Development and validation of a reversed-phase high-performance liquid chromatographic method for the quantitation and stability of α-lipoic acid in cosmetic creams.

OBJECTIVE: To develop and validate a simple reversed-phase HPLC method for the quantitation and evaluation of stability of α-lipoic acid in cosmetics, according to International Conference on Harmonization (ICH) Guidelines. METHODS: The chromatography was performed on a reversed-phase Luna C18, analytical column (150 × 4.6 mm id, 5 µm particle size) with a mobile phase of potassium dihydrogen phosphate (pΗ 4.5; 0.05 M) and acetonitrile (60:40, v/v) and a flow rate of 1.0 mL min-1 with UV detection at 340 nm. Accelerated and long-term stability studies of α-lipoic acid in cosmetic cream were conducted under various degradation conditions including acid, basis, oxidation, and thermal and photolytic degradation, according to European Medicines Agency Guidelines CPMP/ICH/2736/99. RESULTS: The limit of detection (LOD) for the cosmetic cream was 0.9 µg mL-1 and the limit of quantitation (LOQ) was 2.8 µg mL-1 , while the retention time was 7.2 min. The method proved to be linear, precise and accurate. The stability results demonstrated the selectivity of the proposed method to the analysis of α-LA, and the degradation products were determined and evaluated in specific stress conditions in cosmetic creams. The applicability of the method was tested in two different developed cosmetic products (cream with 1.5 % w/w and emulsion with 1.0 % w/w of LA) and proved to be reliable. CONCLUSION: A reversed-phase HPLC-UV method was developed and fully validated for the analysis of α-lipoic acid in cosmetics. It is the first reported application on the quantitation of lipoic acid in cosmetic creams, while at the same time evaluates the stability in forced degradation conditions, in new cosmetic formulations. It proved to be suitable for the reliable quality control of cosmetic products, with a run time of <8 min that allows for the analysis of large number of samples per day.

OBJECTIF: Développer et valider une méthode HPLC (chromatographie en phase liquide à haute performance) simple en phase inversée pour la quantification et l'évaluation de la stabilité de l'acide α-lipoïque dans les cosmétiques, conformément aux Directives de la Conférence internationale sur l'harmonisation (ICH). MÉTHODE: La chromatographie a été réalisée sur une colonne analytique Luna C18 en phase inversée (150 × 4,6 mm id, taille des particules 5 µm) avec une phase mobile de dihydrogénophosphate de potassium (pH 4,5 ; 0,05 M) et d'acétonitrile (60:40, v/v) et un débit de 1,0 ml min−1 avec détection UV à 340 nm. Des études de stabilité accélérée et à longterme de l'acide α-lipoïque dans les crèmes cosmétiques ont été menées dans diverses conditions de dégradation, notamment en milieu acide, basique, par oxydation et dégradation thermique et photolytique, conformément aux lignes directrices de l'Agence européenne des médicaments CPMP/ICH/2736/99. RÉSULTAT: La limite de détection (LD) pour la crème cosmétique était de 0,9 µg ml et la limite de quantification (LQ) était de 2,8 µml−1 , tandis que le temps de rétention était de 7,2 min. La méthode s'est avérée linéaire, précise et exacte. Les résultats de stabilité ont démontré la sélectivité de la méthode proposée pour l'analyse de l'acide α-lipoïque et les produits de dégradation ont été déterminés et évalués dans des conditions de stress spécifiques dans les crèmes cosmétiques. L'applicabilité de la méthode a été testée dans deux produits cosmétiques différents développés (crème avec 1,5 % p/p et émulsion avec 1,0 % p/p d'acide lipoïque) et s'est avérée fiable. CONCLUSION: une méthode HPLC en phase inversée avec détection UV a été développée et entièrement validée pour l'analyse de l'acide α-lipoïque dans les cosmétiques. Il s'agit de la première application signalée concernant la quantification de l'acide lipoïque dans les crèmes cosmétiques et permettant en même temps d'évaluer la stabilité des conditions de dégradation forcée dans les nouvelles formulations cosmétiques. Cette méthode s'est avérée adaptée au contrôle de qualité fiable des produits cosmétiques, avec une durée d'exécution < 8 min qui permet l'analyse d'un grand nombre d'échantillons par jour.

Biomarkers of opiate use.

The interpretation of toxicological findings is critical for the thorough investigation of the use and abuse of psychoactive substances. A positive analytical result for a sample taken could usually result in criminal proceedings and a punitive outcome for the defendant whose sample was analysed. The detection of markers of illicit opiate misuse is important both in the management of substance misuse and in the postmortem identification of illicit opiate use. The aim of this study was to emphasise the role of opiate biomarkers available at the laboratory and in the clinical environment. Urine remains the biological tool of choice for qualitative detection of illicit drug use in a clinical setting, while quantitative accuracy remains strictly the domain of blood. Accurate interpretation of the screening tests within a clinical setting alongside other relevant information remains the key to the usefulness of any test. Moreover, the finding of a morphine/codeine concentration ratio in blood exceeding unity is a strong evidence that the person had used heroin, as opposed to having taken a prescription analgesic drug containing codeine.

Validated GC/MS method for the simultaneous determination of clozapine and norclozapine in human plasma. Application in psychiatric patients under clozapine treatment.

A sensitive and specific GC/MS method for the determination of clozapine (CLZ) and its major metabolite norclozapine (NCLZ), in plasma has been developed, optimized and validated. Specimen preparation includes solid-phase extraction of both analytes using Bond-Elut Certify cartridge and further derivatization with TFAA. Clozapine-d8 was used as internal standard for the determination of CLZ and NCLZ. Limits of detection were 0.45 ng/mL for CLZ and 1.59 ng/mL for NCLZ, while limits of quantification were 1.37 ng/mL for CLZ and 4.8 ng/mL for NCLZ, as calculated by the calibration curves. The calibration curves were linear up to 600 ng/mL for CLZ and NCLZ. Absolute recovery ranged from 82.22% to 95.35% for both analytes. Intra- and interday accuracy was less than 7.13% and --12.52%, respectively, while intra- and interday precision was between 9.47% and 12.07%, respectively, for CLZ and NCLZ. The method covers all therapeutic range and proved suitable for the determination of CLZ and NCLZ not only in psychiatric patients but also in forensic cases with clozapine implication.

Forensic investigation of submersion deaths.

CONTEXT: The diagnosis of the cause and the establishment of the manner of death in submersion cases are routine challenge for forensic pathologists as it presents considerable diagnostic difficulties. OBJECTIVE: To concisely review the main questions during a submersion death investigation and to give the respective documented answers. DATA SOURCES: The search strategy included a literature search of PubMed, Medline and Google Scholar databases, as well as a review of the cited references by the identified studies and a hand search of relevant textbooks and reference works. CONCLUSIONS: A complete autopsy, histopathological examination and full toxicological screening are important to determine whether death indeed followed submersion in the water, or to see whether any natural disease or substance use have contributed or caused death. In ambiguous situations, the co-estimation of circumstantial evidence may be of invaluable importance toward the conclusion concerning the cause and the manner of death. The thorough forensic investigation of the submersion deaths not only serves the justice administration, but it also presents considerable benefits for the public health.

Investigation of the identification point system adaptation in cocaine, benzoylecgonine and ecgonine methyl ester using a single quadrupole mass spectrometer.

At present, no official criteria exist for drug identification using single quadrupole mass spectrometers although the European Union (EU) criteria for compound identification have been adopted. These criteria are evaluated with respect to the confirmation of cocaine and its metabolites by single quadrupole liquid chromatography/mass spectrometry (LC/MS) and problems are highlighted. Spiked samples, proficiency testing samples, certified reference materials and samples from real cases that had screened positive for cocaine derivatives by immunoassay were subjected to confirmation by LC/MS using single ion monitoring with in-source fragmentation. The EU criteria for compound identification were applied for the confirmation of cocaine, benzoylecgonine and ecgonine methyl ester. The use of the identification point (IP) system in spiked, proficiency testing samples and certified reference materials provided acceptable results in all cases while in some cases real positive samples did not provide acceptable results. Failure to meet the EU criteria was attributed to low fragmentation at the lower concentrations and the ion suppression effect while both factors affected compliance with the IP system. The identification of cocaine and its metabolites was considerably improved by using a combination of ammonium formate and formic acid as the LC mobile phase. It appears that poor in-source fragmentation in single quadrupole LC/MS and ion suppression may constitute a problem with drug identification when implementing the IP system in real samples, resulting in false negative results. Further investigation is needed for the use of such IP systems to be suitable for use in LC/MS methods.

Butyrylcholinesterase: biomarker for exposure to organophosphorus insecticides.

Butyrylcholinesterase (BuChE) is routinely measured to assess exposure to or effects of organophosphorus insecticides (OP). As a biomarker, it can be used to clarify the relation between exposure to OP and health impairment. The interpretation of BuChE inhibition data, particularly of small changes in enzymatic activity, sometimes presents significant complexities. These complexities are presented in this short communication and the factors that influence the degree of BuChE inhibition are discussed. Despite the complexities of their interpretation, BuChE measurements remain a mainstay for the fast initial screening of exposure to OP; thus, they are a useful tool in the protection of humans, domestic animals and wildlife from overexposure to these toxic agents.

Health impacts of fire smoke inhalation.

Most fatalities from fires are not due to burns, but are a result of inhalation of toxic gases produced during combustion. Fire produces a complex toxic environment involving flame, heat, oxygen depletion, smoke and toxic gases. As a wide variety of synthetic materials is used in buildings (insulation, furniture, carpeting, and decorative items) the potential for severe health impacts from inhalation of products of combustion during building fires is continuously increasing. In forest fires the burning of biomass leads to smoke emissions, the composition of which, as well as the relative health impacts from their inhalation, depends on the ecosystem's chemical and physical features and the local environmental parameters. In the present review, health problems that appear to people exposed to fire smoke and especially to firefighters are described and suggestions for fire management and prevention are made.

Toxicological investigation of drug-facilitated sexual assaults.

Drug-facilitated sexual assaults have drawn a lot of attention during the last years. The role of the non-forensic doctors in the early recognition and management of such cases is crucial. Diagnosis assisted by samplings must be ascertained without delay, in victims initially brought to the emergency room for a sleepy state, a suspected abuse of drug or a pseudo-alcoholic clinical state. Further, where amnesia is present, it may be prudent to assume that sexual activity has occurred and offer pregnancy and sexually transmitted disease prophylaxis. Forensic doctors should entertain the possibility of poisoning in any presentation involving amnesia. Forensic toxicological laboratories should develop sensitive and specific methods to assure a low threshold of detection of the drugs. The awareness of a delinquent or criminal use of alcohol and/or drugs could permit a better medical recognition, so that proper and timely forensic examination is achieved, and adequate evidence is collected for the establishment of an effective investigation and prosecution.

Rice-body formation and tenosynovitis of the wrist: a case report.

A 61-year-old woman presented with mild pain and swelling on the volar aspect of the wrist, hand, and little finger. Radiography showed a soft-tissue mass shadow, and magnetic resonance imaging showed acute tenosynovitis of the flexor tendons and an inflammatory mass inside the carpal tunnel. Laboratory test results were normal, except for an elevated erythrocyte sedimentation rate (40 mm/h). The patient had an ambiguous Mantoux test result but no history of mycobacterial exposure. Exhaustive investigations for rheumatic disease were negative. Surgical exploration of the lesion revealed rice bodies in the common flexor tendon synovial sheath, extending distally to the tip of the fifth finger. Removal of the rice bodies and thorough excision of the sheath was performed. The patient regained a full and painless range of motion in about 3 months. One-year follow-up revealed no underlying disorder.

Toxicological aspects of fire.

Most fatalities from fires are not due to burns, but are a result of inhalation of toxic gases produced during combustion. Fire produces a complex toxic environment, involving flame, heat, oxygen depletion, smoke and toxic gases. As a wide variety of synthetic materials is used in buildings (insulation, furniture, carpeting, electric wiring covering, decorative items), the potential for poisoning from inhalation of products of combustion is continuously increasing. In the present review, the problems that are present in a fire event, the toxicology of the toxic substances and the specific chemical hazards to firefighters are described. Regulatory toxicology aspects are presented concerning the use of non-flammable building and furnishing materials to prevent fires and decrease of poisonings and deaths resulting from fires.

Occupational exposure to cholinesterase inhibiting pesticides: a Greek case.

The determination of plasma or serum cholinesterase is absolute and it is considered as a reliable index of exposure in workers of organophosphorus pesticides industries. In the present study the plasma cholinesterase of 28 persons working in the packaging of an ortho-thio-phosphate was determined, before and after their exposure to this agent. The results of this study showed a plasma cholinesterase depression of 37%, a decrease which was statistically significant (P<0.001).

Lead content of fresh milk samples from different sites in Athens.

Milk and dairy products constitute a major food, especially for infants and children, and relatively low levels of toxic elements can contribute significantly to dietary intakes and be hazardous for public health. The purpose of this survey was to define the levels of lead in milk samples of different origin, to establish the presence or absence of contamination. The milks from different sites of the metropolitan area of Athens contained negligible traces of lead and thus are safe for the public health.

Immunomodulative effects of aflatoxins and selenium on human natural killer cells.

The possible immunomodulative effects of aflatoxin B1 and selenium (as sodium selenite) at concentrations lower than 5.10(-11) M or 0.05ppb and 10(-5) M, respectively on natural killer (NK) cells from healthy volunteers were evaluated by a 51Cr release natural killer assay. Aflatoxin B1 and selenium separately had an immunosuppressive effect on NK cell activity. When they were studied in combination, they caused a statistically significant increase of the cytotoxic activity of NK cells in the presence of the lowest concentrations of aflatoxin B1 (0.005ppb) and of sodium selenite (10(-8)M).

Analysis of total mercury in human tissues prepared by microwave decomposition using a hydride generator system coupled to an atomic absorption spectrometer.

A fast and efficient procedure has been developed for the analysis of total mercury in human tissues and blood using a hydride vapor generator system coupled to an atomic absorption spectrometer (HVG-AA). Tissue and blood samples were digested in a pressurized microwave decomposition system and the digest diluted prior to formation of free mercury vapor and analysis by atomic absorption. Recovery studies performed on 10 spiked/unspiked pairs of human liver and on 10 spiked/unspiked pairs of human blood samples yielded average recoveries of 99.7% (CV=0.4%) and 101.2% (CV=0.5%), respectively. The method detection limit for liver and blood was 50 microg Hg/kg and 12.5 microg Hg/l, respectively. The "normal" concentrations of mercury in human liver and blood are 33-490 microg Hg/kg and 0.6-59 microg Hg/l, respectively [1]. This method is able to determine mercury poisoning levels and may also be applied to detect mercury near the lower levels of these "normal" ranges, using the standard addition method approach.

The importance of performing in vitro cytotoxicity testing before immunomodulation evaluation.

This study uses the well known sensitive and reproducible human peripheral blood lymphocyte (PBL) assay as a model for the possible discrimination between cytotoxic and/or immunomodulative properties of 5 reference compounds: cadmium chloride (CdCl2): didodecyl-dimethyl ammonium bromide (DDAB); cyclosporin (CsA); dexamethasone (DEXA); and theophylline. Two colorimetric cytotoxicity assays were employed, the thiazolyl blue tetrazolium bromide and neutral red, and the H-thymidine DNA incorporation assay on phytohemagglutinin stimulated or non-stimulated PBL was used, as a parameter of cell mediated immunity. This study demonstrated the importance of performing colorimetric or other cytotoxicity assays before any investigation of mechanisms of action of different substances on immunomodulation and relative human risk assessment. This study showed that CdCl2 and DDAB have a cytotoxic rather than immunomodulative effect on PBL and that CsA, DEXA and to a lesser extent theophylline have immunomodulative rather than cytotoxic effects on PBL within the range of the concentrations studied.

Cytotoxic and immunomodulative effects of flunitrazepam, zipeprol and trihexyphenidyl on human peripheral blood lymphocytes.

The cytotoxic effects of flunitrazepam, zipeprol and trihexyphenidyl on cell viability of peripheral blood mononuclear cells from healthy volunteers were studied at concentrations from 10(-2) to 10(-8) M by 2 colorimetric in vitro assays: the neutral red uptake assay and thiazolyl blue tetrazolium bromide assay. All tested drugs of abuse were non-cytotoxic at concentrations lower than 10(-5) M. Possible immunomodulative effects of these substances were evaluated through phytohemagglutinin induced lymphocyte proliferation ([3H]-thymidine DNA incorporation assay) and by a 51Cr release natural killer assay. The 3 drugs studied produced statistically significant immunomodulative alterations on both immunological parameters.

Delat9-tetrahydrocannabinol content in cannabis plants of greek origin.

The delta9-tetrahydrocannabinol (delta9-THC) content was identified and determined quantitatively using a Gas Chromatography Detector (Gas Chromatography-Electron Ion Detector) instrument in samples of illicit herbal cannabis. Law enforcement authorities sent the samples to the Department of Forensic Medicine and Toxicology, University of Athens, for toxicological analysis. The concentrations of delta9-THC in these samples ranged from 0.08% to 4.41%. Such concentrations suggest that Greece might be at high risk, as an area for the illicit cultivation of "pedigree" cannabis plants. The forensic aspects of cannabis classification are discussed.

An unusual case of lead poisoning in an infant: nursing-associated plumbism.

Lead poisoning remains a common environmental threat for children today. The majority of cases of lead poisoning are caused by oral intake of lead dust or lead-containing paint flakes and thus occur during the second and third years of life. An unusual case of severe lead poisoning in a breast-feeding infant girl is here presented; the source proved to be a nipple shield made of a lead-containing metal. Despite the severity of the intoxication, by the end of treatment and for a year afterwards the infant has been well and her psychokinetic development has been normal.