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Rocha, J. Almeida, R. M. Correia, F. Tosato, N. Madeira, P. R. Filgueiras, V. Lacerda Jr., J. C.C. Freitas and
A. C. Neto, Anal. Methods, 2018, DOI: 10.1039/C7AY03000B.

Volume 8 Number 1 7 January 2016 Pages 1–224 This is an Accepted Manuscript, which has been through the

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 Page 1 of 27 Analytical Methods
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DOI: 10.1039/C7AY03000B

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4 1 Quantification of Cocaine and its Adulterants by Nuclear Magnetic
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7 2 Resonance Spectroscopy without Deuterated Solvent (No-D qNMR)
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11 4 Willy W. F. Rocha,a Júlia de A. Leite,a Radigya M. Correia,a Flávia Tosato,a Natã C. L.

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13 5 Madeira,a Paulo R. Filgueiras,a Valdemar Lacerda Jr.,a Jair C. C. Freitas,a Wanderson
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6 Romão,a,b* Álvaro C. Netoa
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20 8 Universidade Federal do Espírito Santo (UFES), Av. Fernando Ferrari, 514,
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22 9 Goiabeiras, Vitória-ES, CEP: 29075-910, Brasil.
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24 10 b
Instituto Federal do Espírito Santo (IFES), Av. Ministro Salgado Filho, 1000, Soteco,
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26 11 Vila Velha-ES, CEP: 29106-010, Brasil.
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31 13 *Corresponding author. Tel.: +55 02740094512
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33 14 E-mail address: wandersonromao@gmail.com (Wanderson Romão).
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35 15 Instituto Federal do Espírito Santo, 29106-010 Vila Velha – ES, Brazil.
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4 16 Abstract
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9 18 In this study, a new method was developed to quantify cocaine and some adulterants
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11 19 (lidocaine, caffeine, phenacetin, procaine and benzocaine) using the nuclear magnetic

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13 20 resonance spectroscopy without the use of deuterated solvents (No-D qNMR). The No-
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15 21 D qNMR presents as main advantages: low cost (in comparison to the use of deuterated
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17 22 solvents), non-destructive and speed analysis, and able to detect non-volatile
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23 compounds. For the analytical validation achievement, figures of merit such as
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22 24 selectivity and specificity, linearity, quantification limit, detection limit, accuracy,
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24 25 precision and robustness were obtained. The built models presented excellent precision
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26 26 (< 5%), linearity (>0.99), and LOD values which varied between 0.126 and 0.666 mg
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28 27 mL-1. With the purpose to verify the models’ predictive capacity, 34 crack samples
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30 28 seized by the Civil Police of Espírito Santo State – Brazil were analyzed, and the
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32 29 average cocaine content found was around 17.5 wt%, which is in line with expectations
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30 (up to approximately 30 wt%). Moreover, 50 wt% of the samples contained phenacetin,
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37 31 9 wt% caffeine, 3 wt% procaine and lidocaine, while benzocaine wasn’t identified.
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45 35 Keywords: cocaine, adulterants, quantification, no-D qNMR, qNMR.
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4 37 Introduction
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38 The methyl (1S,3S,4R,5R)-3-benzoyloxy-8-methyl-8-azabicyclo[3.2.1]octane-4-
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9 39 carboxylate, also called cocaine, is an tropane alkaloid with stimulant effect extracted
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11 40 from the Erythroxylum coca leaves, popularly known as coca. It’s found mainly in

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13 41 South America, in the Andes region, and it is usually marketed in the salt form (cocaine
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15 42 hydrochloride) or in the form of its free base (crack), Figure 1.1-8
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17 43
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26 44
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28 45 Figure 1. Molecular structure of (a) cocaine hydrochloride and (b) crack.
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47 The cocaine production process varies with the geographical region, where the
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35 48 basic methodology for its obtaining occurs through coca leaves extraction,5 Figure 2.
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37 49 However, it’s not possible to use this route to purify a typical seized cocaine sample
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39 50 because the adulterants added to the drug during the traffic have similar chemical
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41 51 properties to cocaine, for example, pKa, solubility, and reactivity (some adulterants
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43 52 provide positive results in presence of thiocyanate cobalt reagent).1-3
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30 54 Figure 2. Extraction and purification route of cocaine from coca leaves.
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34 56 In the trafficking network, the profit is generated in all phases of drug
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36 57 distribution, therefore, it is common the addition of adulterants and diluents (Table 1) to
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38 58 increase the amount of cocaine, and consequently, the commercial value.5,8-10 Cocaine,
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59 in the hydrochloride form, is usually diluted into carbohydrates like mannitol, lactose or
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43 60 glucose; these diluents are inert substances and serve only to increase the drug volume.
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45 61 Adulterants (non-controlled substances) like local anesthetics and stimulants
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47 62 (levamisole, phenacetin, lidocaine, caffeine, diltiazem, hydroxyzine, procaine,
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49 63 benzocaine) are also added, since they have similar physical characteristics to cocaine.
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51 64 In contrast, crack cannot be easily adulterated for being a beige waxy solid and with a
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65 very characteristic odor, in which, the most cases it have a higher purity.5,7,8
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4 67 Table 1. Major cocaine adulterants.5
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6 Adulterants
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Allobarbital Caffeine Lidocaine Phenacetin
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Paracetamol Diazepam Levamisole Phenobarbital
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Antipyrine Dipyrone Methamphetamine Piracetam
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11 Aspirin Ephedrine Methaqualone Procaine

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12 Atropine Fentanyl Nicotinamide Quinine
13 Benzocaine Flunitrazepam Nitrazepam Tetracaine
14 Benzoic acid Flurazepam Theophylline
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17 69 Adulterants are usually chosen according to their availability. Lidocaine and
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70 procaine are commonly found in Europe and phenacetin in South America. Reports
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22 71 from Brazilian Federal Police (BFP)4 indicate that phenacetin is present mainly in
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24 72 cocaine seizures from Peru and Bolivia, and levamisole is frequently found in the
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26 73 Colombian.
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28 74 Adulterants can maximize cocaine effects and also increase the problems related
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30 75 to health and the risk of intoxication, since they can modify the pharmacological profile
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32 76 of the drug that is being sold.8,11 In this way, such information and classifications are
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77 relevant for national and international authorities, who make great effort to discover the
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37 78 origin and routes of drug trafficking, as well as, to political and governmental decisions
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39 79 on public safety. In a large and diversified country, as the Brazil, it is necessary to
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41 80 generate significant statistic reports about the seizures of cocaine.4,7,12,13 In 2006, the
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43 81 BFP created a program for illicit drugs characterization called PeQui (Drugs Chemical
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45 82 Profile) with the purpose of analyzing drugs in order to assist police investigations.
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83 The techniques employed in forensic analyses are usually high-performance
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50 84 liquid chromatography (HPLC) and gas chromatography (GC) coupled to detectors like
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52 85 a mass spectrometer (MS)4,13,14 or a flame ionization detector (FID).7,12,15,16 These
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54 86 methodologies have been widely reported in the literature with an enormous potential
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56 87 for forensic analyses showing good resolution, accuracy, robustness and sensitivity.
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4 88 In this context, nuclear magnetic resonance (NMR) has advantages such as
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6 89 structural elucidation at molecular level, non-destructive and rapid analysis, not
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8 90 requiring coupling with hyphenated techniques like Liquid Chromatography (LC) or
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91 GC; besides, NMR can detect non-volatile substances.17 The characteristics described
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13 92 above contribute to the expert reports elaboration.7,12,18 Moreover, few analytical
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15 93 methods are capable of distinguish isomeric mixtures of abuse drugs, through the spin-
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17 94 spin coupling. Groombridge et al.7 demonstrated that the 1
H NMR spectra
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19 95 unequivocally differentiated amphetamine from its derivatives. On the other hand, the
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21 96 distinction of this mixture is not easily made by GC-MS from the fragmentation profile
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23 97 of its electron ionization mass spectrometry (EI-MS).19
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98 Studies using high-field NMR have been employed to new structures
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28 99 identification because the method is completely linear and doesn’t require the use of
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30 100 high purity primary standards for quantification experiments. This may be important for
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32 101 abuse drugs analysis, since the reference materials may not be available from
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34 102 commercial suppliers.6,13 In this way, 1H NMR spectroscopy represents one of the most
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36 103 versatile tools in the forensics sciences. Another important characteristic to be cited is
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104 the use of deuterated solvents in the samples’ preparation in order to do not allow that
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41 105 the solvent hydrogens disturb or interfere in the analysis. Although there are certain
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43 106 advantages in this practice, the use of deuterated solvent is not an obligatoriness and its
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45 107 substitution by a conventional solvent reduces the analysis cost.18,20
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47 108 The application of methodology of quantification by NMR (qNMR) is done of
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49 109 the following form: the 1H signal area in a NMR spectrum (Ax) must be directly
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110 proportional to the number of hydrogens (Nx) that absorb the signal radiofrequency, that
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54 111 is:
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56 112 A  = K. N (Eq. 1)
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4 113 where K is the spectrometer constant.29,30
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6 114 For this relation be applicable, the spins need to be in thermal equilibrium, that
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8 115 is, the magnetic pulses must obey the time of at least five times the longest longitudinal
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116 relaxation time (T1).17,21

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13 117 The resolution offered by the Fourier transform enables to choose the best signal
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15 118 to be used for the quantification calculation, this is, a signal having a higher intensity
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17 119 and having no overlap when analyzed in a mixture.17 The relative determination of a
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19 120 quantitative analysis is given by the molar ratio between analyte and an internal
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21 121 standard of known purity and weight:
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23   

24 122 = (Eq. 2)



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27 123 where n is the number of moles, A is the signal area, N is the number of nuclei that
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29 124 absorb in the resonance signal frequency, x is the analyte and PI is the internal
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31 125 standard.17,20
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33 126 Equation 2 is modified to calculate the content of an analyte in a mixture in
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35 127 order to make the calculation by the Equation 3:
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37  .
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38 128 P = P (Eq. 3)


. .
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40 129 where P is the purity, M is the molar weight and m is the sample weight.17
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45 131 With the application of this equation, the construction of a calibration curve is
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47 132 unnecessary since it is possible to obtain analyte purity without the use of the linear
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49 133 equation. This possibility is the great advantage of qNMR when compared to other
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51 134 analytical methods, like chromatography and other spectroscopic techniques, because
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53 135 sometimes it can be difficult to obtain standardized compounds, mainly because of the
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136 excessive cost of them, which depends on the import and availability on the world
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4 137 market.17
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6 138 The quality of the analytical results is essential for the forensic police. For a
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8 139 good analytical performance, it is necessary that it follows statistical reliability by
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140 means of validations, known as "figures of merit". According to the ANVISA

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13 141 Resolution RE nº 899, of May 29th, 2003,22 the following figures of merit are necessary
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15 142 for a quantitative analysis: selectivity and specificity, linearity, quantification limit
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17 143 (LOQ), detection limit (LOD), accuracy, precision and robustness.2,8,21 The objective of
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19 144 this study is to propose a simultaneous quantification methodology based in the 1H
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21 145 NMR technique without deuterated solvent (No-D qNMR) for cocaine and its
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23 146 adulterants (lidocaine, caffeine, phenacetin, procaine and benzocaine) and, posteriorly,
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147 to apply this methodology for seized cocaine samples.
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28 148
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30 149 Experimental
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150 Samples and Reagents
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151 The standards used were: lidocaine hydrochloride monohydrate 99.0% (Vetec
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37 152 Química Fina Ltda., Rio de Janeiro), anhydrous caffeine 98.5% (Bandeirante Brazmo
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39 153 Indústria e Comércio Ltda., São Paulo), phenacetin, procaine and benzocaine 98.0%
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41 154 (Sigma-Aldrich, São Paulo). The cocaine standard was obtained from 600 mg of crack
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43 155 samples from seizures carried out from July to December 2012, provided by the Civil
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45 156 Police of Espírito Santo State – Brazil (PC-ES). After, 34 cocaine hydrochloride
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157 samples (seized by PC-ES from May to June 2012) were applied to constructed models.
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50 158 Methanol with HPLC grade (LiChrosolv®, Merck) was used as solvent in the thin layer
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52 159 chromatography (TLC) and column chromatography (CC) analyses. The internal
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54 160 standard for the 1H NMR analyses was the maleic acid 99.0% dissolved into deuterium
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56 161 oxide 99.9% D (Sigma-Aldrich).
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4 162 Cocaine purification
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6 163 Initially, TLC was performed in order to identify the presence of adulterants
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8 164 (lidocaine, caffeine, phenacetin, procaine and benzocaine) and cocaine in the crack
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165 seized samples through the comparison between the retention factors (Rf) of the
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13 166 analytes. The revealing agents used were Dragendorff, cobalt thiocyanate and vanillin
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15 167 with UV light at 254 nm. The mobile phase that presented the best efficiency in
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17 168 previous studies was methanol/ethyl acetate 9:1 and silica gel was used as stationary
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19 169 phase. Posteriorly, cocaine was obtained from the purification of 600 mg of crack seized
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21 170 samples using chromatographic column with silica gel (0,062-0,2 mm) (70-230 mesh)
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23 171 (Carvalhaes) and the same mobile and stationary phases of TLC.1,2
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172 To confirm the presence of cocaine in the collected fractions, the colorimetric
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28 173 method with cobalt thiocyanate was used. These fractions were analyzed by GC-MS to
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30 174 confirm their purity according to Recommended Methods for the Identification and
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32 175 Analysis of Cocaine in Seized Materials of UNODC.5 The samples were taken to the
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34 176 rotary evaporator to remove the solvent and then they were subjected to the No-D q1H
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36 177 NMR analysis.
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41 179 Method validation
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43 180 Solutions with different concentrations (between 0 and 20 mg.mL-1) of lidocaine,
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45 181 benzocaine, phenacetin, procaine, caffeine and cocaine were prepared. The standards
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47 182 were dissolved into 500 µL of commercial methanol (HPLC grade). The calibration
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49 183 curves were constructed and the figures of merit were determined from these solutions.
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54 185 Linearity
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56 186 The linearity of the calibration curves for each analyte was determined from five
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4 187 calibration levels (measured into triplicates). Table 2 shows the weights of standards
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6 188 used for the model construction. The calibration curves (Eq. 4) were constructed from
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8 189 the ratio between the analyte signal area of each standard and the maleic acid signal as a
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11 190 function of the analyte weight ( xi ). The regression coefficients were calculated by the

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13 191 least squares method and the linearity was determined from the coefficient of
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15 192 determination (R2), Eq. 5.
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193 yˆi = b0 + b1 xi (Eq. 4)
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20 ∑ ( y − yˆ )
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2 i i i
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∑ (y − y)
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24 195 where yi , ŷi and y are, respectively, the ratio between the analyte signal area of each
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27 196 standard and the maleic acid signal as a function of the analyte weight, the predicted
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29 197 values for these ratios and the average value of these ratios. The coefficients of the
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31 198 model b0 and b1 are, respectively, the linear and angular coefficient of the regression.
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34 199
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36 200 Table 2. Weights (in mg) of standards dissolved into 500 µL (5 solutions) used to
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38 201 determine the linearity.
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40 Points Caffeine Lidocaine Benzocaine Phenacetin Procaine Cocaine
41 1 1.0 1.2 1.2 1.0 1.0 2.0
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2 1.9 3.1 2.9 3.1 2.9 4.0
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44 3 3.1 6.0 6.1 6.1 6.1 6.0
45 4 4.1 9.9 10.1 9.9 9.9 15.0
46 5 5.0 15.1 15.1 15.0 15.1 20.0
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49 203 Selectivity and specificity
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51 204 Solutions of the analyte standards and the internal standard were analyzed,
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56 206 the quantification calculation of each component. The selectivity is proven if there is no
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4 207 overlap of signals.
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210 The detection and quantification limits (LOD and LOQ) were determined from
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13 211 the standard deviation ( s ) for all the analytes using the parameters of the equation of
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15 212 the line. LOD and LOQ are calculated from Eqs. 6 e 7.
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17 3s
18 213 LOD = (Eq. 6)
b1
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21 10s
214 LOQ = (Eq. 7)
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25 215
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27 216 Accuracy
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29 217 Accuracy was determined with the points 1, 3 and 5 (Table 2) of the calibration
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31 218 curve, where the quantity of standard added is known using Eq. 8:
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34 219 Accuracy% = . 100 (Eq. 8)

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36 220   is the experimental average of the weights and m is the theoretical weight.21
where m
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222 Precision
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43 223 The spots of low, medium and high concentration were used to check the
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45 224 intermediate precision of the results. The analyses were performed in different days and
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47 225 in triplicate. The intermediate precision was calculated through the relative standard
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49 226 deviation (RSD) calculation in accordance with equation 9:
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54 228 RSD = %& . 100 (Eq. 9)
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8 232 Robustness
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233 The samples of the spots 1 and 5 of the adulterants were stored free of light at -
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13 234 2°C for seven days and then they were analyzed again to determine their stability.
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17 236 Analysis of cocaine seized samples
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19 237 Thirty four cocaine samples seized by the PC-ES were prepared using a
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21 238 concentration of 20 mg. The values of the integrals were applied to the equations of the
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23 239 calibration curves developed from their respective standards, and the weight percentage
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240 (wt %) of each analyte was determined.
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28 241
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30 242 Instrumentation - No-D qNMR
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32 243 The 1H NMR spectra were acquired using a liquid probe of 5 mm BroadBand
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34 244 1
H/19F/X in a 9.4T Varian Agilent® spectrometer, model 400 VNMRS in a temperature
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36 245 of 25°C. The software used was the VNMRj version 4.2. The spectra were analyzing
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246 using the software MestReNova version 6.0.2. The solutions were analyzed with 80 µL
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41 247 of internal standard solution, 15.0 mg of maleic acid dissolved into 1000 µL of
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43 248 deuterium oxide contained in a coaxial insertion tube.
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45 249
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47 250
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49 251 Parameters - No-D qNMR
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252 To ensure that the nuclei integration reading be quantitative, it is necessary to
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54 253 guarantee that the return of the magnetization of these nuclei to the original state be
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56 254 complete. Thus, longitudinal relaxation measurements are required through the 90°
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4 255 inversion-recovery pulse sequence. This sequence shows the longitudinal relaxation
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6 256 constant of each nucleus, in this case, the hydrogens of the molecules under study.
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8 257 Therefore, two samples (of higher and lower weight) of each adulterant standard and of
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258 crack seized samples were used for the longitudinal relaxation time measure (T1). An
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13 259 inversion-recovery pulse sequence was used with a relaxation delay of 20 seconds,
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15 260 number of scans equal to 16, gain equal to 0, spectral width of 6410.3 Hz, coupled NOE
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17 261 with minimum of 0.5 seconds and maximum of 7 seconds and a total experiment time
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19 262 of 0.2 hours. The acquisition time was calculated using the longest time equal to seven
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21 263 times T1.
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23 264 After the determination of the parameters obtained for T1 measures, the 1H NMR
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265 spectra of the cocaine, its adulterants, and of the 34 crack seized samples were obtained
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28 266 using the following parameters: acquisition time of 4.992 s, relaxation time of 43.47 s,
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30 267 pulse of 90°, gain equal to 0 and spectral width of 6410.3 Hz. Subsequently, the baseline
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32 268 correction of 1H NMR spectra was performed automatically and the phase correction,
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34 269 when necessary, manually.
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39 271 Results and Discussion
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273 Cocaine content in the seized samples was determined in order to find the
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46 274 sample of highest purity. GC-MS was used to verify cocaine purity, allowing the
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48 275 qualitative and quantitative determination of cocaine and its adulterants.8 In Fig. 1S,
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50 276 only the pure cocaine signal is observed with an approximately retention time of 10.5
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52 277 min. The EI mass spectrum containing the cocaine fragmentation profile, Fig. 2S,
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54 278 confirms its presence with a high similarity index according to the GC-MS library, Fig.
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4 280 Method validation
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6 281 The quantitative method validation for cocaine and its adulterants was carried
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8 282 out following the norm of ANVISA’s Resolution RE nº 899 – Guide for Validation of
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283 Analytical and Bioanalytical Methods.21 The figures of merit analyzed are:
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13 284
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15 285 Selectivity and specificity
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17 286 To verify the selectivity and specificity of the No-D qNMR method, the 1H
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19 287 NMR spectra of the analytes and the internal standard were overlapped, as well as, their
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21 288 respective chemical structures. The signals related to 1H NMR spectrum of cocaine are
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23 289 located in the regions of 5.56, 4.23, 4.0, 3.58 and 2.79 ppm, Figure 3a. However, the
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290 aromatics’ region (6.0 to 8.7 ppm) was also used to validate the method.8,17,21 Figure 3b
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28 291 shows the No-D qNMR spectrum expansion of all the analytes in the region of interest,
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30 292 between 6.0 and 8.7 ppm (aromatic hydrogens). These one are used because, in this
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32 293 region, there is no overlap of the signals of cocaine and its adulterants. 18 Hays et al.23
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34 294 evaluated the integrated signal area to identify analyte purity, proving that the nucleus
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36 295 observed (the hydrogen proton), in this region, has a molar response coefficient of 1,
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296 assuming that the hydrogen nucleus doesn’t change with the solvent, as long as the
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41 297 instrumental parameters are set correctly. The signals of interest are marked with
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43 298 asterisk and the attributions of the chemical shifts (δ) are in Table 3. From the results, it
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45 299 is possible to verify that the method has sufficient selectivity and specificity for
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47 300 simultaneous quantification and identification of all analytes.
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41 301
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43 302 Figure 3. (a) No-D q(1H)NMR spectra of the analytes ((a) cocaine, (b) caffeine, (c)
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303 procaine, (d) benzocaine, (e) lidocaine and (f) phenacetin) and internal standard (maleic
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48 304 acid) overlapped and their respective chemical structures. (b) Expansion of all
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50 305 compounds in the region between 6,0 and 8,7 ppm. The signals (*) were used for the
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52 306 quantification curve construction.
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4 309 Table 3. Attribution of the 1H chemical shifts (δ) and their respective multiplicities for
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6 310 the cocaine, caffeine, procaine, benzocaine, lidocaine, phenacetin and maleic acid
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8 311 standards.
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Coupling
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12
Constant (J)
13 Ha1 Doublet 8.53
14 Cocaine Ha2 Triplet 8.00 7.5 Hz
15 Ha3 Triplet 8.13
16 Caffeine Hb Singlet 8.43 -
17 Procaine Hc1 Doublet 7.20
18 Hc2 Doublet 8.33
19 8.7 Hz
Hd1 Doublet 7.18
20 Benzocaine
Hd2 Doublet 8.29
21
22 Lidocaine He Long Singlet 7.68 -
23 Hf1 Long Doublet 7.40
Phenacetin 8.9 Hz
24 Hf2 Long Doublet 7.95
25 Maleic acid Hg Singlet 6.27 -
26 312
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28 313 Linearity
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314 The coefficient of determination (R2) obtained from the calibration curve
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33 315 construction (relation between analyte signal integral by internal standard as a function
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35 316 of analyte concentration) was above 0.99 for cocaine and its adulterants (caffeine,
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37 317 procaine, lidocaine, benzocaine and phenacetin, Figure 4). Santos et al.24 and Materazzi
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39 318 et al.25 found R2 values lower than 0.98 for cocaine quantification using other
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41 319 techniques such as: paper spray ionization mass spectrometry and attenuated total
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43 320 reflectance Fourier transform infrared spectroscopy (ATR-FTIR), respectively. Gama et
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321 al.18 found attractive R2 values (0.9994) for cocaine quantification through the No-D
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48 322 NMR technique. After F test being applied, it was observed that there was no significant
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50 323 difference (Fcal (1.0) < Ftab (3.97) at the probability level of 5%) between the
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52 324 methodologies employed by Gama et al. and this one developed in this work.
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54 325 Linearity, in a methodology using the No-D qNMR technique, is something
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56 326 totally expected, since the signals area is always proportional to the number of nuclei
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58 15
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4 327 that absorb the signal radiofrequency, which is, to the concentration.17 Therefore, the
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6 328 constructed analytical curves presented a satisfactory linearity. 31
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39 329
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41 330 Figure 4. Analytical curve constructed from the No-D qNMR data for quantification of
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43 331 (a) cocaine, (b) caffeine, (c) procaine, (d) lidocaine, (e) benzocaine and (f) phenacetin
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332 using the ratio of the analyte signal integral by maleic acid versus the analyte weight.
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48 333
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50 334 LOQ and LOD
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52 335 The LODs and LOQs, Table 4, were statistically calculated through the
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54 336 parameters of the analytical curve obtained. It is observed that the method can quantify
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56 337 and identify low cocaine and its adulterants’ contents. According to LODs values
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4 338 reported in literature, Table 4, the No-D qNMR method has LOD values which vary
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6 339 between 0.126 and 0.666 mg.mL-1, being these values lower than the reported by Gama
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8 340 et al.18 (LOD of 2 mg.mL-1 for cocaine), who used the same analytical technique, as
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341 well as, for the paper chromatography, TLC and PS-MS techniques. However, the LODs
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13 342 values are higher than that obtained from GC-MS, LC-MS and Raman spectroscopy
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15 343 techniques, Table 4. Although GC-MS or GC-FID and LC-MS techniques are
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17 344 commonly used for cocaine quantification, they need sample preparation and require a
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19 345 higher time analysis. This fact doesn’t occur in No-D qNMR analysis. Among all
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21 346 obtained results, benzocaine can be highlighted: it showed the lowest LOD and LOQ
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23 347 values. It is possible to observe that the signal corresponding to the Hd hydrogen from
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348 benzocaine (Fig. 3) was majority in relation to other analytes signals.
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28 349
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30 350 Table 4. LOQ and LOD values for cocaine and its adulterants obtained by No-D qNMR
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32 351 technique. A comparison of LOD is made with other methods reported in literature.
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34 Standard LOD and LOQ LOD by other analytical
35 Analyte
deviation by No-D qNMR methods
36
37
No-D qNMR:18 2 mg.mL-1;
38 Paper chrom.:22 1 mg.mL-1;
0.258 mg.mL-1 -
39 Cocaine 0.0027 PS-MS:24 6.51 µg.mL-1;
0.86 mg.mL-1
40 CG-MS26: 6 ng.mL-1;
41 Raman27: 10 µg.mL-1
42 0.342 mg.mL-1 -
43 Caffeine 0.0031 TLC23: 0.5 mg.mL-1
1.14 mg.mL-1
44 0.666 mg.mL-1 -
45 Procaine 0.0084 -
2.22 mg.mL-1
46
0.060 mg.mL-1- TLC23: 0.5 mg.mL-1
47 Benzocaine 0.0012
0.20 mg.mL-1
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49 PS-MS:24 10 mg.mL-1;
-1
50 0.126 mg.mL - TLC23: 4 mg.mL-1;
Lidocaine 0.0020
51 0.42 mg.mL-1 Paper Chrom.: 0.35 mg.mL-1
52 LC-MS:28 0.35 µg.mL-1
53 0.186 mg.mL-1-
54 Phenacetin 0.0031 0.62 mg. mL-1 TLC23: 0.5 mg.mL-1
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56 352
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4 353 Accuracy
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6 354 Accuracy was calculated using three calibration points (1, 3 and 5), which,
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8 355 presented low, medium and high values of weights, approximately of 2.0, 6.0 and 20.0
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356 mg for cocaine; 1.0, 3.1 and 5.0 mg for caffeine; and 1.2, 6.0 and 15.0 mg for the other
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13 357 adulterants, respectively, Table 5. Accuracy in points 3 and 5 were satisfactory, since
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15 358 they possess an error of approximately 2%. However, the accuracy for point 1 presented
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17 359 variation in results greater than the established by ANVISA. The error source can be
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19 360 derived from the accumulated uncertainty in the weighing of small weights (1 mg).
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21 361
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23 362 Table 5. Accuracy values (in %) for analytes’ low, medium and high concentrations
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363 (curve points).
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28 Analyte Point 1 (%) Point 3 (%) Point 5 (%)
29 Cocaine 91.8 100.3 98.4
30 Caffeine 104.2 102.2 101.0
31 Procaine 121.6 103.1 102.4
32 Benzocaine 114.9 105.3 100.5
33 Lidocaine 127.1 97.6 102.0
34
Phenacetin 119.5 97.7 102.6
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36 364
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38 365 Precision
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40 366 In this study, the intermediate precision for the adulterants was calculated on an
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42 367 interval of 7 days between the analyses, being only evaluated for the cocaine. The
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44 368 results were expressed by the relative standard deviation (RSD), Table 6. The results of
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46 369 points 1, 3 and 5 (low, medium and high concentrations) were used as described in the
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370 accuracy calculation. All deviations showed values lower than established by ANVISA
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51 371 (less than 5%), which indicates a good precision for the method. Miller et al.29 found
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53 372 RSD values between 1.4-23.4% and 8.3-25.4% for intra- and interlaboratory analyses,
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55 373 respectively, using LC-MS method to quantify cocaine in hair samples.
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4 374 Table 6. RSD values (in %) for low (point 1), medium (point 3) and high (point 5)
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6 375 concentrations of the analytes for precision validation.
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8 Analyte Point 1 (%) Point 3 (%) Point 5 (%)
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Cocaine 0.00 0.00 0.66
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Caffeine 2.95 4.47 0.79

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12 Procaine 2.14 4.83 0.43
13 Benzocaine 1.12 1.91 0.54
14 Lidocaine 0.87 3.51 0.11
15 Phenacetin 0.00 4.05 4.70
16 376
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18 377 Robustness
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378 To evaluate method’s robustness, the analytical solution stability was studied.
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23 379 The solutions of points 1 and 5, Table 2, were analyzed and posteriorly stored free of
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25 380 light at -2°C. After seven days, a second analysis was performed, and the results are
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27 381 expressed in Table 7. Under these conditions, the values of the point 1 integrals
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29 382 remained identical, and the values of point 5 resulted into small variations between the
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31 383 analyses (non-significant) which can be attributed to operational errors. Thereby, the
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384 method can be considered robust because when making slight changes in its parameters,
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36 385 there was no significant variation between the results obtained by the original solution
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38 386 and the solution after certain temperature and time conditions. It was also possible to
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40 387 identify that, in this period of time; there was no samples’ degradation. According to
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42 388 Ribani et al.,30 to generate reproductible and trusted results, the samples, standards and
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44 389 reagents used must be stable for a reasonable period of time.
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4 396 Table 7. Values of the integral of the signal areas of solutions 1 and 5 of the respective
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6 397 adulterants in relation to internal standard for robustness validation.
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8 398
9 1st analysis 2nd analysis
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point 1 Point 5 Point 1 Point 5

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12 Caffeine 0.08 0.30 0.08 0.30
13 Procaine 0.11 1.16 0.11 1.17
14 Benzocaine 0.21 1.85 0.21 1.83
15 Lidocaine 0.27 1.55 0.27 1.54
16 Phenacetin 0.44 1.52 0.44 1.67
17 399
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19 400 Quantification of the cocaine seized samples
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401 The 34 seized cocaine samples were submitted to No-D qNMR analyses for
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24 402 cocaine and its adulterants quantification. Using the constructed univariate models, the
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26 403 weights (in mg) of cocaine and its adulterants were obtained, as well as, the cocaine
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28 404 contents (in wt%), as illustrated in Table 8. The benzocaine signal was not identified in
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30 405 any sample and this is the reason why this component is not presented in Table 8.
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32 406 Maldaner et al.31 observed that, for the samples seized in Federal District (from 2010 to
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407 2013), benzocaine was the least adulterant found among those investigated. Herein, the
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37 408 presence of cocaine was not verified in samples 08, 12 and 15, and this result can be
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39 409 related to the storage time of them, since they are of 2012. Samples that showed high
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41 410 purity for cocaine were 9 and 20. In samples 04, 12, 14, 19, 23 and 34, it was possible to
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43 411 identify phenacetin in the mixture, but presenting values close to the LOD of method. In
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45 412 Brazil, phenacetin is the most found adulterant, and normally, with a high content in
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413 cocaine seized samples.32
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4 418 Table 8. Weight (in mg) of cocaine and its adulterants found in the seized samples,
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6 419 calculated from the calibration curve (Fig. 4).
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8 Cocaine Caffeine Procaine Lidocaine Phenacetin Cocaine content
9 Sample
(mg) (mg) (mg) (mg) (mg) (wt%)
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01 3.44 - - - - 17.66
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12 02 3.28 - - - 1.06 14.99
13 03 1.03 - - - 1.06 4.85
14 04 0.39 - - - - 1.50
15 05 3.60 - - - - 16.69
16 06 5.86 - - - - 29.13
17 07 6.18 - - - - 27.21
18 08 - - - - - -
19
09 20.97 - - - - 94.88
20
21
10 1.68 - - - 0.67 6.89
22 11 1.84 - - - 1.36 8.35
23 12 - - - - - -
24 13 6.02 1.22 - - - 28.38
25 14 1.84 - - - - 8.46
26 15 - - - 14.72 - -
27 16 0.55 - - - - 2.56
28
17 1.84 - - - 0.38 8.27
29
30
18 3.60 1.22 - - - 16.92
31 19 0.39 - - - - 1.62
32 20 17.75 - - - - 88.32
33 21 0.87 - - - 0.30 3.98
34 22 3.44 - - - 0.09 15.10
35 23 4.25 - 1,19 - - 16.72
36 24 5.86 - - - - 24.60
37 25 2.32 - - - 1.46 12.07
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26 2.96 0,31 - - 1.06 14.38
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40 27 4.89 - - - - 24.58
41 28 2.48 - - - - 12.15
42 29 2.64 - - - - 11.14
43 30 3.44 - - - - 17.31
44 31 4.89 - - - 3.51 24.09
45 32 4.89 - - - - 24.09
46 33 2.16 - - - 1.16 10.63
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34 1.51 - - - - 7.65
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420
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51 421 Approximately 50% of the seized samples have phenacetin and 38.2% didn’t
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53 422 show adulterants or they weren’t identified in this study (Figure 5). The obtained results
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55 423 for the cocaine content in the seizures are in agreement with the expected, since in the
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4 424 internal traffic trade, the average purity of cocaine reaches up to 30%. The average
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6 425 content found was of 17,5%6,9,16,19 what reveals the impurity of drugs seized in local
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8 426 trade in urban zone.
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427 Among all the works about No-D qNMR applied to forensic analysis in
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13 428 literature, none has reported the determination of figures of merit of cocaine and its
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15 429 adulterants. Gama et al.18 based himself only in linearity and LOD, therefore, this work
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17 430 presents notable contribution for its implementation in forensic analysis.
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31 432 Figure 5. Distribution of the presence of cocaine and its adulterants in the seized
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433 samples.
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36 434
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38 435 Conclusions
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40 436 The No-D qNMR method developed in this work presented excellent linearity,
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437 selectivity, accuracy, precision and robustness for the cocaine and its adulterants’
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45 438 analysis. The described methodology is efficient to quantify cocaine content as well as
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47 439 its adulterants (caffeine, procaine, benzocaine, lidocaine and phenacetin) in seizure
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49 440 samples.
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51 441 The use of the NMR quantification technique is well known, but its application
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53 442 without the use of deuterated solvent with outstanding results for all figures of merit
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443 was not yet reported in literature. In this way, the present work is pioneer in
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4 444 quantification of cocaine and its adulterants, simultaneously, by No-D qNMR. In the
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6 445 future, this technique will be employed to quantify other analytes present in forensic
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8 446 matrices such as the designer drugs.
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13 448 Acknowledgments
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15 449 The authors thank CAPES (23038.007083/2014-40), FAPES (734/2016) and CNPq
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17 450 (445987/2014-6 and 465450/2014-8) for financial support.
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451
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22 452 References
23
24
25 1
F. Tosato, T. Rosa, C. Lelis, A. O. Maldaner, R. S. Ortiz, P. R. Filgueiras, K. M. G.
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32 T. C. Carvalho, F. Tosato, L. M. Souza, H. Santos, B.B. Merlo, R. S. Ortiz, R. R. T.
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48 United Nations office on drugs and crime, Recommended Methods for the
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53 H. P. Rang, M. M. Dale, Farmacologia. 6ª ed. Rio de Janeiro: Elsevier, 2008. 829 p.
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15 M. F. Andreasen, C. Lindholst, E. Kaa, Open Foren Sci. J., 2009, 2, 16-20.
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51 T. R. Hoye, B. M. Eklov, T. D. Ryba, M. Voloshin, L. J. Yao, Org. Lett., 2004, 6, 953-
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DOI: 10.1039/C7AY03000B

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