Unlocking hot trub's potential: a simple method for extracting bitter acids and xanthohumol.

BACKGROUND: Hot trub is a macronutrient- and micronutrient-rich by-product generated in the brewing industry, which is still underrated as a raw material for reprocessing purposes. In this context, this study aimed to investigate the extraction of bitter acids' and xanthohumol from hot trub as well as identify the significance of parameters for the process. The research assessed various extraction parameters, such as pH, ethanol concentration, temperature, and solid-to-liquid ratio, using a Plackett-Burman design. RESULTS: Ethanol concentration and pH were the most significant parameters affecting extraction yield. ß-acids were found to be the principal components of the bitter acids, with a maximum concentration near 16 mg g-1, followed by iso-α-acids and α-acids achieving 6 and 3.6 mg g-1, respectively. The highest yields of bitter acids were observed in the highest ethanol concentration, while pH was relevant to extraction process in treatments with low ethanol ratios. Concerning the xanthohumol extraction, the approach achieved maximum concentration (239 µg g-1) in treatments with ethanol concentration above 30%. Despite their variances, the phytochemicals exhibited comparable extraction patterns, indicating similar interactions with macromolecules. Moreover, the characterization of the solid residues demonstrated that the extraction process did not bring about any alterations to the chemical and total protein profiles. CONCLUSION: Ethanol concentration was found to have the most significant impact on the extraction of bitter acids and xanthohumol, while temperature had no significant effect. The solid remains resulting from the extraction showed potential for use as a protein source. © 2024 Society of Chemical Industry.

Fatal 4-MEC Intoxication: Case Report and Review of Literature.

ABSTRACT: Synthetic cathinones are one of the major pharmacological families of new psychoactive substances and 4-methylethcathinone (4-MEC) has emerged in recent years as a recreational psychostimulant. We report a case of a 35-year-old man found dead and naked at home by his friend. Although no anatomic cause of death was observed at autopsy, toxicological analysis identified 4-MEC and hydroxyzine at therapeutic level (160 ng/mL). 4-Methylethcathinone was quantified in autopsy samples by a validated method consisting in liquid-liquid extraction and gas chromatography coupled to tandem mass spectrometry: peripheral blood, 14.6 µg/mL; cardiac blood, 43.4 µg/mL; urine, 619 µg/mL; vitreous humor, right 2.9 µg/mL and left 4.4 µg/mL; bile, 43.5 µg/mL; and gastric content, 28.2 µg/mL. The cause of death was 4-MEC intoxication and the manner of death could be either accidental or suicidal. The literature concerning 4-MEC was reviewed, focusing on distribution in classical postmortem matrices and 4-MEC metabolism and postmortem redistribution and stability.

Changes in chemical profile of Cascade hop cones according to the growing area.

BACKGROUND: The growing area has a substantial effect on plants, affecting secondary metabolism. For hops, different authors have studied the effect of growing area on the chemical composition of cones with the aim of verifying and understanding the changes in hop characters. Despite the scant literature the subject receives increasing attention by brewers and hop growers. The present study aimed to characterize, using gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography with ultraviolet detection (HPLC-UV), cones of hop (Humulus lupulus L.) cultivar Cascade. Plant material was obtained from nine different areas of Italy and compared with Cascade samples grown in the United States, Germany and Slovenia. RESULTS: Differences in bitter acids and xanthohumol content were observed. Nevertheless, no correlation between bitter acids and xanthohumol production, on the one hand, and rainfall, temperatures and latitude, on the other hand, were observed in our samples. The Slovenia samples were richer in molecules that confer hoppy, woody and flower notes; USA2 samples were more characterized by woody, earthy, grassy and floral aroma, quite different characters if compared to USA1, which had the lowest presence of grassy aromatic compounds. In the Italian samples, TRENTINO was the genotype most characterized by limonene presence. CONCLUSION: The results of this study are indicative of the importance for hop users to know and characterize hops coming from different growing regions. The study pays special attention to the characterization of the differences in chemical characters of Cascade hop in Italy, where hop cultivation has developed only recently, but is in continuous expansion. © 2019 Society of Chemical Industry.

Simultaneous enantioseparation of methcathinone and two isomeric methylmethcathinones using capillary electrophoresis assisted by 2-hydroxyethyl-ß-cyclodextrin.

Methcathinone (ephedrone), 4-methylmethcathinone (mephedrone), and 3-methylmethcathinone (metaphedrone) are toxicologically-important cathinone derivatives used commonly as designer drugs. In this work we show the first method allowing to separate simultaneously all these molecules in a chiral medium, ensuring good resolution between all enantiomers. Eight cyclodextrins have been tested as potential chiral selectors, the best results were obtained with 2-hydroxyethyl-ß-cyclodextrin, unreported so far for efficient separation of cathinones. After optimization, the method was calibrated and validated with and without the use of internal standard. The addition of standard improved an overall repeatability and precision, the use of electrophoretic mobility ratio was especially favorable (RSD < 1%). It was demonstrated that the method may be easily extended by introducing the additional cathinone-related drugs to the sample, maintaining satisfactory separation efficiency.

Research Status of New Designer Drug Methcathinone in Forensic Toxicology.

Methcathinone, a new cathinone designer drug, which is structurally similar to amphetamine analogs, is a central nervous stimulant. Recently, there has been a worldwide rise in its popularity and abuse, and a growing number of cases with disability or even death is reported in several countries, resulting in public concern. The typical symptoms include accelerated heartbeat, high temperature, anxiety, depression, etc. Forensic studies on its toxicity mechanism are rare. This article reviews its toxicological effects, poisoning symptoms, poisoning and addiction mechanisms, and detection methods, to provide theoretical reference for future studies and guidance for related forensic identification.

Patterns of use and toxicity of new para-halogenated substituted cathinones: 4-CMC (clephedrone), 4-CEC (4-chloroethcatinone) and 4-BMC (brephedrone).

OBJECTIVE: This paper aims to present results of the analysis of clephedrone (4-CMC), 4-chloroethcathinone (4-CEC), and brephedrone (4-BMC) on recreational drug markets and a systematic review of all the available information concerning these substances. MATERIAL AND METHODS: Samples collected by the drug checking service of the Spanish harm reduction NGO-Energy Control were analyzed and systematic research was conducted. Between June 2014 and October 2016, 1,471 samples with at least one NPS were analyzed, 397 of which contained cathinones. RESULTS: Clephedrone was found in 29 samples, brephedrone in 8, and both were present in 2 samples. 4-Chloroethcathinone was detected in 5 samples. Eleven out of the 47 purchased samples (23.4%) were tested to contain the substance the user expected. Samples received were mainly sold as 3-MMC, MDMA, ketamine, and other cathinones. No literature on the effects or toxicity of these substances was found; the only information available was on internet fora. On many posts, users exhibit concerns about potential toxicity and side effects of using these substances. CONCLUSION: Since the emergence of these substances could prove to be the next step to the cat-and-mouse game existing between drug producers and legislation, further clinical and epidemiological research should be carried out in order to build evidence to support policy for public health issues.

Fast Sampling, Analyses and Chemometrics for Plant Breeding: Bitter Acids, Xanthohumol and Terpenes in Lupulin Glands of Hops (Humulus lupulus).

INTRODUCTION: The valuable secondary metabolites in hops (bitter acids, xanthohumol, volatile monoterpenes and sesquiterpenes) are sequestered in lupulin glands (extracellular trichomes) which can be collected and analysed with little or no sample preparation. OBJECTIVES: To determine whether high throughput screening of lupulin glands composition, by fast analyses and chemometrics, could be used for breeder selection of hops with key flavour attributes. METHODS: Lupulin glands from 139 plants (39 cultivars/advanced selections) were analysed by Raman and 1 H NMR spectroscopy, and head-space solid-phase microextraction (HS-SPME) GC-FID. The digital X,Y-data were subjected to principal component analysis (PCA) and the results compared with conventional analyses of extracts of whole hops from the same plants. Quantitative 1 H NMR analyses were also done for the bitter acids. RESULTS: Raman spectroscopy rapidly identified hops cultivars with high xanthohumol concentrations and high α:ß bitter acid ratios. 1 H NMR spectroscopy was slower, requiring a solvent extraction, but distinguished cultivars by cohumulone content as well as α:ß acid ratios. HS-SPME-GC rapidly distinguished aroma hops with high myrcene and farnesene contents, and pinpointed a novel selection with unusual sesquiterpenes. The quantitative NMR analyses showed correlations between bitter acid concentrations related to biosynthetic pathways. CONCLUSIONS: Analysis of lupulin glands gave reliable results for the main quality indicators used by hops breeders, potentially avoiding harvesting, drying and solvent extracting whole hops. PCA of digital X,Y-data rapidly discriminated different hops chemotypes, and highlighted plants with potential for new flavourcultivars. Copyright © 2016 John Wiley & Sons, Ltd.

Detection and quantification of new psychoactive substances (NPSs) within the evolved "legal high" product, NRG-2, using high performance liquid chromatography-amperometric detection (HPLC-AD).

The global increase in the production and abuse of cathinone-derived New Psychoactive Substances (NPSs) has developed the requirement for rapid, selective and sensitive protocols for their separation and detection. Electrochemical sensing of these compounds has been demonstrated to be an effective method for the in-field detection of these substances, either in their pure form or in the presence of common adulterants, however, the technique is limited in its ability to discriminate between structurally related cathinone-derivatives (for example: (±)-4'-methylmethcathinone (4-MMC, 2a) and (±)-4'-methyl-N-ethylmethcathinone (4-MEC, 2b) when they are both present in a mixture. In this paper we demonstrate, for the first time, the combination of HPLC-UV with amperometric detection (HPLC-AD) for the qualitative and quantitative analysis of 4-MMC and 4-MEC using either a commercially available impinging jet (LC-FC-A) or custom-made iCell channel (LC-FC-B) flow-cell system incorporating embedded graphite screen-printed macroelectrodes. The protocol offers a cost-effective, reproducible and reliable sensor platform for the simultaneous HPLC-UV and amperometric detection of the target analytes. The two systems have similar limits of detection, in terms of amperometric detection [LC-FC-A: 14.66 µg mL(-1) (2a) and 9.35 µg mL(-1) (2b); LC-FC-B: 57.92 µg mL(-1) (2a) and 26.91 µg mL(-1) (2b)], to the previously reported oxidative electrochemical protocol [39.8 µg mL(-1) (2a) and 84.2 µg mL(-1) (2b)], for two synthetic cathinones, prevalent on the recreational drugs market. Though not as sensitive as standard HPLC-UV detection, both flow cells show a good agreement, between the quantitative electroanalytical data, thereby making them suitable for the detection and quantification of 4-MMC and 4-MEC, either in their pure form or within complex mixtures. Additionally, the simultaneous HPLC-UV and amperometric detection protocol detailed herein shows a marked improvement and advantage over previously reported electroanalytical methods, which were either unable to selectively discriminate between structurally related synthetic cathinones (e.g. 4-MMC and 4-MEC) or utilised harmful and restrictive materials in their design.

Simultaneous determination of twenty-two components in Asari Radix et Rhizoma by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Asari Radix et Rhizoma is a herbal medicine for the treatment of common cold, rhinitis, etc. An ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method has been established for the rapid analysis of 22 components in 27 samples from the raw materials of Asari Radix et Rhizoma and an adulterant. A total of 5 lignans, 5 essential oils, 3 aristolochic acids, 4 alkamides, and 5 flavanoids were identified by co-chromatography of samples extracts and comparison of the retention time, UV spectra, characteristic molecular ions, and fragment ions with those of authentic standards, or tentatively identified by MS/MS determination along with MassFragment software. Moreover, the method was validated for the simultaneous quantification and semi-quantification of 20 components. The samples from Asarum heterotropoides var. mandshuricum differed in the quantity of 2-methoxyl-4,5-methylenedioxypropiophenone and kakuol from those of Asarum sieboldii var. seoulense, and the chemical difference was supported by principal component analysis and orthogonal partial least squared discriminant analysis based on dataset obtained from UHPLC-QTOF/MS. In comparison with the samples from the two medicinal Asarum species mentioned above, those from A. himalaicum differed in the quality and quantity of major compounds and contained higher amounts of aristolochic acid I.

Simultaneous chemo- and enantio-separation of 2-, 3- and 4-chloro-methcatinones by high-performance liquid chromatography-tandem mass spectrometry and its application to oral fluid samples.

A method of analysis was developed for the simultaneous chemo- and enantioseparation of 2-, 3-, and 4-chloromethcathinones by high-performance liquid chromatography tandem mass-spectrometry. The fast method enables the reliable identification of positional isomers of chloromethcathinones in biological samples. In addition, the same method can be used for the enantioselective quantitative determination of one of these compounds and its major phase-1 metabolites in biological fluids. The developed method was applied to oral fluid samples collected by police during routine random traffic control in Belgium from January to November, 2023. It was found that 3-CMC was more frequently abused compared to 4-CMC. Although some differences were observed between the concentrations of enantiomers in OF, most likely the drugs were abused in the racemic form. No abuse of 2-CMC was detected at the timepoint of sample collection.

Challenges encountered in the enantioselective analysis of new psychoactive substances exemplified by clephedrone (4-CMC).

In this study we report on efforts to develop an enantioselective method for the detection of the drug of abuse clephedrone (1-(4-chlorophenyl)-2-(methylamino)-1-propanone (4-chloromethcathinone, also known as 4-CMC or para-chloro-methcathinone)) and its phase-1 metabolites in human biological fluids. The major goal is not to only report results, but primarily to emphasize the various challenges encountered when developing a reliable analytical method for the detection and quantification of novel psychoactive substances (NPS) and their metabolites in the matrix of interest. Such challenges start with the lack of chemical stability of some NPS in biological matrices. Additionally, most often metabolites are unavailable in pure form to serve as analytical standards, just as deuterated standards for native drugs and metabolites are frequently not commercially available. Furthermore, if the NPS is chiral, enantiomerically pure standards with known absolute stereochemistry are required, as well as a stereochemical stability of a drug and its metabolites becomes an issue. In addition, the chirality of a NPS significantly increases the number of species to be detected in the sample and thus challenges the development of an adequate separation method. These issues are shortly addressed, and some solutions offered in this manuscript.

[The optimation of extraction technology of chuanxiong and xiangfu compatibility by SFE-CO2].

OBJECTIVE: To optimize the extraction conditions of Chuanxiong and Xiangfu compatibility by supercritical CO2 and control the quality of the extract. METHODS: The orthogonal design experiments were used to select the optimal conditions and the factors were extraction pressure, extraction temperature, extraction time and flow rate of CO2. The extraction rate was used as inspection index. The contents of alpha-cyperolone and butenyl were detected by HPLC. RESULTS: The optimal conditions were as follows: extraction pressure 25 Mpa, extraction temperature 45 degrees C, extraction time 2 h, flow rate of CO2 20 Kg/h. The average content of alpha-cyperolone, butenyl was 3.26%, 1.50% respectively. CONCLUSION: The method of supercritical CO2 extraction is reliable and feasible and can be used in industry. The quality of the extract is stable and controlled.

Cytotoxicity of the polymerization agent, 2-methyl-4'-(methylthio)-2-morpholinopropiophenone on human monocytes.

This is the first study to detect 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MTMP) from an intravenous injection bag solution by GC-MS. In previous studies, several other photoinitiators were reported to be very cytotoxic. Therefore, we theorized that photoinitiators such as MTMP might also have adverse cellular effects. The purpose of this study was to quantitate the amounts of contaminants from plastic containers such as those used for peripheral parenteral nutrition and to determine the cytotoxicity of such extracts on human monocytes. The sample extraction procedure for GC-MS analysis involved a liquid-phase extraction. The solvent was evaporated under a stream of nitrogen at 50°C. The residue was dissolved in n-hexane and injected into a GC-MS. Normal human peripheral blood mononuclear cells (PBMC), isolated from the buffy coat by centrifugation, were suspended in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cells (1×10(4)) were treated with MTMP for 24 h or 48 h at 37°C. From the GC-MS analysis, 5.62 ± 1.03 µg/mL of MTMP was found in the BFLUID(®) Injection 500 mL solution. In the MTT assay, MTMP decreased cell viability in a dose-dependent manner for both the 24 h and 48 h incubation periods. Our findings suggest that photoinitiators could promote adverse effects in patients. Future studies will clarify the possible health risks of photoinitiator accumulation in human cells.

Screening natural products for inhibitors of quinone reductase-2 using ultrafiltration LC-MS.

Inhibitors of quinone reductase-2 (NQO2; QR-2) can have antimalarial activity and antitumor activities or can function as chemoprevention agents by preventing the metabolic activation of toxic quinones such as menadione. To expedite the search for new natural product inhibitors of QR-2, we developed a screening assay based on ultrafiltration liquid chromatography-mass spectrometry that is compatible with complex samples such as bacterial or botanical extracts. Human QR-2 was prepared recombinantly, and the known QR-2 inhibitor, resveratrol, was used as a positive control and as a competitive ligand to eliminate false positives. Ultrafiltration LC-MS screening of extracts of marine sediment bacteria resulted in the discovery of tetrangulol methyl ether as an inhibitor of QR-2. When applied to the screening of hop extracts from the botanical, Humulus lupulus L., xanthohumol and xanthohumol D were identified as ligands of QR-2. Inhibition of QR-2 by these ligands was confirmed using a functional enzyme assay. Furthermore, binding of xanthohumol and xanthohumol D to the active site of QR-2 was confirmed using X-ray crystallography. Ultrafiltration LC-MS was shown to be a useful assay for the discovery of inhibitors of QR-2 in complex matrixes such as extracts of bacteria and botanicals.

Magnetic mixed hemimicelles solid-phase extraction of xanthohumol in beer coupled with high-performance liquid chromatography determination.

In this study, silica-coated magnetic nanoparticles (Fe(3)O(4)/SiO(2) NPs) modified by cetyltrimethylammonium bromide (CTAB) were synthesized. They were successfully applied for extraction of xanthohumol in beer based on magnetic mixed hemimicelles solid-phase extraction (MMHSPE) coupled with high-performance liquid chromatography-ultraviolet determination. The main factors influencing the extraction efficiency including the surfactant amount, the beer pH, the extraction time, the desorption condition and the maximum extraction beer volume were optimized. Under the optimized conditions, a concentration factor of 60 was achieved by extracting 120 mL beer sample using MMHSPE and the detection limit of xanthohumol is 0.0006 mg/L. The proposed method was successfully applied for determination of xanthohumol in various beer samples with the xanthohumol contents in the range of 0.031-0.567 mg/L. The satisfactory recoveries (90-103%) were obtained in analyzing spiked beer samples.

Human pharmacokinetics of the muscle relaxant, eperisone hydrochloride by liquid chromatography-electrospray tandem mass spectrometry.

Eperisone hydrochloride (4'-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride) is a muscle relaxant agent, widely used in the treatment of patients with muscular contractures, low back pain or spasticity. Because of its mechanism of action (inhibition of gamma-efferent firing and local vasodilatation activity), side effects on central nervous system are rarely observed. A sensitive liquid chromatography-electrospray ionization-mass spectrometry method for determination of eperisone in human plasma has been developed, with a lower limit of quantification of 0.01 ng/mL. The method was applied to a pharmacokinetic study in 12 healthy volunteers given eperisone 100 mg as single dose on day 1 and three times daily on days 2 to 4. Eperisone was rapidly absorbed after oral administration (T (max) = 1.6 h) as it was expected by its fast-onset relaxant activity. Moreover, eperisone underwent a rapid elimination from the body (biological half-life 1.87 h), which was not modified during the repeated dosing as suggested by the C (max) cumulation observed, not different from that expected for a t (1/2) of 1.87 h as suggested by the similar and negligible plasma concentration values (0.063 and 0.067 ng/mL) measured on day 4 before the morning dose and 12 h after evening dose, thus ruling out any potential risk for drug accumulation. Thus, the pharmacokinetic characteristics of eperisone provide further justification for its tolerability in patients with low back pain or spastic palsy, in which the drug is given for periods ranging from few days to several months, respectively.

The complexities associated with new psychoactive substances in influent wastewater: The case of 4-ethylmethcathinone.

Consumption of new psychoactive substances (NPS) is an international problem for health, policing, forensic, and analytical laboratories. The transience of these substances in the community, combined with continual slight structural changes to evade legislation makes the elucidation of NPS an analytical challenge. This is amplified in a matrix as complex as wastewater. For that reason, suspect and non-target methodologies, employing high resolution mass spectrometry are the most appropriate current tool to facilitate the identification of new and existing compounds. In the current work, a qualitative screening method of influent wastewater using liquid chromatography-high resolution mass spectrometry showed a strong signal at m/z 192.1382 - identical to that of two NPS standards that were in our method (pentedrone and 4-methylethcathinone), and with identical fragment ions, but the retention times did not match. This work shows the methodology followed to identify this compound, highlighting the challenges of the identifying "new" compounds in influent wastewater.

Failure Mode Critical Effect Analysis and Design of Experiment-Based Robust Chromatographic Method for Simultaneous Estimation Lornoxicam and Eperisone Hydrochloride.

Failure mode critical effect analysis and design of experiment-based high performance thin layer chromatography (HPTLC) method has been developed for simultaneous estimation of lornoxicam (LOC) and eperisone hydrochloride (EPR). Failure modes were identified on the basis of prior knowledge and experimental data with the help of Ishikawa diagram for the development of method. The criticality of failure mode was assessed by giving risk priority number and criticality rank on the basis of preliminary experimental trials. The identified critical failure modes were analyzed for their effect by design of experiment (DoE)-based Plackett-Burman screening design. From 11 critical factors, the volume of methanol and modifier in mobile phase composition were found as critical failure modes. Critical failure mode was further analyzed by DoE based on central composite design for study of their relationship with resolution of both drugs. Quadratic model suggested by design was further used for failure mode risk control and navigation of design space for a resolution of both drugs more than 1.5. Failure mode risk control strategy was set for robust HPTLC method for simultaneous estimation of both drugs in laboratory mixture. Developed and validated HPTLC method was applied for assay of LOC and EPR in their laboratory mixture and assay values were found in good agreement with a spiked amount of drugs.

The Stability of 4-Chloromethcathinone in Blood and Vitreous Humor.

We present results of our study on the stability of 4-chloromethcathinone (4-CMC) in authentic postmortem peripheral blood and vitreous humor samples. The stability of 4-CMC was determined in postmortem blood samples (for a period of 90 days) and vitreous humor (30 days) at three different temperatures: -15°C, +4°C, and + 23°C. The analyses were carried out using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). In both materials, the lowest 4-CMC stability was demonstrated at room temperature. The blood samples stored in a freezer (-15°C) showed stability for the entire study period (90 days), while in the case of the vitreous humor sample stored at the same temperature the concentration of the substance decreased by 53% after 30 days. The study carried out in authentic postmortem blood and vitreous humor samples confirms the previous reports of 4-CMC instability in biological material. Authors suggest that the biological material should be stored frozen until analyses are carried out as soon as possible after collection of the material.

Univariate and multivariate spectrophotometric methods for simultaneous determination of avobenzone and octinoxate in pure form and in cosmetic formulations: A comparative study.

Simple, economic and precise spectrophotometric and chemometric techniques were used to determine UV filters namely; avobenzone (AV) and octinoxate (OCT) simultaneously in pure form and in cosmetic formulations in concentration range (2-10 µg·mL-1) for both drugs. The spectrophotometric technique includes five different methods; Method (A) is first derivative (D1) spectrophotometry at 380.6 nm for AV and 276.2 nm for OCT, Method (B) is first derivative of ratio spectra (DR1) at 352.8 nm for AV and 312.2 nm for OCT, Method (C) is ratio difference spectrophotometry (RD) at 356 nm and nm 347.2 nm for AV and at 311.6 nm and 281 nm for OCT, Method (D) is mean centering spectrophotometry (MCR) at 356 nm for AV and 301.8 nm for OCT and method (E) is modified Vierordt's method which involves absorbance measurement at 358 nm for AV and 309.2 nm for OCT and determination of the concentration of x and y from the two simultaneous equations. The chemometric technique includes multivariate calibration methods; partial least squares (PLS) and principle component regression (PCR) using the absorption spectra. The proposed methods were applied for determination of (AV) and (OCT) simultaneously in pure form and in cosmetic formulations. These methods were validated according to ICH guidelines.