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Forensic Toxicol (2006) 24:70–74 DOI 10.1007/s11419-006-0017-z

ORIGINAL ARTICLE

Determination of mercury and arsenic in ecstasy tablets by electrochemical methods Inmaculada Fierro · Luis Deban · Rafael Pardo Mariluz Tascón · Dolores Vázquez

Received: 6 October 2006 / Accepted: 23 October 2006 / Published online: 29 November 2006 © Japanese Association of Forensic Toxicology and Springer 2006

Abstract Mercury and arsenic concentrations were determined in ecstasy tablets, which were obtained from different police seizures in Spain, by electrochemical techniques; mercury by differential pulse anodic stripping voltammetry with a rotating gold disk electrode, and arsenic by cathodic stripping voltammetry in the differential pulse mode with a hanging mercury drop electrode. The performance of the procedures was compared with cold-vapor atomic absorption spectrometry for mercury, and with electrothermal atomic absorption spectrometry for arsenic. The procedures were applied to the determination of both elements in nine ecstasy samples; mercury was the element present in higher concentrations, ranging from 0.05 to 1.23 mg/kg, while the range of arsenic concentrations was 0.04–0.49 mg/kg. The described electrochemical techniques for determination of mercury and arsenic in ecstasy tablets should be useful for impurity profiling in forensic analysis practice, because of their low costs and high sensitivity. Keywords Ecstasy · MDMA · Electrochemical methods · Stripping voltammetry · Electrothermal atomic absorption spectrometry · Cold-vapor absorption spectrometry

are strictly controlled by law; they are manufactured underground and sold on black markets. One of the most typical designer drugs is 3,4-methylenedioxymethamphetamine (MDMA), which is well known by the street name of “ecstasy.” Ecstasy tablets are being abused worldwide and cause social problems. In addition to the toxicity of a designer drug itself and the damage it does to the health of its abuser, the hazardous effects of contaminants and impurities included in such tablets should be taken into consideration [1–3]. From the view of criminal investigation, the analysis and profiling of the contaminants may also provide important clues for locating and identifying distribution routes and manufacturing sites of the clandestine tablets. There is extensive literature on adulterants, byproducts, intermediates, and precursors that are present or arise in the process of MDMA synthesis; however, there are very few reports about the determination of metals including metalloids. The analytical methods most frequently used for metals are atomic absorption spectrometry (AAS), inductively coupled plasma (ICP) atomic emission spectrometry, and ICP-mass spectrometry [4]. In this article, electrochemical techniques for the determination of mercury (Hg) and arsenic (As) in ecstasy tablets are proposed.

Introduction Materials and methods Many societies are faced with the continuous appearance of new designer drugs. Most of these materials are derivatives of drugs that affect the nervous system, and

Apparatus and reagents

I. Fierro · L. Deban (*) · R. Pardo · M. Tascón · D. Vázquez Departamento de Química Analítica, Facultad de Ciencias, Universidad de Valladolid, Valladolid 47005, Spain e-mail: [email protected]

The determination of Hg by differential pulse anodic stripping voltammetry (DPASV) was carried out on a Metrohm (Herisau, Switzerland) E746 VA trace analyzer and with a Metrohm 747 VA multimode elec-

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trode equipped with a rotating gold disk electrode (RGDE) as working electrode, a platinum rod as auxiliary electrode, and an Ag/AgCl/KClsat reference electrode. Cold-vapor AAS measurements for Hg were performed on a Perkin-Elmer (Norwalk, CT, USA) flow-injection mercury system (FIMS) instrument equipped with a FIMS-400 unit and a programmable sample dispenser. Differential pulse cathodic stripping voltammetry (DPCSV) for determination of As was carried out on a Metrohm E-506 polarograph connected to a Metrohm E-608 control unit and with a Metrohm 663VA multimode electrode used in the hanging mercury drop electrode (HMDE) mode with a platinum rod as auxiliary electrode and an Ag/AgCl/KClsat reference electrode. Hg used in the HMDE electrode was triply distilled. Electrothermal atomic absorption spectrometry (ETAAS) measurements for As were performed on a Varian (Palo Alto, CA, USA) Spectra AA-800 spectrometer equipped with Zeeman GTA-100 background correction and a PSD-100 programmable sample dispenser. Measurements of pH were made with a Metrohm 654 pH meter. Samples (ecstasy tablets seized by the police) were digested in a Milestone (Sorisole, Italy) MLS 1200 microwave oven. Concentrated nitric acid and 30% (w/v) hydrogen peroxide were used for the digestion. The standard solutions of Hg and As were prepared daily by diluting Panreac (Barcelona, Spain) AAS standards (1000 mg/l). For DPASV reagent, the supporting electrolyte was prepared by mixing 0.448 g of KCl, 0.372 g of Na2EDTA, and 22 ml of HClO4, and then diluting to 1 l with deionized water. For FIMS reagent, tin(II) sulfate solution was prepared by adding 25 g tin(II) sulfate to 250 ml of 0.25 M H2SO4. Hydroxylamine reagent was obtained by dissolving 12 g of sodium chloride and 12 g of hydroxylamine sulfate in water and diluting to 100 ml. For DPCSV reagents, copper(II) chloride (0.1 M) and 5 mM hydrazine sulfate were prepared daily by diluting the respective reagents in ultrapure water; acetate buffer solution (1 M) at pH 5 was prepared by mixing 14.2 ml of 2 M acetic acid with 35.8 ml of 2 M sodium acetate and diluting to 100 ml. Glassware and plasticware were soaked in 2 M nitric acid for 24 h and then rinsed thoroughly with deionized water. Sample digestion A 2-ml volume of concentrated nitric acid and 1 ml of 30% (w/v) hydrogen peroxide were added to 250 mg of a sample. This mixture was placed in a 100-ml Teflon vessel and treated for 20 min in a microwave oven for digestion. The power of the magnetron was linearly increased

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from 250 to 600 W during the process, and the resulting solution was diluted to 100 ml with deionized water. A control reagent blank with deionized water was prepared simultaneously. Hg determination by DPASV To an adequate volume of the digested solution was added 25 ml DPASV reagent (supporting electrolyte), which was then placed in a volumetric cell. The solution was deoxygenated by purging it with water-saturated nitrogen for 10 min. The potential was set at 0.4 V for 180 s, with the solution being stirred at 1900 rpm; then the stirrer was stopped and the electrode potential was set at 0.4 V for another 30 s. Thereafter, the potential was scanned toward more positive values at a scan rate of 20 mV s−1, using a superimposed differential pulse of 50 mV in amplitude. A Hg stripping peak was registered at about 0.60 V (vs Ag/AgCl/KClsat reference electrode) and its current was used as a measure of Hg concentration. The standard addition method was used to test the DPASV sensitivity and to check linearity of the responses. The electrode processes can be written as follows: Deposition on the RGDE at +0.4 V: Hg+2 + Au + 2e− → Hg (Au) Anodic stripping: Hg (Au) → Hg+2 + Au + 2e− Cleaning of the RGDE The surface of the RGDE was cleaned and reactivated after each measurement by polishing the gold disk with alumina powder using a polish cloth. This operation was then repeated without the powder until the electrode showed a mirror-like surface. The electrode was then rinsed with water. To reactivate the electrode surface, the RGDE was immersed in a solution containing the supporting electrolyte and 0.01 M nitric acid, and successive polarization cycles from 2 V (30 s) to 0 V (10 s) were applied for 15 min. Hg determination by FIMS To assess that all Hg was present as Hg(II), 5% (w/v) potassium permanganate was added to the digested solution until the purple color became persistent. The excess of permanganate was removed by adding hydroxylamine reagent until the solution became colorless. Hg(II) was then reduced to the elemental state by addition of 10 ml of the tin(II) reagent and vaporized from the solution in the FIMS closed system. The Hg vapor passed through a cell positioned in the light beam path

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(253.7 nm) of the AAS. Metal contents were obtained from the calibration plots (absorbance vs concentration) by interpolation.

Forensic Toxicol (2006) 24:70–74

lation. Temperature of atomization, limit of detection, chemical modifier, and wavelength used were: 2700°C, 10 µl of 50 ng/ml = 0.2 absorbance, palladium solution (500 µg/ml), and 193.7 nm, respectively.

Arsenic determination by DPCSV The optimal conditions for As determination were partially adapted from Li and Smart [5]. An adequate volume of the digested solution was diluted to 25 ml with deionized water and placed in the voltammetric cell. The solution was then spiked with 250 µl of 0.1 M copper(II) chloride and 250 µl of 5 mM hydrazine sulfate. An excess of hydrazine (final concentration 0.05 mM) was added to the digested sample to reduce the nitrate not consumed in the digestion step; then 5 ml of 2 M HCl was added to the resulting solution, which was then deoxygenated by purging with water-saturated nitrogen for 10 min. A clean mercury drop was formed and the deposition potential was set at −0.4 V for 60 s, with the solution being stirred at 1900 rpm; then the stirrer was stopped and the electrode potential was set at −0.4 V for another 30 s. Thereafter, the potential was scanned toward more negative values at a scan rate of 20 mV s−1 using a superimposed differential pulse of −50 mV in amplitude. The As stripping peak was registered at around −0.75 V, and its current was used as a measure of As concentration. The standard addition method was used to test the DPCSV sensitivity and to check linearity of the responses. The digestion of samples with nitric acid and hydrogen peroxide resulted in the oxidation of As to As5+. However, the addition of hydrazine sulfate and hydrochloric acid caused reduction of As5+ to As3+, and of Cu2+ to Cu+, which formed stable chlorocomplexes. At the imposed potential of −0.4 V, As3+ and Cu+ were further reduced to As0 and Cu0, respectively, in the vicinity of the HMDE. Both elements formed an intermetallic compound that codeposited onto the electrode [5,6]. In the subsequent cathodic scan, As0 was further reduced in acidic medium to arsine. The electrode reaction mechanism involved can be written as follows: Deposition on the HMDE at −0.4 V: As3+ + 3CuCl32− + 6e− → Cu3As + 9Cl− Cathodic stripping: Cu3As + 3H3O+ + 3Hg + 3e− → 3Cu (Hg) + AsH3 + 3H2O Arsenic determination by ETAAS Arsenic contents were obtained from the respective calibration plots (absorbance vs concentration) by interpo-

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Results and discussion Spiked tests Aliquots of 25 ml of digested solutions were spiked with different amounts of Hg and As ranging from 10 to 100 ng; Hg was determined by DPASV and FIMS, and As by DPCSV and ETAAS. The experimental results are shown in Table 1 and were used for regression analysis for each element. Regression analyses Because additions were made on an actual MDMA sample (no blank material available) the underlying model is: Element

found

= Element

sample

+ Element

added

Linear regression analysis of Element found vs Element added gives an estimation of the element present in the original MDMA sample as the intercept; its confidence interval is an indication of the precision of the procedure. The confidence interval of the theoretical slope of the model gives the existence of bias in the determination. The determination coefficient r2 and the square root of the unexplained variance, se, can be also used as indication of the goodness of fit of the theoretical model to the experimental data. Table 2 shows a summary of the results of regression analyses for the four methods.

Table 1 Results of addition tests for Hg and As determinations using electrochemical and nonelectrochemical methods Amount of Hg or As added (ng) 0 10 20 40 60 80 100

Hg found (ng)

As found (ng)

DPASV

FIMS

DPCSV

ETAAS

54 66 72 93 114 136 158

56 68 79 100 119 138 160

123 133 141 160 182 204 226

118 127 135 156 174 193 212

DPASV, differential pulse anodic stripping voltammetry; FIMS, flow-injection mercury system; DPCSV, differential pulse cathodic stripping voltammetry; ETAAS, electrothermal atomic absorption spectrometry

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Table 2 Regression analyses using the addition tests for determination of Hg and As using electrochemical and nonelectrochemical methods Analyte

Mercury Arsenic

Technique

DPASV FIMS DPCSV ETAAS

Parameter Found element (ng)

Confidence interval (ng)

Slope

Confidence interval

r2

se (ng)

53.24 57.60 121.3 117.4

50.40–56.07 55.64–59.56 118.6–124.2 116.2–118.5

1.033 1.021 1.030 0.945

0.982–1.083 0.986–1.056 0.979–1.080 0.924–0.965

0.998 0.999 0.998 0.999

1.797 1.242 1.791 0.738

Table 3 Correlation analyses between the electrochemical and nonelectrochemical methods Analyte

Mercury Arsenic

Correlation

DPASV vs FIMS DPCSV vs ETAAS

Parameter Intercept (ng)

Confidence interval (ng)

Slope

Confidence interval

Fcal

P posterior

−6.52 −6.51

−13.50–0.470 −15.64–2.63

1.021 1.089

0.957–1.086 1.033–1.145

12.14 7.44

0.012 0.032

In the case of Hg, Hg concentrations measured by both DPASV and FIMS methods (Table 2) were well within the range of respective confidence intervals. The FIMS technique gave slightly more precise results with smaller confidence intervals for intercepts and slopes, a smaller se value, and an r2 value closer to 1. In view of the narrow confidence intervals for slopes by both methods, no proportional biases were present in them [7]. On the other hand, in the case of As, As concentrations measured by both DPCSV and ETAAS methods (Table 1) were located at the borders of respective confidence intervals of the intercepts (Table 2). ETAAS gave more precise estimation than DPCSV in view of the r2 and se values. The detection limits for Hg by DPASV and As by DPCSV were 0.04 and 0.5 ppb, respectively.

Correlation analyses between the electrochemical and nonelectrochemical methods Because the study was carried out with actual samples (no blank material being available), no conclusions can be drawn about the accuracy of the procedures. However, the accuracy of the electrochemical procedure with reference to the nonelectrochemical method can be estimated by means of regression analysis applied to the measured concentrations [7] of each element as given in Table 1. The abscissa was used for the element concentrations measured by the nonelectrochemical methods, and the ordinate was assigned to the values measured by the electrochemical procedures. In this case, the underlying model is:

Element

electrochemical

= Element

nonelectrochemical

If both methods yield the same results, the measured concentrations obtained by both methods should coincide; that is, the regression line will have a zero intercept and a slope of unity. However, because both parameters (intercept and slope) are jointly determined, their estimations are not independent; a value of the parameters automatically affects the other. Therefore, it is more convenient to test the joint hypothesis that the intercept is equal to zero and the slope is equal to unity, thus taking into account the correlation existing between the two estimates. The hypothesis is tested by the F-test [8], where F is calculated as follows:

F=

(0 − p)

2

2  x2  + 2x (0 − p)(1 − q ) +  ∑ i  (1 − q )  n 2 s e2 n

where p and q are the estimations of the intercept and the slope, respectively; the x values correspond to the concentrations found by FIMS or ETAAS; se2 is the unexplained variance and n the number of experimental points. Table 3 shows a summary of the results. The null hypothesis must be rejected for both elements; similar results are found for both elements, the cause being the existence of a proportional systematic difference between both methods making the nonelectrochemical methods yield smaller values than electrochemical methods: 2.1% for Hg and 8.9% for As.

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Table 4 Precision (reproducibility) of the DPASV for Hg and DPCSV for As using actual ecstasy samples

Conclusions

Sample no.

The electroanalytical techniques of DPASV and DPCSV can be used as alternatives to ETAAS and FIMS, respectively, to determine low concentrations of the toxic elements As and Hg in ecstasy tablets The main advantage is the lower cost of the instrumentation. The techniques have been applied to different actual samples, showing that the present electrochemical techniques are useful in forensic analysis practice.

1 2 3

Hg (mg/kg)

As (mg/kg)

Mean ± CI

RSD (%)

Mean ± CI

RSD (%)

0.052 ± 0.005 0.121 ± 0.010 0.216 ± 0.016

8.2 6.5 5.8

0.124 ± 0.008 0.069 ± 0.004 0.492 ± 0.035

5.3 4.2 5.7

CI, 95% confidence interval; RSD, relative standard deviation Table 5 Determinations of Hg and As in actual ecstasy samples by electrochemical methods Sample no.

1 2 3 4 5 6 7 8 9

Concentrations in the samples (mg/kg) Hg

As

0.052 0.121 0.216 0.510 0.722 0.966 0.931 1.170 1.226

0.124 0.069 0.492 0.171 0.050 0.038 0.124 0.068 0.313

Precision of the electrochemical methods using actual samples The precision of the electroanalytical procedures was assessed by measuring the concentrations of the element for three different actual samples. Five replicates were carried out and average values, confidence intervals, and relative standard deviation (RSD) values were calculated (Table 4). All RSDs were lower than 10%, thus showing good reproducibility of the procedures. Determinations of Hg and As with actual samples The proposed procedures were applied to nine actual MDMA samples from different sources (Table 5). The values varied widely, with the Hg contents always being higher than the As contents. In any case, the Hg and As values were greater than 0.05 and 0.03 mg/kg, respectively.

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Acknowledgment The authors acknowledge the financial support of this research provided by Junta de Castilla y León (Consejería de Educación y Cultura VA115/04).

References 1. FAO/WHO Joint Expert Committee on Food Additives (1973) Evaluation of certain food additives and the contaminations of mercury, lead and cadmium. Technical Report Series No. 505. World Health Organization, Geneva 2. FAO/WHO Joint Expert Committee on Food Additives (1999) Food additives and contaminants 53rd Meeting, Rome, 1–10 June 3. Johannesson M (2002) A review of risks associated to arsenic, cadmium, lead, mercury, and zinc (Appendix A, p 62). In: Johannesson M et al (eds) The market implication of integrated management for heavy metals flows for bioenergy use in the European Union. Kalmar (Sweden), Kalmar University Department of Biology and Environmental Science 4. Comment S, Lock E, Zingg C, Jakob A (2001) The analysis of ecstasy tablets by ICP/MS and ICP/AES. Probl Forensic Sci 46:131–146 5. Li H, Smart RB (1996) Determination of sub-nanomolar concentration of arsenic (III) in natural waters by square wave cathodic stripping voltammetry. Anal Chim Acta 325:25–32 6. Kotoucek M, Vasicova J, Ruzicka J (1993) Determination of arsenic by cathodic stripping voltammetry at a hanging mercury drop electrode. Mikrochim Acta 111:55–62 7. Massart DL, Vandeginste BGM, Buydens LMC, De Jong S, Lewi PI, Smeyers-Verbeke J (1997) Handbook of chemometrics and qualimetrics: part A. Elsevier, Amsterdam, p 410 8. Massart DL, Vandeginste BGM, Buydens LMC, De Jong S, Lewi PI, Smeyers-Verbeke J (1997) Handbook of chemometrics and qualimetrics: part A. Elsevier, Amsterdam, p 193