Simultaneous determination of nine UV filters and four preservatives in suncare products by high-performance liquid chromatography

HPLC method for quantitative determination of four preservatives and nine UV filters worldwide authorized in commercial suncare product was developed and validated, and then 101 samples of commercial suncare products were analyzed for the UV filters and preservatives using the proposed method. The mobile phase was acetonitrile-water containing 0.5% acetic acid using a gradient elution at a flow rate of 0.9 mL/min and UV measurements were carried out at 320 nm for UV filters and 254 nm for preservatives. The correlation coefficients of each calibration curves were mostly higher than 0.999. The percent relative standard deviations (%RSD) ranged from 0.97% to 6.1% for five sample aliquots. The recoveries from the spiked solutions were 98-102%. 2-ethylhexyl-p-methoxycinnamate (EHMC) was detected in 96 of 101 commercial suncare products and the concentration was in the range of 3.08-8.16% and 18 samples were found to exceed the 7.5% which has been defined as the maximum allowed concentration in Korea. Methyl paraben was detected in 81 of 101 samples and the next-most often detected preservatives were propyl paraben (25), ethyl paraben (18), and butyl paraben (4). Three samples of 101 suncare products exceeded the maximum allowed concentration (i.e., 0.58-0.79%). The proposed HPLC method allows efficient and simultaneous analysis of preservatives and UV filters suitable for quality control assays of commercial suncare products.     

Simultaneous electrochemical determination of three sunscreens using cetyltrimethylammonium bromide

This paper proposes a methodology based on electrochemical reduction for the simultaneous determination of three sunscreen agents, namely 4-methylbenzylidene camphor (MBC), benzophenone-3 (BENZO) and 2-ethylhexyl-4-methoxycinnamate (EHMC) by differential-pulse polarography (DPP). The highest peak currents and optimal separation of reduction peaks were obtained by using a supporting electrolyte consisted of Britton-Robinson buffer-methanol (8:2) solution at pH 4.0 and cationic surfactant 3.0 x 10(-4)mol L(-1) cetyltrimethylammonium bromide (CTAB). The methodology was validated using four commercial sunscreen preparation as a sample and the results showed high recovery rates. The efficiency of the proposed methodology was demonstrated by comparing the results obtained by DPP with those obtained by the high-performance liquid chromatography (HPLC) method.     

Unexpected photolysis of the sunscreen octinoxate in the presence of the sunscreen avobenzone

A major concern raised about photostability studies of sunscreen products is that the photodegradation of sunscreens does not readily translate into changes in product performance. This study examines the correlation between photochemical degradation of sunscreen agents and changes in protection provided by sunscreen films. Films of a commercial sunscreen product containing avobenzone, oxybenzone and octinoxate were irradiated using a fluorescent UV-A phototherapy lamp with additional UV-B blocking filter. Periodically, during irradiation the transmittances of the films were measured and samples collected for chemical analysis of the sunscreen agents using high-performance liquid chromatography techniques. The results show that UV-induced changes in UV transmittance of sunscreen films correlate with changes in concentration of sunscreen agents. In a parallel experiment, we also irradiated a thin film of the same product in the cavity of an electron spin resonance (ESR) spectrometer. We report the concomitant photolysis of avobenzone and octinoxate that predominates over expected E/Z photoisomerization and that irradiation of a film of this product produced free radicals detected by ESR spectroscopy that persisted even after exposure had ended.     

Neutral desorption extractive electrospray ionization mass spectrometry for fast screening sunscreen agents in cream cosmetic products

High throughput analysis of sunscreen agents present in cream cosmetic has been demonstrated, typically 2 samples per minute, using neutral desorption extractive electrospray ionization mass spectrometry (ND-EESI-MS) without sample pretreatment. For the targeted compounds such as 4-Aminobenzoic acid and oxybenzone, ND-EESI-MS method provided linear signal responses in the range of 1-100 ppb. Limits of detection (LOD) of the method were estimated at sub-ppb levels for the analytes tested. Reasonable relative standard deviation (RSD = 8.4-16.0%) was obtained as a result of 10 independent measurements for commercial cosmetics samples spiked with each individual sunscreen agents at 1-10 ppb. Acceptable recoveries were achieved in the range of 87-116% for direct analysis of commercial cream cosmetic samples. The experimental data demonstrate that ND-EESI-MS is a useful tool for high throughput screening of sunscreen agents in highly viscous cream cosmetic products, with the capability to obtain quantitative information of the analytes.     

Laser Flash Photolysis Studies of the UVA Sunscreen Mexoryl SX

Abstract The results of a nanosecond laser flash photolysis investigation of the UVA sunscreen Mexoryl* SX in various solvent environments and within a commercial sunscreen formulation are reported. To the best of our knowledge this is the first laser flash photolysis study of a commercial suncare formulation. In each of these environments kinetic UV-visible absorption measurements following nanosecond 355 nm laser excitation reveals a short-lived species with a solvent-dependent absorption maximum around 470–500 nm and a solvent-dependent lifetime of 50–120 ns. This transient absorption is attributed to the triplet state of Mexoryl* SX on the basis that it is quenched by molecular oxygen leading to the formation of singlet oxygen in acetonitrile. The singlet oxygen quantum yield (φ_Δ), determined by comparative time-resolved near-infrared luminescence measurements and extrapolated to the limit of complete triplet state quenching, is estimated as 0.09 ± 0.03 in acetonitrile. In aqueous solution the shorter triplet state lifetime combined with lower ambient oxygen concentrations precludes significant triplet state quenching. For the commercial sunscreen formulation there was no observable difference in the measured triplet lifetime between samples exposed to oxygen or argon, suggesting that the singlet oxygen quantum yield in such environments is likely to be orders of magnitude lower than that measured in acetonitrile.     

Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy

Raman spectroscopy has been used for the quantitative determination of the conversion efficiency at each step in the production of ethanol from biomass. The method requires little sample preparation; therefore, it is suitable for screening large numbers of biomass samples and reaction conditions in a complex sample matrix. Dilute acid or ammonia-pretreated corn stover was used as a model biomass for these studies. Ammonia pretreatment was suitable for subsequent measurements with Raman spectroscopy, but dilute acid-pretreated corn stover generated a large background signal that surpassed the Raman signal. The background signal is attributed to lignin, which remains in the plant tissue after dilute acid pretreatment. A commercial enzyme mixture was used for the enzymatic hydrolysis of corn stover, and glucose levels were measured with a dispersive 785 nm Raman spectrometer. The glucose detection limit in hydrolysis liquor by Raman spectroscopy was 8 g L⁻¹. The mean hydrolysis efficiency for three replicate measurements obtained with Raman spectroscopy (86 ± 4%) was compared to the result obtained using an enzymatic reaction with UV-vis spectrophotometry detection (78 ± 8%). The results indicate good accuracy, as determined using a Student's t-test, and better precision for the Raman spectroscopy measurement relative to the enzymatic detection assay. The detection of glucose in hydrolysis broth by Raman spectroscopy showed no spectral interference, provided the sample was filtered to remove insoluble cellulose prior to analysis. The hydrolysate was further subjected to fermentation to yield ethanol. The detection limit for ethanol in fermentation broth by Raman spectroscopy was found to be 6 g L⁻¹. Comparison of the fermentation efficiencies measured by Raman spectroscopy (80 ± 10%) and gas chromatrography-mass spectrometry (87 ± 9%) were statistically the same. The work demonstrates the utility of Raman spectroscopy for screening the entire conversion process to generate lignocellulosic ethanol.     

Ultraviolet resonance Raman spectroscopy as a robust spectroscopic tool for in situ sunscreen analysis

Current techniques being used for sunscreen analysis are incapable of direct determination of the active ingredients in sunscreen formulations. Therefore, the development of methodologies for rapid in situ analysis of sunscreens is desirable. This paper describes the application of ultraviolet resonance Raman spectroscopy (UVRRS) to the direct in situ analysis of sunscreen formulations. High-quality UV resonance Raman spectra were obtained for five sunscreen active ingredients (AIs), mixtures of the AIs and real sunscreen formulation samples. The spectra from the sunscreen formulations gave distinct spectral signatures indicative of the sunscreen AIs in each sample, with essentially no interference from the complex sunscreen matrix. Also, despite the fact that many of the AIs are fluorescent, no fluorescence interferences in the resonance Raman spectra were observed. Excitation wavelength-dependent studies throughout the 244-275 nm region demonstrate that the best discrimination of the AIs was achieved at an excitation wavelength of 244 nm. Thus, by tuning the excitation wavelength within the absorption bands of the AIs, complete identification of these analytes can be achieved in situ without any sample pretreatment or separation. The limit of detection found for a common AI in situ with this technique is 0.23% (w/w), the limit of quantitation is 0.78% (w/w), while the dynamic range is between 0.8% and 50% (w/w). The technique is fast, robust, lacks any major interference, and can be adapted for routine online quality control.     

The feasibility of using fluorescence spectroscopy as a rapid, non-invasive method for evaluating sunscreen performance

We have carried out ex vivo studies to examine the feasibility of using fluorescence spectroscopy as an in vivo quantitative technique to assess sunscreen substantivity in terms of skin surface thickness and/or photoprotection. We found that the majority of sunscreens produced insufficient natural fluorescence and so we have attempted to increase the fluorescent signal by adding various fluorescing agents to the sunscreens. However, none of these substances is ideal; either they do not bind sufficiently strongly to sunscreen products, or their fluorescence is quenched by the active ingredients contained within sunscreens. The feasibility of using fluorescence spectroscopy for in vivo quantitative assessments of sunscreen substantivity therefore remains unproved and is dependent on a suitable fluorescent agent being found. Such an agent would have to be non-toxic, mix readily with sunscreens and be excited by visible wavelengths.     

高效液相色谱法测定化妆品中的12种紫外吸收剂

建立了高效液相色谱同时测定化妆品中12种紫外吸收剂含量的方法。样品用甲醇提取,高速离心,过滤,以SB-C8柱(250 mm×4.6 mm, 5 μm)为分离色谱柱,甲醇和0.1%(v/v)甲酸水溶液为流动相,梯度洗脱,以311 nm为检测波长进行定性、定量分析。该方法前处理简单、易操作,12种紫外吸收剂分离效果良好;在1.0～500 mg/L范围内呈线性关系,相关系数大于0.9995;方法检出限为0.002～0.1 mg/L;实际样品中的加标回收率为97.4%～107.5%,相对标准偏差为1.54%～4.98%。该方法简便、准确,能够满足化妆品中紫外吸收剂的检测要求。     

FT-Raman spectroscopic studies of guarana and some extracts

The Raman spectrum of guarana, an important product of the Amazonian rain forest, has been investigated; the therapeutic properties of guarana and it's extracts have been realised for some time and have been attributed to guaranine, which could be a complex of caffeine and tannins or to a new xanthine natural product. The purpose of this study is two-fold: firstly, to provide molecular structural information about guarana seeds and their extracts and, secondly, to test the viability of using the technique as a method of verification of genuine guarana extracts from synthetic composites. Raman spectroscopy shows that the composition of the guarana is very similar for the whole seed and for the outer and inner portions of the dissected seed, which are closely similar also to the ground commercial powders produced in the Amazon for the distributors. The results indicate that Fourier-transform Raman spectroscopy could be used for the monitoring of quality control of guarana products in the phytopharmaceutical industry.     

Validation of a direct non-destructive quantitative analysis of amiodarone hydrochloride in Angoron formulations using FT-Raman spectroscopy

Raman spectroscopy was applied for the direct non-destructive analysis of amiodarone hydrochloride (ADH), the active ingredient of the liquid formulation Angoron^®. The FT-Raman spectra were obtained through the un-broken as-received ampoules of Angoron^®. Using the most intense vibration of the active pharmaceutical ingredient (API) at 1568 cm⁻¹, a calibration model, based on solutions with known concentrations, was developed. The model was applied to the Raman spectra recorded from three as-purchased commercial formulations of Angoron^® having nominal strength of 50 mg ml⁻¹ ADH. The average value of the API in these samples was found to be 48.56 ± 0.64 mg ml⁻¹ while the detection limit of the proposed technique was found to be 2.11 mg ml⁻¹. The results were compared to those obtained from the application of HPLC using the methodology described in the European Pharmacopoeia and found to be in excellent agreement. The proposed analytical methodology was also validated by evaluating the linearity of the calibration line as well as its accuracy and precision. The main advantage of Raman spectroscopy over HPLC method during routine analysis is that it is considerably faster and no solvent consuming. Furthermore, Raman spectroscopy is non-destructive for the sample. However, the detection limit for Raman spectroscopy is much higher than the corresponding for the HPLC methodology.     

Quantitative analysis of thymine with surface-enhanced Raman spectroscopy and partial least squares (PLS) regression

Silver sol surface-enhanced Raman spectroscopy (SERS) was considered as a technique in the quantitative analysis of low-concentration thymine. Because of the poor stability and reproducibility of SERS signal, a polymer of polyacrylic acid sodium was selected as a stable medium to add into silver sol in order to obtain a surface-enhanced Raman spectroscopy signal. Assignments of Raman shift for solid thymine, SERS of thymine, and SERS of thymine containing stable medium were given. The comparison of Raman peaks between them showed that the addition of stable medium had a little influence on the SERS of thymine and is suitable for the quantitative analysis of low-concentration thymine.     

Raman Spectroscopy for Intracellular Monitoring of Carotenoid in <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

In the present study, we explore the feasibility of Raman spectroscopy for intracellular monitoring of carotenoid in filamentous fungi Blakeslea trispora. Although carotenoid production from this fungus has been extensively studied through various chromatographic methods and ultraviolet-visible spectroscopy, no intracellular monitoring has been demonstrated until now. The intensity of the Raman spectrum, and more conveniently that of the strongest ν ₁ carotenoid band at ∼1,519 cm⁻¹, exhibits a good linear correlation with the carotenoid content of the sample as determined by high-performance liquid chromatography (HPLC) and ultraviolet-visible (UV-Vis) spectroscopy. Our results suggest that Raman spectroscopy can serve as an alternative method for the study and quantification of carotenoid in batch-mated submerged cultivations of B. trispora and similar organisms. Although not as accurate as HPLC, it allows a rapid sampling and analysis, avoiding the prolonged and tedious classical isolation procedures required for carotenoid determination by HPLC and UV-Vis spectroscopy.     

Degradation pathway of homoserine lactone bacterial signal molecules by halogen antimicrobials identified by liquid chromatography with photodiode array and mass spectrometric detection

The degradation pathway of acylated homoserine lactone bacterial signaling molecules by oxidizing hypochlorite and stabilized hypobromite antimicrobials has been characterized. A reversed-phase HPLC separation using a cyano column was developed to detect the parent lactones, lactone-hydrolysis products, and halogenation products. Elucidation of the structures of the reaction products was done with the aid of online photodiode array UV spectroscopy and atmospheric pressure chemical ionization mass spectrometry. Quantitative output of the HPLC method was also used to estimate the kinetics of the degradation pathway. The results of this work found that only beta-keto-amide signal molecules are halogenated, where normal amide signals are not, and may represent one possible mechanism for control of industrial biofilms.     

The Photostabilizing Effect of Grape Seed Extract on Three Common Sunscreen Absorbers

The photostabilizing ability of grape seed extract on three common sunscreen absorbers, 2‐ethylhexyl‐p‐methoxycinnamate (EHMC), benzophenone‐3 (BP3) and tert‐butylmethoxy dibenzoylmethane (BMDBM), was investigated. Samples were exposed to simulated solar radiation and monitored by spectrophotometric and chromatographic methods. The chemical composition of the grape seed extract was determined by GC‐MS and HPLC‐MS, and the major secondary metabolites were found to be epicatechin and catechin. Exposure of the extract to UV radiation increased the UV absorption capacity of the extract. All sunscreens showed an improved photostability in the extract. The inherent photo‐instability of BMDBM when exposed to UV radiation was almost eliminated in the presence of grape seed extract. A mixture of all three sunscreens in the extract showed very high photostability and a red shift covering the entire UVB and UVA regions, thereby improving the broad‐spectrum protection. The incorporation of grape seed extract in sunscreen and other cosmetic formulations for topical application boosts photoprotection by stabilizing the UV filters and enhancing broad‐spectrum coverage. This in turn helps in reducing the amounts of absorbers and other additives incorporated in a sunscreen product and consequently lowers the risk of an unprecedented buildup of photoproducts whose toxicities are currently unknown.     

Application of liquid chromatography-two-dimensional nuclear magnetic resonance spectroscopy using pre-concentration column trapping and liquid chromatography-mass spectrometry for the identification of degradation products in stressed commercial amlodipine maleate tablets

Application of the HPLC hyphenated techniques of LC-two-dimensional (2D) NMR using pre-concentration column trapping and LC-MS was demonstrated by the identification of two major degradation products, DP-1 and DP-2, in stressed commercial tablets of amlodipine maleate. The molecular formulas were estimated by LC-MS. Sample pre-concentration by column trapping was conducted to obtain adequate 2D-NMR signals by reducing the peak widths of the degradation products and making sure that the maximum amount of each component was inside the flow cell for NMR detection. Double-quantum filtered correlation spectroscopy (DQF-COSY) was applied to identify DP-1 as beta-N-lactosylamlodipine by suppressing the residual water signal without affecting the sample signal and by measuring the coupling constant of the lactose anomeric proton. Heteronuclear multiple bond coherence spectroscopy (HMBC) was applied to characterize DP-2 as an aspartic acid derivative of amlodipine by detecting long-range CH correlations. The chemical structures of the degradation products could be successfully elucidated unambiguously without an isolation process.     

高效液相色谱法同时测定防晒类化妆品中15种紫外线吸收剂

建立了一种反相高效液相色谱同时测定防晒类化妆品中15种紫外线吸收剂的分析方法。化妆水、乳液、膏霜和蜡质样品中首先加入四氢呋喃(含2 g/L氢氧化铵),涡旋、振荡、混匀(若蜡质样品仍分散不完全,可超声振荡加热至50 ℃),再加入80%(v/v)甲醇水溶液振荡混匀、超声提取、离心、过滤后,采用XTerra MS C₁₈柱分离,经水(含0.1%(v/v)甲酸)和甲醇(含0.1%(v/v)甲酸)梯度洗脱,以二极管阵列检测器检测,检测波长为280 nm和311 nm,外标法定量。实验中对不同基质类型样品的前处理条件(样品分散溶剂、萃取溶剂和萃取时间等)进行了重点优化。结果表明,15种紫外线吸收剂在各自的线性范围内呈良好的线性关系(r²≥0.9991),方法的定量限为1.2~5.1 μg/g,在低、中、高3个添加水平下的回收率为84.2%~100.7%,相对标准偏差(RSD)为0.9%~9.5%。该分析方法分离效果好、灵敏度高、定量准确,可用于防晒类化妆品的实际检测。     

Rapid monitoring of antibiotics using Raman and surface enhanced Raman spectroscopy

Comparatively few studies have explored the ability of Raman spectroscopy for the quantitative analysis of microbial secondary metabolites in fermentation broths. In this study we investigated the ability of Raman spectroscopy to differentiate between different penicillins and to quantify the level of penicillin in fermentation broths. However, the Raman signal is rather weak, therefore the Raman signal was enhanced using surface enhanced Raman spectroscopy (SERS) employing silver colloids. It was difficult by eye to differentiate between the five different penicillin molecules studied using Raman and SERS spectra, therefore the spectra were analysed by multivariate cluster analysis. Principal components analysis (PCA) clearly showed that SERS rather than the Raman spectra produced reproducible enough spectra to allow for the recovery of each of the different penicillins into their respective five groups. To highlight this further the first five principal components were used to construct a dendrogram using agglomerative clustering, and this again clearly showed that SERS can be used to identify which penicillin molecule was being analysed, despite their molecular similarities. With respect to the quantification of penicillin G it was shown that Raman spectroscopy could be used to quantify the amount of penicillin present in solution when relatively high levels of penicillin were analysed (>50 mM). By contrast, the SERS spectra showed reduced fluorescence, and improved signal to noise ratios from considerably lower concentrations of the antibiotic. This could prove to be advantageous in industry for monitoring low levels of penicillin in the early stages of antibiotic production. In addition, SERS may have advantages for quantifying low levels of high value, low yield, secondary metabolites in microbial processes.     

Detection of counterfeit stevia products using a handheld Raman spectrometer

Stevia is a highly appreciated natural sweetener because it can be consumed by diabetic patients. Due to the increasing popularity of stevia during the last years, counterfeit products have been making their way into the market. Raman spectroscopy is a versatile analytical technique that can be used for control tasks and handheld modern devices expand its possible applications to instant in situ measurements. The Raman spectra of six commercial stevia products (five purchased in Bolivia and one in Germany) were recorded and compared to the spectra of standards of rebaudioside A and stevioside as well as the spectra of the artificial sweeteners sodium cyclamate and sodium saccharin. Based on the Raman spectroscopic data, it was verified that three of the Bolivian products were counterfeit products and another one was rich in maltodextrin. The Raman spectra of one Bolivian product and the German one revealed rebaudioside A and stevioside as major components. Raman spectroscopy was capable of detecting contents as low as 5% (w/w) of sodium cyclamate during measurements of stevia-sodium cyclamate mixtures. The results show that Raman spectroscopy can successfully be used to detect counterfeit stevia and underline its high potential for the detection of food adulteration.     

HPLC analysis of thimerosal and its degradation products in ophthalmic solutions with electrochemical detection

A reverse-phase high-performance liquid chromatographic assay using amperometric detection on a glassy-carbon electrode has been developed for analysis of thimerosal and its main degradation products, thiosalicylic acid and dithiodibenzoic acid, in ophthalmic formulations. A potential value of 0.9 V vs. Ag/AgCl was chosen for simultaneous detection of thimerosal and thiosalicylic acid, obtaining limits of detection of 1.0 and 0.2 ng injected, respectively. A potential value of 1.2V was applied for simultaneous determination of all three compounds studied, obtaining in this case limits of detection of 3,4 and 4 ng injected for thimerosal, thiosalicylic acid and dithiodibenzoic acid, respectively. The results obtained reveal the utility of the HPLC method in quality control of commercial products containing thimerosal with good detectability.