Evaluation of operational parameters for clinical evaluation of skin hydration by corneometry methodAbstract
The Corneometer CM® 825 from Courage & Khazaka electronic GmbH is widely utilized in clinical efficacy testing of cosmetics. Assessing methodological variables is crucial for ensuring the reliability of the process. The aim of this study was to evaluate the operational and instrumental parameters of the Corneometer CM® 825 in the clinical assessment of skin hydration levels. Precision and accuracy of the measurements were assessed by measuring hydration levels on 10 participants in specific areas of the forearm and face. Three different skin conditions (dry, normal, and hydrated) resulting from the use or non-use of moisturizing cosmetics and a drying promoter were evaluated within 120 minutes of their application. The electrical capacitance method demonstrated good repeatability, with coefficients of variation mostly below 10%. However, higher variability was observed in inter-observer readings. The equipment exhibited high sensitivity and accurately discriminated the differences between hydrated and dry skin. Operational training and standardization of the methodology are essential to ensure accurate, reproducible, and reliable results in studies utilizing the Corneometer equipment for assessing skin hydration levels.
Keywords:
Skin hydration; Non-invasive methods; Cosmetics; Corneometer
INTRODUCTION
Important protective function and appearance of the skin depend on the integrity and hydration of the stratum corneum, influenced by its flexibility and elasticity properties. Therefore, the water content of the stratum corneum is crucial for maintaining healthy-looking skin (Chaudhuri, Bojanowski, 2017; Crowther, 2016).
Consequently, several cosmetic formulations have been developed to contribute to the health, repair, and hydration of the skin (Korać, Krajiłnik, Milić, 2015). Evaluating the efficacy of these products is essential to demonstrate their objectives and benefits (Gonçalves, Campos, 2009).
Non-invasive methods for assessing cosmetic efficacy allow for the quantitative measurement of the biological, functional, and mechanical characteristics of the skin. These biophysical techniques enable the measurement of various skin parameters without causing pain or discomfort to the participants, and they can be evaluated under real conditions of product use (Gonçalves, Campos, 2009; Heinrich et al., 2003; Rogiers et al., 1999).
Among the different instrumental methods, the Corneometer CM® 825 equipment (Courage & Khazaka electronic GmbH), which measures the water content of the stratum corneum, has various cosmetic applications (Clarys et al., 2011). This method is based on electrical capacitance, allowing for quick measurements in different skin conditions with high reproducibility and easy handling (Clarys et al., 2011; Heinrich et al., 2003).
To obtain reliable and reproducible results, it is crucial to standardize and critically evaluate the study conditions and the protocol used in clinical analyses involving non-invasive measurements. Factors such as environmental conditions, inclusion and exclusion criteria, acclimatization period, and duration and frequency of product applications should be considered (Heinrich et al., 2003; Gonçalves, Campos, 2009).
Calibrating the instruments is also an essential procedure for ensuring result precision and accuracy. The calibration process involves a set of operations that establish the correspondence between the values measured by the equipment and the pre-established reference values (Phillips et al., 2001).
Therefore, this study aimed to evaluate the operational and instrumental parameters of the corneometry method for its application in clinical studies assessing skin hydration.
MATERIAL AND METHODS
Measurement principle
The measurements of stratum corneum hydration were conducted using the Corneometer® CM 825 equipment, manufactured by Courage & Khazaka electronic GmbH. This equipment utilizes the electrical capacitance method, employing a low frequency current (40-75 Hz) (Anthonissen et al., 2014; Barel, Clarys, 2013; Berardesca, 1997; Wilhelm, 1998).
To perform the readings, a probe consisting of gold plates separated by a glass slide is used. The probe is covered with a material that has a low dielectric constant. It is placed in contact with the skin surface, applying a constant pressure of 3.5 N. An electric field is then applied, penetrating the stratum corneum. The electric field’s formation depends on the electrodes’ geometry, the dielectric material covering the electrodes (constant capacitance), and the skin capacitance in contact with the electrode surface (variable capacitance). This capacitor system reacts to changes in the dielectric constant caused by the water content present in the stratum corneum. The results are displayed on an arbitrary scale ranging from 0 to 120 arbitrary units (A.U), with higher readings indicating higher levels of hydration (Barel, Clarys, 2013; Berardesca, 1997). Hydration measurements were performed in quintuplicate.
Environmental conditions
Environmental conditions play a significant role in the measurement of stratum corneum hydration, as mentioned in the literature. Air temperature and relative humidity can impact the water content of the stratum corneum. Therefore, in order to ensure reproducible measurements, a constant temperature of 20±2 °C and relative humidity of 50±5% RH were maintained (Anthonissen et al., 2014; Barel, Clarys, 2013; Berardesca, 1997; Wilhelm, 1998).
Prior to instrumental measurements, subjects were left at determined temperature and humidity values for 20 minutes, ensuring that the tested areas were adjusted to the room conditions (Berardesca, 1997; Wilhelm, 1998).
Precision and Accuracy Tests: Experimental Design
This was a comparative, open, randomized and controlled clinical study carried out in an air-conditioned room at the Clinical trial Laboratory of the Cosmetic and Dermatological Science Specialty Center, located at the Faculty of Pharmaceutical Sciences at University of Campinas. To evaluate the precision and accuracy of the equipment, a total of 10 healthy subjects between the ages of 18 and 34, with Fitzpatrick phototypes II, III, and IV (Fitzpatrick et al., 1974), were recruited. Exclusion criteria included cognitive limitations that could hinder the subjects’ understanding of the study objectives and guidelines, as well as a known history of allergic reactions to the components of the formulations.
For operational training of the research group members, in vivo tests of Accuracy and Precision (Intra- and interobserver) were conducted. Additionally, the equipment calibration was verified using an in vitro method (Barel, Clarys, 2013).
Accuracy
The accuracy of a method is the proximity between results obtained and a reference value accepted as true (ANVISA, 2017). For this, measurements were taken under three different conditions: measurements in the area without product application (control area), measurements in the area with application of a drying promoter product (produces superficial skin dryness - Dry area) and measurements in the area with cosmetic application moisturizer (Hydrated area).
Drying promoter product had as constituents: Distilled Water, Aluminum Hydrochloride, Butylated Hydroxytoluene, DMDM Hydantoin, Glycerin, Stearyl Ether, PPG-15-Stearyl Ether, Polyethylene Glycol and Stearyl Alcohol Polymers, Cyclopentasiloxane and Hydroxypropyl almidon phosphate. The moisturizing formulation was composed of: Distilled Water, caprylic/ capric triglyceride, Acrylates - C10-30 alkyl acrylate crosspolymer, Dicapryl Ether, Disodium EDTA, Glycerin, Olus Oil, Sodium Acrylate Copolymer, Tocopheryl Acetate, Phenoxyethanol, Sodium Hydroxide, Stearyl Alcohol, Xanthan Gum and Polyglyceryl 3-Caprylate (commercially available formulation).
To investigate this criterion, a qualitative comparison was conducted between the equipment measurements and the components of each formulation. The purpose was to determine the conformity of the results based on the hydration degree scale provided by the manufacturer (Courage & Khazaka, 2019) as shown in Table I.
Hydration readings were performed before (Baseline time - T0) and after 15 minutes (T15), 30 minutes (T30), 60 minutes (T60) and 120 minutes (T120) product application.
Precision
Precision of an analytical method is indeed defined as the proximity between a series of measurements obtained from the same sample (ANVISA, 2017). It demonstrates the method capability to yield consistent results with low data variability (Ravichandran et al., 2010).
In this study, precision was evaluated at two levels: intraobserver and interobserver.
Intraobserver Precision: Intraobserver precision assesses the analytical procedure under the same operating conditions, with the same analyst and instrumentation, within a short period of time (ANVISA, 2017). The analysis was performed in the areas where the drying promoter product and the moisturizing cosmetic were applied, as well as in the area without any product application. Measurements were taken at the time intervals described in section 5.3.1.
Interobserver Precision: For the evaluation of interobserver precision, measurements taken by two different operators were compared in different anatomical areas (forearm, malar, and forehead) without any product application. This comparison involved 10 subjects from the research group (Anthonissen et al., 2014).
By assessing both intraobserver and interobserver precision, the study aimed to evaluate the consistency and reliability of the method across different conditions and operators. This analysis provides insights into the method’s ability to generate reproducible results and minimize measurement variability.
In vitro calibration check
The calibration check was conducted using an in vitro test with the aid of filter paper placed on a low dielectric plastic sheet, as illustrated in Figure 1. The materials were kept at a temperature of 20º±2°C and a relative humidity of 45% RH - 55% RH. The calibration check involved simulating high hydration measurements by applying the probe to a cellulose filter saturated with a NaCl solution (Figure 1A). Similarly, low hydration values were obtained by taking readings on a polyurethane film (15μm) placed on the cellulose filter impregnated with a saline solution (Figure 1B and 1C). The equipment calibration is considered valid when the measurements remain within the range of 120±5 AU (high value check) and 20±5 AU (low value check) (Courage & Khazaka, 2019).
in vitro calibration method preparation illustrating the saturation of cellulose filter with saline solution (A), positioning of the polyurethane film (B) and probe readings (C).
Areas delimitation
Three areas (20cm²) were delimited on the anterior forearm of the participants as shown in Figure 2. Same distance was measured from the cubital fossa and the hands to avoid contact with veins, tendons or other elements that could interfere with the perpendicular positioning of the probe on skin and potentially result in inaccurate readings. Areas with hair also were avoided (Berardesca, 1997).
To assess the interobserver precision, measurements were also taken on the malar and on the frontal region of the face (Figure 3) with the same probe positioning precautions described for the forearm.
Products application
Products were applied at 2mg/cm² in previously randomized areas.
The application was carried out with standardized movements for 30 seconds, as shown in Figure 4, to obtain a homogeneous distribution in the marked areas. Participants were instructed not to wash the skin test region for at least 2 hours before the measurements (Berardesca, 1997).
Statistical analysis
Statistical analysis was performed respecting the assumptions determined by the results, as well as the guidelines described in ANVISA Guide No. 10 of 2017 entitled “GUIDE FOR STATISTICAL TREATMENT OF ANALYTICAL VALIDATION”. The analyses were performed using GraphPad Prism8 (GraphPad, San Diego, LA, USA).
Accuracy
To evaluate the accuracy of the developed formulations, the means of hydration measurements were compared by the two-way analysis of variance (ANOVA), with post-hoc Tukey. A confidence level of 95% was considered for the analysis.
Percentage difference of means for this parameter was also calculated using the formula:
Qt=measurements taken in a time after product application; Q0=measurements taken before product application (T0); Ap=Area with product application and Ac=Area without product application (control region).
Precision
The coefficient of variation (CV) is a measure of data variability commonly used to express the precision of a method. It is calculated using the following equation (ANVISA, 2017):
RESULTS
Accuracy
The effect of applying moisturizing and drying promoter products on capacitance measurements is shown in Figures 5 and 6. The data was analyzed based on the ingredients present in the formulations and the degree of hydration scale (Table I).
Electrical capacitance values measured in control, hydrated, and dry areas of the participants’ forearms (n=10) at time zero (T0), 15 minutes (T15), 30 minutes (T30), 60 minutes (T60), and 120 minutes (T120) after the application of the products.
Percentage difference in the skin hydration value (%) hydration values obtained in dry and hydrated areas in relation to the control site and participants’ baseline values (n=10) at all time points.
The results clearly demonstrate that the equipment was able to differentiate between the moisturizing and drying promoter products. Significant differences were observed between these products and the control area (skin without product).
When the moisturizing product was applied, hydration measurements higher than 40 AU were obtained. There was a significant percentage increase of 61.84%, 54.26%, 55.18%, and 50.77% at T15, T30, T60, and T120, respectively, compared to the initial time. These findings indicate a high level of skin hydration according to the scale.
On the other hand, the application of the drying promoter product resulted in hydration measurements lower than 30 AU Significant percentage decreases of -51.20%, -52.77%, -43.37%, and -34.45% were observed at T15, T30, T60, and T120, respectively, compared to the baseline time (T0). These results indicate low levels of skin hydration in the dry area according to the scale. These findings highlight the accuracy of the capacitance measurements in detecting and quantifying the effects of different products on skin hydration. The results align with the expected outcomes based on the formulation ingredients and provide valuable information for evaluating the efficacy of the tested products.
Intraobserver precision
This parameter analysis through the coefficient of variation in the three skin conditions (dry, normal and hydrated) is shown in Table II.
Means (n=10) of capacitance measurements (AU) and their respective Coefficients of Variation (CV) in the forearm areas at times zero (T0 - baseline time), 15 minutes (T15), 30 minutes (T30), 60 minutes (T60) and 120 minutes (T120)
Table II shows that the measurements of electrical capacitance (in arbitrary units - AU) obtained at all time intervals in the control and hydrated areas and at times T0 and T120 of dry area resulted in a coefficient of variation lower than 10%. However, in the dry area where the drying promoter was applied, the coefficient of variation values were higher than 10% at T15 (12.70%) and T30 (11.90%). This indicates a relatively higher degree of variability in the capacitance measurements in the presence of the drying promoter.
The coefficient of variation values of the electrical capacitance measurements can also vary depending on the hydration level of the stratum corneum, as shown in Figure 7. In Figure 7 indicates that there is a negative correlation between the coefficient of variation and the electrical capacitance values in the normal, dry, and hydrated areas at times T15, T30, and T60. The correlation coefficients (r) obtained for areas A, B, and C were -0.61, -0.60, and -0.42, respectively.
Coefficient of Variation (%) calculated based on Eletrical Capacitance Value (A.U.) of the participants (n=10) at T15 (A), T30 (B), T60 (C) and T120 (D).
It is verified that less negative correlation values (r) are obtained with time as capacitance levels increase in the dehydrated area (Table III). At T4, a weak positive correlation was observed (r=0.13) and thus, a smaller dispersion of the coefficients of variation, as shown in Figure 7D.
Coefficients of variation (%) of measurements obtained by two different operators in the forearm, malar and forehead regions of 10 participants (P)
Interobserver Precision
The interobserver precision evaluation through the coefficient of variation analysis is shown in Table III.
Most coefficients of variation obtained by measurements of two operators on the forearm, malar and forehead remained below 10%. It is also inferred that the data variability obtained in the interobserver precision was higher than in the intraobserver study.
In vitro method: calibration check
In this study, calibration check of Corneometer® CM 825 probe was performed as described by Courage & Khazaka (2019). A second check was performed consecutively using the same filter and polyurethane film as the first check. The results are shown in Table IV.
Electrical capacitance values obtained in the first check remained within the limit established by the manufacturer, indicating that the probe is suitable for use. However, the measurements obtained in the second check were below the reference range: 120±5 AU (high value check) and 20±5 AU (low value check).
DISCUSSION
Efficiency evaluation of a method serves several purposes, such as verifying the laboratory’s capacity to produce consistent results comparable to certified laboratories, assessing modified or newly acquired equipment, and incorporating new techniques (Amarante et al., 2001; Pum, 2019). This process is crucial in laboratory routines to prevent unnecessary repetition and testing, thus saving valuable resources and time. To achieve this, it is essential to identify relevant parameters, establish acceptance criteria, select an appropriate methodology, and consider the analyst’s expertise in all the factors being studied (Ravichandran et al., 2010).
Several parameters can be evaluated during the process, including specificity, linearity, calibration curve, precision, accuracy, limit of detection and quantification, robustness, and sensitivity. The selection of each parameter depends on the type of method and its intended application (Amarante et al., 2001; Ravichandran et al., 2010).
According to the literature, the Corneometer® CM 825 has been shown to exhibit high reproducibility and sensitivity in measuring skin hydration levels under various skin conditions (Barel, Clarys, 2013; Gonçalves, Campos, 2009).
Therefore, this method was employed to assess the accuracy and precision of quantifying the water content of the stratum corneum after the application of different cosmetic products. Moisturizing cosmetics are an integral part of a skincare routine aimed at enhancing skin smoothness, hydration, and overall appearance. These products can function as occlusives and/or humectants. Occlusive substances, such as hydrocarbons, silicones, or vegetable and animal fats, are designed to create a barrier on the skin’s surface, preventing the loss of water through the epidermis. On the other hand, humectant ingredients act as moisture-attracting agents due to their hydroxyl groups, which possess hygroscopic properties and help attract and retain water molecules (Draelos, 2018; Møss, 1996; Nolan, Marmur, 2012).
Thus, it is observed that the presence of emollient and humectant components in the product, such as caprylic/capric triglyceride and glycerol, contributed to the significant increase in electrical capacitance values after 15, 30, 60 and 120 minutes of its application.
The method’s accuracy was further verified by quantifying low hydration levels. In this case, a drying promoter formulation containing aluminum hydrochloride was applied to these specific areas. Aluminum hydrochloride is an active ingredient commonly used in antiperspirants, working by blocking the sweat gland ducts and forming a plug composed of precipitated salts and damaged cells (Darbre, 2005; Piérard et al., 2003). However, despite its widespread use in such formulations, aluminum salts can constrict pores by denaturing proteins, potentially sensitizing the skin (Kirtschig, Schaefer, 2015). As a response to the topical sensitizer’s effects, the skin’s barrier function may become compromised, leading to increased transepidermal water loss and dehydration of the stratum corneum (Proksch, Brandner, Jensen, 2008). The precision study evaluated the proximity of different measurements. The majority of coefficient of variation (CV) values were below 10%, indicating low variability between measurements and, consequently, good repeatability of the equipment (Anthonissen et al., 2014; Barel, Clarys, 2013). However, higher CV values were observed at T15 and T30 in areas where the drying promoter was applied.
This increased variability could be attributed to the non-absorption of the product after 15 and 30 minutes, resulting in the formation of a residual layer of white powder, as illustrated in Figure 8. As described in the literature, substances such as non-absorbed dipolar components present between the probe and the skin can interfere with equipment readings and should be taken into account during result analysis. According to the manufacturer’s guidelines, measurements should ideally be performed at least 60 minutes after product application. Within a shorter time interval, the equipment may measure the water content of the formulation rather than the water content of the stratum corneum (Courage & Khazaka, 2019). Hence, the combination of non-absorption of the product and inadequate reading time may have contributed to the observed variability in the measurements of the area with the negative control.
However, according to the study conducted by Barel and Clarys (2013), the significant reduction in coefficient of variation (CV) values observed in higher capacitance measurements may be attributed to the lower sensitivity of the equipment towards high hydration levels. As a result, the equipment may detect differences with less precision in these readings. Figure 7D demonstrates a low correlation (r=0.13) between the CV values of dry, hydrated, and control areas (Table II).
The assessment of measurement variations performed by two or more operators on the same sample or subject, known as interobserver precision, is another important aspect to consider when evaluating the suitability of the method. Tests examining this parameter are often influenced by differences in knowledge and experience among laboratory participants regarding the methods and equipment used (ANVISA, 2017).
In such analyses, higher CV values are expected due to the presence of interobserver variability. This variability combines error sources observed in the intraobserver analysis with variations resulting from measurements taken by different operators (Anthonissen et al., 2014). These deviations can be caused by factors like slight differences in probe positioning on the test area or the influence of pressure applied to the probe (operator effect). Therefore, variations arising from interobserver measurements can be minimized through operator training (Anthonissen et al., 2014; Clarys, Clijsen, Barel, 2011). Furthermore, it is worth noting that the coefficients of variation (CV) values were slightly higher in the forehead region compared to the forearm and cheekbones. Previous studies have demonstrated that different anatomical regions exhibit varying degrees of hydration, with the forehead region typically displaying higher electrical capacitance values (Berardesca, 1997; Marrakchi, Maibach, 2007; Wilhelm, 1998). While a lower variability in measurements was expected in this area, as discussed earlier, other factors such as the presence of hair, heterogeneity of facial topography, and individual characteristics may have made it challenging to position the probe perpendicular to the surface or maintain consistent contact with the skin, potentially leading to higher inter-observer variation coefficient values (Lodén, Hagforsen, Lindberg, 1995; Wilhelm, 1998).
Therefore, based on the results reported in the scientific literature (Anthonissen et al., 2015; Barel, Clarys, 2013), the coefficients of variation obtained from the precision analysis described in this study were very similar to those found in other reputable research institutes. This similarity indicates that the corneometry method is being accurately applied by the members of the research group.
In addition to operational variables, instrumental factors such as calibration checks should be considered to ensure reliable readings. In an in vitro calibration evaluation using a model, Barel and Clarys (1997) observed a high correlation between electrical capacitance measurements and the amount of water adsorbed on the filter. This correlation exhibited linearity even for smaller amounts of water, with a plateau value being reached at high degrees of hydration. Furthermore, maximum electrical capacitance values were obtained through measurements on paper filters with varying compositions and thicknesses (ranging from 70 to 1000 pm) saturated with water or saline solutions of ionic or non-ionic character.
Using this method, Clarys et al. (2011) conducted tests to determine the depth reached by the field formed by the equipment’s probe, measuring hydration values in 0 to 5 layers of polyurethane films. In their study, they observed a stray field penetration of up to 45 μm, which exceeded the limit reported by the manufacturer (10-20 μm).
Currently, this methodology is employed to verify equipment calibration. However, in this study, it was observed that the results of the second calibration check did not align with the reference parameters. This discrepancy could be attributed to the reuse of the material employed in the calibration procedure. According to the manufacturer’s recommendations, the polyurethane sheet should ideally be replaced with each calibration check, as repeated measurements can cause the plastic film to become porous.
Therefore, it is crucial to ensure the cleanliness and quality of the material before performing the calibration check to avoid inaccurate results (Courage & Khazaka, 2019).
Thus, this study underscores the significance of establishing a protocol to validate the quality and reliability of the results, considering various variables associated with the equipment, its usage, and the individual anatomical characteristics of the subjects under evaluation. The proposed parameters can be applied to assess the methodology in clinical studies with diverse objectives that utilize the corneometry method.
CONCLUSION
Operational training and the comprehensive assessment of multiple parameters during tests are crucial to guarantee accurate, reproducible, and reliable results in clinical trials that utilize the Corneometer equipment for skin hydration assessment.
ACKNOWLEDGEMENT
This work received support from the São Paulo Research Foundation (FAPESP-2020/12443-8) and the National Council for Scientific and Technological Development (CNPq).
REFERENCES
- Amarante O, Caldas E, Brito N, Santos T, Vele M. Validação de métodos analíticos: uma breve revisão. Cad Pesq. 2001; 12: 116-131.
- Anthonissen M, Daly D, Peeters R, Van Brussel M, Fieuws S, Moortgat P, et al. Reliability of Repeated Measurements on Post-Burn Scars with Corneometer CM 825®. Skin Res Technol. 2015; 21: 302-12.
- ANVISA - Agência Nacional De Vigilância Sanitária (Brasil). Resolução nº 166, de 24 de julho de 2017. Dispõe sobre a validação de métodos analíticos e dá outras providências. Diário Oficial da União, 25 jun 2017.
- Barel A, Clarys P. Skin Capacitance. In: Berardesca E, Maibach H, Wilhelm K, editors. Non Invasive Diagnostic Techniques In Clinical Dermatology. 1st ed. Springer Berlin, Heidelberg. 2013. p. 357-366.
- Berardesca E. EEMCO guidance for the assessment of stratum corneum hydration: electrical methods. Skin Res Technol. 1997; 3: 126-32.
- Chaudhuri R, bojanowski K. Improvement of hydration and epidermal barrier function in human skin by a novel compound isosorbide dicaprylate. Int J Cosmet Sci. 2017; 39: 518-526.
- Clarys P, Clijsen R, Taeymans J, Barel AO. Hydration measurements of the stratum corneum: comparison between the capacitance method (digital version of the Corneometer CM 825®) and the impedance method (Skicon-200EX®). Skin Res Technol. 2011; 18: 316-23.
- Clarys P, Clijsen R, Barel AO. Influence of probe application pressure on in vitro and in vivo capacitance (Corneometer CM 825®) and conductance (Skicon 200 EX®) measurements. Skin Res Technol. 2011; 17: 445-50.
-
Courage & Khazaka Eletronic Gmbh. Manual Corneometer® CM 825. Germany, 2019. Available from: https://www.courage-khazaka.de/en/downloads-en/item/prospekt-cm825-e
» https://www.courage-khazaka.de/en/downloads-en/item/prospekt-cm825-e - Crowther J. Understanding effects of topical ingredients on electrical measurement of skin hydration. Int J Cosmet Sci. 2016; 38: 589-598.
- Darbre PD. Aluminium, antiperspirants and breast cancer. J inorg biochem. 2005; 99: 1912-9.
- Draelos ZD. The science behind skin care: Moisturizers. J Cosmetic Dermatol. 2018; 17: 138-44.
- Fitzpatrick TB, Pathar M, Parrish J. Protection of human skin against the effects of the sunburn ultraviolet (290-320 nm). In: Fitzpatrick, TB, editor. Sunlight and man, normal and abnormal photobiological responses. Tokyo: Etials, 1974. p. 751-765.
- Gonçalves GMS, Campos PMBGM. Aplicação de métodos de biofísica no estudo da eficácia de produtos dermocosméticos. Braz J Pharm Sci. 2009; 45: 1-10.
- Heinrich U, Koop U, Leneveu-Duchemin M-C, Osterrieder K, Bielfeldt S, Chkarnat C, et al. Multicentre comparison of skin hydration in terms of physical-, physiological- and product-dependent parameters by the capacitive method (Corneometer CM 825). Int J Cosmet Sci. 2003; 25: 45-53.
- Kirtschig G, Schaefer C. Dermatological medications and local therapeutics. Drugs During Pregnancy and Lactation. 2015: 467-92.
- Korać R, Krajišnik D, Milić J. Sensory and instrumental characterization of fast inverting oil-in-water emulsions for cosmetic application. Int J Cosmet Sci. 2015; 38: 246-56
- Lodén M, Hagforsen E, Lindberg M. The presence of body hair influences the measurement of skin hydration with the Corneometer. Acta Derm Venereol. 1995; 75(6): 449-50.
- Marrakchi S, Maibach HI. Biophysical parameters of skin: map of human face, regional, and age-related differences. Contact Derm. 2007; 57(1): 28-34.
- Møss J. The effect of 3 moisturisers on skin surface hydration: Electrical conductance (Skicon-200), capacitance (Corneometer CM420), and transepidermal water loss (TEWL). Skin Res Technol. 1996; 2(1): 32-36.
- Nolan K, Marmur E. Moisturizers: reality and the skin benefits. Dermatol Ther. 2012; 25(3): 229-33.
- Phillips SD, Estler WT, Doiron T, Eberhardt KR, Levenson MS. A Careful Consideration of the Calibration Concept. J Res Natl Inst Stand Technol. 2001; 106(2): 371-9.
- Piérard GE, Elsner P, Marks R, Masson P, Paye M, EEMCO Group. EEMCO guidance for the efficacy assessment of antiperspirants and deodorants. Skin Pharmacol Appl Skin Physiol. 2003; 16(5): 324-42.
- Proksch E, Brandner JM, Jensen JM. The skin: an indispensable barrier. Exp Dermatol. 2008; 17(12): 1063-72.
- Pum J. A practical guide to validation and verification of analytical methods in the clinical laboratory. Adv Clin Chem. 2019; 90: 215-281.
- Ravichandran V, Shalini S, Sundram K, Harish R. Validation of analitical methods -strategies and importance. Int J Pharm Pharm. 2010; 2(3): 18-22.
- Rogiers V, Balls M, Basketter D, Berardesca E, Edwards C, Elsner P, et al. The potential use of non-invasive methods in the safety assessment of cosmetic products. Altern Lab Anim. 1999; 27(4): 515-37.
- Wilhelm K-P. Possible Pitfalls in Hydration Measurements. Curr Probl Dermatol. 1998; 223-34.
