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No-Deuterium Proton NMR (No-D NMR): A Simple, Fast and PowerfulMethod for Analyzes of Ilegal Drugs

Lucas A. Gama, Bianca M. Bortolini, Valdemar L. Junior, WandersonRomao, Alvaro C. Neto

PII: S0026-265X(14)00147-7DOI: doi: 10.1016/j.microc.2014.07.014Reference: MICROC 2008

To appear in: Microchemical Journal

Received date: 15 July 2014Accepted date: 28 July 2014

Please cite this article as: Lucas A. Gama, Bianca M. Bortolini, Valdemar L. Junior,Wanderson Romao, Alvaro C. Neto, No-Deuterium Proton NMR (No-D NMR): A Simple,Fast and Powerful Method for Analyzes of Ilegal Drugs, Microchemical Journal (2014),doi: 10.1016/j.microc.2014.07.014

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No-Deuterium Proton NMR (No-D NMR): A Simple, Fast

and Powerful Method for Analyzes of Ilegal Drugs.

Lucas A. Gama,a Bianca M. Bortolini,b Valdemar L. Junior,a Wanderson Romão*

,a,c Alvaro C. Netoa*

aDepartamento de Química, Universidade Federal do Espírito Santo, Avenida

Fernando Ferrai, 514, Goiabeiras, Vitória – ES, CEP: 29075-910.

b Laboratório de Química Legal, Superintendência de Polícia técnico-científica

da Polícia Civil do Estado do Espírito Santo, Rua José Farias s/ n, Santa Lúcia,

Vitória – ES, CEP: 29045-300.

c Instituto Federal do Espírito Santo, Av. Ministro Salgado Filho, Soteco, Vila

Velha- ES, CEP: 29106-010.

* Corresponding author: alvarocunhaneto@gmail.com and

wandersonromao@gmail.com

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ABSTRACT

The Nuclear Magnetic Resonance (NMR) has been employed as an excellent

analytical technique for analyzing mixtures. For the use of NMR as a routine

experimentation, new methodologies have been developed without the use of

deuterated solvents also known as No-D NMR, which keeps the intrinsic

advantages of NMR as a fast, non-invasive and non-destructive technique. In

this study, we used No-D NMR as a tool for analysis in 71 samples of cocaine,

one of ecstasy and one of metilona seized by Police in the State of Espírito

Santo. During the analysis of cocaine seizure samples, we can observe a

tendency of trafficking through its adulterants such as caffeine, phenacetin and

lidocaine. The No-D NMR technique was also applied in order to develop a

methodology for the analysis of illicit drugs and possibly become a routine in

NMR laboratories. In these analyses, we can observe the high resolution power

of the No-D technique combined with lower operating costs.

Keywords: No-D NMR, illicit drugs, cocaine.

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1. INTRODUCTION

Cocaine is an alkaloid extracted from the leaves of shrubs of genus

Erytroxylum, also known as coca, where organic solvents, acids and bases are

employed. Cocaine can be marketed in the hydrochloride form, termed as

cocaine and in free base form, also called the pasta base, free base cocaine,

crack or merla. Both the hydrochloride and the free base originate at different

stages in the extraction and purification process of cocaine. The crack is the

form with greater market expansion, especially in population groups with lower

purchasing power [1-4].

Coca cultivation in the world had a total area of 155,600 hectares in 2011,

representing an area 30% smaller than in 2000. This decrease was

accompanied by a decrease of cocaine use in many South American countries,

however, the Brazil consumption has increased substantially. Due to its

geographical location, Brazil is one of the places that play an important role in

the global illicit market for cocaine, bordering the largest producers of cocaine in

the world. Bolivia alone accounted for 54.0% of the cocaine seized in Brazil in

2011, followed by Peru and Colombia with 38.0 and 7.5%, respectively.

Moreover, Brazil has a large coastal area facing the Atlantic Ocean, which

allows drug distribution to Europe and Africa. Cocaine exported from Brazil is

destined for the Portuguese-speaking countries, mainly Portugal [5,6]. The illicit

trade in cocaine use two types of substances: adulterants — pharmacologically

active compounds to potentiate, minimize or mask it; and diluents — inactive

compounds that only increase the weight of the sample. The main contaminants

found in police seizure samples are benzocaine, phenacetin, caffeine, benzoic

acid, procaine, ketamine, and lidocaine. The diluents used are sugars and

derivatives, as well as inorganic compounds [4,6-8].

Brazilian police use from non-instrumental methods such as volumetric

and/or colorimetric reactions to the chromatographic instrumental methods such

as gas chromatography (GC) for the study of drugs. The forensic laboratories

often use the the Scott Ruybal test, that employs a reagent kit, to develop a

blue color for identification of cocaine and crack. These tests display, however,

poor specificity, and may sometimes provide false-positives or false-negatives,

specially for the more complex mixtures or impure samples and for common

adulterants such as lidocaine and ketamine [8]. On other hand, GC’s major

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limitation is the need for the sample to be volatile or thermally stable or be

chemically derived, making it a more time-consuming analysis.

The need for use of noninvasive techniques has made the Nuclear Magnetic

Resonance (NMR) one of the most important techniques for structural

elucidation of drugs and for allowing their identification and quantification along

with their fillers, diluents, and other metabolites. The absence of derivatization

steps and the possibility of using benchmarks that do not have high purity, or

the use of electronic signals for quantification, highlight the importance of NMR.

This technique shows to be a versatile and effective tool for the forensic areas

because it is nondestructive, fast, selective and sensitive, allowing the

identification of various drugs and mixtures thereof, besides the logistics of

trafficking [1,9].

The application of NMR techniques are usually performed with the use of

deuterated solvent [10]. However, there is no need for this type of solvent,

which results in lower analytical costs. NMR spectroscopy without the use of

deuterated solvent, also known as No-D NMR, was a method developed for

determining the concentration of reagents and monitoring of reactions based on

NMR spectra obtained for samples dissolved in common laboratory solvents.

The spectra have a signal/noise (S/N) ratio and appropriate resolutions for

experimental routines, which can be used to obtain quantitatively reliable

values. This technique has economic advantages with lower cost when we use

non-deuterated solventes; scale advantages as the automation of experiments;

operational advantages due to the use of solvent itself from a chemical reaction;

and less time spent to obtain a spectrum due to elimination the solubilization

step for some substances [11,12].

Hoye et al. demonstrate the utility of the No-D NMR for monitoring reactions.

For Fischer esterification of acetic acid with methanol, they monitored the

progress of the reaction with rise of signals corresponding to the ethyl ester

formed until reaction balance. Monitoring was also conducted similarly for the

decomposition of n-BuLi in tetrahydrofuran at room temperature, in which there

was decrease in tetrahydrofuran signal and the appearance and increase in

intensity of the ethylene and the lithium enolate-acetaldehyde: decomposition

products [11]. Hoye et al. also demonstrate titration methodologies using No-D

NMR. [11,12]. Besides the methods, they show comparisons of alkyl lithium

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reagent (in the form n-, s- and t-) and Grignard titrations through No-D NMR and

colorimetric titration, which presented excellent compliance with the standards

for both. NMR is also a faster technique [12].

In this study, samples of cocaine seized by Espírito Santo state police

were studied by employing the technique of No-D NMR so that this

methodology can be used routinely in the analysis of cocaine seized. In the No-

D NMR was also applied to other drugs of abuse, such as ecstasy and

metilona.

2. MATERIALS AND METHODS

2.1 Materials

Samples of 71 cocaine seizure carried out from January to May 2012,

one seizure sample of ecstasy and one of metilona — besides theond the

standards of cocaine, phenacetin, caffeine, lidocaine — were used in this study.

Commercial methanol (Vetec Fine Chemicals LTDA, Brazil) was used in

samples solution preparation for No-D NMR analysis, which was previously

dried under anhydrous Na2SO4, [13]. The deuterated methanol (Sigma-Aldrich,

USA) was used in the acquisition of both: conventional ¹H-NMR and No-D NMR

spectra, however, in the last, it was used in a coaxial insertion tube.

2.2 Samples

550 μL of solution 0.1 mol.L-1 of cocaine, lidocaine, and phenacetin

standards were prepared as well as 0.05 mol L-1 of caffeine standard. The

solutions of the 71 samples seized were prepared at a concentration of 0.3 mol

L-1. The metilona sample was prepared at a concentration of 0.04 mol L-1 and

for the sample of ecstasy, a concentration of 0.06 mol L-1 was used.

All solutions for the No-D NMR were prepared in commercial methanol

and placed in tubes of 5 mm NMR together with a coaxial insertion tube

containing deuterated methanol (CD3OD).

To assess the limit of detection (LOD) of the methodology, successive

dilutions of the cocaine standard sample were performed from 55 to ≈ 2 mg mL-

1 and the spectrum of No-D NMR obtained.

2.2 Instrumentation

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The ¹H NMR spectra were acquired using a liquid probe of 5mm

BroadBand 1H/19F/X in a 9.4 T Varian Agilent ® spectrometer model 400

VNMRS to a temperature of 25 °C. NMR spectra were obtained with acquisition

time of 20 seconds, standby time of 5 s, 45° pulse (7.6 μs) with 8 transients and

spectral window of 6410.3 Hz. The No-D NMR spectra were also obtained with

acquisition time of 20 seconds, standby time of 20 s, 22° pulse (3.8 μs) with the

same spectral window, as many transients, as mentioned by Hoye et al. [12,14].

3. RESULTS AND DISCUSSION

The use of an insertion tube with deuterated solvent is proposed due to

signal quality when performing automatic mode lock and shimming, unlike what

is proposed by Hoye [11]. The No-D NMR is proposed to be carried out without

frequency lock (unlocked). However, in this study we observed an improvement

in the quality of the signals when the lock is held.

For the standard of cocaine, spectra were performed employing

conventional NMR (Figure 1a) and also No-D NMR (Figure 1b). A comparison

of the two spectra in Figure 1 shows that, even though solvent signal is major

to No-D NMR, the expansion of the aromatic region is able to identify

characteristic signals for standard cocaine sample. Cocaine for example, shows

three signals in the region of aromatic hydrogens, in which a doublet at 8.02

ppm referring to hydrogens "a", a triplet at 7.71 ppm referring to hydrogen "c",

and another triplet at 7.57 ppm for hydrogens "b". The other signs related to

cocaine are the multiplets at 5.56; 4.23; 4.03; 3.59; 2.90 and 2.50 ppm referring

to hydrogens "f", "i", "h", "e", "j" and "g", respectively, and the singlet at 3.66

ppm referring to hydrogens "d "(Figure 1) [9].

Figure 1.

In order to study the No-D NMR, we used the aromatic hydrogens region

because it has no overlapping signals of cocaine and its main adulterants,

represented in Figure 2. The main adulterants are lidocaine (Figure 2b),

caffeine (Figure 2c) and phenacetin (Figure 2d). Lidocaine has a signal with

chemical shift at 7.15 ppm representing the three hydrogens (H2) of the

benzene ring. Caffeine has singlet referring to a hydrogen (H3) of the pyrimidine

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ring with chemical shift at 7.92 ppm. In turn, phenacetin has two pairs of

benzene hydrogens: H4a shows a doublet at 7.46 ppm; and H4b hydrogens,

another doublet at 6.91 ppm, both related to two equivalent hydrogens (Figure

2d).

Figure 2.

Figure 3 shows two spectra of a cocaine seizure sample with expansion

in the aromatic hydrogen region, where Figure 3a shows the spectrum obtained

by dissolving in commercial methanol and Figure 3b dissolution in the

deuterated solvent. The identification of signals in the aromatic hydrogen region

of the main cocaine adulterants (caffeine, phenacetin and lignocaine) was not

affected, which shows the good resolution of the spectra and that the analyses

of No-D NMR can replace conventional NMR in identifying of cocaine and its

adulterants. This can assist in the establishment of trafficking trends. The region

of aliphatic hydrogens is presented with many signals that hinder a reliable

identification of cocaine and its adulterants.

Figure 3.

The limit of detection (LOD) of cocaine was tested for the of No-D NMR

method by successively diluting the initial solution of cocaine standard. Figure 4

shows the expansion of aromatic hydrogen signals obtained for the 55 mg mL-1

sample, Figure 4a, and its dilutions, Figure 4b-g. The No-NMR method is able

to detect cocaine to a limit concentration of ≈ 2 mg mL-1 (1 mg dissolved in 550

L), Figure 4g, where it can be seen that the corresponding cocaine hydrogen

H1c signal is impaired by spectral noise as well defined as LOD. The No-D

NMR method is also able to quantify the cocaine from simple univariate linear

calibration, Figure 5. The ratio of aromatic hydrogen region areas (H1a + H1b +

H1c) by methanol hydrogen region (HCH3OH) versus cocaine (mass in mg

dissolved in 550 μL of methanol) were plotted, and the following equations 1

can be used to obtain quantitative information on cocaine:

y = 0.0009x +8.10-5 (equation 1)

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where y = ratio of hydrogen region areas and x = cocaine concentration.

Figure 4.

Figure 5.

The No-D NMR spectra of 71 cocaine seizure samples were obtained,

and from them it was possible to carry out the distribution of adulterants in the

State of Espírito Santo (Figure 6). The metropolitan area of the state (Area 3,

Figure 6) has 28 seizure samples, which is the largest amount of police seizure

samples and the highest in adulterations with caffeine, lidocaine and

phenacetin, thus proving to be the most diverse. The amount of seizure

samples and adulterations indicate that many seizure samples have more than

one adulterant. Adulterations caffeine, lidocaine and phenacetin are shown

predominantly in the metropolitan area, which indicates that the seizure

samples from other areas may produce samples of adulterated cocaine in the

metropolitan area. Phenacetin in the southern area (Area 4, Figure 6) does not

have great representation. It may be coming from any other area. Seizure

samples without adulterants show a trend of cocaine entry coming from other

producing countries through the northern state area (Area 1, Figure 6). This is

the area with the largest amounts of unadulterated cocaine seizure samples,

and a possible subsequent distribution to the central (Area 2, Figure 6) and

metropolitan areas. Other adulterants such as benzocaine (3 seizure samples)

were also found, but in insignificant amounts.

Figure 6.

Other Illicit Drugs

No-D NMR can also be used to analyze other illicit drugs as seen in

Figure 7, showing the utility of this technique in Forensic Chemistry. The

aromatic hydrogen region shows to be able to identify ecstasy and metilona

structures that do not have overlapping signals for adulterants of these seizure

samples, as it is for cocaine. For the expansion of the aromatic hydrogen region

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for the ecstasy spectrum (Figure 7a), we identified one doublet at 6.68 ppm

referring to hydrogen (H3); one at 6.65 ppm doublet referring to hydrogen (H2)

and at 6.60 ppm one doublet of doublet referring to hydrogen (H4); and finally, a

singlet at 5.80 ppm corresponding to two hydrogens (H1). For the expansion of

metilona spectrum (Figure 7b) we have doublet of doublet at 7.67 ppm referring

to hydrogen (H4); at 7.46 ppm, a doublet one referring to one hydrogen (H2); at

6.98 ppm, a doublet referring to hydrogen H3 and at 6.09 ppm, a singlet

referring to hydrogen H1.

Figure 7.

4. Conclusion

The use of deuterated solvent by conventional NMR is one of the

drawbacks for this technique to be used in routine analysis, because of its price.

However, with No-D NMR methodology it is possible to reduce costs by using

commercial solvent for dissolving the samples.

The No-D NMR is an excellent tool for routine analysis because, besides

providing data on new drugs, standard purity and correlating drugs and

adulterants, it can determine trends in the illegal market. The technique of No-D

NMR showed to be even simple and faster than the GC methodologies, which

are used by the Brazilian Police, thus showing that it can be routinely used in

NMR laboratories, especially when automated equipment is used.

5. Acknowledgements

The authors are grateful to the Civil Police in the State of Espírito Santo,

Brazil, the Center of Competence in Petroleum Chemistry (NCQP) and

development agencies FAPES, CAPES and CNPq.

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Figure Captions

Figure 1. Hydrogens spectrum for cocaine standard (a), dissolved in deuterated

methanol and (b), dissolved in comercial methanol.

Figure 2. Expansion of the aromatic region of spectra No-D NMR-¹H for

cocaine (a), lidocaine (b) caffeine (c) and phenacetin (d).

Figure 3. Expansion of the aromatic region of the spectra of solubilized seizure

samples of cocaine in (a) commercial methanol and (b) deuterated methanol.

Figure 4. Expansion of the region of aromatic hydrogens of the No-D NMR-¹H

spectrum for dilutions of cocaine standard. (A: 30 mg of cocaine, B: 15.0 mg C:

10.0 mg D: 4.0 mg, E: 3.0 mg, F: G and 2.0 mg: 1.0 mg). All samples were

dissolved in 550 L of commercial methanol.

Figure 5. Evaluation of the relationship between the ratio of integrals of

aromatic signals of cocaine with the solvent signal with cocaine mass dissolved.

Figure 6. Espírito Santo State áreas (1 - North, 2 - Central, 3 - Metropolitan and

4 - South) and evaluation of adulterants per area.

Figure 7. No-D NMR-¹H spectra expanded in the region of aromatic hydrogens

for (a) Ecstasy and (b) Metilona.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

Figure 6.

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Figure 7.

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Highlights

Nuclear Magnetic Resonance has been employed without the use of

deuterated solvents (No-D NMR) for analyzes of illicit drugs;

No-D NMR spectra were acquired for 71 samples of cocaine, one of

ecstasy and one of metilona;

No-D NMR is able to detect and quantify street cocaine samples and

distinguish from its adulterants: caffeine, phenacetin and lidocaine;

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