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.

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.

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.

Validación de un método rápido en orina por lC/MS/MS para la búsqueda de compuestos empleados en sumisión química y su aplicación a muestras reales / Validation of a fast screening method in urine by lC/MS/MS for compounds used in drug-facilitated crime (DFC) and its application to real samples

La Oficina de Naciones Unidas contra la Droga y el Delito (UNODC) en 2011 señala que "El delito facilitado por drogas (DFD) es una expresión general que abarca la violación y otras agresiones sexuales, el robo con violencia o intimidación, la extorsión de dinero y los malos tratos deliberados de ancianos o niños bajo la influencia de sustancias sicotrópicas". En este trabajo se validó un método cualitativo y rápido a partir de muestras de orina por LC/MS/MS para 39 compuestos comprendidos en los listados de sumisión química. El objetivo fue alcanzar un límite de detección un 50 % por debajo de la concentración propuesta como "Límites mínimos de funcionamiento exigidos (MRPL)" por la UNODC, para poder ser aplicado a muestras reales.

The United Nations Office on Drugs and Crime (UNODC) in 2011, states that "The Drug-facilitated crime (DFC) is a general term that includes rape or other sexual assault, robbery, money extortion, as well as the deliberate maltreatment of the elderly or children under the influence of psychotropic substances". In this work we validated a qualitative and fast method from urine samples by LC/MS/MS for 39 compounds included in the Drug-facilitated crime lists. The aim was to reach a detection limit of 50% below the proposed concentration as "minimum required performance limits (MRPL)" by UNODC in order to be applied in real samples.

Possibilities for discrimination between chewing of coca leaves and abuse of cocaine by hair analysis including hygrine, cuscohygrine, cinnamoylcocaine and cocaine metabolite/cocaine ratios.

Contrary to the illegal use of any form of manufactured cocaine, chewing of coca leaves and drinking of coca tea are allowed and are very common and socially integrated in several South American countries. Because of this different legal state, an analytical method for discrimination between use of coca leaves and abuse of processed cocaine preparations is required. In this study, the applicability of hair analysis for this purpose was examined. Hair samples from 26 Argentinean coca chewers and 22 German cocaine users were analysed for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), cuscohygrine (CUS) and hygrine (HYG) by hydrophilic interaction liquid chromatography (HILIC) in combination with triplequad mass spectrometry (MS/MS) and hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS). The following concentrations (range, median, ng/mg) were determined in hair of the coca chewers: COC 0.085-75.5, 17.0; NC 0.03-1.15, 0.12; BE 0.046-35.5, 6.1; EME 0.014-6.0, 0.66; CE 0.00-13.8, 0.38; CIN 0.005-16.8, 0.79; TRO 0.02-0.16, 0.023; CUS 0.026-26.7, 0.31. In lack of a reference substance, only qualitative data were obtained for HYG, and two metabolites of CUS were detected which were not found in hair of the cocaine users. For interpretation, the concentrations of the metabolites and of the coca alkaloids in relation to cocaine were statistically compared between coca chewers and cocaine users. By analysis of variance (ANOVA) significant differences were found for all analytes (α = 0.000 to 0.030) with the exception of TRO (α = 0.218). The ratios CUS/COC, CIN/COC and EME/COC appeared to be the most suitable criteria for discrimination between both groups with the means and medians 5-fold to 10-fold higher for coca chewers and a low overlap of the ranges between both groups. The same was qualitatively found for HYG. However, these criteria cannot exclude cocaine use in addition to coca chewing. In this regard screening for typical cutting agents can be helpful and led to the detection of levamisole (21×), lidocaine (6×) and paracetamol (3×) in the 22 samples from German cocaine users, whereas no levamisole, lidocaine (3×) and paracetamol (1×) were found in hair from the Argentinean coca chewers. These criteria have to be confirmed for South American cocaine consumers including smokers of coca paste and may be different because of different composition of the drug and other use habits.