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.

An evaluation of dental methods by Lamendin and Prince and Ubelaker for estimation of adult age in a sample of modern Greeks.

Teeth can be used as accurate tools in age-at-death estimation in forensic cases. No previous data exist on estimating age from teeth in a modern Greek population. The aim of this study was to evaluate Lamendin's and Prince and Ubelaker's ageing methods on a modern Greek skeletal sample. In total, 1436 single-rooted teeth from 306 adult individuals (161 males and 145 females) were examined. Only measurements of periodontosis and translucency showed positive correlation with age. Results showed a bias - an overestimation for ages under 40 years and an underestimation over this age. However, the use of wider age groups proved to be more appropriate. Low values of error were observed for the group of middle-aged individuals. In conclusion, both methods can be considered accurate in estimating age-at-death of middle-aged individuals. This study provides more information about the accuracy and applicability of these dental methods on modern European populations.

Analysis of volatile organic compounds released from the decay of surrogate human models simulating victims of collapsed buildings by thermal desorption-comprehensive two-dimensional gas chromatography-time of flight mass spectrometry.

Field experiments were devised to mimic the entrapment conditions under the rubble of collapsed buildings aiming to investigate the evolution of volatile organic compounds (VOCs) during the early dead body decomposition stage. Three pig carcasses were placed inside concrete tunnels of a search and rescue (SAR) operational field terrain for simulating the entrapment environment after a building collapse. The experimental campaign employed both laboratory and on-site analytical methods running in parallel. The current work focuses only on the results of the laboratory method using thermal desorption coupled to comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (TD-GC×GC-TOF MS). The flow-modulated TD-GC×GC-TOF MS provided enhanced separation of the VOC profile and served as a reference method for the evaluation of the on-site analytical methods in the current experimental campaign. Bespoke software was used to deconvolve the VOC profile to extract as much information as possible into peak lists. In total, 288 unique VOCs were identified (i.e., not found in blank samples). The majority were aliphatics (172), aromatics (25) and nitrogen compounds (19), followed by ketones (17), esters (13), alcohols (12), aldehydes (11), sulfur (9), miscellaneous (8) and acid compounds (2). The TD-GC×GC-TOF MS proved to be a sensitive and powerful system for resolving the chemical puzzle of above-ground "scent of death".

Sex determination by tooth size in a sample of Greek population.

Sex assessment from tooth measurements can be of major importance for forensic and bioarchaeological investigations, especially when only teeth or jaws are available. The purpose of this study is to assess the reliability and applicability of establishing sex identity in a sample of Greek population using the discriminant function proposed by Rösing et al. (1995). The study comprised of 172 dental casts derived from two private orthodontic clinics in Athens. The individuals were randomly selected and all had clear medical history. The mesiodistal crown diameters of all the teeth were measured apart from those of the 3rd molars. The values quoted for the sample to which the discriminant function was first applied were similar to those obtained for the Greek sample. The results of the preliminary statistical analysis did not support the use of the specific discriminant function for a reliable determination of sex by means of the mesiodistal diameter of the teeth. However, there was considerable variation between different populations and this might explain the reason for lack of discriminating power of the specific function in the Greek population. In order to investigate whether a better discriminant function could be obtained using the Greek data, separate discriminant function analysis was performed on the same teeth and a different equation emerged without, however, any real improvement in the classification process, with an overall correct classification of 72%. The results showed that there were a considerably higher percentage of females correctly classified than males. The results lead to the conclusion that the use of the mesiodistal diameter of teeth is not as a reliable method as one would have expected for determining sex of human remains from a forensic context. Therefore, this method could be used only in combination with other identification approaches.

Forensic investigation of suicide cases in major Greek correctional facilities.

INTRODUCTION: According to Greek legislation the medico-legal investigation of deaths occurring in prisons is mandatory. Furthermore, in cases of suicide or of suspected suicide the contribution of medico-legal investigation is of grave importance. The current paper addresses the medico-legal investigation of suicide cases in Greek correctional facilities and aims to describe the current situation. MATERIALS & METHODS: Our study consists of the meticulous research in the data records of major Greek correctional facilities, for the time period 1999-2010. Official permission was obtained by the Hellenic Ministry of Justice, which provided us the access to the records. Data was also collected from the Piraeus Forensic Service, from the Department of Pathological Anatomy of the University of Athens and finally from our own records. Measures were taken to respect the anonymity of the cases. Data was collected for the social, penal, medical history as well as for the medico-legal investigation. RESULTS-CONCLUSIONS: It appears that 85.7% of suicide cases were transferred to the Prisoner's Hospital (p < 0.0001), the forensic pathologist who conducted the PME did not perform scene investigation in none of the 70 suicide cases. In a total of 70 cases, histopathological examination, was requested only in 30 cases (42.9%). Hanging was the preferred method for those who committed suicide, followed by the poisoning due to psychoactive substances. Understanding the mistakes made during the forensic investigation of suicide cases inside correctional facilities is necessary, in order to prevent them from occurring again in the future, by implementing appropriate new policies and guidelines.

The social profile of victims of suicide in major Greek correctional facilities.

INTRODUCTION: Suicide rates in correctional institutions have been increasing during the last decades. The reasons for this increase remain unclear, yet a lot of contradictory explanations were stated: the increase might be due to mass incarceration and overcrowding of small cells resulting in high psychosocial stress, changes in psychiatric health policy which might have transferred the care for patients from mental hospitals to custodial institutions, or legislation changes that might have led to a selection of offenders at higher risk (e.g. offenders who committed high violent offences or suffered from mental disorders without being referred to psychiatric hospitals). In Greece the situation is not described in details, at least during the last few years. By law, every death of prisoner is subject to medicolegal investigation. MATERIALS & METHODS: Our study consists of the meticulous research of the data records of major Greek correctional facilities, for the time period 1999-2010. An official permission was obtained from the Hellenic Ministry of Justice, which gave us access to these restricted records. Data was also collected from the Piraeus Forensic Service, from the Department of Pathological Anatomy of the University of Athens and finally from our own records. Measures were taken to respect the anonymity of the cases. Data was collected for the social, penal, medical history as well as for the medicolegal investigation. RESULTS - CONCLUSIONS: A total of 339 cases were collected, only 259 of which had available full data records, due to weaknesses in the prison records. From the 259 cases, 70 incidents of suicide were collected. Victims of suicide in major Greek correctional facilities, appear to fulfill the expected profile, as in the general population.

Physiology and biochemistry of human subjects during entrapment.

A classification of various categories of entrapped people under the ruins of collapsed buildings after earthquakes, technical failures or explosions is proposed. Type and degree of injury at the moment of building collapse and duration of entrapment are the two basic parameters in this classification. The aim is to provide sources and types of volatile organic compounds (VOCs) that can be used for establishing a new method for locating entrapped victims based on human chemical signatures. Potential target compounds, among others, are ammonia, acetone, isoprene, dimethylsulfide, dimethyldisulfide and trimethylamine. In this context, the possible neuroendocrine, metabolic and physical responses of potential victims during the different types of entrapment are correlated with the sources of VOCs such as expired air, urine, blood and sweat. The proposed classification scheme was developed as part of an integrated research project which investigates the use of combined audio, video and chemical methods for the early location of entrapped people under the ruins of collapsed buildings.

Combined chemical and optical methods for monitoring the early decay stages of surrogate human models.

As the body decays shortly after death, a variety of gases and volatile organic compounds (VOCs) constantly emanate. Ethical and practical reasons limit the use of human corpses in controlled, time-dependent, intervening experiments for monitoring the chemistry of body decay. Therefore the utilization of pig carcasses serves as a potential surrogate to human models. The aim of this work was to study buried body decay in conditions of entrapment in collapsed buildings. Six domestic pigs were used to study carcass decay. They were enclosed in plastic body bags after being partially buried with rubbles, resembling entrapment in collapsed buildings. Three experimental cycles were performed, employing two pig carcasses in each cycle; VOCs and inorganic gases were measured daily, along with daily visible and thermal images. VOCs were collected in standard sorbent tubes and subsequently analyzed using a Thermal Desorption/Gas Chromatograph/high sensitivity bench-top Time-of-Flight Mass Spectrometer (TD/GC/TOF-MS). A comprehensive, stage by stage, detailed information on the decay process is being presented based on the experimental macroscopic observations, justifying thus the use of pig carcasses as surrogate material. A variety of VOCs were identified including almost all chemical classes: sulfur, nitrogen, oxygen compounds (aldehydes, alcohols, ketones, acids and esters), hydrocarbons, fluorides and chlorides. Carcasses obtained from a pig farm resulted in more sulfur and nitrogen cadaveric volatiles. Carbon dioxide was by far the most abundant inorganic gas identified along with carbon monoxide, hydrogen sulfide and sulfur dioxide. Visual monitoring was based on video captured images allowing for macroscopic observations, while thermal camera monitoring which is mostly temperature dependent, resulted in highlighting the local micro-changes on the carcasses, as a result of the intense microbial activity. The combination of chemical and optical methods proved very useful and informative, uncovering hidden aspects of the early stages of decay and also guiding in the development of combined chemical and imaging methods for the detection of dead bodies.

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.

Fundamental issues in forensic semen detection / Problemas fundamentales en la detección forense de semen

Sperm detection can be an important factor in confirming sexual assault in cases of rape. A large number of cases received in a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains. The objective of this paper is to provide an overview of the most important methods and tests used in the identification of spermatozoa or constituents ofseminal fluid during the investigation ofalleged sexual assault cases in forensic medical practice. Furthermore, this paper focusses on the basic knowledge that is necessary to the graduate students who wish to specialize in forensic sciences.

La detección de esperma puede ser un factor importante a la hora de confirmar un ataque sexual en los casos de violación. Un gran número de los casos recibidos en el laboratorio forense tienen relación con ofensas sexuales, lo cual hace necesario examinar muestras de presencia de manchas seminales. El objetivo de este trabajo es proporcionar una apreciación global de los métodos y pruebas más importantes usados en la identificación de espermatozoos o constituyentes del fluido seminal durante la investigación de supuestos casos de ataque sexual, en la práctica médica forense. Además, este trabajo presta atención al conocimiento básico necesario para los estudiantes graduados que desean especializarse en las ciencias forenses.

Self-injurious behavior among Greek male prisoners: prevalence and risk factors.

BACKGROUND: Self-harm among prisoners is a common phenomenon. This study aims to estimate the prevalence of self-injurious behavior (SIB) among Greek male prisoners, record their motives and determine independent risk factors. METHODS: A self-administered, anonymous questionnaire was administered to 173 male prisoners in the Chalkida prison, Greece. The questionnaire included items on self-harm/SIB, demographic parameters, childhood history, family history, physical and mental disease, lifestyle and smoking habits, alcohol dependence (CAGE questionnaire), illicit substance use, aggression (Buss-Perry Aggression Questionnaire [BPAQ] and Lifetime History of Aggression [LTHA]), impulsivity (Barrat Impulsivity Scale-11) and suicidal ideation (Spectrum of Suicidal Behavior Scale). Univariate nonparametric statistics and multivariate ordinal logistic regression were performed. RESULTS: Of all the participants, 49.4% (95% CI: 41.5-57.3%) disclosed self-harm (direct or indirect). The prevalence of SIB was equal to 34.8% (95% CI: 27.5-42.6%). Most frequently, SIB coexisted with indirect self-harm (80.7%). The most common underlying motives were to obtain emotional release (31.6%) and to release anger (21.1%). At the univariate analysis, SIB was positively associated with a host of closely related factors: low education, physical/sexual abuse in childhood, parental neglect, parental divorce, alcoholism in family, psychiatric condition in family, recidivism, age, sentence already served, impulsivity, aggression, alcohol dependence, self-reported diagnosed psychiatric condition and illicit substance use. Childhood variables were particularly associated with the presence of diagnosed psychiatric condition. At the multivariate analysis, however, only three parameters were proven independent risk factors: self-reported diagnosed psychiatric condition, illicit substance use and aggression (BPAQ scale). CONCLUSION: The prevalence of SIB is particularly high. Psychiatric condition, illicit substance use and aggression seem to be the most meaningful risk factors; childhood events seem only to act indirectly.

Fundamental issues in forensic semen detection.

Sperm detection can be an important factor in confirming sexual assault in cases of rape. A large number of cases received in a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains. The objective of this paper is to provide an overview of the most important methods and tests used in the identification of spermatozoa or constituents of seminal fluid during the investigation of alleged sexual assault cases in forensic medical practice. Furthermore, this paper focusses on the basic knowledge that is necessary to the graduate students who wish to specialize in forensic sciences.

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.

Health care provision in prisons: a review on European and international guidelines.

Health care provision in prisons is an area of increasing international concern. The spectrum of health problems which prisoners may bring to prison is wide and in many cases prevalence is greater than in the general population.The profound knowledge of delicate issues such as medical confidentiality and informed consent of the patient, with special refer to infectious disease, hunger strike and prison research is necessary in the prison working environment. In the following paragraphs the European and International conventions and guidelines on the matter are discussed.

Human exposure to endocrine disruptors and breast milk.

Endocrine system is one of the most sensitive communication networks of the human body which influences all aspects of human health and well-being, including reproductive potential, cognitive functions, thyroid and metabolism, digestion and hormonal balance. In recent years basic laboratory research has been focused on the potential relationship between environmental contaminants and cellular endocrine function. Environmental contaminants are ubiquitous in the environment, alter endocrine physiology and produce endocrine disruption without acting as classic toxicants. These endocrine disruptors (EDCs) are lipophilic and stored for long periods of time in the adipose tissue. Maternal exposure to EDCs during pregnancy and lactation has as a result the exposure of the fetus and neonate through placenta and breast milk. It has been recognized that human milk is the best natural food for neonates providing immunologic, developmental and practical advantages throughout childhood. However, contamination of human milk by the presence of environmental toxicants is widespread through the past decades due to inadequately controlled pollution. Persistent pesticides, chemical solvents and others tend to invade slowly the environment, to bioaccumulate in the food chain and to have long half-lives in animals and humans. During the past fifteen years, the scientific interest has been focused on xenoestrogens, i.e.,environmental chemicals with estrogen disrupting activity. Certain adverse health and reproductive outcomes are attributed to these chemicals in wildlife, in laboratory animals, as well as in humans. Although most toxic agents are hazardous in high doses, the human health risks associated with EDCs concern exposure to low doses. The human health risks that may be associated with these low-level but constant exposures are still largely unknown and highly controversial. In this paper, we review available data on environmental chemicals present in breast milk that may affect child health through breastfeeding. Specifically, we focused on the breast-feeding pharmacokinetic aspects related to infant exposure of chemical pollutants that have estrogen and antiandrogen activities, such as environmental estrogen disruptors or xenoestrogens.

Do clinically insignificant tumors of the prostate exist?

BACKGROUND: The discrepancy between minimal disease on biopsy and disease found in the subsequent prostatectomy specimen, in terms of the size and grade of tumor, extracapsular extension or positive margins, led several authors to dispute the existence of clinically insignificant impalpable tumors of the prostate. However, considering that prostate-specific antigen (PSA) is an indicator of prostate malignancy and since many impalpable prostatic carcinomas (PCs) are detected by a combination of PSA, transurethral ultrasound and needle biopsy (T1c), in the era of PSA screening, it is expected that most of the impalpable tumors found incidentally at transurethral resection of the prostate (stage T1a/b), could be clinically insignificant. AIM: The aim of this study was to identify the characteristics of latent, impalpable PCs and to analyze the incidence of clinically insignificant PCs among hypothetical stage T1 prostate cancers in tumors found incidentally at postmortem examination. METHODS: We examined 40 cases of impalpable PCs found in 212 prostate autopsy specimens of men between 30 and 98 years of age who died of diseases other than carcinoma of the prostate and related conditions. RESULTS: Most of T1 histological PCs (57.5%) had a Gleason score between 2 and 4, while 30% had Gleason score between 5 and 6. Only 5 (12.5%) had a Gleason score above 7. Twenty-nine of 40 stage T1 histological cancers (67.5%) had volume of <1 cm(3). The highest volume tumors were those of intermediate and high grade (Gleason sums 5-8). Among tumors with volumes of <1 cm(3), 96.55% were confined within the prostatic capsule. CONCLUSIONS: The majority of impalpable PCs were low-volume, well-differentiated tumors corresponding to clinically insignificant neoplasms. Similar characteristics could be attributed to most of the impalpable carcinomas detected after prostatectomy in clinical practice.