In this study positive ESI tandem mass spectra of the [M ϩ H] ϩ ions of morphinan alkaloids obtained using an ion trap MS were compared with those from a triple quadrupole MS. This allows to assess the differences of the tandem-in-time versus the tandem-in-space principle, often hampering the development of ESI MS/MS libraries. Fragmentation pathways and possible fragment ion structures were discussed. In order to obtain elemental composition, accurate mass measurements were performed. According to the MS/MS fragmentation pathway, the investigated compounds can be grouped into 4 subsets: (1) morphine and codeine, (2) morphinone, codeinone, and neopinone, (3) thebaine and oripavine, (4) -7] or CE/ESI-MS [8,9], mostly from a forensic point of view. Frequently fragmentation by CID is described and applied as LC/MS/MS. Two reviews highlight the field [10,11]. Some systematic approaches to the setup of libraries have been undertaken, including data for morphine and codeine [12][13][14]. However, none of these studies compared data of ion trap and triple quad systems. Most of the morphinan type substances have been investigated before by electron impact ionization MS [15][16][17]. Tandem MS instrumentation and the principles of operation have been extensively discussed in the literature [18]. In contrast to magnetic sector instruments, triple quadrupole, quadrupole ion trap, and quadrupole time-of-flight are all considered to perform low collision energy fragmentation. Nevertheless, the obtained tandem mass spectra can look remarkably different. Although the structure elucidation potential of CID was early recognized [19], the setup of ESI-MS/MS libraries was hampered by lacking reproducibility. This article provides comparable ion trap and triple quadrupole MS/MS data of all discussed substances. Accurate mass data of selected morphinans were obtained by quadrupole time-of-flight and FT-ICR mass spectrometry, respectively.

Ceramides are sphingolipids consisting of sphingoidbases, which are amide-linked to fatty acids. In the stratum corneum, they represent the major constituent of the free extractable intercellular lipids and play a significant role in maintaining and structuring the water permeability barrier of the skin. Using thin layer chromatography, which represents the method of the first choice in analyzing the stratum corneum ceramides, at least seven classes can be distinguished. Each ceramide class contains various species, which have the same head group and different chain lengths. As in many other skin disorders, atopic dermatitis and psoriasis show derangements in content and profile of the ceramides. Such derangements were reported for both the lesional involved as well as for the normal-appearing uninvolved skin. In this study, we focused on investigating the stratum corneum ceramides of the uninvolved skin in atopic dermatitis and psoriasis patients compared to healthy skin. The aim of the investigations was to explore possible significant and specific differences which can be accomplished for purposes of early diagnostics. The skin lipids were collected by means of an in vivo topical extraction procedure using an extraction mixture consisting of n-hexane and ethanol, (2:1). An automated multiple development-high performance thin layer chromatography (AMD-HPTLC) method with photodensitometric detection were applied to separate the ceramides and to estimate their contents. For studying their molecular profile within each ceramide class, a new method of normal phase HPLC with atmospheric pressure chemical ionization mass spectrometry were used. The results obtained by AMD-HPTLC exposed no significant alterations regarding the relative composition of the major stratum corneum lipids and primarily the ceramides. In addition, the mass spectrometric profiles within each ceramide class were similar in the patients and the healthy control subjects. In conclusion, this study revealed that the normal-appearing uninvolved skin of atopic dermatitis and psoriasis patients does not prove significant or specific deficiencies with respect to the free extractable major stratum corneum lipids and mainly the ceramides, when compared to healthy skin. Thus, they cannot be used for diagnostic purposes. Furthermore, our data are not consistent with the concept that impairments in the ceramide composition represent an obligate etiologic factor for both diseases.

The ceramides of the stratum corneum are critical to maintaining the epidermal barrier function of the skin. A number of skin diseases and disorders are known to be related to impairments of the ceramide pattern. Therefore, obtaining mass spectrometric profiles of the nine ceramide classes known to exist aids our understanding of the underlying molecular mechanisms, which should eventually lead to new diagnostic opportunities: for example, the mass spectrometric profiles of patients suffering from serious skin diseases such as atopic dermatitis and psoriasis can be compared to those of healthy controls. Previous work on mass spectrometric analysis of ceramides relied mostly on GC/MS after hydrolysis and derivatization. The introduction of ESI-MS and LC/ESI-MS has provided new options for directly analyzing intact ceramides. However, some of the ceramide classes are not accessible to ESI-MS. However, as shown in this work, these limitations of GC/MS and ESI-MS can be overcome using a new approach based on normal phase LC interfaced with APCI-MS. Separation and online detection of the stratum corneum ceramide classes became possible in one run. Ceramide species with C26 and/or C28 fatty acid chains were the most abundant ones in Cer [NP], Cer [NH], Cer [AP], and Cer [AH]. The main component of Cer [AS] was C16. The omega-esterified ceramide classes Cer [EOS], Cer [EOP] and Cer [EOH] contained mostly species with fatty acids >C30. This was also the case for Cer [NS], suggesting an analogy to the omega-esterified ceramides. In addition, evidence for a new ceramide class Cer [NdS] was found.

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The molecular mechanisms of skin adaptation to the environmental stress are poorly understood. The aryl hydrocarbon receptor nuclear translocator (Arnt) lies at the intersection of several crucial adaptive pathways. Nevertheless, its role in adaptation of the skin to environmental stress has just begun to be unraveled. Here we show that Arnt is expressed in human and mouse skin in a developmentally dependent manner. Targeted K14-driven deletion of Arnt in the mouse epidermis resulted in early postnatal death, associated with a failure of epidermal barrier function. Gene expression profiling of Arnt-null mouse epidermis revealed upregulation of genes of the epidermal differentiation complex on mouse chromosome 3, including S100a genes (S100a8, S100a9, S100a10) and genes coding for small proline-rich proteins (Sprr1a, Sprr2i, Sprr2j, Sprrl1). HPTLC analysis showed significant accumulation of Cer[NS] and Cer[NH] ceramide species in Arnt-null epidermis, suggesting alterations in lipid metabolism. Continuous retention of corneosomes in Arnt-null epidermis that resulted in an abnormally dense corny layer and impaired desquamation was associated with upregulation of Slpi, an inhibitor of stratum corneum chymotryptic enzyme (SCCE) that plays a key role in corneosome degradation. The functional defects in Arnt-null mouse epidermis underscore the crucial role of Arnt in the maintenance of epidermal homeostasis, especially during the perinatal transition to the ex utero environment.