Discrimination and quantification of two isomeric antineoplastic drugs by rapid and non-invasive analytical control using a handheld Raman spectrometer.

Raman spectroscopy is a rapid, non-destructive and non-invasive method that is a promising tool for real-time analytical control of drug concentrations. This study evaluated a handheld Raman device to discriminate and quantify two isomeric drugs used to treat cancer. Doxorubicin (DOXO) and epirubicin (EPIR) samples were analyzed at therapeutic concentrations from 0.1 to 2mg/mL (n=90) and 0.08-2mg/mL (n=90) by non-invasive measurements using a portable Raman spectrometer. The discrimination of these two molecules was demonstrated for all concentrations (n=180) by qualitative analysis using partial least square discriminant analysis (PLS-DA) with 100% classification accuracy, sensitivity and specificity and 0% error rate. For each molecule, quantitative analyses were performed using PLS regression. The validity of the model was evaluated using root mean square error of cross validation (RMSECV) and prediction (RMSEP) that furnished 0.05 and 0.02mg/mL for DOXO and 0.17 and 0.16mg/mL for EPIR after pretreatment optimization. Based on the accuracy profile, the linearity range was from 1.256 to 2.000mg/mL for DOXO (R2=0.9988) and from 0.553 to 2.000mg/Ml for EPIR (R2=0.9240) and repeatability (CV% max of 1.8% for DOXO and 3.2% for EPIR) and intermediate precision (CV% max of 2.8% for DOXO and 4.5% for EPIR) were both acceptable. Despite the narrow validated concentration range for quantitative analysis, this study shows the potential of a handheld Raman spectrometer coupled to chemometric approaches for real-time quantification of cytotoxic drugs, as well for discriminating between two drugs with similar UV absorption profiles. Finally, the use of a handheld spectrometer with the possibility of a direct measurement of substances in containers is a potentially valuable tool for combining patient safety with security of healthcare workers.

Examination of the effect of Stratum Corneum isolation process on the integrity of the barrier function: a confocal Raman spectroscopy study.

BACKGROUND: Stratum Corneum (SC) is the most superficial layer of the epidermis. It plays the main barrier role against water loss and the aggression of external chemical and biological substances. Thermal treatment in warm purified water followed by trypsin incubation of excised human skin is a well-established in vitro method for SC removal. Different protocols can be found in literature, but little is described about the effect of temperature and trypsin during isolation process on its barrier function. METHODS: In this study, we have examined the epidermis and SC matrix structural change upon isolation by means of confocal Raman spectroscopy. RESULTS: Several spectral features, i.e. in-depth and planar lateral packing, conformational order and secondary structure have been investigated to reveal modifications in the structural properties of the lipids and proteins in the SC. Thermal treatment at 60°C leads to a losing in compactness and a steeper concentration of the lipid and protein descriptors while the trypsinization step modifies the organization of the proteins and of the lipid barrier, leading to a domain organization. CONCLUSIONS: The present study improves the knowledge of the effects on the barrier function of SC removal protocol.

Age-dependent changes in stratum corneum barrier function.

BACKGROUND/PURPOSE: The Stratum Corneum (SC) barrier function mainly depends on the SC structure at the tissue level, its composition, and the organization of intercellular lipidic cement at the molecular level. The goal of this study was to assess the age-dependent changes of the SC barrier function and the associated physiological parameters. METHODS: This study was conducted on 40 French women divided into four groups of age. Measurements were done on three sites: cheek, protected, and exposed arm sites. SC composition (water, lipid/protein ratio, cholesterol, and ceramides) was measured using Raman confocal microspectroscopy, skin surface hydration using skin conductance, and barrier function through transepidermal water loss (TEWL) measurements. RESULTS: Transepidermal water loss decreases slightly with age, which is partially explained by the age-dependent increase in SC thickness. This decrease is faster for the face compared to both arm sites. The lipid to protein ratio and lipid compactness decrease significantly with age only for the arm sites. Water concentration profiles only decrease very close to the skin surface. At all ages tested, the SC on the cheek showed significantly higher TEWL, water and lipid content and less thickness compared to the arm sites. Comparison of the exposed to unexposed arm site showed difference only for the lipid compactness at the older group studied. CONCLUSION: Skin aging, body site and environmental exposure can affect the SC barrier function, its structure, and its lipid content. The thickening of the SC with age compensates for the decrease of the quantity and ordering of the lipidic cement.

Vibrational spectroscopy coupled to classical least square analysis, a new approach for determination of skin moisturizing agents' mechanisms.

BACKGROUND: Skin dryness is an omnipresent symptom in various types of skin disorders. Thereby, a large panel of skin care products is developed for therapeutic purposes. However, there is still a lack of non-invasive methods to determine the mechanisms of action of moisturizers at the molecular level. METHODS: In the present study, confocal Raman spectroscopy coupled to classical least square analyses and ATR-FTIR were used to investigate the effect of different molecules on the stratum corneum (SC) hydration degree and barrier state. First, hygroscopic property was determined by analyzing samples at 90% RH; secondly, the water barrier function was evaluated after the dehydration process (4% RH). The molecules penetration kinetics across SC were also studied for 2 h. RESULTS: Using the present approach, glycerin and propylene glycol were found to be humectants; lanoline showed occlusive action, lactic acid has both humectant and barrier enhancer properties, and ethylhexyl palmitate and caprylic/capric acid triglyceride seemed to be emollients. These observations are in accordance with literature. CONCLUSION: The present method non-invasively characterizes the mechanism of action of tested molecules. This may improve knowledge of new molecules' structure-activity relationship and help make an effective therapeutic concept dealing with the various skin dysfunctions.

Development of a percutaneous penetration predictive model by SR-FTIR.

This work focused on developing a new evaluation criterion of percutaneous penetration, in complement to Log Pow and MW and based on high spatial resolution Fourier transformed infrared (FTIR) microspectroscopy with a synchrotron source (SR-FTIR). Classic Franz cell experiments were run and after 22 h molecule distribution in skin was determined either by HPLC or by SR-FTIR. HPLC data served as reference. HPLC and SR-FTIR results were compared and a new predictive criterion based from SR-FTIR results, named S(index), was determined using a multi-block data analysis technique (ComDim). A predictive cartography of the distribution of molecules in the skin was built and compared to OECD predictive cartography. This new criterion S(index) and the cartography using SR-FTIR/HPLC results provides relevant information for risk analysis regarding prediction of percutaneous penetration and could be used to build a new mathematical model.

Physical and chemical perturbations of the supramolecular organization of the stratum corneum lipids: in vitro to ex vivo study.

BACKGROUND: FTIR spectroscopy is classically used to study the supramolecular organization of the stratum corneum lipids. Exposure to UV(A) is responsible for a small decrease in packing observed on cutaneous lipid films. METHODS: Lipid films and human skin biopsies were either exposed to UV(A) irradiation of 120 J/cm(2), UV(B) irradiation of 0.15 J/cm(2) or put in contact with ethanol. Using FTIR in vitro and IR microspectroscopy ex vivo provided information on: i) the precise localisation of the stratum corneum in the skin, ii) its thickness, and iii) the organization of its constituted lipids. RESULTS: Different action modes were observed for UV irradiation and the contact with ethanol with a certain destabilisation of the lipidic layer. Ethanol was also found to be responsible for the creation of pores. The destabilisation of the lipid cement was mainly observed ex vivo. CONCLUSION: The barrier function of the skin is affected by the action of physical and chemical external agents at the molecular level. The increased laxity of the lipid packing could enable the percutaneous penetration velocity of actives.

Spectral monitoring of photoirradiated skin lipids: MS and IR approaches.

An investigation of the effects of UV(A) irradiation on the stratum corneum lipids was carried out in vitro on films. The modifications of their conformational order were studied by FTIR and the formation of new entities was detected by mass spectroscopy. The results show not only differences in behaviour of the three lipid classes (fatty acids (FA), ceramides (CER), and cholesterol), but also variation within a class, depending on the molecules structure. Upon UV(A) irradiation, beta scission occurs on all the components, saturated and unsaturated. Moreover, unsaturated FA or CER having a double bond on their FA moiety may become saturated or may be transformed into their free radical form. Unsaturated FA are more sensitive to UV(A) and lead more easily to oxygenated components than unsaturated CER. The chemical effects are irradiation dose dependent but do not deeply influence the supramolecular organisation of these lipids. The global conformation of the lipids stays in an orthorhombic state, a decrease of the packing density however is observed.

Nuclear magnetic resonance spectrometry and liquid chromatography of two bile acid epimers: ursodeoxycholic and chenodeoxycholic acid.

A specific reverse-phase high-performance liquid chromatographic (HPLC) technique is described for the analysis of bile acids and their conjugates in human serum. Precise quantitation is obtained using UV detection. 13C-NMR spectrometry suggests a structural explanation for the different HPLC retention times of chenodeoxycholic acid, its epimer (ursodeoxycholic acid), and their methyl esters.

[Not Available]. / Identification et dosage des sels biliaires et de leurs derives conjugues en chromatographie liquide.

Detection and determination of bile acids and their glycine- and taurine-conjugated derivatives are realized by reversed-phase liquid chromatography either directly or after ion-pair formation. Operating conditions and the relation between capacity factors and structure are investigated. The determination of bile acids extracted from biological samples is possible by these techniques.