Overview of skin whitening agents with an insight into the illegal cosmetic market in Europe.

Lightening skin tone is an ancient and well-documented practice, and remains common practice among many cultures. Whitening agents such as corticosteroids, tretinoin and hydroquinone are medically applied to effectively lighten the skin tone of hyperpigmented lesions. However, when these agents are used cosmetically, they are associated with a variety of side-effect. Alternative agents, such as arbutin and its derivatives kojic acid and nicotinamide have been subsequently developed for cosmetic purposes. Unfortunately, some cosmetics contain whitening agents that are banned for use in cosmetic products. This article provides an overview of the mode of action and potential side-effects of cosmetic legal and illegal whitening agents, and the pattern of use of these types of products. Finally, an EU analysis of the health problems due to the presence of illegal products on the market is summarized.

A strategy for the identification of plants in illegal pharmaceutical preparations and food supplements using chromatographic fingerprints.

The detection of regulated and forbidden herbs in pharmaceutical preparations and nutritional supplements is a growing problem for laboratories charged with the analysis of illegal pharmaceutical preparations and counterfeit medicines. This article presents a feasibility study of the use of chromatographic fingerprints for the detection of plants in pharmaceutical preparations. Fingerprints were developed for three non-regulated common herbal products--Rhamnus purshiana, Passiflora incarnata L. and Crataegus monogyna--and this was done by combining three different types of detection: diode-array detection, evaporative light scattering detection and mass spectrometry. It is shown that these plants could be detected in respective triturations of the dry extracts with lactose and three different herbal matrices as well as in commercial preparations purchased on the open market.

Chromatographic impurity fingerprinting of genuine and counterfeit Cialis® as a means to compare the discriminating ability of PDA and MS detection.

Public health is threatened worldwide by counterfeit medicines. Their quality, safety and efficacy cannot be guaranteed since no quality control is performed during and/or after the manufacturing process. Characterization of these products is a very important topic. During this study a High Performance Liquid Chromatography-Photodiode Array (HPLC-PDA) and a High Performance Liquid Chromatography - Mass Spectrometry (HPLC-MS) method were developed to analyse both genuine and counterfeit samples of Cialis®. The obtained PDA and MS fingerprints were explored and modelled using unsupervised Principal Component Analysis (PCA) and supervised Partial Least Squares and its discriminant variant (PLS, PLS-DA) as well the classification methods including Soft Independent Modelling of Class Analogy (SIMCA) and the k Nearest Neighbour classifier (kNN). Both MS1 and MS2 data and data measured at 254 nm and 270 nm were used with the aim to test the potential complementarity of PDA and MS detection. First, it was checked if both groups of fingerprints can support differentiation between genuine and counterfeit medicines. Then, it was verified if the obtained multivariate models could be improved by combining information present in MS and PDA fingerprints. Survey of the models obtained for the 254 nm data, 270 nm data and 254_270 nm data combination showed that a tendency of discrimination could be observed with PLS. For the 270 nm data and 254_270 nm data combination a perfect discrimination between genuine and counterfeit medicines is obtained with PLS-DA and SIMCA. This shows that 270 nm alone performs equally well compared to 254_270 nm. For the MS1 and MS1_MS2 data perfect models were obtained using PLS-DA and kNN, indicating that the MS2 data do not provide any extra useful information to acquire the aimed distinction. When combining MS1 and 270 nm perfect models were gained by PLS-DA and SIMCA, which is very similar to the results obtained for PDA alone. These results show that both detectors have a potential to reveal chemical differences between genuine and counterfeit medicines and thus enable the construction of diagnostic models with excellent recognition. However, if a larger sample set, including more possible sources of variation, is analysed more sophisticated techniques such as MS might be necessary.

Development of a Stationary Phase Optimised Selectivity Liquid Chromatography based screening method for adulterations of food supplements for the treatment of pain.

Illegally adulterated dietary supplements are an increasing problem worldwide. One of the important groups of often adulterated products are the dietary supplements, sold for the treatment of pain. These often contain analgesics, a heterogeneous group of molecules, containing both hydrophilic and hydrophobic compounds. The development of a screening method for these components, especially when mass spectrometric detection is not available, necessitates chromatographic separation, difficult to achieve with traditional chromatographic columns. In this paper Stationary Phase Optimised Selectivity Liquid Chromatography was used for the development of a screening method for nine analgesics, codeine and caffeine, often present in this type of dietary supplements. The method shows a good separation of all the compounds, allowing the screening to be performed with diode array detection and is fully compatible with mass spectrometry. The method was validated for its selectivity following the guidelines as described for the screening of pesticide residues and residues of veterinary medicines in food.

HS-GC-MS method for the analysis of fragrance allergens in complex cosmetic matrices.

Potential allergenic fragrances are part of the Cosmetic Regulation with labelling and concentration restrictions. This means that they have to be declared on the ingredients list, when their concentration exceeds the labelling limit of 10 ppm or 100 ppm for leave-on or rinse-off cosmetics, respectively. Labelling is important regarding consumer safety. In this way, sensitised people towards fragrances might select their products based on the ingredients list to prevent elicitation of an allergic reaction. It is therefore important to quantify potential allergenic ingredients in cosmetic products. An easy to perform liquid extraction was developed, combined with a new headspace GC-MS method. The latter was capable of analysing 24 volatile allergenic fragrances in complex cosmetic formulations, such as hydrophilic (O/W) and lipophilic (W/O) creams, lotions and gels. This method was successfully validated using the total error approach. The trueness deviations for all components were smaller than 8%, and the expectation tolerance limits did not exceed the acceptance limits of ± 20% at the labelling limit. The current methodology was used to analyse 18 cosmetic samples that were already identified as being illegal on the EU market for containing forbidden skin whitening substances. Our results showed that these cosmetic products also contained undeclared fragrances above the limit value for labelling, which imposes an additional health risk for the consumer.

ATR-FTIR spectroscopy and chemometrics: An interesting tool to discriminate and characterize counterfeit medicines.

Counterfeit medicines pose a huge threat to public health worldwide. High amounts of counterfeit pharmaceuticals enter the European market and therefore detection of these products is essential. Attenuated Total Reflection Fourier-Transform infrared spectroscopy (ATR-FTIR) might be useful for the screening of counterfeit medicines since it is easy to use and little sample preparation is required. Furthermore, this approach might be helpful to customs to obtain a first evaluation of suspected samples. This study proposes a combination of ATR-FTIR and chemometrics to discriminate and classify counterfeit medicines. A sample set, containing 209 samples in total, was analyzed using ATR-FTIR and the obtained spectra were used as fingerprints in the chemometric data-analysis which included Principal Component Analysis (PCA), k-Nearest Neighbours (k-NN), Classification and Regression Trees (CART) and Soft Independent Modelling of Class Analogy (SIMCA). First it was verified whether the mentioned techniques are capable to distinguish samples containing different active pharmaceutical ingredients (APIs). PCA showed a clear tendency of discrimination based on the API present; k-NN, CART and SIMCA were capable to create suitable prediction models based on the presence of different APIs. However k-NN performs the least while SIMCA performs the best. Secondly, it was tested whether these three models could be expanded to discriminate between genuine and counterfeit samples as well. k-NN was not able to make the desired discrimination and therefore it was not useful. CART performed better but also this model was less suited. SIMCA, on the other hand, resulted in a model with a 100% correct discrimination between genuine and counterfeit drugs. This study shows that chemometric analysis of ATR-FTIR fingerprints is a valuable tool to discriminate genuine from counterfeit samples and to classify counterfeit medicines.

Optimization and validation of a micellar electrokinetic chromatographic method for the analysis of several angiotensin-II-receptor antagonists.

We have optimized a micellar electrokinetic capillary chromatographic method for the separation of six angiotensin-II-receptor antagonists (ARA-IIs): candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, and valsartan. A face-centred central composite design was applied to study the effect of the pH, the molarity of the running buffer, and the concentration of the micelle-forming agent on the separation properties. A combination of the studied parameters permitted the separation of the six ARA-IIs, which was best carried out using a 55-mM sodium phosphate buffer solution (pH 6.5) containing 15 mM of sodium dodecyl sulfate. The same system can also be applied for the quantitative determination of these compounds, but only for the more stable ARA-IIs (candesartan, eprosartan mesylate, losartan potassium, and valsartan). Some system parameters (linearity, precision, and accuracy) were validated.

Gel filtration of urine: a method to detect nephrotoxicity in rats.

Sephadex G-100 gel filtration of urine from male Wistar rats revealed 3 protein peaks: peak I (eluted with the void volume of the column), peak II (Mr between 67 000 and 43 000), and peak III (Mr about 13 700). Nephrotoxic compounds were given as a single i.p. injection. Peak I, and especially peak II were significantly increased in 24-h urine samples from rats receiving mercuric acetate (1.58 mg/kg), mercuric trifluoroacetate (MTFA) (2.22 mg/kg), sodium ethylmercurithiosalicylate (EMTSA) (20.2 mg/kg), sodium tetrathionate (250 mg/kg), ammonium fluoride (18.5 mg/kg), paramomycin sulfate (800 mg/kg), ochratoxin A (5 mg/kg) and cis-platinum (4 mg/kg). It is concluded that gel filtration of urine can be used as a method to detect nephrotoxicity in rats.

Indications for the presence of sulfate esters as considerable metabolites of sunset yellow FCF and orange GGN in rat faeces.

Male Wistar rats received by stomach intubation 500 mg/kg of the azo dyes Sunset Yellow FCF and Orange GGN. Previous work revealed that in the Sunset Yellow FCF as well as in the Orange GGN aqueous faeces extracts a considerable orange metabolite is present, eluting as a strong peak before the unchanged dye after ion-pair reverse-phase liquid chromatography (RP-IP-HPLC). Incubation of both aqueous faeces extracts with arylsulphatase followed by high-performance liquid chromatography (HPLC) analysis, resulted in a significant orange metabolite peak height decrease together with a noticeable unchanged dye peak height increase. By means of semi-preparative HPLC, combined with cation-exchange purification, the orange Sunset Yellow FCF faeces metabolite could be isolated. It was identified by means of FT-1HMR spectroscopy as a Sunset Yellow FCF structurally related compound with the same 1HMR pattern, which is therefore modified on the free aromatic hydroxyl group.

Impact of dilution on the pharmacokinetic behavior of acetaminophen in rabbits after oral administration.

Seven rabbits received two different acetaminophen solutions by gavage in a randomized crossover fashion. In one administration the dose was given in a concentrated small volume of an ethanol-glycerol-water mixture (preparation I). In another administration an identical dose was given in a 10-fold water-diluted volume of the same mixture (preparation II). Three rabbits also received the same dose in a 10-fold original mixture volume (preparation III). The acetaminophen concentrations were measured by HPLC in plasma samples collected for 3.5 h after gavage. The lag times ranged from 2.5 to 23 min for preparation I and from 2.2 to 29 min for preparation II. The mean peak plasma concentrations (12.38 micrograms/mL for preparation I and 9.14 micrograms/mL for preparation II) and the mean time-to-peak concentrations (26.57 min for preparation I and 36.57 min for preparation II) were significantly different. The total area under the plasma concentration curve and the absorption and elimination half-lives did not, however, differ significantly. For the three rabbits receiving the acetaminophen doses in the 10-fold ethanol-glycerol-water mixture volumes (preparation III), the total area under the plasma concentration curve obviously was increased.

Experimental design on liquid chromatographic parameters in the analysis of tetracycline on poly(styrene-divinylbenzene).

A previously described isocratic liquid chromatographic (LC) method for the analysis of tetracycline on poly(styrene-divinylbenzene) allows the complete separation and resolution of tetracycline (TC), 4-epitetracycline (ETC), anhydrotetracycline (ATC) and 4-epianhydrotetracycline (EATC). By means of a half-fraction factorial design, the importance of the individual chromatographic parameters and parameter interactions of this LC method was examined. The influence on the retention of ETC, TC and EATC is measured. A mathematical regression model was derived which predicts retention times with good reliability. Both the retentions of ETC and EATC are strongly affected by chromatographic parameters and parameter interactions, which only slightly affect the retention of TC. Adequate combination of the most relevant of these parameters enables optimization of the chromatographic separation if necessary.

Experimental design for the rapid selection of separation conditions for methyl and propyl parahydroxybenzoate, phenylephrine hydrochloride and chlorphenamine maleate by ion-pair liquid chromatography.

Methyl and propyl parahydroxybenzoate (MPHB, PPHB), phenylephrine hydrochloride (PE) and chlorphenamine maleate (CPM) are often combined as ingredients in cough-syrups. Due to distinct chemical structures, pKa values among other chemical properties are different. This may result in a particular chromatographic behaviour on ion-pair reversed-phase liquid chromatographic (LC) systems. A face-centred central composite design was applied to study the impact of four LC mobile phase parameters and parameter interactions on the retention of these four compounds. The mobile phase parameters studied were the concentration of methanol as organic modifier, the concentration of sodium dioctylsulphosuccinate (SDSS) as counter-ion, the concentration of dimethyloctylamine (DMOA) as competitive base and the pH. By means of the proposed design, mathematical regression models and response surface plots were calculated, which could predict the compounds' retention times with good statistical reliability. Adequate combination of the most relevant of these mobile phase parameters enabled complete chromatographic separations within short times of analysis.

Measurement uncertainty from validation and duplicate analysis results in HPLC analysis of multivitamin preparations and nutrients with different galenic forms.

An approach to calculate the measurement uncertainty in the HPLC analysis of several hydro- and liposoluble vitamins in multivitamin preparations with different galenic composition and properties is described. In the first instance it is examined if duplicate analysis results, obtained with a fully validated analysis method on different lots of an effervescent tablet preparation spread over several points of time, might contribute to calculate the measurement uncertainty of the HPLC method used and if the established uncertainty is acceptable in the assessment of compliance with the legal content limits. Analysis of variance (ANOVA) and precision calculations, based on the ISO 5725-2 norm are applied on the analysis results obtained to estimate precision components, necessary to derive the measurement uncertainty. In the second instance it is demonstrated to which extent the fully validated method of analysis for effervescent tablets is applicable to other galenic forms as e.g. capsules with oily emulsions, tablets, coated tablets, oral solutions, em leader and which specific modifications in the analysis steps are involved. By means of duplicate analysis results, acquired from a large series of real samples over a considerable period of time and classified according to their similarity in content, galenic forms and matrices, estimations of measurement uncertainty calculations are shown.

Half-fraction and full factorial designs versus central composite design for retention modelling in reversed-phase ion-pair liquid chromatography.

In a previous paper (J. O. De Beer, C. V. Vandenbroucke and D. L. Massart, J. Pharm. Biomed. Anal., 12, (1994) 1379-1396) liquid chromatographic (LC) retention modelling of the cough-syrup compounds methyl para-hydroxybenzoate (MPHB) and propyl para-hydroxybenzoate (PPHB), phenylephrine hydrochloride (PE) and chlorphenamine maleate (CPM) was studied using a face-centred central composite design. It is examined whether smaller half-fractional and full factorial designs with fewer experiments tend to reliably predict retention times of the latter compounds as well. Simplified regression modelling, however, neglecting more first-order and interactive effects and disregarding pure second-order effects, has to be set up. These smaller designs finally satisfy the prediction of the retention of MPHB, PPHB and PE also. Retention prediction of CPM is much less accurate. CPM has a pKa value of 4.0, which is encompassed by the examined mobile phase pH limits 3.0 and 5.0. Since the largest retention shifts occur near the pKa value, retention prediction in this area becomes more complex. CPM retention modelling from a full factorial design is useful if the mobile phase pH is fixed at 5.0 for methanol as well as for acetonitrile as organic modifers. The full factorial design, applied with acetonitrile as organic modifer, enables the selection of suitable LC parameter combinations for fast and complete separation of the four compounds in cough-syrup analysis.

Detection of whitening agents in illegal cosmetics using attenuated total reflectance-infrared spectroscopy.

Cosmetic products containing illegal whitening agents are still found on the European market. They represent a considerable risk to public health, since they are often characterised by severe side effects when used chronically. The detection of such products at customs is not always simple, due to misleading packaging and the existence of products containing only legal components. Therefore there is a need for easy to use equipment and techniques to perform an initial screening of samples. The use of attenuated total reflectance-infrared (ATR-IR) spectroscopy, combined with chemometrics, was evaluated for that purpose. It was found that the combination of ATR-IR with the simple chemometric technique k-nearest neighbours gave good results. A model was obtained in which a minimum of illegal samples was categorised as legal. The correctly classified illegal samples could be attributed to the illegal components present.

Comparative dissolution study on counterfeit medicines of PDE-5 inhibitors.

Counterfeit medicines are a growing problem in both developing and industrialised countries. In general the evaluation of these medicines is limited to the identification and the dosage of the active ingredients. In this study in vitro dissolution tests were conducted on two sets of counterfeit medicines containing PDE-5 inhibitors (sildenafil citrate and tadalafil). The dissolution profiles were statistically compared to the ones of the genuine products using the f2-method and a comparison at each time point using the Cochran test. The results showed low equivalences between counterfeit and genuine products as well as higher variations around the mean dissolution value at the different time points for the counterfeit products.

Headspace-gas chromatographic fingerprints to discriminate and classify counterfeit medicines.

Counterfeit medicines are a global threat to public health. These pharmaceuticals are not subjected to quality control and therefore their safety, quality and efficacy cannot be guaranteed. Today, the safety evaluation of counterfeit medicines is mainly based on the identification and quantification of the active substances present. However, the analysis of potential toxic secondary components, like residual solvents, becomes more important. Assessment of residual solvent content and chemometric analysis of fingerprints might be useful in the discrimination between genuine and counterfeit pharmaceuticals. Moreover, the fingerprint approach might also contribute in the evaluation of the health risks different types of counterfeit medicines pose. In this study a number of genuine and counterfeit Viagra(®) and Cialis(®) samples were analyzed for residual solvent content using headspace-GC-MS. The obtained chromatograms were used as fingerprints and analyzed using different chemometric techniques: Principal Component Analysis, Projection Pursuit, Classification and Regression Trees and Soft Independent Modelling of Class Analogy. It was tested whether these techniques can distinguish genuine pharmaceuticals from counterfeit ones and if distinct types of counterfeits could be differentiated based on health risks. This chemometric analysis showed that for both data sets PCA clearly discriminated between genuine and counterfeit drugs, and SIMCA generated the best predictive models. This technique not only resulted in a 100% correct classification rate for the discrimination between genuine and counterfeit medicines, the classification of the counterfeit samples was also superior compared to CART. This study shows that chemometric analysis of headspace-GC impurity fingerprints allows to distinguish between genuine and counterfeit medicines and to differentiate between groups of counterfeit products based on the public health risks they pose.

Detection of sibutramine in adulterated dietary supplements using attenuated total reflectance-infrared spectroscopy.

Sibutramine is one of the most occurring adulterants encountered in dietary supplements with slimming as indication. These adulterated dietary supplements often contain a herbal matrix. When customs intercept these kind of supplements it is almost impossible to discriminate between the legal products and the adulterated ones, due to misleading packaging. Therefore in most cases these products are confiscated and send to laboratories for analysis. This results inherently in the confiscation of legal, non-adulterated products. Therefore there is a need for easy to use equipment and techniques to perform an initial screening of samples. Attenuated total reflectance-infrared (ATR-IR) spectroscopy was evaluated for the detection of sibutramine in adulterated dietary supplements. Data interpretation was performed using different basic chemometric techniques. It was found that the use of ATR-IR combined with the k-Nearest Neighbours (k-NN) was able to detect all adulterated dietary supplements in an external test set and this with a minimum of false positive results. This means that a small amount of legal products will still be confiscated and analyzed in a laboratory to be found negative, but no adulterated samples will pass the initial ATR-IR screening.

Characterization of suspected illegal skin whitening cosmetics.

An important group of suspected illegal cosmetics consists of skin bleaching products, which are usually applied to the skin of the face, hands and décolleté for local depigmentation of hyper pigmented regions or more importantly, for a generalized reduction of the skin tone. These cosmetic products are suspected to contain illegal active substances that may provoke as well local as systemic toxic effects, being the reason for their banning from the EU market. In that respect, illegal and restricted substances in cosmetics, known to have bleaching properties, are in particular hydroquinone, tretinoin and corticosteroids. From a legislative point of view, all cosmetic products containing a prohibited whitening agent are illegal and must be taken off the EU market. A newly developed screening method using ultra high performance liquid chromatography-time off flight-mass spectrometry allows routine analysis of suspected products. 163 suspected skin whitening cosmetics, collected by Belgian inspectors at high risk sites such as airports and so-called ethnic cosmetic shops, were analyzed and 59% were classified as illegal. The whitening agents mostly detected were clobetasol propionate and hydroquinone, which represent a serious health risk when repeatedly and abundantly applied to the skin.