Evaluation of an Oral Fluid Collection Device and a Solid-Phase Extraction Method for the Determination of Coca Leaf Alkaloids by Gas Chromatography-Mass Spectrometry.

Some South American countries have ancient traditions that may pose legal problems, such as the consumption of coca leaves, as this can provide positive results for cocaine use after the analysis of biological samples. For this reason, it is necessary to find specific markers that help differentiate legal from illegal consumption, such as tropacocaine, cinnamoylcocaine, and especially hygrine and cuscohygrine. In this work, two techniques for collecting biological samples are compared: the Quantisal® Oral Fluid collection device and passive drooling. Once the samples were collected, they were subjected to solid-phase extraction for subsequent injection into GC-MS. Different validation parameters included in international guides have been studied to evaluate whether the proposed method is valid for the defined purpose, placing special emphasis on the study of the matrix effect and little value on GC-MS analyses. With respect to this parameter, an increase in the signal was found for CUS and t-CIN, but it was not significant for the rest of the substances studied. The recoveries have varied significantly depending on the way of working, being higher when working with standardized areas. After carrying out work with the oral fluid samples collected from laboratory volunteers, the method was applied to two real samples. The results obtained support the need for further research to overcome certain limitations presented by the device.

Detection of coca alkaloids in oral fluid from coca leaf (tea) consumers: using solid phase extraction to improve validation parameters and widen the detection window.

Hygrine and cuscohygrine, two coca leaf alkaloids, have been previously proposed as markers to differentiate legal and illegal cocaine consumption. This is a very common problem in some countries of South America, where the consumption of coca leaves has a long tradition. Analytical methods focusing on the assessment of coca leaf alkaloids, such as cuscohygrine, hygrine, tropacocaine and t-cinnamoylcocaine, in oral fluid are virtually non-existent in forensic toxicology laboratories worldwide due to their lack of application. However, the problem of differentiating legal and illegal cocaine use in criminal justice, DUID (drug-impaired driving) and WDT (workplace drug testing) programs is growing. Therefore, researchers are obliged to develop methods to measure coca leaf alkaloids (cuscohygrine, hygrine and t-cinnamoylcocaine) in biological matrices for further validation for routine analyses in forensic toxicology laboratories. This work aims to optimize a previously published separation method by protein precipitation in oral fluid by using solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in multiple reaction monitoring (MRM) mode. The use of SPE allowed the matrix effect and the background to be reduced in the chromatograms due to the obtained cleaner extracts. Consequently, improved detection and quantification limits were reached. Findings showed that the detection windows for coca leaf alkaloids were longer than three hours in real oral fluid samples from volunteers who drank a cup of coca tea. These detection windows are quite higher than those previously obtained when using the method based on separation by protein precipitation.

Personal Doses of Cocaine and Coca Paste are Adulterated in Cartagena de Indias (Colombia).

Knowledge of drug composition consumed on the streets and the identification and quantification of their adulterants is essential for understanding unexpected side effects, tracking routes, and drug profiling. Therefore, this work aimed to determine the purity and to identify and quantify the main adulterants found in personal doses of cocaine (perico) and coca paste (bazuco) in Cartagena de Indias (Colombia). The data collected in this study describe a first attempt to introduce the qualitative and quantitative analyses of adulterants present in street drugs in Cartagena de Indias to improve surveillance. Through gas chromatography coupled to mass spectrometry (GC-MS), the purity and adulterants were quantified in 45 personal doses of cocaine powder and coca paste. 100% of the personal doses in the city were adulterated; caffeine, phenacetin, and levamisole were the main adulterants identified in cocaine. Besides the above, lidocaine was also found in coca paste. The purity of cocaine varied from 8% to almost 70%, with caffeine ranging from 6% to 42%. In the case of coca paste, the maximum content of cocaine found was 60%, while some samples contained as little as 14%. The results are consistent with other research in terms of the widespread use of caffeine as an adulterant, but they also follow the growing trend of the use of levamisole and phenacetin. The wide range of cocaine content in samples sold in the illicit market could cause undesirable effects on cocaine users who do not know the exact intended dose for consumption; so, this study intends to make these results available not only to academic, public health, and national security agencies but also to tourists entering Cartagena de Indias, so that they are aware of what they are consuming and the risks to which they are exposed.

Analysis of Erythroxylum coca Leaves by Imaging Mass Spectrometry (MALDI-FT-ICR IMS).

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can determine the chemical identity and spatial distribution of several molecules in a single analysis, conserving its natural histology. However, there are no specific studies on the spatial distribution of alkaloids in Erythroxylum coca leaves by MALDI IMS, preserving the histology of the monitored compounds. Therefore, in this work, positive-ion mode MALDI Fourier-transform ion cyclotron resonance imaging mass spectrometry (MALDI(+)FT-ICR IMS) was applied to identify and analyze the distribution of alkaloids on the surface of coca leaves, evaluating the ionization efficiency of three matrices (α-cyano-4-hydroxycinnamic acid (CHCA), 2-mercaptobenzothiazole (MBT), and 2,5-dihydroxybenzoic acid (DHB)). The last was chosen as the best matrix in this study, and it was studied in five concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 mg·mL-1), where 2 mg·mL-1 was the most efficient. The washing of coca leaves with the organic solvents (acetonitrile, methanol, toluene, and dichloromethane) tested did not improve the performance of the ionization process. Finally, a tissue section, 50 µm thick, was used to study the inner part of the leaf tissue, where alkaloids and flavonoid molecules were detected.

Development of a Reliable Method for Assessing Coca Alkaloids in Oral Fluid by HPLC-MS-MS.

A reliable method based on high-performance liquid chromatography-tandem mass spectrometry has been developed for the assessment of coca alkaloids/metabolites [cocaine (COC), benzoylecgonine (BE), cocaethylene (CE), ecgonine methyl ester (EME), anhydroecgonine methyl ester (AEME), tropococaine (TRO), transcinnamoylcocaine (trCIN), cuscohygrine (CUS) and hygrine (HYG)] in oral fluid samples from cocaine abusers and from coca leaves consumers (coca leaves chewers and coca tea drinkers). Oral fluid samples were collected by the passive drool technique (spitting), and after centrifugation the supernatant was treated for protein removal by adding acidified acetonitrile. The developed method was fully validated according to the international criteria and good results have been obtained (intraday and inter-day precisions were lower than ±20%, intraday and inter day accuracy was within the 75-116% range, and LODs/LOQs was lower and close to cut-off values for COC and BE). The proposed method has been successfully applied to oral fluid samples from cocaine abusers, and also from coca leave chewers and coca tea drinkers. CUS and HYG were only found in oral fluid from people who chewed coca leaves and drank coca tea and were not detected in cocaine abusers. Both CUS and HYG could be good markers in oral fluid for distinguishing people who consume coca leaves legally (coca leave chewers and coca tea drinkers) from those who consume illegal cocaine.

Using evidence from hair and other soft tissues to infer the need for and receipt of health-related care provision.

The Bioarchaeology of Care approach developed by Tilley is usually applied to skeletalized human remains, given the usual constraints of preservation bias that are seen with archaeological assemblages. However, other tissues, such as hair are sometimes preserved and can provide a wealth of information that can supplement the skeletal data. Archaeological hair has been analysed for drug compounds for almost thirty years. This article integrates data from hair analyses for coca metabolites, stable light isotope analysis and aDNA to expand the potential of the Bioarchaeology of Care approach using the example of a spontaneously mummified adult female from northern Chile.

Identification and Quantification of Cocaine and Active Adulterants in Coca-Paste Seized Samples: Useful Scientific Support to Health Care.

Adulteration is a common practice in the illicit drugs market, but the psychoactive and toxic effects provided by adulterants are clinically underestimated. Coca-paste (CP) is a smokable form of cocaine which has an extremely high abuse liability. CP seized samples are sold adulterated; however, qualitative and quantitative data of CP adulteration in forensic literature is still scarce. Besides, it is unknown if adulterants remain stable when CP is heated. This study was designed to report the chemical content of an extensive series of CP seized samples and to demonstrate the stability (i.e., chemical integrity) of the adulterants heated. To achieve this goal, the following strategies were applied: (1) a CP adulterated sample was heated and its fume was chemically analyzed; (2) the vapor of isolated adulterants were analyzed after heating; (3) plasma levels of animals exposed to CP and adulterants were measured. Ninety percent of CP seized samples were adulterated. Adulteration was dominated by phenacetin and caffeine and much less by other compounds (i.e., aminopyrine, levamisole, benzocaine). In the majority of CP analyzed samples, both cocaine and caffeine content was 30%, phenacetin 20% and the combination of these three components reached 90%. Typical cocaine pyrolysis compounds (i.e., BA, CMCHTs, and AEME) were observed in the volatilized cocaine and CP sample but no pyrolysis compounds were found after isolated adulterants heating. Cocaine, phenacetin, and caffeine were detected in plasma. We provide current forensic data about CP seized samples and demonstrated the chemical integrity of their adulterants heated.

Behaviour of hygrine and cuscohygrine in illicit cocaine production establishes their use as markers for chewing coca leaves in contrast with cocaine abuse.

Hygrine (HYG) and cuscohygrine (CUS) are natural alkaloids of coca leaves but are not found in illicit cocaine seizures. Therefore, they were proposed as markers for coca chewing in contrast to cocaine abuse in urine and hair testing. In order to examine at which step of the illegal cocaine production these compounds are lost, coca leaves were processed according to an authentic procedure by extraction with lime and kerosene, re-extraction with sulphuric acid, and precipitation of coca paste with ammonia. Non-extracted and extracted coca leaves, acidic extract and coca paste were analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for cocaine, ecgonine methyl ester (EME), cinnamoylcocaine (CIN), HYG, and CUS. It follows from the results that under these conditions, HYG and CUS are extracted only to a minor extent by kerosene and are not precipitated from the acidic re-extract in the coca paste. Due to this behaviour in illegal cocaine production, they fulfil the conditions as markers for coca chewing in an optimal way. However, for unambiguous discrimination between coca chewing and cocaine abuse in human samples, additional markers of manufactured cocaine are required. Copyright © 2016 John Wiley & Sons, Ltd.

Ex Vivo Treatment with a Polyphenol-Enriched Cocoa Extract Ameliorates Myocardial Infarct and Postischemic Mitochondrial Injury in Normotensive and Hypertensive Rats.

Our objective was to determine the effects of a polyphenol-enriched cocoa extract (PCE) on myocardial postischemic alterations in normotensive (Wistar rats, W) and spontaneously hypertensive rats (SHR). Isolated hearts were submitted to 110 min of perfusion or 20 min stabilization, 30 min global ischemia, and 60 min reperfusion (R). Other hearts were treated with PCE at the onset of R. Infarct size, the reduced glutathione (GSH), and the expression of phospho-Akt, P-GSK-3ß, and P-eNOS were assessed. In isolated mitochondria, the Ca(2+)-mediated response of mitochondrial permeability transition pore (mPTP), membrane potential (Δψm), and superoxide production were determined. PCE decreased infarct size, partly preserved GSH, increased the P-Akt, P-GSK-3ß, and P-eNOS contents, improved mPTP response to Ca(2+), decreased the superoxide production, and restored Δψm. These data show that PCE decreases the cardiac postischemic damage in W rats and SHR and suggest that Akt/GSK-3ß/eNOS dependent pathways are involved.

Geographically Sourcing Cocaine's Origin - Delineation of the Nineteen Major Coca Growing Regions in South America.

Previously, geo-sourcing to five major coca growing regions within South America was accomplished. However, the expansion of coca cultivation throughout South America made sub-regional origin determinations increasingly difficult. The former methodology was recently enhanced with additional stable isotope analyses ((2)H and (18)O) to fully characterize cocaine due to the varying environmental conditions in which the coca was grown. An improved data analysis method was implemented with the combination of machine learning and multivariate statistical analysis methods to provide further partitioning between growing regions. Here, we show how the combination of trace cocaine alkaloids, stable isotopes, and multivariate statistical analyses can be used to classify illicit cocaine as originating from one of 19 growing regions within South America. The data obtained through this approach can be used to describe current coca cultivation and production trends, highlight trafficking routes, as well as identify new coca growing regions.

Coca: High Altitude Remedy of the Ancient Incas.

The use of coca leaf for medicinal purposes is a centuries-old tradition of the native peoples of South America. Coca products are thought by many laypersons to provide risk-free benefits to users participating in strenuous activities at high altitude. Physiologic studies of coca have increased understanding of its possible mechanism of action as well as its potential impact on high altitude activities. This present work explores the role of coca throughout the history of the Andean peoples and explores whether this ancient remedy has a place in modern medicine. A focused summary of research articles with particular relevance to the field of wilderness medicine is also included to better provide the reader with lessons not only from history but also from another culture.

Theobromine inhibits differentiation of 3T3-L1 cells during the early stage of adipogenesis via AMPK and MAPK signaling pathways.

Obesity is characterized by hypertrophy and/or by the differentiation or adipogenesis of pre-existing adipocytes. In this study, we investigated the inhibitory effects of theobromine, a type of alkaloid in cocoa, on adipocyte differentiation of 3T3-L1 preadipocytes and its mechanisms of action. Theobromine inhibited the accumulation of lipid droplets, the expression of PPARγ and C/EBPα, and the mRNA expression of aP2 and leptin. The inhibition of adipogenic differentiation by theobromine occurred primarily in the early stages of differentiation. In addition, theobromine arrested the cell cycle at the G0/G1 phase and regulated the expressions of CDK2, p27, and p21. Theobromine treatment increased AMPK phosphorylation and knockdown of AMPKα1/α2 prevented the ability of theobromine to inhibit PPARγ expression in the differentiating 3T3-L1 cells. Theobromine reduced the phosphorylation of ERK and JNK. Moreover, the secretion and the mRNA level of TNF-α and IL-6 were inhibited by theobromine treatment. These data suggest that theobromine inhibits adipocyte differentiation during the early stages of adipogenesis by regulating the expression of PPARγ and C/EBPα through the AMPK and ERK/JNK signaling pathways in 3T3-L1 preadipocytes.

Application of hygrine and cuscohygrine as possible markers to distinguish coca chewing from cocaine abuse on WDT and forensic cases.

The objectives of present work are twofold. First, we want to verify that hygrine and cuscohygrine are good markers to distinguish between chewing coca leaves and cocaine abuse. Secondly, we try to develop a quick and easy qualitative method to determine the two mentioned markers. We analyzed two kinds of urine samples: the first group consisted of twenty-four (24) subjects: urine samples were obtained from various types of workers (e.g. doctors, chemists, nurses, technicians, painters, contractors, employees and some retired persons) who admitted chewing coca leaves. Frequency of the habit of chewing coca leaves was variable. They practiced "coqueo" between two (2) and forty-four (44) years. Sixteen (16) of them used alkaline substances to enhance the extraction of cocaine from the leaves The second group of urine samples consisted on thirty-eight (38) cocaine abusers, from forensic cases from Spain and Argentina. A GC/MS qualitative method, performed after liquid-liquid extraction, was developed and validated (the parameters studied were selectivity/specificity, LOD and stability), and then applied to the urine samples. Hygrine and cuscohygrine are good markers to distinguish between chewing coca leaves and cocaine abuse, and the qualitative method presented can be used successfully in workplace drug testing and forensic cases.

The biosynthesis of hydroxycinnamoyl quinate esters and their role in the storage of cocaine in Erythroxylum coca.

Complexation of alkaloids is an important strategy plants utilize to facilitate storage in vacuoles and avoid autotoxicity. Previous studies have implicated hydroxycinnamoyl quinate esters in the complexation of purine alkaloids in Coffea arabica. The goal of this study was to determine if Erythroxylum coca uses similar complexation agents to store abundant tropane alkaloids, such as cocaine and cinnamoyl cocaine. Metabolite analysis of various E. coca organs established a close correlation between levels of coca alkaloids and those of two hydroxycinnamoyl esters of quinic acid, chlorogenic acid and 4-coumaroyl quinate. The BAHD acyltransferase catalyzing the final step in hydroxycinnamoyl quinate biosynthesis was isolated and characterized, and its gene expression found to correlate with tropane alkaloid accumulation. A physical interaction between chlorogenic acid and cocaine was observed and quantified in vitro using UV and NMR spectroscopic methods yielding similar values to those reported for a caffeine chlorogenate complex in C. arabica. These results suggest that storage of cocaine and other coca alkaloids in large quantities in E. coca involves hydroxycinnamoyl quinate esters as complexation partners.

Reform by subtraction: the path of denunciation of international drug treaties and reaccession with reservations.

Almost all countries are parties to the international drug conventions of 1961, 1971 and 1988. These strongly bind parties with respect to their domestic regulation of controlled substances, including requirements that possession, growing or use be a criminal offense and that any regulated market in the substances be limited to use only for medical or scientific purposes. Even where countries have argued they have "wiggle room", reform within the bounds of the conventions has often resulted in "net-widening" which nullifies the intent of the reform. Among the options for effective reform, probably the most immediately viable is the route of denunciation and reaccession with reservations--the route which Bolivia has now taken in order to legalise a regulated domestic market in coca leaves for chewing. The paper considers the existing record of reservations (by more than 30 parties to each of the conventions). Also discussed are the options for response to the reservations by other parties, which vary between the treaties, and how pursuing the option of denunciation and reaccession with reservation might potentially play out.

The use of coca: prehistory, history, and ethnography.

The purpose of this article is to review the use of the natural mild stimulant coca, which is a story that originates with the prehistory of coca, evolves through its following historical uses, and leads up to the eventual development of cocaine. This discussion will begin with the botanical background of the coca plant, followed by a review of some of the prehistoric, historic and ethnographic evidence of coca use, which indicates the extensive antiquity and pervasiveness of coca use in South and Central America. The diverse roles that coca played among the Inca and other indigenous peoples led to the early adoption of coca in the West and, in turn, to the resultant discovery of cocaine and its assorted early applications, particularly for medicinal purposes.

Detección de Benzoilecgonina en orina de consumidores de té de coca mediante métodos de inmunoensayo / Detection of Benzoilecgonine in urine of consumers of coca tea by immunoassay methods

Con el fin de detectar la presencia de benzoilecgonina en orina de consumidores de té de coca, se realizó un estudio piloto analizando muestras de orina a 10 voluntarios sanos, no consumidores de cocaína, antes de ingerir la infusión de té de coca (Nasa Esh´s Coca Nasa), y las recolectadas hasta las 48 horas después de la ingestión, de una toma única de 100 mL el mismo día. El análisis se realizó por métodos de inmunoensayo, cualitativo, mediante pruebas rápidas Acu-check, y semicuantitativo AxSYM Cocaine Metabolite, basado en In munoensayo de Fluorescencia Polarizada (FPIA). Antes de la ingestión de la infusión por los métodos cualitativos y semicuantitativos las muestras recolectadas resultaron negativas, después de haber ingerido la infusión, por ambos métodos se detectó concentraciones de benzoilecgonina desde la primera hasta las 48 horas con diversas variaciones entre las muestras, observándose excelente concordancia entre los métodos para la determinación de benzoilecgonina en orina. En este estudio, se concluyó que existe la presencia de benzoilecgonina en muestras de orina de consumidores de té de coca. Los métodos utilizados sólo proporcionan resultados preliminares, se recomienda utilizar unos más específicos a fin de encontrar parámetros que permitan discriminar individuos que hayan ingerido infusión de té de coca, de aquellos que son adictos a la cocaína. Además, es importante prevenir a los consumidores de té de coca sobre el riesgo de detección de benzoilecgonina en orina dentro de las primeras 24 a 48 horas y las implicaciones que trae consigo tales hallazgos de laboratorio.

In order to detect the presence of benzoylecgonine in urine of consumers of coca tea, a pilot study was conducted by analyzing urine samples from 10 volunteers not cocaine users, be fore drinking the coca tea infusion, and collected until 48 hours after ingestión of a single shot (100 mL) the same day, The analysis was performed by immunoassay qualitative methods, using fast Acu-check tests, and semi quantitative automated equipment Abbott Axsym System, based on fluorescence polarization immunoassay (FPIA). Before ingestion of the infusion for qualitative and semiquantitative methods for samples collected were negative, and after drinking the tea, for both methods, concentrations of benzoylecgonine was detected from the first to 48 hours with several variations between samples, observed excellent agreement between the methods for the determination of benzoilecgonine in urine. In this study, we concluded that there is the presence of benzoylecgonine in urine samples from consumers of coca tea. According to the methods used provide only preliminary results, we recommend using a more specific in order to find parameters to discriminate individuals who have ingested coca tea infusions of dose that are cocaine addicts, In turn, it is important to prevent cosumers coca tea on the risk of detection of benzoylecgonine in urine within the first 24 to 48 hours and the implications it brings such laboratory findings.

Coca-paste seized samples characterization: chemical analysis, stimulating effect in rats and relevance of caffeine as a major adulterant.

Coca-paste (CP) is a drug of abuse that so far has not been extensively characterized. CP is an intermediate product of the cocaine alkaloid extraction process from coca leaves, hence it has a high content of cocaine base mixed with other chemical substances (impurities) and it is probably adulterated when it reaches the consumers. Despite its high prevalence and distribution through South America, little is known about its effects on the central nervous system. In the present study, a chemical analysis of CP samples from different police seizures was performed to determine the cocaine base content and the presence and content of impurities and adulterants. Some CP representative samples were selected to study the effects on the locomotor activity induced after acute systemic administration in rats as a measure of its stimulant action. The behavioral response was compared to equivalent doses of cocaine. As expected, cocaine was the main component in most of the CP samples assayed. Caffeine was the only active adulterant detected. Interestingly, several CP samples elicited a higher stimulant effect compared to that observed after cocaine when administered at equivalent doses of cocaine base. Combined treatment of cocaine and caffeine, as surrogate of different CP samples mimicked their stimulant effect. We demonstrated that cocaine and caffeine are the main components responsible for the CP-induced stimulant action while the contribution of the impurities was imperceptible.

Can coca leaves contribute to improving the nutritional status of the Andean population?

BACKGROUND: Coca leaves (Erythroxylum coca) have been promoted as a food that could address the dietary deficiencies of the Andean population, but this is based on nutrient analyses of a small sample of leaves. OBJECTIVE: We assessed the nutritional potential of eight samples of coca leaves grown in different regions of Peru. METHODS: We used AOAC techniques to measure nutrients, nutrient inhibitors (phytate, polyphenols, oxalic acid, and fiber), and alkaloid concentrations, all expressed per 100 g dry weight (DW) of the ground leaves. Minerals were measured by inductively coupled lasma- mass spectrometry in n twondependent laboratories. RESULTS: The leaves contained protein, , 20.28 g/1 0DW with lysine as the limiting amino acid; n-cbetarotene, 3.51 mg/100gDW ; vitamin E, 16.72 mg/100gDW ; trace amounts of vitamin D; calcium, 990.18 and 1033.17 mg/100 gDW at two different laboratories; iron, 29.16 and 29.16 mg/100 gDW; zinc, 2.71 and 2.63 mg/100 gDW; and magnesium, 225.19 and 196.69 mg/l001gDW Cocaine was the principal alkaloid, with a concentration of 0.56 g/100 gDW; other alkaloids were also identified. The results were compared with those for other edible leaves. The nutrient contributions of coca powder (5 g) and bread made with coca were compared with those of normal portions of alternative foods. CONCLUSIONS: Two spoonfuls of coca leaf flour would satisfy less than 10% of dietary intakes for schoolchildren and adults for critical commonly deficient nutrients in the diet. Coca leaves do not provide nutritional benefits when eaten in the recommended quantities, and the presence of absorbable cocaine and other alkaloids may be potentially harmful; hence coca leaves cannot be recommended as a food.