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Thin layer chromatography coupled to paper spray ionization mass spectrometry for cocaine and its adulterants analysis
Accepted Manuscript Title: Thin Layer Chromatography coupled to Paper Spray Ionization Mass Spectrometry for Cocaine and its Adulterants Analysis Author: Thays C. De Carvalho Flavia Tosato Lindamara M. Souza Heloa Santos Bianca B. Merlo Rafael S. Ortiz Rayza R.T. Rodrigues Paulo R. Filgueiras Hildegardo S. Franc¸a Rodinei Augusti Wanderson Rom˜ao Boniek G. Vaz PII: DOI: Reference:
S0379-0738(16)30061-5 http://dx.doi.org/doi:10.1016/j.forsciint.2016.02.039 FSI 8346
To appear in:
FSI
Received date: Revised date: Accepted date:
28-7-2015 13-2-2016 22-2-2016
Please cite this article as: T.C. De Carvalho, F. Tosato, L.M. Souza, H. Santos, B.B. Merlo, R.S. Ortiz, R.R.T. Rodrigues, P.R. Filgueiras, H.S. Franc¸a, R. Augusti, W. Rom˜ao, B.G. Vaz, Thin Layer Chromatography coupled to Paper Spray Ionization Mass Spectrometry for Cocaine and its Adulterants Analysis, Forensic Science International (2016), http://dx.doi.org/10.1016/j.forsciint.2016.02.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Thin Layer Chromatography coupled to Paper Spray Ionization
ip t
Mass Spectrometry for Cocaine and its Adulterants Analysis
Thays C. De Carvalho1, Flavia Tosato,2 Lindamara M. Souza,2 Heloa Santos,2
cr
Bianca B. Merlo,3 Rafael S. Ortiz,4 Rayza R. T. Rodrigues,2 Paulo R. Filgueiras,2
Instituto de Química, Universidade Federal de Goiás, 74001-970 Goiânia, GO,
an
1
us
Hildegardo S. França,5 Rodinei Augusti,6 Wanderson Romão,2,5† Boniek G. Vaz1,2±
Brazil
Laboratório de Petroleômica e Química Forense, Departamento de Química,
M
2
Universidade Federal do Espírito Santo, 29075-910, Vitória, ES, Brazil Laboratório de Química Legal, Superintendência de Polícia Técnico-Científica da
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3
Superintendência de Polícia Federal no Estado do Rio Grande do Sul, 90160-
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4
te
Polícia Civil do Estado do Espírito Santo, 29045-300, Vitória, ES, Brazil
093, Porto Alegre, RS, Brazil 5
Instituto Federal do Espírito Santo, 29106-010, Vila Velha, ES, Brazil
6
Departamento de Química, Universidade Federal de Minas Gerais, 31270-901,
Belo Horizonte – MG, Brazil
Corresponding Author: †
W.R: [email protected] / Phone: +55-27-3149-0833
±
B.G.V: [email protected]/Phone: +55 62 3521-1016 R 261
Page 1 of 39
Abstract
Thin Layer Chromatography (TLC) is a simple and inexpensive type of
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chromatography that is extensively used in forensic laboratories for drugs of abuse analysis. In this work, TLC is optimized to analyze cocaine and its adulterants
cr
(caffeine, benzocaine, lidocaine and phenacetin) in which the sensitivity (visual
us
determination of LOD from 0.5 to 14 mg mL-1) and the selectivity (from the study of three different eluents: CHCl3:CH3OH:HCOOHglacial (75:20:5 v %), (C2H5)2O:CHCl3
an
(50:50 v %) and CH3OH:NH4OH (100:1.5 v %)) were evaluated. Aiming to improve these figures of merit, the TLC spots were identified and quantified (linearity with
M
R2 > 0.98) by the paper spray ionization mass spectrometry (PS-MS), reaching
d
now lower LOD values (> 1.0 g mL-1). The method developed in this work open
te
up perspective of enhancing the reliability of traditional and routine TLC analysis employed in the criminal expertise units. Higher sensitivity, selectivity and rapidity
Ac ce p
can be provided in forensic reports, besides the possibility of quantitative analysis. Due to the great simplicity, the PS(+)-MS technique can also be coupled directly to other separation techniques such as the paper chromatography and can still be used in analyses of LSD blotter, documents and synthetic drugs.
Keywords: cocaine, TLC, Paper spray ionization, PS-MS.
Page 2 of 39
Introduction
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Cocaine is mainly consumed as a salt (cocaine hydrochloride) or in a freebase form (crack). Cocaine hydrochloride is a water-soluble salt obtained as a
cr
powder, and it can be administered via aspiration or intravenously. Crack, however, appears as a rock and is slightly soluble, but it is easily volatilized when heated
us
because of its low melting point (approximately 95 ºC) and can be administered via
(IUPAC),
cocaine
is
an
smoke.i According to the International Union of Pure and Applied Chemistry a
[1R-(exo,exo)-3-(benzoyloxy)–8–methyl–8–
M
azabicyclo[3.2.1]octane-2-carboxylic acid methyl esther. Its molecular formula is
96–98 ºC, and its pKb = 5.4.ii,iii
d
C17H21NO4, and its molar mass is 303.4 g mol-1. Cocaine’s melting point range is
te
In the last few years, the purity of “commercial” cocaine has been
Ac ce p
decreasing. To increase drug volume and therefore drug trafficking profits, chemical additives are blended to the alkaloid as adulterants and/or diluents. Among the chemical additives, psychoactive substances, commonly anesthetics, are used to mimic or increase the drug effect for users.iv,v Figure 1a-d shows the chemical structures of some adulterants found in cocaine samples seized in the Brazilian illicit market. They are: benzocaine, phenacetin, caffeine and lidocaine.iii
Figure 1
Expert methodologies for cocaine analysis are mostly qualitative. At least two
Page 3 of 39
examinations are performed: a preliminary test using wet chemistry and an analytical test with higher selectivity. Colorimetric tests or pre-tests have the advantages of rapidity, low cost and ease of execution and interpretation.
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However, they feature low specificity. Among the colorimetric tests used to determine the presence of alkaloids, is usually employed the test using a solution
cr
of cobalt thiocyanate.v,vi Nevertheless, positive results can be found when some
us
adulterants or diluents are present, such as lidocaine, powdered milk and promethazine.v
an
Other analytical methodologies are routinely applied for cocaine analysis, including thin layer chromatography (TLC),vii-x gas chromatography coupled with
M
mass spectrometry (GC-MS)xi and gas chromatography coupled with flame ionization detector (GC-FID).xii,xiii Among them, TLC is a more simple and cheap
te
d
separation technique, being extensively used in the most Brazilian forensic laboratories for drugs of abuse analysis (cocaine, crack, marijuana, and ecstasy
Ac ce p
tablets).v,xiv TLC is based on the differences of affinities of analyte between the stationary and mobile phases.xiv TLC has also been explored for cocaine detection in urine and other biological matrixes.xv The compounds separated on the TLC plate form spots which are usually
detected with UV light, iodine vapor, or other visualization reagents. However, some spots may be missed in visualization. In addition, the visualization methods are not capable of identifying the nature of the separated compounds.
xiv
To
overcome this limitation, mass spectrometric methods have been developed for identifying the compounds separated on the TLC plates.xvi
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Recently, the use of new techniques of ambient mass spectrometry, that allow desorption, ionization and characterization of analytes via mass spectrometry directly from their natural matrixes, becomes an attractive alternative in forensic
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problems. These ionization/desorption techniques require no sample preparation or pre-separation.i,vii,xvii,xviii
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Paper spray ionisation (PS), introduced in 2010 by Wang et al,xix is a new
us
ambient mass spectrometry technique for qualitative and quantitative analysis of complex mixtures. PS-MS involves directly loading the sample
an
onto a triangular-shaped paper, which is wetted with a solvent and placed in front of the mass spectrometer inlet. The spray of the charged micro-droplets
M
is formed by application (usually 3-5 kV) in the opposite side of the paper tip and the desolvation occurs without any sheath gas.xx The PS-MS
process.
te
d
mechanism of ions formations in the gaseous phase is similar to ESI
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The PS-MS has been explored in various applications, mainly to analyze directly complex samples such as illicit substances in raw urine,xx pharmaceuticals in whole blood,xxi biological tissue,xxii contaminants in foodstuffs,xxiii and other applications.xxiv Herein, the TLC system is optimized and has its figures of merit improved (detection limit, sensitivity and selectivity) from coupling to PSI-MS method. This analytical methodology has been employed to detection and quantification of cocaine and its adulterants (benzocaine, phenacetin, caffeine and lidocaine).
Experimental Section
Page 5 of 39
Reagents and Solutions A reference standard sample of cocaine provided by PC-ES (Civil Police of Espirito Santo State, Brazil) and standards of phenacetin, benzocaine (both
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provided by Sigma-Aldrich, Sao Paulo), lidocaine and anhydrous caffeine (Vetec Quimica Fina Ltda, Rio de Janeiro and Bandeirante Brazmo Industria e Comercio
cr
Ltda, Sao Paulo, respectively) were used to prepare stock solutions of 12, 14 and
us
20 mg mL-1.
The reagents used in the TLC and PS-MS analysis were methanol HPLC
an
grade (J.T.Baker), formic acid 99% RPE ACS PA (Carlo Erba reagentes), ammonium hydroxide (Sigma Aldrich), Milli-Q water, chloroform, glacial acetic acid
d
M
and ethyl ether (Vetec Química Fina Ltda, Rio de Janeiro).
te
TLC
Initially, an optimization of the TLC system was performed by varying the
Ac ce p
application volume (5, 10, 15, 20 and 25 L), the form of application (micropipette, silica capillary and microsyringe) and the analyte concentration (4, 6, 8, 10, 14 and 16 mg mL-1). In this step, cocaine was used as analyte, silica gel as stationary phase with support of aluminum and the methanol:NH4OH mixture in the proportion 99.5:0.5 v % as eluent. The TLC system was eluted and revealed in the ultraviolet (UV) chamber at 254 nm. After the optimization of application volume (10 L), concentration (10 mg·mL-1) and form of application, three different eluents were evaluated to analyze cocaine and its adulterants (lidocaine, caffeine, benzocaine and phenacetin) by
Page 6 of 39
TLC. The eluents were: i) CHCl3:CH3OH:HCOOHglacial 75:20:5 v %; ii) (C2H5)2O:CHCl3 50:50 v %; and iii) CH3OH:NH4OH 100:1.5 v %. These three systems are commonly recommended by the UNODC (United Nations Office on
ip t
Drugs and Crime) manual.xxv The histogram shown in Figure 2 illustrates the experimental procedure. The last system composed by CH3OH:NH4OH, were
cr
tested in various proportions: 99.5:0.5; 99:1; 97.5:2,5; 97:3.0 and 96.5:3.5 v %
us
aiming to improve the chromatographic resolution by varying the pH of the medium, Figure 2. After the optimization of the mobile phase, the limit-of-detection
an
(LOD) was visually determined for all active ingredients, in the concentration range 0.5-14 mg·mL-1. The solutions between 0.5 and 10 mg mL-1 were prepared from
M
the 20 mg mL-1 stock solution.
All TLC plates were revealed in the UV chamber at 254 nm. The retention
te
d
factors (Rf) were calculated as the ratio of the distance eluted by sample (da) and
Ac ce p
the distance eluted by solvent (ds) according to Equation 1: R f da ds
(Eq. 1)
Figure 2
PS-MS
PS-MS analyses were performed in the mass spectrometer LTQ XL (Thermo Scientific, Bremem, Germany) with PS source coupled in the positive ionization mode, PS(+).
Page 7 of 39
In general, a piece of paper (Whatman Grade 1, GE Healthcare, USA) was cut in a triangular geometry (base and high of 1 cm) and fixed to an alligator clip, connected to 0.5 mm wire linked to the mass spectrometer.xxvi,xxvii Afterwards, each
ip t
TLC spot containing the analyte was resuspended in methanol acidified with HCOOH (0.1 v/v %) and 10 µL of the resultant solution was applied to the PS(+)-
cr
MS paper. The PS source prototype assembly was performed according to the
us
description of Yang et al.xix By using the mass spectrometer LTQ XL there is no need of an external source of high voltage, as the PS source is fed by a potential
an
generated from the ESI interface catheter, Figure 3.
M
Figure 3
Figures of merit
te
d
The relationship between the signal intensity (y) and the concentration of
Ac ce p
cocaine or adulterant (x) is modeled by a first order equation: y b0 b1 x (Eq. 2)
where b0 and b1 are intercept and slope, respectively. From the calibration curve the LOD for each substance was determined using the equation 3:
LOD k s b1
(Eq. 3)
where factor k is chosen number according to the desirable degree of confidence (usually k = 3 is accepted), b1 is determined from calibration curve and s is the standard deviation of ten white analyzes. An important parameter to be evaluated when ambient ionization mass spectrometry techniques are applied to forensic analysis is related to its precision
Page 8 of 39
(repeatability and reproducibility). Repeatability measures the error of an individual determination and is important criteria for judging the performance of an analytical
equation 4:xxviii (Eq. 4)
cr
ˆ repeatability R d 2
ip t
procedure. The standard deviation of repeatability ( ˆ repeatability ) was estimated by
where R is the average of the ratios of cocaine/benzoylecgonine compounds (m/z
us
304/290) at 10 mg mL-1 of a cocaine solution. For this study, nine analyzes (or
an
replicates) were performed by PS(+)MS using ever the same solution during five consecutive days. The d 2 is a factor that depends on the size of the sample.xxix In
M
this study was used d 2 = 2.970, that is related to number of replicates (n = 9). Then
te
repeatability of analyzes.
d
an ANOVA was performed to evaluate the influence of the "day" factor on the
TLC
Ac ce p
Results and discussions
To evaluate the best methodology to apply the analyte in the TLC system,
the spot form and its Rf values were evaluated according to the type of applicator (Figure 4a, micropipette, silica capillary, and microsyringe); variation of application volume (L) (Figure 4b); and analyte concentration (mg mL-1, Figure 4c). Monitoring initially the system of application, well defined, homogeneous and uniform spots were observed in all cases, Figure 4a. Thus, any of the three systems can be employed for TLC analysis in forensic routines. When evaluating the application volume, the spot area becomes biggest with cocaine volumes
Page 9 of 39
higher or equal to 15 L. This behavior can be harmful to seized street sample analysis, interfering in the chromatographic resolution due to coelution problems. Therefore, a volume equal to 10 L was defined for all analytes. Despite the TLC
cr
mL-1), the adopted concentration in this work was 10 mg mL-1.
ip t
technique being sensible for all concentrations evaluated (between 4 and 16 mg
us
Figure 4
then
evaluated
according
to
an
The TLC efficiency for detecting cocaine and adulterants standards was three
different
eluent
systems
M
(CHCl3:CH3OH:CH3COOHglacial, 75:20:5 v %, CH3CH2OCH2CH3:CHCl3, 50:50 v %); and CH3OH:NH4OH 100:1.5 v %), Figure 5a-c. All standards were prepared at 10
d
mg mL-1 in methanol, and were applied in the TLC system via silica capillary. In the
te
first eluent (CHCl3:CH3OH:CH3COOHglacial, 75:20:5 v %, Figure 5a) spots tailing
Ac ce p
were observed for all standards, and they showed similar Rf values (Rf = 0.710.76, Table 1). This result differs from the reported by Sabino et al.vii For the (CH3CH2)2O:CHCl3, 50:50 v % (Figure 5b) system, a better performance of the TLC system is observed when compared to Figure 5a, in which well defined and resolved spots can be identified. In this case, however, the majority of compounds showed similar Rf values (Rf from 0.71 to 0.76, Table 1). The cocaine standard that shows Rf = 0.63 is a unique exception, Figure 5b. Finally, the best results were obtained for the TLC system when the eluent CH3OH:NH4OH (100:1.5 v %) was employed, in which distinct Rf values were observed for the compounds (Rf
Page 10 of 39
varying from 0.69 to 0.81), Figure 5c and Table 1. A similar TLC system was also reported by Nicola et al.xxx employing CH3Cl/CH3OH/NH4OH (100:20:1 v %).
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Figure 5 Table 1
cr
Aiming to increase the interval of values of Rf observed among standards,
us
the ratio of CH3OH:NH4OH eluent was varied, with NH4OH concentration ranging between 0.5 and 3.5 % producing the systems 99.5:0.5; 99:1; 97.5:2.5; 97:3; and
an
96.5:3.5 v %, respectively. The TLC system and standards Rf values are showed on Figure 6 and Table 2. Note that the best TLC system was obtained when low
M
concentrations of NH4OH were used (< 1 v %). At this situation, the interaction between cocaine and the stationary phase increases. This is related to the fact that
d
NH4OH alters the acid/base equilibrium of the cocaine molecule, which the cocaine
te
in the chlorhydrate form is predominant when NH4OH concentration is reduced in
Ac ce p
the system. Similarly, caffeine, which has high pka and polarity, also interact mainly with the stationary phase. Conversely, lidocaine, benzocaine and phenacetin have lower pka values (pKA ≤ 8) and higher Rf values, preferably thus interacting with the mobile phase. Therefore, the CH3OH:NH4OH system in the ratio 99.5:0.5 v % was defined for this work.
Figure 6 Table 2
Aiming to evaluate the LOD of the TLC system, the analyte concentration was varied in the range of 0.5 to 14 mg mL-1 using CH3OH:NH4OH at 99.5:0.5 v %
Page 11 of 39
as mobile phase. Note that phenacetin, benzocaine, cocaine and caffeine showing low LOD (0.5 mg mL-1), while lidocaine had LOD of 4.0 mg mL-1. Figure 7
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Table 3
cr
TLC plus PS-MS
Aiming to increase the selectivity of the TLC technique, the standards spots
us
were also analyzed by the PS(+)MS technique. For it, the revealed spots were
an
extracted and resuspended in acidified methanol (0.1 v % of HCOOH). Then the supernatant was deposited on the surface of the triangular shaped paper with
M
paraffin channel. After the drying, the analytes were analyzed by the PS(+)-MS technique according to its concentration (0.5, 4, 10 and 14 mg mL-1), Figure 8.
d
Figure 8 illustrates the PS(+) mass spectra of the extracts containing the
te
standards of cocaine, benzocaine, caffeine, lidocaine and phenacetin. In general
Ac ce p
benzocaine, caffeine and lidocaine are detected as protonated molecules, [M+H]+ cations with m/z 166, 195 and 235, respectively, while phenacetin is detected as [M+H]+ cation with m/z 180 and sodium adduct, [M+Na]+, with m/z 202. For the cocaine standard, [M+H]+ cation with m/z 304 is identified, besides a cocaine derivative, benzoylecgonine, [M +H]+ ion with m/z 290, which is a degradation product. The structure and chemical connectivity of the analytes were confirmed by experiments of collision induced dissociation (CID) and the PS(+)MS/MS spectra are illustrated on Figure 9.
Figure 8
Page 12 of 39
The cocaine (m/z 304), Figure 9a, is characterized for the loss of C7H6O2 leading to the formation of the ion with m/z 182. Benzocaine (m/z 166), Figure 9b,
ip t
shows two neutral losses. The first, the elimination of C2H2 leads the formation of the ion with m/z 138, and second, a loss of H2O generates the ion with m/z 120.
cr
The PS(+)MS/MS spectrum of caffeine, Figure 9c, is characterized by the loss of
us
H2O forming the ion with m/z 177, which subsequently loses a CO creating the ion with m/z 149. The loss of HCN from the ion with m/z 177 leads the formation of the
an
ion with m/z 150. It can also be observed a minor consecutive loss of CHO forming the ion with m/z 163 followed by the ion with m/z 133. Lidocaine, Figure 9d, is
M
characterized by the fragmentation of C9H10NO forming the ion with m/z 86. Phenacetin, Figure 9e, is characterized by the loss of H2O forming the ion with m/z
te
d
162, and by the loss of C2H4 forming the ion with m/z 152. The main fragmentation route of phenacetin is the loss of C2H2O forming the ion with m/z 138, which loses
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an ethylene forming the ion with m/z 110. Finally, the benzoylecgnonine, hydrolysis product of cocaine, Figure 9f, is characterized by the loss of H2O forming the ion with m/z 272 and by a loss similar to cocaine, C7H6O2, that generated the ion with m/z 168.
Figure 9
The PS(+)-MS technique ability to quantify cocaine and its adulterants was evaluated by the construction of curves as function of analyte concentration, in which the linearity and the LOD were reported, Figure 10 and Table 3. The calibration curves built for the standards shows excellent linearity (benzocaine, (R2
Page 13 of 39
= 0,9984), caffeine (R2 = 0,9999), phenacetin (R2 = 0,9989), lidocaine (R2 = 0,9998) and cocaine (R2 = 0,9993) and LOD lower than that of the TLC technique. The PS(+)-MS technique can also be used to identify and quantify the active
ip t
ingredients present in the TLC spots from cocaine and crack samples seized by the forensic police, thus eliminating false-positive results. Besides that, due to the
cr
great simplicity, the PS(+)-MS technique can be coupled directly to other
us
separation techniques such as paper chromatography and for the analysis of LSD
an
blotters and synthetic drugs.
M
Figure 10
Repeatability
te
d
To monitor the precision of the method, we consider the ratio of relative intensity of cocaine/benzoylecgonine (m/z 304/290) detected in the nine analyzes. Table 3
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shows these values, where the estimated value of the repeatability standard deviation was 0.065. Table 4 describes the ANOVA tests performed in Excel. Note that if the variance of the results in the five days is tiny, an insignificant change in results among analysis is taken on the same day. Therefore, the PS(+)-MS technique shows a good repeatability. ANOVA measures the total variation of a given system and compares it with the total variation to determine whether such measurement is feasible. Table 5 illustrates the variability of this method is mostly due to the difference among the samples, as F calculated (8.98) being higher than the critical F (2.60). Table 4
Page 14 of 39
Table 5
Conclusion and Perspectives
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This paper allows the detection of cocaine and its adulterants by TLC with LOD varying from 0.5 to 2 mg mL-1. The identification and rapid quantification of
cr
components revealed in each PS-MS spot is also reported. The method developed
us
in this work opens up perspectives of enhancing the reliability of traditional and routine TLC analyses employed in the criminal expertise units, thus eliminating
an
false positive results. Higher sensitivity, selectivity and rapidity can be provided in the forensic report, besides enabling quantitative analysis. Due to its great
M
simplicity, the PSI(+)-MS technique can also be coupled directly to other separation
i
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te
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d
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(a) S. Berkov, J. Bastida, M. Nikolova, F. Viladomat, C. Codina, Rapid TLC/GC-
MS identification of acetylcholinesterase inhibitors in alkaloid extracts. Phytochem. Anal. 19 (2008) 411-419; (b) F.L. Hsu, C.H. Chen, C.H. Yuan, C. H. Shiea, Interfaces to connect thin-layer chromatography with electrospray ionization mass spectrometry, J. Anal. Chem. 75 (2003) 2493-2498; (c) K.G. Asano, M.J. Ford, B.A. Tomkins, G.J. Van Berkel, Self-Aspirating Atmospheric Pressure Chemical Ionization Source for Direct Sampling of Analytes on Surfaces and in Liquid Solutions, Rapid Commun. Mass Spectrom. 19 (2005) 2305-2312; (d) G.W. Caldwell, J.A. Masucci, W.J. Jones, Indirect thin-layer chromatography-fast atom bombardment and chemical ionization mass spectrometry determination of carbohydrates utilizing simple and rapid microtransfer techniques. J. Chromatogr. A 514 (1990), 377-382; (e) A.J. Kubis, K.V. Somayajula, A.G. Sharkey, D.M.
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Hercules, Laser Mass Spectrometric Analysis of Compounds Separated by ThinLayer Chromatography. Anal. Chem. 61 (1989), 2516-2523; (f) Y. Han, P. Levkin, I. Abarientos, H. Liu, F. Svec, J.M.J. Fréchet,
Monolithic Superhydrophobic
ip t
Polymer Layer with Photopatterned Virtual Channel for the Separation of Peptides Using Two-Dimensional Thin Layer Chromatography-Desorption Electrospray Süß,
cr
Ionization Mass Spectrometry, Anal. Chem. 82 (2010), 2520-2528; (g) B. Fuchs, R. A. Nimptsch, J. Schiller, MALDI-TOF-MS Directly combined with TLC: A
J. Shiea,
us
Review of the current state. Chromatogr. Suppl. 69 (2009), 95-105; (h) Y. C. Chen, J. Sunner, Thin-Layer Chromatography/Mass Spectrometry Using
an
Activated Carbon, Surface-assisted Laser Desorption/Ionization" J. Chromatogr. A 826 (1998), 77-86; (i) J. K. Wu,Y. C. Chen, A novel approach of combining thinlayer chromatography with surface-assisted laser desorption/ionization (SALDI) xvii
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time-of-flight mass spectrometry, J. Mass Spectrom. 37 (2002) , 85-90. W. Romão, P.M. Lalli, M.F. Franco, G. Sanvido, N.V. Schwab, R. Lanaro, J.L.
d
Costa, B.D. Sabino, M.I.M.S Bueno, G. F. Sa, R.J. Daroda, V. Souza, M.N. Eberlin, Chemical profile of meta-chlorophenylpiperazine (m-CPP) in ecstasy tablets by
te
easy ambient sonic-spray ionization, X-ray fluorescence, ion mobility mass spectrometry and NMR, Analytical and Bioanalytical Chemistry, 400 (2011) 3053xviii
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3064.
B.D. Sabino, M.L. Sodré, E.A. Alves, H.F. Rozembaum, F.O.M. Alonso, D.N.
Correa, M.N. Eberlin, W. Romão, Analysis of Street Ecstasy Tablets by Thin Layer Chromatography Coupled To Easy Ambient Sonic-Spray Ionization Mass Spectrometry. Brazilian Journal of Analytical Chemistry 1 (2010) 2010. xix
(a) H. Wang, J. Liu, R. G. Cooks, Z. Ouyang,Paper spray for direct analysis of
complex mixtures using mass spectrometry. Angew. Chem., 122 (2010) 889-892. (b) J. Liu, H. Wang, N. E. Manicke, J. M. Lin, R. G. Cooks, Z. Ouyang, Development, characterization, and application of paper spray ionization. Analytical Chemetry 82 (2010) 2463–2471.
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N. E. Manicke, Q. Yang, H. Wang, S. Oradu, Z. Ouyang, R. G. Cooks,
Assessment of paper spray ionization for quantitation of pharmaceuticals in blood spots, International Journal of Mass Spectrometry 300 (2011) 123–129. H. Wang, N. E. Manicke, Q. Yang, L. Zheng, R. Shi, R. G. Cooks, Z. Ouyang,
ip t
xxi
Direct analysis of biological tissue by paper spray mass spectrometry. Analytical
xxii
cr
Chemistry 83 (2011) 1197– 1201.
Z. Zhang, R. G. Cooks, Z. Ouyang, Paper spray: a simple and efficient means of
us
analysis of different contaminants in foodstuffs. Analyst 137 (2012) 2556–2558. xxiii
D. Taverna, L. Di Donna, F. Mazzotti, B. Policicchio, G. Sindona, High-
an
throughput determination of sudan azo-dyes whitin powdered chili pepper by paper spray mass spectrometry, Journal of Mass Spectrometry 48 (2013) 544–547. xxiv
(a) J. Deng, Y. Yang, Chemical fingerprint analysis for quality assessment and
M
control of Bansha herbal tea using paper spray mass spectrometry, Analytica Chimica Acta 785 (2013) 82–90; (b) E. Sokol, R.J. Noll, R.G. Cooks, L.W. Beegle,
d
H.I. Kim, I. Kanik, Miniature mass spectrometer equipped with electrospray and
te
desorption electrospray ionization for direct analysis of organics from solids and solutions. Internation Journal of Mass Spectrometry, 306 (2011) 187–195. Recommended Methods for the Identification and Analysis of Cocaine in Seized
Ac ce p
xxv
Materials”, UNODC, ST/NAR/7Rev1, Março, 2012. xxvi
T.C. Carvalho, I.F. Oliveira, L.V. Tose, G. Vanini, L.M. Souza, A.C. Neto, L.F.
Machado, J.C.L. Ambrosio, V. Lacerda Jr., B.G. Vaz,
W. Romão, Qualitative
Analysis of Designer Drugs by Direct Blotter Paper Spray Ionisation Mass Spectrometry (PSI-MS), Anal Methods, 8 (2016), 614-620. xxvii
T. C. Colletes, P. T. Garcia, R. B. Campanha, P. V. Abdelnur, W. Romão, W. K.
T. Coltro, B. G. Vaz, A new insert sample approach to paper spray mass spectrometry: paper substrate with paraffin barriers, Analyst, 2016, in press, doi: 10.1039/C5AN01954K. xxviii
R. C. L. Pereira, R. C. Simas, Y. E. Corilo, B. G. Vaz, C. F. Klitzke, E. M.
Schmidt, M. A. Pudenzi, R. M. C. F. Silva, E. T. Moraes, W. L. Bastos, M. N. Eberlin, H. D. L. Nascimento, Precision in Petroleomics via Ultrahigh Resolution
Page 19 of 39
Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Energy Fuels, 27 (2013),7208-7216. xxix
ip t
D. C. Montgomery, Introduction to statistical quality control. 2 ed. New York, John Wiley, 1991. xxx A. J. Nicola, A.I. Gusev, D.M. Hercules, Direct Quantitative Analysis from ThinLayer Chromatography Plates Using Matrix-Assisted Laser Desorption/Ionization
Ac ce p
te
d
M
an
us
cr
Mass Spectrometry, Applied Spectroscopy, 50 (1996) 1479-1482.
Page 20 of 39
Tables Table 1
Benzocaine Lidocaine
0.76 0.76
0.76 0.71
CH3OH:NH4OH (100:1.5 v %) 0.79 0.69 0.74
ip t
(CH3CH2)2O (50:50 v %) 0.76 0.74 0.63
cr
Phenacetin Caffeine Cocaine
CHCl3:CH3OH:CH3COOH (75:20:5 v %) 0.74 0.74 0.71
0.78 0.81
Ac ce p
te
d
M
an
us
Standards
Page 21 of 39
Table 2 99.5:0.5
99:1
97.5:2.5
97:3
96.5:3.5
Phenacetin Caffeine Cocaine
0.84 0.66 0.63
0.82 0.67 0,67
0.63 0.60 0.62
0.70 0.67 0.71
0.73 0.72 0.75
Benzocaine Lidocaine
0.86 0.81
0.82 0.78
0.68 0.67
0.72 0.71
0.76 0.76
Ac ce p
te
d
M
an
us
cr
ip t
Compound
Page 22 of 39
Table 3 TLC (mg mL-1)
PSI(+)-MS (g mL-1)
Cocaine
0.5 mg mL-1
6.0
Caffeine
0.5 mg mL-1
4.0
Phenacetin
0.5 mg mL-1
80.0
Benzocaine
0.5 mg mL-1
1.0
Lidocaine
4.0 mg mL-1
cr
ip t
Standard
Ac ce p
te
d
M
an
us
90.0
Page 23 of 39
Table 4 1
2
3
4
5
6
7
8
9
1
0.09
0.05
0.05
0.06
0.02
0.04
0.07
0.02
0.02
2
0.37
0.46
0.25
0.08
0.05
0.13
0.13
0.55
0.21
3
0.08
0.03
0.09
0.26
0.03
0.03
0.18
0.09
0.15
4
0.01
0.02
0.01
0.12
0.04
0.07
0.02
0.02
0.11
5
0.04
0.04
0.02
0.07
0.02
0.01
0.03
cr
ip t
Day/Analysis
0.03
Ac ce p
te
d
M
an
us
0.07
Page 24 of 39
n
Sum Average Variance
1
9
0.44
0.05
0.0006
2
9
2.24
0.25
0.0306
3
9
0.94
0.10
0.0060
4
9
0.42
0.05
0.0018
5
9
0.34
0.04
0.0005
Source
SQa
DFb
MQc
Between Groups
0.28
4
0.07
Within Groups
0.32
40
Total
0.6
44
us
cr
Day
ip t
Table 5
F critical
8.98
2.60
an
F
M
0.008
SQ = sum of squares. bDF = degrees of freedom, cMQ = mean square.
Ac ce p
te
d
a
Page 25 of 39
Figures and Tables Captions
ip t
Figure 1. Main adulterants seized in Brazilian illicit cocaine market.
Figure 2. Systems of TLC elution evaluated for identification of cocaine and its
cr
adulterants: phenacetin, lidocaine, benzocaine and caffeine.
us
Figure 3. Illustration of the experimental design. Each TLC spot was transferred to a vial with methanol and 0.1 v/v% HCOOH. The resultant solution was then
an
transferred to triangular paper and subjected to spray ionization and the spot
M
contents are analyzed by mass spectrometry.
Figure 4. Optimization of TLC system varying (a) application methods
te
concentration (mg mL-1)
d
(micropipette, silica capillary, and microsyringe); (b) volume (L); and (c) analyte
Ac ce p
Figure 5. TLC system using as mobile phases: (a) CHCl3:CH3OH:CH3COOHglacial (75:20:5 v %); (b) CH3CH2OCH2CH3:CHCl3 (50:50 %); and (c) CH3OH:NH4OH (100:1.5 v %).
Figure 6. TLC with CH3OH:NH4OH system at (a) 99.5:0.5 v %; (b) 99:1.0 v %; (c) 97.5:2.5 v %; (d) 97:3 v %; (e) 96.5:3.5 v %.
Figure 7. TLC with CH3OH:NH4OH system at 99.5:0.5 v % for compounds varying the concentration from 0.5 to 14 mg mL-1.
Figure 8. PS(+)-MS spectra of the benzocaine, caffeine, lidocaine, phenacetin and cocaine standards according to concentration: 0.5; 4.0; 10.0 and 14.0 mg mL-1.
Page 26 of 39
Figure 9. PS(+)-MS/MS for (a) cocaine, (b) benzocaine, (c) caffeine, (d) lidocaine,
ip t
(e) phenacetin and (f) benzoylecgonine.
Figure 10. Calibration curves for benzocaine, caffeine, phenacetin, lidocaine and
us
cr
cocaine obtained from the PS(+) mass spectra.
Table 1. Rf values for phenacetin, caffeine, cocaine, benzocaine and lidocaine
M
an
standards.
Table 2. Rf values of standards for different ratios (in volume %) of the
te
d
CH3OH:NH4OH mobile phase.
MS.
Ac ce p
Table 3 – LOD observed for each standard studied with TLC and TLC plus PS(+)-
Table
4.
Values
of
signals
ratio
of
m/z
304/290
corresponding
to
cocaine/benzoylecgonine obtained by PS(+)MS analysis from nine replicates of five different days Table 5.
Analysis of Variance (ANOVA) of individual results obtained on five
different days by PS(+)MS from cocaine/benzoylecgonine ratio.
Page 27 of 39
5. Acknowledgments The
authors
would
like
to
thank
FAPES
(65921380/2013),
CAPES
Ac ce p
te
d
M
an
us
cr
ip t
(23038.007083/2014-40) and CNPq (445987/2014-6) for their financial support.
Page 28 of 39
Page 29 of 39
d
te
Ac ce p us
an
M
cr
ip t
Highlights
ip t
TLC system was optimized for analysis of cocaine and its adulterants; The sensitivity (LOD from 0.5 to 14 mg mL-1) and the selectivity were evaluated;
cr
The TLC spots were quantified by the PS-MS (R2 > 0.98 and LOD > 1 g mL-1);
Ac ce p
te
d
M
an
us
The sensitivity and selectivity of TLC was improved from coupling with PS-MS;
Page 30 of 39
Ac ce p
te
d
M
an
us
cr
ip t
Figures
Figure 1
Page 31 of 39
ip t cr us an M d te Ac ce p
Figure 2
Page 32 of 39
ip t cr us
Ac ce p
te
d
M
an
Figure 3
Page 33 of 39
ip t cr us an M d te Ac ce p
Figure 4
Page 34 of 39
ip t cr us
Ac ce p
te
d
M
an
Figure 5
Page 35 of 39
Ac ce p
te
d
M
an
us
cr
ip t
Figure 6
Figure 7
Page 36 of 39
b) Benzocaine
a) Cocaine [C16H19NO4 + H]+ [C17H21NO4 + H]+
290
%
%
50
0 100
14,0
290
50 270
280
290
300 m/z
310
320
330
340
350
[C8H10N4O2 + H]+
100 50
110
120
130
140
0,5
50 0 100
4,0
195
%
10,0
195
50
170
180
[C14H22N2O + H+]
0 100
190
200
0,5
4,0
an
0 100
160
235
50
50
150 m/z
235
100
195
0 100
235
10,0
235
14,0
50
0 100
195
14,0
0 100 50
M
50
0
150
160
170
180
190
200 m/z
210
220
230
240
250
200
210
220
230
240
250 m/z
260
270
280
290
300
e) Phenacetin
+ [C10H13NO2 + H]+ [C10H13NO2 + Na]
50
180
te
0 100
0,5
d
100
202
4,0
180
%
50
0 100
Ac ce p
%
100
d) Lidocaine
c) Caffeine
0
0
14,0
us
260
10,0
166
50
304 250
166
50
304
0
166
0 100
10,0
290
0 100
4,0
50
304
0 100
166
0 100
4,0
50
0,5
50
304
290
0 100
0,5
ip t
50
[C9H11NO2 + H]+
100
cr
100
202 180
10,0
202
50
0 100
180
14,0
50
0 150
160
170
180
190
202 200 210 m/z
220
230
240
250
Figure 8
Page 37 of 39
304 100
182
100
- C2H4
-H2O
b)Benzocaine
a) Cocaine
166 138 He
%
%
He
ip t
- C7H7O2
166
304
120
0 60
80
100
m/z
d) Lindocaine
-H2O 195 133 149150163 177
110
90
130 150 m/z
170
190
80
138
235
168
100
-C7H5O2 290
d
-C2H4
180
-C2H2O
He
100 120 140 160 180 200 220 m/z
f) Benzoylecgnonine
e) Phenacetine 100
%
Ac ce p
-C2H4
60
80
100
120 m/z
152
140
He
%
te
He
110
0
0 60
M
70
235
-C9H10NO
an
-HCN -CHO -CO
0 50
86
100 He
%
%
-CHO
110
160
us
-C2H3NO
-CO 138
100
140
m/z
195
c)Caffeine
120
cr
080 100 120 140 160 180 200 220 240 260 280 300
-H2O 290
-H2O 180
162 160
272 180
0
80
120
160 200 m/z
240
280
Figure 9
Page 38 of 39
b) Benzocaine 25
5 10 Concentration (mg/mL)
15 10 y = 1.4738x - 0.7985 R² = 0.9984
5 0 0
15
c) Caffeine
5 10 Concentration (mg/mL)
15
d) Lidocaine
10 y = 1.5013x + 1.207 R² = 0.9999
5
us
15
5 4 3 2
an
20
Intensity (106)
25 Intensity (104)
ip t
0
20
cr
y = 0.8849x - 0.158 R² = 0.9993
Intensity (105)
Intensity (105)
a) Cocaine 14 12 10 8 6 4 2 0
1
y = 0.2411x + 0.6911 R² = 0.9998
0
0 5 10 Concentration (mg/mL)
15
0
5 10 Concentration (mg/mL)
15
M
0
d
te
Intensity (105)
e) Phenacetin 6 5 4 3 2 1 0
Ac ce p
0
y = 0.2912x + 0.7203 R² = 0.9989
5 10 Concentration (mg/mL)
15
Figure 10
Page 39 of 39
S0379-0738(16)30061-5 http://dx.doi.org/doi:10.1016/j.forsciint.2016.02.039 FSI 8346
To appear in:
FSI
Received date: Revised date: Accepted date:
28-7-2015 13-2-2016 22-2-2016
Please cite this article as: T.C. De Carvalho, F. Tosato, L.M. Souza, H. Santos, B.B. Merlo, R.S. Ortiz, R.R.T. Rodrigues, P.R. Filgueiras, H.S. Franc¸a, R. Augusti, W. Rom˜ao, B.G. Vaz, Thin Layer Chromatography coupled to Paper Spray Ionization Mass Spectrometry for Cocaine and its Adulterants Analysis, Forensic Science International (2016), http://dx.doi.org/10.1016/j.forsciint.2016.02.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Thin Layer Chromatography coupled to Paper Spray Ionization
ip t
Mass Spectrometry for Cocaine and its Adulterants Analysis
Thays C. De Carvalho1, Flavia Tosato,2 Lindamara M. Souza,2 Heloa Santos,2
cr
Bianca B. Merlo,3 Rafael S. Ortiz,4 Rayza R. T. Rodrigues,2 Paulo R. Filgueiras,2
Instituto de Química, Universidade Federal de Goiás, 74001-970 Goiânia, GO,
an
1
us
Hildegardo S. França,5 Rodinei Augusti,6 Wanderson Romão,2,5† Boniek G. Vaz1,2±
Brazil
Laboratório de Petroleômica e Química Forense, Departamento de Química,
M
2
Universidade Federal do Espírito Santo, 29075-910, Vitória, ES, Brazil Laboratório de Química Legal, Superintendência de Polícia Técnico-Científica da
d
3
Superintendência de Polícia Federal no Estado do Rio Grande do Sul, 90160-
Ac ce p
4
te
Polícia Civil do Estado do Espírito Santo, 29045-300, Vitória, ES, Brazil
093, Porto Alegre, RS, Brazil 5
Instituto Federal do Espírito Santo, 29106-010, Vila Velha, ES, Brazil
6
Departamento de Química, Universidade Federal de Minas Gerais, 31270-901,
Belo Horizonte – MG, Brazil
Corresponding Author: †
W.R: [email protected] / Phone: +55-27-3149-0833
±
B.G.V: [email protected]/Phone: +55 62 3521-1016 R 261
Page 1 of 39
Abstract
Thin Layer Chromatography (TLC) is a simple and inexpensive type of
ip t
chromatography that is extensively used in forensic laboratories for drugs of abuse analysis. In this work, TLC is optimized to analyze cocaine and its adulterants
cr
(caffeine, benzocaine, lidocaine and phenacetin) in which the sensitivity (visual
us
determination of LOD from 0.5 to 14 mg mL-1) and the selectivity (from the study of three different eluents: CHCl3:CH3OH:HCOOHglacial (75:20:5 v %), (C2H5)2O:CHCl3
an
(50:50 v %) and CH3OH:NH4OH (100:1.5 v %)) were evaluated. Aiming to improve these figures of merit, the TLC spots were identified and quantified (linearity with
M
R2 > 0.98) by the paper spray ionization mass spectrometry (PS-MS), reaching
d
now lower LOD values (> 1.0 g mL-1). The method developed in this work open
te
up perspective of enhancing the reliability of traditional and routine TLC analysis employed in the criminal expertise units. Higher sensitivity, selectivity and rapidity
Ac ce p
can be provided in forensic reports, besides the possibility of quantitative analysis. Due to the great simplicity, the PS(+)-MS technique can also be coupled directly to other separation techniques such as the paper chromatography and can still be used in analyses of LSD blotter, documents and synthetic drugs.
Keywords: cocaine, TLC, Paper spray ionization, PS-MS.
Page 2 of 39
Introduction
ip t
Cocaine is mainly consumed as a salt (cocaine hydrochloride) or in a freebase form (crack). Cocaine hydrochloride is a water-soluble salt obtained as a
cr
powder, and it can be administered via aspiration or intravenously. Crack, however, appears as a rock and is slightly soluble, but it is easily volatilized when heated
us
because of its low melting point (approximately 95 ºC) and can be administered via
(IUPAC),
cocaine
is
an
smoke.i According to the International Union of Pure and Applied Chemistry a
[1R-(exo,exo)-3-(benzoyloxy)–8–methyl–8–
M
azabicyclo[3.2.1]octane-2-carboxylic acid methyl esther. Its molecular formula is
96–98 ºC, and its pKb = 5.4.ii,iii
d
C17H21NO4, and its molar mass is 303.4 g mol-1. Cocaine’s melting point range is
te
In the last few years, the purity of “commercial” cocaine has been
Ac ce p
decreasing. To increase drug volume and therefore drug trafficking profits, chemical additives are blended to the alkaloid as adulterants and/or diluents. Among the chemical additives, psychoactive substances, commonly anesthetics, are used to mimic or increase the drug effect for users.iv,v Figure 1a-d shows the chemical structures of some adulterants found in cocaine samples seized in the Brazilian illicit market. They are: benzocaine, phenacetin, caffeine and lidocaine.iii
Figure 1
Expert methodologies for cocaine analysis are mostly qualitative. At least two
Page 3 of 39
examinations are performed: a preliminary test using wet chemistry and an analytical test with higher selectivity. Colorimetric tests or pre-tests have the advantages of rapidity, low cost and ease of execution and interpretation.
ip t
However, they feature low specificity. Among the colorimetric tests used to determine the presence of alkaloids, is usually employed the test using a solution
cr
of cobalt thiocyanate.v,vi Nevertheless, positive results can be found when some
us
adulterants or diluents are present, such as lidocaine, powdered milk and promethazine.v
an
Other analytical methodologies are routinely applied for cocaine analysis, including thin layer chromatography (TLC),vii-x gas chromatography coupled with
M
mass spectrometry (GC-MS)xi and gas chromatography coupled with flame ionization detector (GC-FID).xii,xiii Among them, TLC is a more simple and cheap
te
d
separation technique, being extensively used in the most Brazilian forensic laboratories for drugs of abuse analysis (cocaine, crack, marijuana, and ecstasy
Ac ce p
tablets).v,xiv TLC is based on the differences of affinities of analyte between the stationary and mobile phases.xiv TLC has also been explored for cocaine detection in urine and other biological matrixes.xv The compounds separated on the TLC plate form spots which are usually
detected with UV light, iodine vapor, or other visualization reagents. However, some spots may be missed in visualization. In addition, the visualization methods are not capable of identifying the nature of the separated compounds.
xiv
To
overcome this limitation, mass spectrometric methods have been developed for identifying the compounds separated on the TLC plates.xvi
Page 4 of 39
Recently, the use of new techniques of ambient mass spectrometry, that allow desorption, ionization and characterization of analytes via mass spectrometry directly from their natural matrixes, becomes an attractive alternative in forensic
ip t
problems. These ionization/desorption techniques require no sample preparation or pre-separation.i,vii,xvii,xviii
cr
Paper spray ionisation (PS), introduced in 2010 by Wang et al,xix is a new
us
ambient mass spectrometry technique for qualitative and quantitative analysis of complex mixtures. PS-MS involves directly loading the sample
an
onto a triangular-shaped paper, which is wetted with a solvent and placed in front of the mass spectrometer inlet. The spray of the charged micro-droplets
M
is formed by application (usually 3-5 kV) in the opposite side of the paper tip and the desolvation occurs without any sheath gas.xx The PS-MS
process.
te
d
mechanism of ions formations in the gaseous phase is similar to ESI
Ac ce p
The PS-MS has been explored in various applications, mainly to analyze directly complex samples such as illicit substances in raw urine,xx pharmaceuticals in whole blood,xxi biological tissue,xxii contaminants in foodstuffs,xxiii and other applications.xxiv Herein, the TLC system is optimized and has its figures of merit improved (detection limit, sensitivity and selectivity) from coupling to PSI-MS method. This analytical methodology has been employed to detection and quantification of cocaine and its adulterants (benzocaine, phenacetin, caffeine and lidocaine).
Experimental Section
Page 5 of 39
Reagents and Solutions A reference standard sample of cocaine provided by PC-ES (Civil Police of Espirito Santo State, Brazil) and standards of phenacetin, benzocaine (both
ip t
provided by Sigma-Aldrich, Sao Paulo), lidocaine and anhydrous caffeine (Vetec Quimica Fina Ltda, Rio de Janeiro and Bandeirante Brazmo Industria e Comercio
cr
Ltda, Sao Paulo, respectively) were used to prepare stock solutions of 12, 14 and
us
20 mg mL-1.
The reagents used in the TLC and PS-MS analysis were methanol HPLC
an
grade (J.T.Baker), formic acid 99% RPE ACS PA (Carlo Erba reagentes), ammonium hydroxide (Sigma Aldrich), Milli-Q water, chloroform, glacial acetic acid
d
M
and ethyl ether (Vetec Química Fina Ltda, Rio de Janeiro).
te
TLC
Initially, an optimization of the TLC system was performed by varying the
Ac ce p
application volume (5, 10, 15, 20 and 25 L), the form of application (micropipette, silica capillary and microsyringe) and the analyte concentration (4, 6, 8, 10, 14 and 16 mg mL-1). In this step, cocaine was used as analyte, silica gel as stationary phase with support of aluminum and the methanol:NH4OH mixture in the proportion 99.5:0.5 v % as eluent. The TLC system was eluted and revealed in the ultraviolet (UV) chamber at 254 nm. After the optimization of application volume (10 L), concentration (10 mg·mL-1) and form of application, three different eluents were evaluated to analyze cocaine and its adulterants (lidocaine, caffeine, benzocaine and phenacetin) by
Page 6 of 39
TLC. The eluents were: i) CHCl3:CH3OH:HCOOHglacial 75:20:5 v %; ii) (C2H5)2O:CHCl3 50:50 v %; and iii) CH3OH:NH4OH 100:1.5 v %. These three systems are commonly recommended by the UNODC (United Nations Office on
ip t
Drugs and Crime) manual.xxv The histogram shown in Figure 2 illustrates the experimental procedure. The last system composed by CH3OH:NH4OH, were
cr
tested in various proportions: 99.5:0.5; 99:1; 97.5:2,5; 97:3.0 and 96.5:3.5 v %
us
aiming to improve the chromatographic resolution by varying the pH of the medium, Figure 2. After the optimization of the mobile phase, the limit-of-detection
an
(LOD) was visually determined for all active ingredients, in the concentration range 0.5-14 mg·mL-1. The solutions between 0.5 and 10 mg mL-1 were prepared from
M
the 20 mg mL-1 stock solution.
All TLC plates were revealed in the UV chamber at 254 nm. The retention
te
d
factors (Rf) were calculated as the ratio of the distance eluted by sample (da) and
Ac ce p
the distance eluted by solvent (ds) according to Equation 1: R f da ds
(Eq. 1)
Figure 2
PS-MS
PS-MS analyses were performed in the mass spectrometer LTQ XL (Thermo Scientific, Bremem, Germany) with PS source coupled in the positive ionization mode, PS(+).
Page 7 of 39
In general, a piece of paper (Whatman Grade 1, GE Healthcare, USA) was cut in a triangular geometry (base and high of 1 cm) and fixed to an alligator clip, connected to 0.5 mm wire linked to the mass spectrometer.xxvi,xxvii Afterwards, each
ip t
TLC spot containing the analyte was resuspended in methanol acidified with HCOOH (0.1 v/v %) and 10 µL of the resultant solution was applied to the PS(+)-
cr
MS paper. The PS source prototype assembly was performed according to the
us
description of Yang et al.xix By using the mass spectrometer LTQ XL there is no need of an external source of high voltage, as the PS source is fed by a potential
an
generated from the ESI interface catheter, Figure 3.
M
Figure 3
Figures of merit
te
d
The relationship between the signal intensity (y) and the concentration of
Ac ce p
cocaine or adulterant (x) is modeled by a first order equation: y b0 b1 x (Eq. 2)
where b0 and b1 are intercept and slope, respectively. From the calibration curve the LOD for each substance was determined using the equation 3:
LOD k s b1
(Eq. 3)
where factor k is chosen number according to the desirable degree of confidence (usually k = 3 is accepted), b1 is determined from calibration curve and s is the standard deviation of ten white analyzes. An important parameter to be evaluated when ambient ionization mass spectrometry techniques are applied to forensic analysis is related to its precision
Page 8 of 39
(repeatability and reproducibility). Repeatability measures the error of an individual determination and is important criteria for judging the performance of an analytical
equation 4:xxviii (Eq. 4)
cr
ˆ repeatability R d 2
ip t
procedure. The standard deviation of repeatability ( ˆ repeatability ) was estimated by
where R is the average of the ratios of cocaine/benzoylecgonine compounds (m/z
us
304/290) at 10 mg mL-1 of a cocaine solution. For this study, nine analyzes (or
an
replicates) were performed by PS(+)MS using ever the same solution during five consecutive days. The d 2 is a factor that depends on the size of the sample.xxix In
M
this study was used d 2 = 2.970, that is related to number of replicates (n = 9). Then
te
repeatability of analyzes.
d
an ANOVA was performed to evaluate the influence of the "day" factor on the
TLC
Ac ce p
Results and discussions
To evaluate the best methodology to apply the analyte in the TLC system,
the spot form and its Rf values were evaluated according to the type of applicator (Figure 4a, micropipette, silica capillary, and microsyringe); variation of application volume (L) (Figure 4b); and analyte concentration (mg mL-1, Figure 4c). Monitoring initially the system of application, well defined, homogeneous and uniform spots were observed in all cases, Figure 4a. Thus, any of the three systems can be employed for TLC analysis in forensic routines. When evaluating the application volume, the spot area becomes biggest with cocaine volumes
Page 9 of 39
higher or equal to 15 L. This behavior can be harmful to seized street sample analysis, interfering in the chromatographic resolution due to coelution problems. Therefore, a volume equal to 10 L was defined for all analytes. Despite the TLC
cr
mL-1), the adopted concentration in this work was 10 mg mL-1.
ip t
technique being sensible for all concentrations evaluated (between 4 and 16 mg
us
Figure 4
then
evaluated
according
to
an
The TLC efficiency for detecting cocaine and adulterants standards was three
different
eluent
systems
M
(CHCl3:CH3OH:CH3COOHglacial, 75:20:5 v %, CH3CH2OCH2CH3:CHCl3, 50:50 v %); and CH3OH:NH4OH 100:1.5 v %), Figure 5a-c. All standards were prepared at 10
d
mg mL-1 in methanol, and were applied in the TLC system via silica capillary. In the
te
first eluent (CHCl3:CH3OH:CH3COOHglacial, 75:20:5 v %, Figure 5a) spots tailing
Ac ce p
were observed for all standards, and they showed similar Rf values (Rf = 0.710.76, Table 1). This result differs from the reported by Sabino et al.vii For the (CH3CH2)2O:CHCl3, 50:50 v % (Figure 5b) system, a better performance of the TLC system is observed when compared to Figure 5a, in which well defined and resolved spots can be identified. In this case, however, the majority of compounds showed similar Rf values (Rf from 0.71 to 0.76, Table 1). The cocaine standard that shows Rf = 0.63 is a unique exception, Figure 5b. Finally, the best results were obtained for the TLC system when the eluent CH3OH:NH4OH (100:1.5 v %) was employed, in which distinct Rf values were observed for the compounds (Rf
Page 10 of 39
varying from 0.69 to 0.81), Figure 5c and Table 1. A similar TLC system was also reported by Nicola et al.xxx employing CH3Cl/CH3OH/NH4OH (100:20:1 v %).
ip t
Figure 5 Table 1
cr
Aiming to increase the interval of values of Rf observed among standards,
us
the ratio of CH3OH:NH4OH eluent was varied, with NH4OH concentration ranging between 0.5 and 3.5 % producing the systems 99.5:0.5; 99:1; 97.5:2.5; 97:3; and
an
96.5:3.5 v %, respectively. The TLC system and standards Rf values are showed on Figure 6 and Table 2. Note that the best TLC system was obtained when low
M
concentrations of NH4OH were used (< 1 v %). At this situation, the interaction between cocaine and the stationary phase increases. This is related to the fact that
d
NH4OH alters the acid/base equilibrium of the cocaine molecule, which the cocaine
te
in the chlorhydrate form is predominant when NH4OH concentration is reduced in
Ac ce p
the system. Similarly, caffeine, which has high pka and polarity, also interact mainly with the stationary phase. Conversely, lidocaine, benzocaine and phenacetin have lower pka values (pKA ≤ 8) and higher Rf values, preferably thus interacting with the mobile phase. Therefore, the CH3OH:NH4OH system in the ratio 99.5:0.5 v % was defined for this work.
Figure 6 Table 2
Aiming to evaluate the LOD of the TLC system, the analyte concentration was varied in the range of 0.5 to 14 mg mL-1 using CH3OH:NH4OH at 99.5:0.5 v %
Page 11 of 39
as mobile phase. Note that phenacetin, benzocaine, cocaine and caffeine showing low LOD (0.5 mg mL-1), while lidocaine had LOD of 4.0 mg mL-1. Figure 7
ip t
Table 3
cr
TLC plus PS-MS
Aiming to increase the selectivity of the TLC technique, the standards spots
us
were also analyzed by the PS(+)MS technique. For it, the revealed spots were
an
extracted and resuspended in acidified methanol (0.1 v % of HCOOH). Then the supernatant was deposited on the surface of the triangular shaped paper with
M
paraffin channel. After the drying, the analytes were analyzed by the PS(+)-MS technique according to its concentration (0.5, 4, 10 and 14 mg mL-1), Figure 8.
d
Figure 8 illustrates the PS(+) mass spectra of the extracts containing the
te
standards of cocaine, benzocaine, caffeine, lidocaine and phenacetin. In general
Ac ce p
benzocaine, caffeine and lidocaine are detected as protonated molecules, [M+H]+ cations with m/z 166, 195 and 235, respectively, while phenacetin is detected as [M+H]+ cation with m/z 180 and sodium adduct, [M+Na]+, with m/z 202. For the cocaine standard, [M+H]+ cation with m/z 304 is identified, besides a cocaine derivative, benzoylecgonine, [M +H]+ ion with m/z 290, which is a degradation product. The structure and chemical connectivity of the analytes were confirmed by experiments of collision induced dissociation (CID) and the PS(+)MS/MS spectra are illustrated on Figure 9.
Figure 8
Page 12 of 39
The cocaine (m/z 304), Figure 9a, is characterized for the loss of C7H6O2 leading to the formation of the ion with m/z 182. Benzocaine (m/z 166), Figure 9b,
ip t
shows two neutral losses. The first, the elimination of C2H2 leads the formation of the ion with m/z 138, and second, a loss of H2O generates the ion with m/z 120.
cr
The PS(+)MS/MS spectrum of caffeine, Figure 9c, is characterized by the loss of
us
H2O forming the ion with m/z 177, which subsequently loses a CO creating the ion with m/z 149. The loss of HCN from the ion with m/z 177 leads the formation of the
an
ion with m/z 150. It can also be observed a minor consecutive loss of CHO forming the ion with m/z 163 followed by the ion with m/z 133. Lidocaine, Figure 9d, is
M
characterized by the fragmentation of C9H10NO forming the ion with m/z 86. Phenacetin, Figure 9e, is characterized by the loss of H2O forming the ion with m/z
te
d
162, and by the loss of C2H4 forming the ion with m/z 152. The main fragmentation route of phenacetin is the loss of C2H2O forming the ion with m/z 138, which loses
Ac ce p
an ethylene forming the ion with m/z 110. Finally, the benzoylecgnonine, hydrolysis product of cocaine, Figure 9f, is characterized by the loss of H2O forming the ion with m/z 272 and by a loss similar to cocaine, C7H6O2, that generated the ion with m/z 168.
Figure 9
The PS(+)-MS technique ability to quantify cocaine and its adulterants was evaluated by the construction of curves as function of analyte concentration, in which the linearity and the LOD were reported, Figure 10 and Table 3. The calibration curves built for the standards shows excellent linearity (benzocaine, (R2
Page 13 of 39
= 0,9984), caffeine (R2 = 0,9999), phenacetin (R2 = 0,9989), lidocaine (R2 = 0,9998) and cocaine (R2 = 0,9993) and LOD lower than that of the TLC technique. The PS(+)-MS technique can also be used to identify and quantify the active
ip t
ingredients present in the TLC spots from cocaine and crack samples seized by the forensic police, thus eliminating false-positive results. Besides that, due to the
cr
great simplicity, the PS(+)-MS technique can be coupled directly to other
us
separation techniques such as paper chromatography and for the analysis of LSD
an
blotters and synthetic drugs.
M
Figure 10
Repeatability
te
d
To monitor the precision of the method, we consider the ratio of relative intensity of cocaine/benzoylecgonine (m/z 304/290) detected in the nine analyzes. Table 3
Ac ce p
shows these values, where the estimated value of the repeatability standard deviation was 0.065. Table 4 describes the ANOVA tests performed in Excel. Note that if the variance of the results in the five days is tiny, an insignificant change in results among analysis is taken on the same day. Therefore, the PS(+)-MS technique shows a good repeatability. ANOVA measures the total variation of a given system and compares it with the total variation to determine whether such measurement is feasible. Table 5 illustrates the variability of this method is mostly due to the difference among the samples, as F calculated (8.98) being higher than the critical F (2.60). Table 4
Page 14 of 39
Table 5
Conclusion and Perspectives
ip t
This paper allows the detection of cocaine and its adulterants by TLC with LOD varying from 0.5 to 2 mg mL-1. The identification and rapid quantification of
cr
components revealed in each PS-MS spot is also reported. The method developed
us
in this work opens up perspectives of enhancing the reliability of traditional and routine TLC analyses employed in the criminal expertise units, thus eliminating
an
false positive results. Higher sensitivity, selectivity and rapidity can be provided in the forensic report, besides enabling quantitative analysis. Due to its great
M
simplicity, the PSI(+)-MS technique can also be coupled directly to other separation
i
Ac ce p
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te
blotter and synthetic drugs.
d
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ip t
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Ac ce p
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cr
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Page 20 of 39
Tables Table 1
Benzocaine Lidocaine
0.76 0.76
0.76 0.71
CH3OH:NH4OH (100:1.5 v %) 0.79 0.69 0.74
ip t
(CH3CH2)2O (50:50 v %) 0.76 0.74 0.63
cr
Phenacetin Caffeine Cocaine
CHCl3:CH3OH:CH3COOH (75:20:5 v %) 0.74 0.74 0.71
0.78 0.81
Ac ce p
te
d
M
an
us
Standards
Page 21 of 39
Table 2 99.5:0.5
99:1
97.5:2.5
97:3
96.5:3.5
Phenacetin Caffeine Cocaine
0.84 0.66 0.63
0.82 0.67 0,67
0.63 0.60 0.62
0.70 0.67 0.71
0.73 0.72 0.75
Benzocaine Lidocaine
0.86 0.81
0.82 0.78
0.68 0.67
0.72 0.71
0.76 0.76
Ac ce p
te
d
M
an
us
cr
ip t
Compound
Page 22 of 39
Table 3 TLC (mg mL-1)
PSI(+)-MS (g mL-1)
Cocaine
0.5 mg mL-1
6.0
Caffeine
0.5 mg mL-1
4.0
Phenacetin
0.5 mg mL-1
80.0
Benzocaine
0.5 mg mL-1
1.0
Lidocaine
4.0 mg mL-1
cr
ip t
Standard
Ac ce p
te
d
M
an
us
90.0
Page 23 of 39
Table 4 1
2
3
4
5
6
7
8
9
1
0.09
0.05
0.05
0.06
0.02
0.04
0.07
0.02
0.02
2
0.37
0.46
0.25
0.08
0.05
0.13
0.13
0.55
0.21
3
0.08
0.03
0.09
0.26
0.03
0.03
0.18
0.09
0.15
4
0.01
0.02
0.01
0.12
0.04
0.07
0.02
0.02
0.11
5
0.04
0.04
0.02
0.07
0.02
0.01
0.03
cr
ip t
Day/Analysis
0.03
Ac ce p
te
d
M
an
us
0.07
Page 24 of 39
n
Sum Average Variance
1
9
0.44
0.05
0.0006
2
9
2.24
0.25
0.0306
3
9
0.94
0.10
0.0060
4
9
0.42
0.05
0.0018
5
9
0.34
0.04
0.0005
Source
SQa
DFb
MQc
Between Groups
0.28
4
0.07
Within Groups
0.32
40
Total
0.6
44
us
cr
Day
ip t
Table 5
F critical
8.98
2.60
an
F
M
0.008
SQ = sum of squares. bDF = degrees of freedom, cMQ = mean square.
Ac ce p
te
d
a
Page 25 of 39
Figures and Tables Captions
ip t
Figure 1. Main adulterants seized in Brazilian illicit cocaine market.
Figure 2. Systems of TLC elution evaluated for identification of cocaine and its
cr
adulterants: phenacetin, lidocaine, benzocaine and caffeine.
us
Figure 3. Illustration of the experimental design. Each TLC spot was transferred to a vial with methanol and 0.1 v/v% HCOOH. The resultant solution was then
an
transferred to triangular paper and subjected to spray ionization and the spot
M
contents are analyzed by mass spectrometry.
Figure 4. Optimization of TLC system varying (a) application methods
te
concentration (mg mL-1)
d
(micropipette, silica capillary, and microsyringe); (b) volume (L); and (c) analyte
Ac ce p
Figure 5. TLC system using as mobile phases: (a) CHCl3:CH3OH:CH3COOHglacial (75:20:5 v %); (b) CH3CH2OCH2CH3:CHCl3 (50:50 %); and (c) CH3OH:NH4OH (100:1.5 v %).
Figure 6. TLC with CH3OH:NH4OH system at (a) 99.5:0.5 v %; (b) 99:1.0 v %; (c) 97.5:2.5 v %; (d) 97:3 v %; (e) 96.5:3.5 v %.
Figure 7. TLC with CH3OH:NH4OH system at 99.5:0.5 v % for compounds varying the concentration from 0.5 to 14 mg mL-1.
Figure 8. PS(+)-MS spectra of the benzocaine, caffeine, lidocaine, phenacetin and cocaine standards according to concentration: 0.5; 4.0; 10.0 and 14.0 mg mL-1.
Page 26 of 39
Figure 9. PS(+)-MS/MS for (a) cocaine, (b) benzocaine, (c) caffeine, (d) lidocaine,
ip t
(e) phenacetin and (f) benzoylecgonine.
Figure 10. Calibration curves for benzocaine, caffeine, phenacetin, lidocaine and
us
cr
cocaine obtained from the PS(+) mass spectra.
Table 1. Rf values for phenacetin, caffeine, cocaine, benzocaine and lidocaine
M
an
standards.
Table 2. Rf values of standards for different ratios (in volume %) of the
te
d
CH3OH:NH4OH mobile phase.
MS.
Ac ce p
Table 3 – LOD observed for each standard studied with TLC and TLC plus PS(+)-
Table
4.
Values
of
signals
ratio
of
m/z
304/290
corresponding
to
cocaine/benzoylecgonine obtained by PS(+)MS analysis from nine replicates of five different days Table 5.
Analysis of Variance (ANOVA) of individual results obtained on five
different days by PS(+)MS from cocaine/benzoylecgonine ratio.
Page 27 of 39
5. Acknowledgments The
authors
would
like
to
thank
FAPES
(65921380/2013),
CAPES
Ac ce p
te
d
M
an
us
cr
ip t
(23038.007083/2014-40) and CNPq (445987/2014-6) for their financial support.
Page 28 of 39
Page 29 of 39
d
te
Ac ce p us
an
M
cr
ip t
Highlights
ip t
TLC system was optimized for analysis of cocaine and its adulterants; The sensitivity (LOD from 0.5 to 14 mg mL-1) and the selectivity were evaluated;
cr
The TLC spots were quantified by the PS-MS (R2 > 0.98 and LOD > 1 g mL-1);
Ac ce p
te
d
M
an
us
The sensitivity and selectivity of TLC was improved from coupling with PS-MS;
Page 30 of 39
Ac ce p
te
d
M
an
us
cr
ip t
Figures
Figure 1
Page 31 of 39
ip t cr us an M d te Ac ce p
Figure 2
Page 32 of 39
ip t cr us
Ac ce p
te
d
M
an
Figure 3
Page 33 of 39
ip t cr us an M d te Ac ce p
Figure 4
Page 34 of 39
ip t cr us
Ac ce p
te
d
M
an
Figure 5
Page 35 of 39
Ac ce p
te
d
M
an
us
cr
ip t
Figure 6
Figure 7
Page 36 of 39
b) Benzocaine
a) Cocaine [C16H19NO4 + H]+ [C17H21NO4 + H]+
290
%
%
50
0 100
14,0
290
50 270
280
290
300 m/z
310
320
330
340
350
[C8H10N4O2 + H]+
100 50
110
120
130
140
0,5
50 0 100
4,0
195
%
10,0
195
50
170
180
[C14H22N2O + H+]
0 100
190
200
0,5
4,0
an
0 100
160
235
50
50
150 m/z
235
100
195
0 100
235
10,0
235
14,0
50
0 100
195
14,0
0 100 50
M
50
0
150
160
170
180
190
200 m/z
210
220
230
240
250
200
210
220
230
240
250 m/z
260
270
280
290
300
e) Phenacetin
+ [C10H13NO2 + H]+ [C10H13NO2 + Na]
50
180
te
0 100
0,5
d
100
202
4,0
180
%
50
0 100
Ac ce p
%
100
d) Lidocaine
c) Caffeine
0
0
14,0
us
260
10,0
166
50
304 250
166
50
304
0
166
0 100
10,0
290
0 100
4,0
50
304
0 100
166
0 100
4,0
50
0,5
50
304
290
0 100
0,5
ip t
50
[C9H11NO2 + H]+
100
cr
100
202 180
10,0
202
50
0 100
180
14,0
50
0 150
160
170
180
190
202 200 210 m/z
220
230
240
250
Figure 8
Page 37 of 39
304 100
182
100
- C2H4
-H2O
b)Benzocaine
a) Cocaine
166 138 He
%
%
He
ip t
- C7H7O2
166
304
120
0 60
80
100
m/z
d) Lindocaine
-H2O 195 133 149150163 177
110
90
130 150 m/z
170
190
80
138
235
168
100
-C7H5O2 290
d
-C2H4
180
-C2H2O
He
100 120 140 160 180 200 220 m/z
f) Benzoylecgnonine
e) Phenacetine 100
%
Ac ce p
-C2H4
60
80
100
120 m/z
152
140
He
%
te
He
110
0
0 60
M
70
235
-C9H10NO
an
-HCN -CHO -CO
0 50
86
100 He
%
%
-CHO
110
160
us
-C2H3NO
-CO 138
100
140
m/z
195
c)Caffeine
120
cr
080 100 120 140 160 180 200 220 240 260 280 300
-H2O 290
-H2O 180
162 160
272 180
0
80
120
160 200 m/z
240
280
Figure 9
Page 38 of 39
b) Benzocaine 25
5 10 Concentration (mg/mL)
15 10 y = 1.4738x - 0.7985 R² = 0.9984
5 0 0
15
c) Caffeine
5 10 Concentration (mg/mL)
15
d) Lidocaine
10 y = 1.5013x + 1.207 R² = 0.9999
5
us
15
5 4 3 2
an
20
Intensity (106)
25 Intensity (104)
ip t
0
20
cr
y = 0.8849x - 0.158 R² = 0.9993
Intensity (105)
Intensity (105)
a) Cocaine 14 12 10 8 6 4 2 0
1
y = 0.2411x + 0.6911 R² = 0.9998
0
0 5 10 Concentration (mg/mL)
15
0
5 10 Concentration (mg/mL)
15
M
0
d
te
Intensity (105)
e) Phenacetin 6 5 4 3 2 1 0
Ac ce p
0
y = 0.2912x + 0.7203 R² = 0.9989
5 10 Concentration (mg/mL)
15
Figure 10
Page 39 of 39