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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. vol.57 no.3 Concepción  2012

http://dx.doi.org/10.4067/S0717-97072012000300012 

J. Chil. Chem. Soc., 57, No 3 (2012), págs.: 1253-1255

 

VALIDATION OF A METHOD TO DETECT COCAINE AND BENZOYLECGONINE IN HUMAN WHOLE BLOOD BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY AND APPLICATION IN BODY PACKERS AND STUFFERS CASES IN THE NORTH OF CHILE

 

FELIPE BRAVO1,2,3, MARÍA CECILIA CONTZEN2, JULIO MOLLO1, PEDRO BUC CALDERON1,3,4 and JULIO BENITES1,3*

1Departamento de Ciencias Químicas y Farmacéuticas, Universidad Arturo Prat, Casilla 121, Iquique, Chile.
2Laboratorio Referencial Norte. Servicio Médico Legal. Iquique, Chile.
3
Instituto de EtnoFarmacología (IDE), Universidad Arturo Prat. Iquique. Chile. Avenida Arturo Prat 2120. Casilla 121. Iquique. Chile.
4
Toxicology and Cancer Biology Research Group, Louvain Drug Research Institute (LDRI), Université Catholique de Louvain, Brussels, Belgium


ABSTRACT

The present paper describes a method for the simultaneous determination of cocaine and benzoylecgonine in human whole blood. The drugs were extracted with phosphate buffer at pH 6, followed by solid-phase extraction and quantification by GC/MS with electron impact ionization using helium as carrier gas. Quantification was performed using cocaine-d3 as internal standard in selected ion monitoring mode. The method is very simple, rapid and sensitive. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The limits of detection were 3.6 ng/mL for cocaine and 6.8 ng/mL for benzoylecgonine. The method was applied to blood samples removed at autopsy from body packers and stuffers cases in the I - IV and XV regions of Chile during 2008-2010.

Keywords: Benzoylecgonine, cocaine, gas chromatography, mass selective detector, solid-phase extraction, body packers-stuffers.


INTRODUCTION

Cocaine (COC) is one of the most widely abused drugs in the world and a potent central nervous system stimulant, and it is therefore highly important to identify and quantify its level in forensic toxicology. COC is a tropane alkaloid extracted from the leaves of Erythroxylon coca, originating from South America, mainly Bolivia, Colombia and Peru.1

In the human body, COC is rapidly metabolized to the inactive benzoylecgonine (BZE), the main metabolite in both blood and urine.2 COC is a powerful substance used as drug of abuse in many countries because it produces increased alertness and euphoria.3,4 Acute COC use causes very pleasant feelings of well-being, hyperactivity, restlessness, increased blood pressure, increased heart rate, and euphoria. It suppresses fatigue, hunger and thirst stimuli. Side effects can include twitching, paranoia and impotence, which usually increase with frequent use. Excessive dosage of COC can produce hallucinations, paranoid delusions, a marked elevation of blood pressure, tachycardia and tachyarrhythmia. Toxicity results in seizures, followed by respiratory and circulatory depression of medullar origin. This may lead to death from respiratory failure, stroke, cerebral haemorrhage, or heart failure.5

Several analytical methods for COC and BZE detection have been reported in different biological matrices such as whole blood6,7, brain6, plasma8, nails9, saliva10,11, urine12, hair.13,14 They include GC/MS, HPLC-APCI-MS/MS, GC/ NPD, and HPLC-MS/MS techniques.

COC abuse has increased during the last decades in our country, especially in the north of Chile. According to the Servicio Médico Legal-Iquique, during the 2008-2010 period the number of deaths related to COC abuse in the I - IV and XV regions of Chile was 2353. Of these cases, 341 (14.5%) were positive for COC, the most abused drug detected in postmortem cases in that period. Of these 341 deaths, 8 correspond to two distinct groups of individuals, "body packers" and "body-stuffers" that swallow packets containing illicit drugs in an attempt to avoid detection by law enforcement agencies. "Body-packers" are international smugglers who ingest packages of drugs in order to transport them and subsequently retrieve them in a foreign country.15 "Body-stuffers" are individuals who ingest drugs at or around the time of their arrest in order to "swallow the evidence." 16

In the context of the development and validation of chromatographic methods to characterize drug abuse17,18, we show in this work the first report of body packers and stuffers cases in the I - IV and XV regions of Chile.

MATERIALS AND METHODS

Chemicals and reagents

Cocaine, benzoylecgonine, and cocaine-d3 were purchased from Cerilliant (TX, USA). Ethyl acetate (HPLC grade) was purchased from Fisher (USA). Acetic acid, ammonia, potassium hydroxide, and potassium dihydrogen phosphate (analytical reagent grade) were purchased from Merck (Germany). Methanol, isopropanol and dichloromethane (HPLC grade) were purchased from Sigma-Aldrich (USA). N,O-Bis(trimethylsilyl)trifluoroacetamide:trimet hylchlorosilane was purchased from Supelco (USA).

Biological samples

Samples of outdated whole blood were obtained from the blood bank of "Dr. Ernesto Torres Galdames" Hospital, and stored at -30 °C until analysis.

Sodium fluoride was added to the blood samples removed at autopsy from body packers and stuffers cases, and they were stored at -30 °C until analysis.

Sample preparation

Cocaine and benzoylecgonine sample preparation

A 2 mL portion of human whole blood was shaken for 1 min and then homogenized thoroughly. To the homogenate was added 20 vL of a 20 ng/ mL solution of cocaine-d3 as internal standard (IS), and 8 mL of 100 mM pH 6.0 phosphate buffer. The sample solution was vortexed for 1 min and then sonicated at room temperature for 30 min. After centrifugation at 3500 rpm for 10 min, the supernatant was introduced in the extraction column.

The concentrations of the analytes were calculated using calibration curves from of spiked human whole blood sample (the ranges were 25 -1500 ng/mL for both analytes). Linear regression lines were obtained by plotting the peak area ratios (the compound's peak area divided by that of the internal standard)

Solid-phase extraction (SPE)

The solid-phase extraction was performed using a Bond Elut Certify column (Agilent, USA-Weisser Analytical). The solid-phase extraction cartridges were preconditioned with 2 mL of methanol and 2 mL of 100 mM pH 6.0 phosphate buffer, all under vacuum (no more than 3 mm Hg). The prepared samples were then applied and allowed to pass through the column at a rate of 1 to 1.5 mL/min. The sorbent was washed with 2 mL of water and 0.5 mL of 0.05 M acetic acid. The vacuum was maintained at 15 mm Hg for 10 min to dry the column completely. Finally, the analytes were eluted with 3 mL of dichloromethane:isopropanol:ammonia (78:20:2 v/v/v) and 1 mL of ethyl acetate:ammonia (98:2 v/v) into amber collection tubes. The solvent was evaporated under a gentle stream of nitrogen and the residue was derivatized with 50 vL of bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane (99:1) and 50 mL of ethyl acetate for 25 min at 70 °C.

GC-MS

Chromatographic analysis was carried out on an Agilent 6890N series system (Agilent, USA - Weisser Analytical) equipped with a 7683 series Automatic Sampler linked with injector programmed temperature volatilization (PTV) and DB-5MS capillary columns (50 m x 0.22 mm, 0.33 mm film thickness). The injection volume was 5 mL in solvent vent mode. A selective mass detector together with a Chemstation software suite (Agilent, USA-Weisser Analytical) version A.09 was used for data processing and instrument control. The temperature of the PTV injector in solvent vent mode was set at 50 °C and the flow rate was kept at 1.2 mL/min using helium as carrier gas. The oven temperature was programmed as follows: the initial temperature was set at 50 °C, held for 4 min, and ramped at 35 °C/min to 180 °C, where it was held for 1 min and ramped at 25 °C/min to 250 °C and held for 1.24 min, and again ramped at 20 °C/min to 315 °C and held for 4 min.

The GC-MS analysis for identification of COC and BZE was carried out in a gas chromatograph equipped with an Agilent 5975 mass selective detector operated in electron impact mode (Agilent USA- Weisser Analytical). The temperatures of the quadrupole, ion source, and mass selective detector interface were 150, 230 and 280 °C, respectively.


Figure 1. Structures of cocaine and benzoylecgonine.

Validation of the method

The validation of the method was performed by establishing recovery value, linearity, intra- and inter-assay precision, limits of detection and quantification. The analyses intra- and inter-assay precisions were performed in ten replicates for each day.

The LOQ is the lowest concentration that can be measured on the standard curves with acceptable reproducibility. The LOQ determined for the tested analytes allowed the measurement of subtherapeutic and toxic concentrations of these compounds. LOQ values were 10.9 ng/mL for COC and 20.6 ng/mL for BZE. Although they were below the therapeutic or abuse concentrations, they were low enough for this method to be used in routine screening and quantitation of COC and BZE drugs in autopsy and clinical samples.

The solid phase extraction was carried out in triplicate at three concentrations (low, medium and high) of each compound in human whole blood. As shown in Table 1, the solid-phase extraction efficiency was more than 95% and 92%, respectively, for COC and BZE.


Table 1. Linearity, relative standard deviation (RSD), limit of detection (LOD), limit of quantitation (LOQ), and solid-phase extraction efficiency in human whole blood. (mass selective detector.
a r2 = Square of correlation coefficient with a weighting factor of 1/concentration.

Table 2 shows the results obtained for intra־assay and inter־assay precision calculations and selected ions used for qualification and quantification of all the analytes. Inter-day and intra-day precision were <2.3% for all analytes. Precision of analytes under investigation at reported concentrations satisfactorily met the internationally established acceptance criteria.20


Table 2. Intra־ and inter־day precision, and selected ions for cocaine and benzoylecgonine.

Comparison of this method with different chromatographic methods previously reported in the literature shows clearly that the LODs obtained here are essentially at the same level or better.7,8,10,12

A representative chromatogram obtained by GC/MS following the extraction of COC and BZE and cocaine-<i3 in SIM mode is shown in Fig. 2. Using this method, all the analytes of interest were resolved in <18 min.


Figure 2. Chromatographic spectrum of cocaine, benzoylecgonine and cocaine־d3 in SIM mode.

RESULTS AND DISCUSSION

The development of a GC/MS analytical procedure for COC and BZE detection in human whole blood is reported. The optimization of this method was first carried out using cocaine-<5B as IS and then progressed to using a blank human whole blood sample spiked with each of the two analytes. The development and validation of an analytical method to quantify central nervous system stimulant drugs such as COC and BZE offers several advantages. They include the ability to maximize the information in cases with limited sample availability19 as well as the ability to relate the concentrations found in human whole blood with the clinical symptoms of the exposed individual, and whether these concentrations are connected to the cause of death.

The SPE has some merits for sample preparation compared with liquid-liquid extraction: (1) very simple extraction procedure; (2) small solvent volume; (3) high throughput performance and feasibility of treating many samples at one time; and (4) minimization of differences among individuals. We confirmed the usefulness of the Bond Elut Certify column for extraction of abused drugs from biological fluids.17,18

Table 1 shows the linearity, relative standard deviation, limit of detection (LOD), limit of quantitation (LOQ), and solid-phase extraction efficiency in human whole blood using GC/MS selected ion monitoring. Linearity is the ability of the method to be directly proportional to the concentration of the analytes within a given range. Linearity was assessed using an internal standard. Under these conditions, concentrations in human whole blood ranged between 25 and 1500 ng/mL for both analytes. Correlation coefficients were r2 > 0.998 for human whole blood obtained for each compound. Relative standard deviations for the calibration curve were 2.78 and 3.18%, respectively, for COC and BZE. The ranges of linearity were satisfactory with respect to the subtherapeutic and toxic range for forensic and clinical purposes.

The LODs were calculated based on the standard deviation (SD) of the response and the slope (S) of the calibration curve(s) at levels approximating the LOD according to the formula: LOD = 3.3(SD/S); the LODs were 3.6 ng/ mL for COC and 6.8 ng/mL for BZE. The LOD values clearly indicated that this method is quite sensitive for COC and BZE analysis in human whole blood samples.

Of 2,353 autopsies, which were classified as illicit drugs in the I ־ IV and XV regions of Chile during the 2008-2010 period, 341 cases (14.5%) were positive for COC. Among them, 8 cases were classified as body packers and stuffers (Table 3). No gender difference was observed in death by COC drug, indeed 50% were male and 50% were female cases. These values, obtained from real autopsy samples, were determined by the GC/MS method, and drug concentrations ranged between 87 ng/mL - 7500 ng/mL. Since the international literature reports that COC values above 1000 ng/mL are toxic doses,21 it should be noted that in 2 cases, COC concentrations largely exceeded such toxic doses. These cases correspond to 2 body packers with signs of overdose due to drug leakage. On the other hand, it may be argued that 3 cases shown in Table 3 have subtherapeutic COC doses, so death might be due to other means. The probable reason for this discrepancy is likely due to blood sampling; indeed, in the Calama (94 ng/mL) and Antofagasta (470 ng/mL) cases, the analyses were made on peripheral blood and not on blood from the femoral vein. Regarding the Iquique case (87 ng/mL), although the blood sampling was made from the femoral vein, it must be underlined that this patient was subjected to a hem diluting procedure, thus modifying the COC concentration. In this case, death by drug intoxication was certified by histological analysis.


Table 3. Body packers and stuffers cases in which cocaine was detected in human whole blood samples (I - IV and XV regions in the north of Chile during (2008-2010)
* Plastic bag
** Unreported

CONCLUSION

The present study shows the validation of this GC/MS method for the analysis of COC and BZE in whole blood samples. The main advantage of this method is its selectivity and sensitivity for clinical and postmortem toxicological analysis. The reliability of identification was achieved by monitoring fragment ions and the use of relative retention time. The current method is being applied by the Laboratorio Referencial Norte of the Servicio Medico Legal, Iquique, in forensic cases such as body packers and stuffers in the north of Chile.

ACKNOWLEDGEMENTS

We thank Laboratorio Referencial Norte of the Servicio Medico Legal, Iquique, Conicyt-MEC 80100002, and Universidad Arturo Prat for financial support of this study.

 

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(Received: December 6, 2011 - Accepted: April 24, 2012)

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