Optimized precursor ion selection for labile ions in a linear ion trap mass spectrometer and its impact on quantification using selected reaction monitoring

The fragmentation of fragile ions during the application of an isolation waveform for precursor ion selection and the resulting loss of isolated ion intensity is well‐known in ion trap mass spectrometry (ITMS). To obtain adequate ion intensity in the selected reaction monitoring (SRM) of fragile precursor ions, a wider ion isolation width is required. However, the increased isolation width significantly diminishes the selectivity of the channels chosen for SRM, which is a serious problem for samples with complex matrices. The sensitive and selective quantification of many lipid molecules, including ceramides from real biological samples, using a linear ion trap mass spectrometer is also hindered by the same problem because of the ease of water loss from protonated ceramide ions. In this study, a method for the reliable quantification of ceramides using SRM with near unity precursor ion isolation has been developed for ITMS by utilizing alternative precursor ions generated by in‐source dissociation. The selected precursor ions allow the isolation of ions with unit mass width and the selective analysis of ceramides using SRM with negligible loss of sensitivity. The quantification of C18:0‐, C24:0‐ and C24:1‐ceramides using the present method shows excellent linearity over the concentration ranges from 6 to 100, 25 to 1000 and 25 to 1000 nM, respectively. The limits of detection of C18:0‐, C24:0‐ and C24:1‐ceramides were 0.25, 0.25 and 5 fmol, respectively. The developed method was successfully applied to quantify ceramides in fetal bovine serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultraperformance convergence chromatography‐high resolution tandem mass spectrometry for lipid biomarker profiling and identification

Lipids represent biologically ubiquitous and highly dynamic molecules in terms of abundance and structural diversity. Whereas the potential for lipids to inform on disease/injury is promising, their unique characteristics make detection and identification of lipids from biological samples analytically demanding. We report the use of ultraperformance convergence chromatography (UPC²), a variant of supercritical fluid chromatography, coupled to high‐resolution, data‐independent tandem mass spectrometry for characterization of total lipid extracts from mouse lung tissue. The UPC² platform resulted in lipid class separation and when combined with orthogonal column chemistries yielded chromatographic separation of intra‐class species based on acyl chain hydrophobicity. Moreover, the combined approach of using UPC² with orthogonal column chemistries, accurate mass measurements, time‐aligned low‐ and high‐collision energy total ion chromatograms, and positive and negative ion mode product ion spectra correlation allowed for confident lipid identification. Of great interest was the identification of differentially expressed ceramides that were elevated 24 h post whole thorax lung irradiation. The identification of lipids that were elevated 24 h post‐irradiation signifies a unique opportunity to investigate early mechanisms of action prior to the onset of clinical symptoms in the whole thorax lung irradiation mouse model.     

A Practical Approach for the In Vitro Safety and Efficacy Assessment of an Anti-Ageing Cosmetic Cream Enriched with Functional Compounds

(1) Background: Cosmeceuticals are topical products applied to human skin to prevent skin ageing and maintain a healthy skin appearance. Their effectiveness is closely linked to the compounds present in a final formulation. In this article, we propose a panel of in vitro tests to support the efficacy assessment of an anti-ageing cream enriched with functional compounds. (2) Methods: biocompatibility and the irritant effect were evaluated on reconstructed human epidermis (RHE) and corneal epithelium (HCE) 3D models. After a preliminary MTT assay, normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT) were used to evaluate the extracellular matrix (ECM) protein synthesis, and interleukin-6 (IL-6) and metalloproteinase-1 (MMP-1) production. (3) Results: data collected showed good biocompatibility and demonstrated the absence of the irritant effect in both 3D models. Therefore, we demonstrated a statistical increase in collagen and elastin productions in NHDF cells. In HaCaT cells, we highlighted an anti-inflammatory effect through a reduction in IL-6 levels in inflammatory stimulated conditions. Moreover, the reduction of MMP-1 production after UV-B radiation was demonstrated, showing significant photo-protection. (4) Conclusion: a multiple in vitro assays approach is proposed for the valid and practical assessment of the anti-ageing protection, anti-inflammatory and biocompatible claims that can be assigned to a cosmetic product containing functional compounds.     

High-throughput screening assay for the quantification of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 in HepG2 cells using RapidFire mass spectrometry

Ceramides are known to be involved in various biological processes with their physiological levels elevated in various disease conditions such as diabetes, Alzheimer's, atherosclerosis. To facilitate the rapid screening of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 inhibition in HepG2 cells, a RapidFire coupled to tandem mass spectrometry (RF–MS/MS) method has been developed. The RF platform provides an automated solid-phase extraction system that gave a throughput of 12.6 s per sample to an MS/MS system using electrospray ionization under the positive ion mode. Chromatographic separation of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 was achieved using a ternary gradient on C₈ type E cartridge. The MS/MS ion transitions monitored were 538.2 → 264.2, 650.7 → 264.2, 648.6 → 264.2, 566.4 → 264.2, 510.4 → 264.2, 594.4 → 264.2, 622.5 → 264.2, and 552.3 → 250.2 for Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, d18:1/22:0, and the internal standard (Cer d17:1/18:0), respectively. The RF–MS/MS methodology showed an excellent performance with an average Z′ value of 0.5–0.7. This is the first report of an RF–MS/MS assay for screening of ceramides which is amenable for high-throughput screening.     

Determination of the serine palmitoyl transferase inhibitor myriocin by electrospray and Q‐trap mass spectrometry

Myriocin is a potent inhibitor of serine‐palmitoyl‐transferase, the first and rate‐determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC–MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14‐OH–myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid‐phase extraction, separated by gradient reversed‐phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14‐OH‐myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r ² = 0.9996). The intra‐ and between‐day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra‐tracheal delivery. Measured levels ranged from 4.11 (median; 2.3–7.4 IQR, n = 4) to 11.7 (median; 7.6–22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.     

Isolation,structural determination and cytotoxic activity of two new ceramides from the root of <Emphasis Type="Italic">Isatis indigotica</Emphasis>

Two new ceramides were isolated from the 95% EtOH extract of traditional Chinese medicinal plant Isatis indigotica. Their structures were elucidated as 1-O-β-D-glucopyranosyl-(2S, 3R)-N-(2′-hydroxype-ntacosanoyl)-octadeca-11E-sphingenine (1) and 1-O-β-D-glucopyranosyl-(2S,3R)-N-(2′-hydroxyhe xacosanoyl)-octadeca-11E-sphingenine (2) on the basis of spectroscopic data. Their cytotoxic effects were evaluated by using MTT method.     

Isolation,structural determination and cytotoxic activity of two new ceramides from the root of Isatis indigotica

Two new ceramides were isolated from the 95% EtOH extract of traditional Chinese medicinal plant Isatis indigotica. Their structures were elucidated as 1-O-β-D-glucopyranosyl-(2S, 3R)-N-(2'-hydroxypentacosanoyl)-octadeca-11E-sphingenine (1) and 1-O-β-D-glucopyranosyl-(2S,3R)-N-(2'-hydroxyhe xacosanoyl)-octadeca-11E-sphingenine (2) on the basis of spectroscopic data. Their cytotoxic effects were evaluated by using MTT method.     

Optical Control of Cytokine Production Using Photoswitchable Galactosylceramides

α-Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro-inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN-7000, the activity of which can be modulated with light. Based on established structure–activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro-inflammatory cytokines, such as IFN-γ. The azobenzene derivative α-GalACer-4 proved to be more potent than KRN-7000 itself when activated with 370 nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

Synthesis and stereochemistry of ceramide B, (2S,3R,4E,6R)-N-(30-hydroxytriacontanoyl)-6-hydroxy-4-sphingenine, a new ceramide in human epidermis

(2S,3R,4E,6R)-N-(30-Hydroxytriacontanoyl)-6-hydroxy-4-sphingenine (1) and its (6S)-isomer (1′) were synthesized by starting from pentadecan-15-olide, the enantiomers of 1-pentadecyn-3-ol, and (S)-Garner's aldehyde. Comparison of the ¹H NMR spectra of the tetraacetyl derivatives of 1 and 1′ with that of ceramide B, a new protein-bound ceramide in human stratum corneum, revealed it to be (2S,3R,4E,6R)-1.