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.

Perfil de alcaloides de la hoja de coca en el fluido oral de un mascador de hoja de coca y un bebedor de té de coca: Estudio preliminar / Coca alkaloids profile in oral fluid from people chewing coca leaves and drinking coca tea: Preliminary study

Actualmente el fluido oral (FO) es aceptado como una matriz biológica alternativa para detectar drogas en toxicología clínica y forense. En países como Argentina donde el uso de hojas de coca (mascar hojas de coca o beber té de coca) es legal son necesarios procedimientos adecuados para logar una clara diferenciación entre los individuos que usan las hojas de coca de manera legal de aquéllos que usan cocaína en forma ilegal. Poca es la información que hay en la literatura sobre el perfil de los alcaloides de la hoja de coca en FO de personas que mascan hojas de coca o toman té de coca y hasta el presente trabajo no se hallaron datos sobre el perfil en FO de la higrina (HIG) y cuscohigrina (CUS). De este estudio preliminar participaron dos voluntarios. Los resultados mostraron que la CUS e HIG siguieron siendo positivas después que la cocaína (COC) y benzoilecgonina (BE) cayeron por debajo de los valores cut- off propuestos por las guías internacionales para FO en casos de screening (15 a 20 ng/ mL) y de confirmación (8 a 10 ng/mL) en el caso del mascador de coca. En el participante que tomó una taza de té de coca, en el último punto examinado (1 h) resultó ser positivo para la COC y BE y también para la CUS e HIG. El FO podría ser una muestra útil para confirmar el uso legal de la hoja de coca, aun cuando futuros estudios son necesarios para corroborar estos primeros datos.

Nowadays oral fluid (OF) is accepted as an alternative biological sample for detecting drugs in clinical and forensic toxicology. In countries like Argentina, where the use of coca leaves (coca leaves chewing and coca tea drinking) is legal, adequate procedures are required to allow a clear differentiation between people who use coca leaves (legal practice) and those who use cocaine (illicit practice). There is scarce literature regarding coca leaf alkaloids profile in OF from people who chew coca leaves and drink coca tea. Until now, coca leaf alkaloids profile of hygrine (HYG) and cuscohygrine (CUS) in OF were not described in the literature. The current preliminary study was performed with two healthy volunteers. In this research CUS and HYG have been found to be positive (detectable) even when cocaine (COC) and benzoylecgonine (BE) are dropped below the cut-off values proposed by international guidelines for screening (15 to 20 ng/mL), and confirmation (8 to 10 ng/mL) in OF. In addition, CUS and HYG were also found to be positive at the same time of the last detection of COC and BE after coca tea consumption. The OF would be a useful sample to confirm the legal use of coca leaf, even when more researches are therefore needed.

Discrimination between chewing of coca leaves or drinking of coca tea and smoking of "paco" (coca paste) by hair analysis. A preliminary study of possibilities and limitations.

BACKGROUND: Hair analysis is a suitable way to discriminate between coca chewers and consumers of manufactured cocaine using the coca alkaloids hygrine (HYG) and cuscohygrine (CUS) as markers. In the present preliminary study it was examined whether CUS and HYG can be detected in hair of occasional and moderate coca chewers or coca tea drinkers, whether CUS and HYG appear in hair of PACO consumers (smoking coca paste waste), and whether anhydroecgonine methyl ester (AEME) is a useful cocaine smoking marker in this context. METHOD: Three groups were included: 10 volunteers from Buenos Aires with occasional or moderate chewing of coca leaves or drinking coca tea, 20 Argentinean PACO smokers and 8 German cocaine users. The hair samples (1-4 segments) were analyzed by a validated LC-MS/MS method for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), AEME, CUS and HYG. For comparison, eight samples of coca leaves or coca tea were analyzed. RESULTS: Only low concentrations of COC were found in hair of seven occasional users of coca leaves or coca tea (0.010-0.051 ng/mg). For three moderate chewers of coca leaves all compounds were detected including AEME but except TRO. The hair samples of PACO smokers contained much higher concentrations of COC (0.027-341 ng/mg, mean 37.4 ng/mg) and its metabolites. CUS was not found in these samples but traces of HYG were seen in 8 of 37 hair segments. AEME as a marker for coca smoking was detected in hair of 15 smokers. In comparison to COC, the concentrations of EME and CIN were higher for PACO smokers than for German cocaine consumers. AEME (56 ± 20 µg/g) was detected in all coca leave and coca tea samples which explains the detection of this substance in hair of coca chewers. Therefore, its use for differentiation between coca chewers and PACO smokers is limited. CONCLUSION: CUS remains to be the most suitable marker in hair for chewing coca leaves or drinking coca tea more frequently than two times per month since it does not appear in hair of Argentinean PACO smokers and German cocaine users. Contrary to a previous proposal, the ratios CIN/COC and EME/COC appeared not to be applicable as criteria for this purpose because of the higher concentration of these alkaloids in hair of PACO smokers. More research is needed to assess the value of AEME in hair of South American coca leave or cocaine users.

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.