saxitoxin ( 7 samples ) . penetration of the sodium channel blocker compounds tested 
 in human 
 skin of the same donor using ethanol as the vehicle . chemical structures 
 are shown for ( a ) saxitoxin , ( d ) lidocaine , ( g ) aconitine , and ( j ) 
 btx - a . positive ion mode desi - ms ion images of the compounds are shown 
 for human skin in which ( b ) saxitoxin , ( e ) lidocaine , ( h ) aconitine , 
 and ( k ) btx - a were topically applied . optical images of cross sections 
 of the same tissue sections imaged by desi after h&e stain are 
 shown in ( c ) for saxitoxin , ( f ) for lidocaine , ( i ) for aconitine , 
 and ( l ) for btx - a . penetration of novel 
 synthetic analogue tested in human skin of 
 the same donor . positive ion mode desi - ms ion images of the compound are shown for 
 human skin in which stx - ge was topically applied using ( b ) ethanol 
 and ( d ) dmso as vehicles . optical images of the same tissue sections 
 imaged by desi after h&e stain are shown in ( c ) and ( f ) , respectively . desi mass spectra of selected skin regions outlined with a small black 
 box in the optical images in ( c ) and ( f ) are shown for stx - ge ( d ) 
 in ethanol and ( g ) in dmso . a synthetic derivative of saxitoxin bearing a long chain , 
 glycerol 
 ether group ( stx - ge ) was also examined in human skin desi - ms imaging 
 experiments . the attachment of a lipophilic 
 chain to saxitoxin was expected to facilitate transdermal penetration 
 of this compound . detection of stx - ge was observed in the positive 
 ion mode with protonation and loss of h2o at m / z 681.4652 ( mass error of 0.56 ppm ) and in its 
 protonated form m / z 699.4760 ( mass 
 error of 0.55 ppm ) . an endogenous lipid , ubiquitously distributed 
 throughout the skin tissue , was detected at the same nominal m / z 699.4 of stx - ge . this lipid ( measured 
 at m / z 699.4082 ) could be completely 
 resolved from toxin derivative using high mass resolution desi - msi , 
 thereby enabling accurate mapping of the exogenous compound in tissue 
 ( figure s2 ) . the desi - ms ion image for 
 stx - ge is shown in figure 2b . as is noted in 
 figure 2b , despite the hydrocarbon appendage , 
 stx - ge does not penetrate human skin , similar to the results with 
 saxitoxin ( ethanol as vehicle , 6 samples ) . this compound was only 
 detected outside of the tissue section , and only lipids were identified 
 in the mass spectra of the epidermis and superficial dermis of the 
 skin ( figure 2d ) . in an attempt to improve 
 the transdermal penetrability of stx - ge , dmso is a well - known skin penetration enhancer that interacts with 
 lipids in the stratum cornea and epidermis and induces conformational 
 changes in keratin . our desi - msi results 
 show that some degree of skin penetration by stx - ge is achieved when 
 dmso is employed ( figure 2e ) . figure 2 g shows the mass spectra of the superficial layer 
 of the skin dermis in which stx - ge can be clearly detected together 
 with the complex phospholipids that characterize this layer of tissue . an average penetration depth of 0.62 mm was measured for stx - ge using 
 dmso ( 3 samples ) . in the clinic , eutectic mixture of local anesthetics 
 ( emla ) cream 
 is prescribed as a dermal anesthetic . emla cream is an oil phase emulsion 
 containing a eutectic mixture of lidocaine and prilocaine in a ratio 
 of 1:1 by weight . we have investigated the skin penetration of these 
 compounds in emla cream applications to human skin in addition to 
 samples in which aconitine , btx - a , saxitoxin , and stx - ge were each 
 added . desi - ms ion images of lidocaine and prilocaine ( m / z 221.1651 , mass error of 0.3 ppm ) show complete 
 transverse skin penetration to the hypodermis as well as diffuse lateral 
 distribution within the skin section in all 14 samples analyzed ( figure 3 ) . when aconitine is included to the emla cream 
 formulation , similar penetration behavior was observed ( average depth 
 2.49 mm , 4 samples ) ; however , lateral spread of the compound was far 
 less pronounced ( figure 3a ) . btx - a also shows 
 complete skin penetration ( average depth 1.10 mm , 4 samples ) , albeit 
 not to the same depth as lidocaine / prilocaine ( figure 3b ) ; no penetration was observed for saxitoxin ( 4 samples ) . stx - ge showed partial skin penetration , with permeation observed through 
 the skin epidermis reaching the superficial dermis , similar to results 
 obtained using dmso as the vehicle ( figure 3c ) . in a few skin sections in which less compound was detected , complete 
 penetration of the stx - ge compound up to the superficial dermis was 
 seen ( average penetration depth of 0.45 mm for 4 samples ) ( figure s3 ) . topical application of analgesics 
 has been used for the treatment 
 of dermal pain caused by skin injuries such as sunburn . to test if 
 the penetrability and diffusivity of small molecules in human skin 
 is altered due to uv burn injuries , we have performed desi - ms experiments 
 on a burn injury model by applying uv radiation ( 300450 nm ) 
 to ex vivo human skin . following exposure 
 to uv light , compounds of interest were applied as ethanol solutions . uv radiation of human skin sufficient to induce dna damage , as measured 
 by increase in growth arrest and dna - damage - inducible gadd45a protein , 
 did not significantly alter skin penetration of the five compounds 
 tested ( figure s4 ) when compared to our 
 earlier findings ( see figures 1 and 2 ) . desi - ms ion images showing the penetration of ( a ) aconitine , 
 ( b ) 
 btx - a and ( c ) stx - ge tested in human skin of the same donor using 
 emla cream . ion images showing the penetration of lidocaine and prilocaine , 
 compounds with are both present in the emla cream formulation , are 
 also presented for the three sections analyzed . we have also evaluated the penetration behavior of 
 aconitine , lidocaine , and btx - a at different application times ( 1 , 
 4 , and 10 h ) in ex vivo human skin . interestingly , aconitine was the 
 only compound that showed a pronounced change in penetration depth 
 with application time , with decreased depth penetration occurring 
 at 1 h of application in comparison to 4 and 10 h ( figure s5 ) . transdermal permeation 
 experiments using live murine subjects were performed for comparative 
 purposes against ex vivo data . solutions of aconitine and stx - ge in 
 either ethanol or dmso were applied to the shaved back of male sprague dawley 
 rats under general anesthesia ( see supporting 
 information for experimental details ) . the animals were sacrificed , their skin removed , sectioned , and 
 analyzed following the same experimental procedures used for ex vivo 
 experiments . pathologic evaluation of the tissue sections revealed 
 that in all samples analyzed , besides skin epidermis , dermis , and 
 hypodermis layers , muscle tissue was also present . complete skin penetration 
 of aconitine was noted using both ethanol and dmso as vehicles , and 
 in both cases the compound reached the muscle layer of the tissue 
 section . interestingly , many samples showed a different spatial distribution 
 of analyte within the sectioned tissue , which varied depending on 
 the choice of vehicle ( figure 4 ) . ion images 
 of aconitine applied in ethanol to rat skin suggest that transverse 
 diffusion of this compound is facilitated through hair follicles , 
 an important pathway for the penetration of topically administered 
 substances . the same experiment performed 
 with dmso reveals a very different ion map that is much more reminiscent 
 of the ex vivo data . pathologic evaluation of the h&e stained 
 tissue sections in comparison with the desi - ms ion images confirms 
 that aconitine is colocalized with the regions of hair follicles within 
 the tissue section . in the case of stx - ge , complete penetration of 
 this compound occurs using both ethanol and dmso , reaching the deep 
 dermis layer of the skin ( figure s6 ) . the 
 results from ethanol experiments stand in contrast to those obtained 
 with human skin . overall , deeper tissue penetration of both aconitine 
 and stx - ge was achieved in vivo using dmso as vehicle ( 1.90 mm in 
 ethanol for 7 samples versus 2.60 mm for 3 samples in dmso for aconitine ; 
 0.90 mm in ethanol for 5 samples versus 1.12 mm for 7 samples in dmso 
 for stx - ge ) ( table s2 ) . desi - ms ion images showing 
 the penetration of aconitine tested 
 in in vivo rat skin using ( a ) ethanol and ( b ) dmso as vehicles . optical 
 images of the same tissue sections imaged by desi after h&e stain 
 are also shown . our 
 findings demonstrate that desi - msi is an operationally simple 
 yet sensitive and powerful tool for investigating transdermal permeation 
 of small molecules . we have successfully employed high mass resolution 
 desi - msi to visualize the permeation of topically applied sodium channel 
 modulators on ex vivo human skin samples and on live animal skin . these results lead the way for subsequent animal behavioral studies 
 to assess the local antinociceptive effect of these agents when applied 
 in different vehicles . we believe desi - msi methodology has the potential 
 to play a guiding role in topical drug development and formulations 
 research .
determining 
 permeability of a given compound through human skin 
 is a principal challenge owing to the highly complex nature of dermal 
 tissue . 
 we describe the application of an ambient mass spectrometry 
 imaging method for visualizing skin penetration of sodium channel 
 modulators , including novel synthetic analogs of natural neurotoxic 
 alkaloids , topically applied ex vivo to human skin . 
 our simple and label - free approach enables 
 successful mapping of the transverse and lateral diffusion of small 
 molecules having different physicochemical properties without the 
 need for extensive sample preparation .